Molecular Rotors: Fluorescent Sensors for Microviscosity and Conformation of Biomolecules.

The viscosity and crowding of biological environment are considered vital for the correct cellular function, and alterations in these parameters are known to underly a number of pathologies including diabetes, malaria, cancer and neurodegenerative diseases, to name a few. Over the last decades, fluorescent molecular probes termed molecular rotors proved extremely useful for exploring viscosity, crowding, and underlying molecular interactions in biologically relevant settings. In this review, we will discuss the basic principles underpinning the functionality of these probes and will review advances in their use as sensors for lipid order, protein crowding and conformation, temperature and non-canonical nucleic acid structures in live cells and other relevant biological settings.

Viscosity-Sensitive Membrane Dyes as Tools To Estimate the Crystalline Structure of Lipid Bilayers.

Lipid membranes are crucial for cellular integrity and regulation, and tight control of their structural and mechanical properties is vital to ensure that they function properly. Fluorescent probes sensitive to the membrane's microenvironment are useful for investigating lipid membrane properties; however, there is currently a lack of quantitative correlation between the exact parameters of lipid organization and a readout from these dyes. Here, we investigate this relationship for "molecular rotors", or microviscosity sensors, by simultaneously measuring their fluorescence lifetime to determine the membrane viscosity, while using X-ray diffraction to determine the membrane's structural properties. Our results reveal a phase-dependent correlation between the membrane's structural parameters and mechanical properties measured by a BODIPY-based molecular rotor, giving excellent predictive power for the structural descriptors of the lipid bilayer. We also demonstrate that differences in membrane thickness between different lipid phases are not a prerequisite for the formation of lipid microdomains and that this requirement can be disrupted by the presence of line-active molecules. Our results underpin the use of membrane-sensitive dyes as reporters of the structure of lipid membranes.

Directly imaging emergence of phase separation in peroxidized lipid membranes.

Lipid peroxidation is a process which is key in cell signaling and disease, it is exploited in cancer therapy in the form of photodynamic therapy. The appearance of hydrophilic moieties within the bilayer's hydrocarbon core will dramatically alter the structure and mechanical behavior of membranes. Here, we combine viscosity sensitive fluorophores, advanced microscopy, and X-ray diffraction and molecular simulations to directly and quantitatively measure the bilayer's structural and viscoelastic properties, and correlate these with atomistic molecular modelling. Our results indicate an increase in microviscosity and a decrease in the bending rigidity upon peroxidation of the membranes, contrary to the trend observed with non-oxidized lipids. Fluorescence lifetime imaging microscopy and MD simulations give evidence for the presence of membrane regions of different local order in the oxidized membranes. We hypothesize that oxidation promotes stronger lipid-lipid interactions, which lead to an increase in the lateral heterogeneity within the bilayer and the creation of lipid clusters of higher order.

Modulating membrane fusion through the design of fusogenic DNA circuits and bilayer composition.

Membrane fusion is a ubiquitous phenomenon linked to many biological processes, and represents a crucial step in liposome-based drug delivery strategies. The ability to control, ever more precisely, membrane fusion pathways would thus be highly valuable for next generation nano-medical solutions and, more generally, the design of advanced biomimetic systems such as synthetic cells. In this article, we present fusogenic nanostructures constructed from synthetic DNA which, different from previous solutions, unlock routes for modulating the rate of fusion and making it conditional to the presence of soluble DNA molecules, thus demonstrating how membrane fusion can be controlled through simple DNA-based molecular circuits. We then systematically explore the relationship between lipid-membrane composition, its biophysical properties, and measured fusion efficiency, linking our observations to the stability of transition states in the fusion pathway. Finally, we observe that specific lipid compositions lead to the emergence of complex bilayer architectures in the fusion products, such as nested morphologies, which are accompanied by alterations in biophysical behaviour. Our findings provide multiple, orthogonal strategies to program lipid-membrane fusion, which leverage the design of either the fusogenic DNA constructs or the physico/chemical properties of the membranes, and could thus be valuable in applications where some design parameters are constrained by other factors such as material cost and biocompatibility, as it is often the case in biotechnological applications.

Mapping cisplatin-induced viscosity alterations in cancer cells using molecular rotor and fluorescence lifetime imaging microscopy.

