Subphthalocyanine-flipper dyads for selective membrane staining.

The design, synthesis and evaluation of a subphthalocyanine-flipper (SubPc-Flipper) amphiphilic dyad is reported. This dyad combines two fluorophores that function in the visible region (420-800 nm) for the simultaneous sensing of both ordered and disordered lipidic membranes. The flipper probes part of the dyad possesses mechanosensitivity, long fluorescence lifetimes (τ = 3.5-5 ns) and selective staining of ordered membranes. On the other hand, subphthalocyanines (SubPc) are short-lifetime (τ = 1-2.5 ns) fluorophores that are insensitive to membrane tension. As a result of a Förster Resonance Energy Transfer (FRET) process, the dyad not only retains the mechanosensitivity of flippers but also demonstrates high selectivity and emission in different kinds of lipidic membranes. The dyad exhibits high emission and sensitivity to membrane tension (Δτ = 3.5 ns) when tested in giant unilamellar vesicles (GUVs) with different membrane orders. Overall, the results of this study represent a significant advancement in the applications of flippers and dyads in mechanobiology.

Passive coupling of membrane tension and cell volume during active response of cells to osmosis.

During osmotic changes of their environment, cells actively regulate their volume and plasma membrane tension that can passively change through osmosis. How tension and volume are coupled during osmotic adaptation remains unknown, as their quantitative characterization is lacking. Here, we performed dynamic membrane tension and cell volume measurements during osmotic shocks. During the first few seconds following the shock, cell volume varied to equilibrate osmotic pressures inside and outside the cell, and membrane tension dynamically followed these changes. A theoretical model based on the passive, reversible unfolding of the membrane as it detaches from the actin cortex during volume increase quantitatively describes our data. After the initial response, tension and volume recovered from hypoosmotic shocks but not from hyperosmotic shocks. Using a fluorescent membrane tension probe (fluorescent lipid tension reporter [Flipper-TR]), we investigated the coupling between tension and volume during these asymmetric recoveries. Caveolae depletion and pharmacological inhibition of ion transporters and channels, mTORCs, and the cytoskeleton all affected tension and volume responses. Treatments targeting mTORC2 and specific downstream effectors caused identical changes to both tension and volume responses, their coupling remaining the same. This supports that the coupling of tension and volume responses to osmotic shocks is primarily regulated by mTORC2.

Microwave Detection of Wet Triacetone Triperoxide (TATP): Non-Covalent Forces and Water Dynamics.

The water adducts of triacetone triperoxide (TATP) have been observed by using broadband rotational spectroscopy. This work opens a new way for the gas-phase detection of this improvised explosive. The observed clusters exhibit unusual water dynamics and rarely observed multicenter interactions. TATP-H2 O is formed from the D3 symmetry conformer of TATP with water lying close to the C3 axis. Water rotation around this axis with a very low barrier gives rise to the rotational spectrum of a symmetric top. The main interaction of the monohydrate is a four-center trifurcated donor Ow -H⋅⋅⋅O hydrogen bond, not observed previously in the gas phase, reinforced by a weak four-center trifurcated acceptor C-H⋅⋅⋅Ow interaction. Surprisingly, all structural signatures show the weakness of these interactions. The complex TATP-(H2 O)2 is formed from the monohydrated TATP by the self-association of water.

Influence of core extension and side chain nature in targeting G-quadruplex structures with perylene monoimide derivatives.

A structure-activity relationship (SAR) study in terms of G-quadruplex binding ability and antiproliferative activity of six fluorescent perylenemonoimide (PMIs) derivatives is reported. A positive charge seems to be the key to target G4. This study also reveals the importance of the element substitution in the potential biological activity of PMIs, being the polyethylene glycol (PEG) chains in the peri position responsible for their antiproliferative activity. Among them, the cationic PMI6 with two PEG chains is the most promising compound since its fluorescence is enhanced in the presence of G-quadruplex structures. Moreover, PMI6 binds to the human telomeric G-quadruplex hTelo with high affinity and displays a high antiproliferative potential towards HeLa (cervical adenocarcinoma), A549 (lung adenocarcinoma) and A2780 (ovarian adenocarcinoma) cells. Its fate can be followed inside cells thanks to its fluorescent properties: the compound is found to accumulate in the mitochondria.

Synthesis of Fluorogenic Arylureas and Amides and Their Interaction with Amines: A Competition between Turn-on Fluorescence and Organic Radicals on the Way to a Smart Label for Fish Freshness.