SIGNIFICANCE: Despite the importance of the cell membrane in regulation of drug activity, the influence of drug treatments on its physical properties is still poorly understood. The combination of fluorescence lifetime imaging microscopy (FLIM) with specific viscosity-sensitive fluorescent molecular rotors allows the quantification of membrane viscosity with high spatiotemporal resolution, down to the individual cell organelles. AIM: The aim of our work was to analyze microviscosity of the plasma membrane of living cancer cells during chemotherapy with cisplatin using FLIM and correlate the observed changes with lipid composition and cell's response to treatment. APPROACH: FLIM together with viscosity-sensitive boron dipyrromethene-based fluorescent molecular rotor was used to map the fluidity of the cell's membrane. Chemical analysis of membrane lipid composition was performed with time-of-flight secondary ion mass spectrometry (ToF-SIMS). RESULTS: We detected a significant steady increase in membrane viscosity in viable cancer cells, both in cell monolayers and tumor spheroids, upon prolonged treatment with cisplatin, as well as in cisplatin-adapted cell line. ToF-SIMS revealed correlative changes in lipid profile of cisplatin-treated cells. CONCLUSIONS: These results suggest an involvement of membrane viscosity in the cell adaptation to the drug and in the acquisition of drug resistance.

Imaging non-classical mechanical responses of lipid membranes using molecular rotors.

Lipid packing in cellular membranes has a direct effect on membrane tension and microviscosity, and plays a central role in cellular adaptation, homeostasis and disease. According to conventional mechanical descriptions, viscosity and tension are directly interconnected, with increased tension leading to decreased membrane microviscosity. However, the intricate molecular interactions that combine to build the structure and function of a cell membrane suggest a more complex relationship between these parameters. In this work, a viscosity-sensitive fluorophore ('molecular rotor') is used to map changes in microviscosity in model membranes under conditions of osmotic stress. Our results suggest that the relationship between membrane tension and microviscosity is strongly influenced by the bilayer's lipid composition. In particular, we show that the effects of increasing tension are minimised for membranes that exhibit liquid disordered (Ld) - liquid ordered (Lo) phase coexistence; while, surprisingly, membranes in pure gel and Lo phases exhibit a negative compressibility behaviour, i.e. they soften upon compression.

Enhancement of the Upconversion Emission by Visible-to-Near-Infrared Fluorescent Graphene Quantum Dots for miRNA Detection.

We developed a sensor for the detection of specific microRNA (miRNA) sequences that was based on graphene quantum dots (GQDs) and ssDNA-UCNP@SiO2. The proposed sensor exploits the interaction between the sp(2) carbon atoms of the GQD, mainly π-π stacking, and the DNA nucleobases anchored on the upconversion nanoparticles (UCNPs). This interaction brings the GQD to the surface of the ssDNA-UCNP@SiO2 system, enhancing the upconversion emission. On the other hand, hybridization of the single-stranded DNA (ssDNA) chains anchored on the nanoparticles with their complementary miRNA sequences blocks the capacity of the UCNPs to interact with the GQD through π-π stacking. That gives as result a reduction of the fluorescent enhancement, which is dependent on the concentration of miRNA sequences. This effect was used to create a sensor for miRNA sequences with a detection limit of 10 fM.

[Prevalence of metabolic syndrome in the adult population of Yecla (Murcia). Degree of agreement between three definitions of it]. / Prevalencia del síndrome metabólico en la población adulta de Yecla (Murcia). Grado de acuerdo entre tres definiciones.

OBJECTIVES: To determine the prevalence of metabolic syndrome (MS), its components and insulin resistance (IR) in the adult population of Yecla. To study the variability between 3 definitions of the syndrome and IR. To identify the variables that predict the presence of IR and to verify the diagnostic validity of several strategies for predicting it. DESIGN: Descriptive, cross-sectional study. SETTING: Primary care, Yecla (Murcia), Spain. PARTICIPANTS: We studied 317 persons (292 with analysis) out of 424 selected by stratified (age and sex) random sampling from 18,059 people > or = 30 years old and possessing a health card. MAIN MEASUREMENTS: We used WHO-98, NCEP III, and EGIR criteria for diagnosing MS, and WHO-99 for defining DM2, impaired basal glucose and impaired glucose tolerance. The following variables were collected: social, demographic and personal details, plasma lipid, glycosylated haemoglobin, microalbuminuria, and insulin levels. IR was defined by the HOMA method at > or = 3.8 or as the highest quartile of basal insulinemia in normoglycaemic persons. RESULTS: MS prevalence was NCEP 20.2% (95% CI, 15.6-24.8), WHO 35.3% (95% CI, 29.8-40.8), EGIR 24% (95% CI, 19.1-28.9), and IR was 27.7% (95% CI, 22.6-32.8). The sensitivity and specificity of NCEP, WHO, and EGIR criteria for detecting IR were (46% and 90%), (78% and 81%), and (73% and 95%), respectively. Insulin resistance was associated significantly with age, basal glycaemia, triglycerides, and waist circumference. CONCLUSIONS: Metabolic syndrome is common in Yecla (more so in men). There is disagreement between several diagnostic criteria for the syndrome, with NCEP criteria less sensitive in determining IR. A generally accepted definition is needed.