We describe the synthesis of fluorogenic arylureas and amides and their interaction with primary or secondary amines under air and light in organic-aqueous mixtures to give rise to a new class of persistent organic radicals, described on the basis of their electron paramagnetic resonance (EPR), as well as UV-vis, fluorescence, NMR, and quantum mechanics calculations, and their prospective use as multi-signal reporters in a smart label for fish freshness.

Flipper Probes for the Community.

This article describes four fluorescent membrane tension probes that have been designed, synthesized, evaluated, commercialized and applied to current biology challenges in the context of the NCCR Chemical Biology. Their names are Flipper-TR®, ER Flipper-TR®, Lyso Flipper-TR®, and Mito Flipper-TR®. They are available from Spirochrome.

Fluorescent Membrane Tension Probes for Super-Resolution Microscopy: Combining Mechanosensitive Cascade Switching with Dynamic-Covalent Ketone Chemistry.

We report the design, synthesis, and evaluation of fluorescent flipper probes for single-molecule super-resolution imaging of membrane tension in living cells. Reversible switching from bright-state ketones to dark-state hydrates, hemiacetals, and hemithioacetals is demonstrated for twisted and planarized mechanophores in solution and membranes. Broadband femtosecond fluorescence up-conversion spectroscopy evinces ultrafast chalcogen-bonding cascade switching in the excited state in solution. According to fluorescence lifetime imaging microscopy, the new flippers image membrane tension in live cells with record red shifts and photostability. Single-molecule localization microscopy with the new tension probes resolves membranes well below the diffraction limit.

Synthesis and Application of Ruthenium(II) Alkenyl Complexes with Perylene Fluorophores for the Detection of Toxic Vapours and Gases.

A series of new ruthenium(II) vinyl complexes has been prepared incorporating perylenemonoimide (PMI) units. This fluorogenic moiety was functionalised with terminal alkyne or pyridyl groups, allowing attachment to the metal either as a vinyl ligand or through the pyridyl nitrogen. The inherent low solubility of the perylene compounds was improved through the design of poly-PEGylated (PEG=polyethylene glycol) units bearing a terminal alkyne or a pyridyl group. By absorbing the compounds on silica, vapours and gases could be detected in the solid state. The reaction of the complexes [Ru(CH=CH-PerIm )Cl(CO)(py-3PEG)(PPh3 )2 ] and [Ru(CH=CH-3PEG)Cl(CO)(py-PerIm )(PPh3 )2 ] with carbon monoxide, isonitrile or cyanide was found to result in modulation of the fluorescence behaviour. The complexes were observed to display solvatochromic effects and the interaction of the complexes with a wide range of other species was also studied. The study suggests that such complexes have potential for the detection of gases or vapours that are toxic to humans.

Manipulation of Transmembrane Transport by Synthetic K+ Ionophore Depsipeptides and Its Implications in Glucose-Stimulated Insulin Secretion in ß-Cells.

The cyclic depsipeptide cereulide toxin it is a very well-known potassium electrogenic ionophore particularly sensitive to pancreatic beta cells. The mechanistic details of its specific activity are unknown. Here, we describe a series of synthetic substituted cereulide potassium ionophores that cause impressive selective activation of glucose-induced insulin secretion in a constitutive manner in rat insulinoma INS1E cells. Our study demonstrates that the different electroneutral K+ transport mechanism exhibited by the anionic mutant depsipeptides when compared with classical electrogenic cereulides can have an important impact of pharmacological value on glucose-stimulated insulin secretion.

Solvent-Free Off-On Detection of the Improvised Explosive Triacetone Triperoxide (TATP) with Fluorogenic Materials.

A fluorogenic perylenediimide-functionalized polyacrylate capable of generating color and fluorescence changes in the presence of triacetone triperoxide TATP), an improvised explosive used in terrorist attacks, under solvent-free, solid-state conditions has been developed. The material works by accumulating volatile TATP until it reaches a threshold; therefore, triggering colorimetric and fluorescent responses.

Detection of Contaminants of High Environmental Impact by Means of Fluorogenic Probes.

This personal account describes our contribution to the design of selective fluorogenic probes for contaminants of high environmental impact. For this purpose, we have developed a new family of highly versatile fluorogenic reagents that were able to show large differences in their fluorescence in the presence of selected analytes. They were used in the preparation of fluorogenic probes for the detection of contaminants of high environmental impact which currently have no good solutions: phosphorylating agents, such as chemical weapons; methyl mercury(II); the cyanide anion; amino-acid metabolites, such as doping substances; and biogenic amine mimics, such as drugs of abuse and recreational drugs. The development of new materials for specific sensing was achieved by anchoring selected probes to silica nanomaterials, suitable for the selective detection of organic analytes in water for immediate application to toxicological or environmental purposes.

Structural Conformers of (1,3-Dithiol-2-ylidene)ethanethioamides: The Balance between Thioamide Rotation and Preservation of Classical Sulfur-Sulfur Hypervalent Bonds.

The reaction of N-(2-phthalimidoethyl)-N-alkylisopropylamines and S2Cl2 gave 4-N-(2-phthalimidoethyl)-N-alkylamino-5-chloro-1,2-dithiol-3-thiones that quantitatively cycloadded to dimethyl or diethyl acetylenedicarboxylate to give stable thioacid chlorides, which in turn reacted with one equivalent of aniline or a thiole to give thioanilides or a dithioester. Several compounds of this series showed atropisomers that were studied by a combination of dynamic NMR, simulation of the signals, conformational analysis by DFT methods, and single crystal X-ray diffraction, showing a good correlation between the theoretical calculations, the experimental values of energies, and the preferred conformations in the solid state. The steric hindering of the crowded substitution at the central amine group was found to be the reason for the presence of permanent atropisomers in this series of compounds and the cause of a unique disposition of the thioxo group at close-to-right angles with respect to the plane defined by the 1,3-dithiole ring in the dithiafulvene derivatives, thus breaking the sulfur-sulfur hypervalent bond that is always found in this kind of compounds.

Single-Molecule Localization Microscopy and Tracking with a Fluorescent Mechanosensitive Probe.

A milestone in optical imaging of mechanical forces in cells has been the development of the family of flipper fluorescent probes able to report membrane tension noninvasively in living cells through their fluorescence lifetime. The specifically designed Flipper-CF3 probe with an engineered inherent blinking mechanism was recently introduced for super-resolution fluorescence microscopy of lipid ordered membranes but was too dim to be detected in lipid disordered membranes at the single-molecule level (García-Calvo, J. J. Am. Chem. Soc. 2020, 142(28), 12034-12038). We show here that the original and commercially available probe Flipper-TR is compatible with single-molecule based super-resolution imaging and resolves both liquid ordered and liquid disordered membranes of giant unilamellar vesicles below the diffraction limit. Single probe molecules were additionally tracked in lipid bilayers, enabling to distinguish membranes of varying composition from the diffusion coefficient of the probe. Differences in brightness between Flipper-CF3 and Flipper-TR originate in their steady-state absorption and fluorescence properties. The general compatibility of the Flipper-TR scaffold with single-molecule detection is further shown in super-resolution experiments with targetable Flipper-TR derivatives.

Nanocrystalline Cellulose to Reduce Superplasticizer Demand in 3D Printing of Cementitious Materials.

One challenge for 3D printing is that the mortar must flow easily through the printer nozzle, and after printing, it must develop compressive strength fast and high enough to support the layers on it. This requires an exact and difficult control of the superplasticizer (SP) dosing. Nanocrystalline cellulose (CNC) has gained significant interest as a rheological modifier of mortar by interacting with the various cement components. This research studied the potential of nanocrystalline cellulose (CNC) as a mortar aid for 3D printing and its interactions with SPs. Interactions of a CNC and SP with cement suspensions were investigated by means of monitoring the effect on cement dispersion (by monitoring the particle chord length distributions in real time) and their impact on mortar mechanical properties. Although cement dispersion was increased by both CNC and SP, only CNC prevented cement agglomeration when shearing was reduced. Furthermore, combining SP and CNC led to faster development of compressive strength and increased compressive strength up to 30% compared to mortar that had undergone a one-day curing process.

Silica Nanoparticle/Fluorescent Dye Assembly Capable of Ultrasensitively Detecting Airborne Triacetone Triperoxide: Proof-of-Concept Detection of Improvised Explosive Devices in the Workroom.

We describe the proof of concept of a portable testing setup for the detection of triacetone triperoxide (TATP), a common component in improvised explosive devices. The system allows for field testing and generation of real-time results to test for TATP vapor traces in air by simply using circulation of the air samples through the sensing mechanism under the air conditioning system of an ordinary room. In this way, the controlled trapping of the analyte in the chemical sensor gives reliable results at extremely low concentrations of TATP in air under real-life conditions, suitable for daily use in luggage storage for airlines or a locker room for a major sporting event. The reported fluorescent methodology is very sensitive and selective, allowing for the trapping of triacetone triperoxide in the chemical sensor to give reliable results at very low concentrations in air under ambient conditions, by comparing the fluorescence of the material before and after exposition to TATP traces in air.