Prevalencia del síndrome metabólico en la población adulta de Yecla (Murcia). Grado de acuerdo entre tres definiciones / Prevalence of metabolic syndrome in the adult population of Yecla (Murcia). Degree of agreement between three definitions of it

Objetivos. Detectar la prevalencia del síndrome metabólico (SM), sus componentes y la resistencia a la insulina (RI) en la población adulta de Yecla. Estudiar la concordancia de 3 definiciones del SM entre sí y con la RI. Identificar variables que puedan predecir la presencia de RI y comprobar la validez diagnóstica de varias estrategias para predecirla. Diseño. Estudio descriptivo, transversal. Emplazamiento. Población de Yecla (Murcia). Ámbito de atención primaria. Participantes. Estudiamos a 317 personas (292 aportaron analítica) de 424 seleccionadas mediante muestreo aleatorio estratificado (edad y sexo) de 18.059 con tarjeta sanitaria y edad é 30 años. Mediciones principales. Utilizamos los criterios NCEP III, OMS-98 y EGIR (Grupo Europeo de Estudio de la Resistencia a la Insulina) para diagnosticar el SM y OMS-99 para definir la diabetes mellitus no insulinodependiente, la glucemia basal alterada y la tolerancia alterada a la glucosa. Recogimos variables sociodemográficas y antropométricas, y determinamos la presencia de lípidos, microalbuminuria, HbA1c e insulinemia; definimos RI si el índice HOMA é 3,8 o como cuartil más alto de insulinemia basal en normoglucémicos. Resultados. La prevalencia del SM fue, según los criterios NCEP, del 20,2% (intervalo de confianza [IC] del 95%, 15,6-24,8), OMS del 35,3% (IC del 95%, 29,8-40,8), EGIR del 24% (IC del 95%, 19,1-28,9) y RI del 27,7% (IC del 95%, 22,6-32,8). La sensibilidad y la especificidad de NCEP, OMS y EGIR para detectar RI fueron del 46 y el 90%, del 78 y el 81% y del 73 y el 95%, respectivamente. La edad, la glucemia basal, los triglicéridos y el perímetro de la cintura se asocian significativamente con RI. Conclusiones. Hay una alta prevalencia de SM en el área (mayor en los varones). Hay diferencias entre los diferentes criterios diagnósticos del síndrome, y los de NCEP son menos sensible para determinar la RI. Es necesario establecer una definición universalmente aceptada del SM

Objectives. To determine the prevalence of metabolic syndrome (MS), its components and insulin resistance (IR) in the adult population of Yecla. To study the variability between 3 definitions of the syndrome and IR. To identify the variables that predict the presence of IR and to verify the diagnostic validity of several strategies for predicting it. Design. Descriptive, cross-sectional study. Setting. Primary care, Yecla (Murcia), Spain. Participants. We studied 317 persons (292 with analysis) out of 424 selected by stratified (age and sex) random sampling from 18 059 people >=30 years old and possessing a health card. Main measurements. We used WHO-98, NCEP III, and EGIR criteria for diagnosing MS, and WHO-99 for defining DM2, impaired basal glucose and impaired glucose tolerance. The following variables were collected: social, demographic and personal details, plasma lipid, glycosylated haemoglobin, microalbuminuria, and insulin levels. IR was defined by the HOMA method at >=3.8 or as the highest quartile of basal insulinemia in normoglycaemic persons. Results. MS prevalence was NCEP 20.2% (95% CI, 15.6-24.8), WHO 35.3% (95% CI, 29.8-40.8), EGIR 24% (95% CI, 19.1-28.9), and IR was 27.7% (95% CI, 22.6-32.8). The sensitivity and specificity of NCEP, WHO, and EGIR criteria for detecting IR were (46% and 90%), (78% and 81%), and (73% and 95%), respectively. Insulin resistance was associated significantly with age, basal glycaemia, triglycerides, and waist circumference. Conclusions. Metabolic syndrome is common in Yecla (more so in men). There is disagreement between several diagnostic criteria for the syndrome, with NCEP criteria less sensitive in determining IR. A generally accepted definition is needed