Development of Lightweight Mortars Using Sustainable Low-Density Glass Aggregates from Secondary Raw Materials.

In this study, different lightweight expanded glass aggregates (LEGAs) were produced from glass cullet and various carbonated wastes, through a thermal impact process. The effects of LEGA microstructure and morphology on both the adherence to the cement paste and the mechanical properties of mortars after 28 days of curing were studied. The properties of lightweight mortars made of either LEGAs or expanded clay aggregates were compared. The results demonstrated the feasibility of using LEGAs to produce glass lightweight aggregate mortar, with flexural and compressive strength values ranging from 5.5 to 8.2 MPa and from 28.1 to 47.6 MPa, respectively. The differences in mechanical properties were explained according to the microstructures of the fracture surfaces. Thus, arlite-type ceramic aggregates presented surface porosities that allowed mortar intrusion and the formation of an interconnected interface; although the surfaces of the vitreous aggregates were free from porosity due to their vitreous nature, the mortars obtained from different wastes presented compressive and flexural strengths in the range of lightweight mortars.

HydroFlipper membrane tension probes: imaging membrane hydration and mechanical compression simultaneously in living cells.

HydroFlippers are introduced as the first fluorescent membrane tension probes that report simultaneously on membrane compression and hydration. The probe design is centered around a sensing cycle that couples the mechanical planarization of twisted push-pull fluorophores with the dynamic covalent hydration of their exocyclic acceptor. In FLIM images of living cells, tension-induced deplanarization is reported as a decrease in fluorescence lifetime of the dehydrated mechanophore. Membrane hydration is reported as the ratio of the photon counts associated to the hydrated and dehydrated mechanophores in reconvoluted lifetime frequency histograms. Trends for tension-induced decompression and hydration of cellular membranes of interest (MOIs) covering plasma membrane, lysosomes, mitochondria, ER, and Golgi are found not to be the same. Tension-induced changes in mechanical compression are rather independent of the nature of the MOI, while the responsiveness to changes in hydration are highly dependent on the intrinsic order of the MOI. These results confirm the mechanical planarization of push-pull probes in the ground state as most robust mechanism to routinely image membrane tension in living cells, while the availability of simultaneous information on membrane hydration will open new perspectives in mechanobiology.

The primary dipole of flipper probes.

Despite their growing popularity in biology to image membrane tension, central design principles of flipper probes have never been validated. Here we report that upon deletion of their primary dipole, from electron-poor and electron-rich dithienothiophenes, absorptions blue-shift, lifetimes shorten dramatically, and mechanosensitivity in cells vanishes not partially, but completely.

Residual Strength and Drying Behavior of Concrete Reinforced with Recycled Steel Fiber from Tires.

Fiber reinforcement of concrete is an effective technique of providing ductility to concrete, increasing its flexural residual strength while reducing its potential for cracking due to drying shrinkage. There are currently a wide variety of industrial fibers on the market. Recycled steel fibers (RSF) from tires could offer a viable substitute of industrialized fibers in a more sustainable and eco-friendly way. However, mistrust exists among users, based on fear that the recycling process will reduce the performance, coupled with the difficulty of characterization of the geometry of the RSF, as a consequence of the size variability introduced by the recycling process. This work compares the behavior of RSF from tires compared with industrialized steel or polypropylene fibers, evaluating the fresh state, compressive strength, flexural residual strength, and drying behavior. The concept of Equivalent Fiber Length (EFL) is also defined to help the statistical geometrical characterization of the RSF. A microstructural analysis was carried out to evaluate the integration of the fiber in the matrix, as well as the possible presence of contaminants. The conclusion is reached that the addition of RSF has a similar effect to that of industrialized fibers on concrete's properties when added at the same percentage.

Self-Assembly Hydrosoluble Coronenes: A Rich Source of Supramolecular Turn-On Fluorogenic Sensing Materials in Aqueous Media.

Water-soluble coronenes, that form nanoparticles by self-association, work as new fluorescent materials by complexation with cucurbit[7]uril, as well as selective turn-on fluorogenic sensors for nitroaromatic explosives with remarkable selectivity, by using only water as solvent.