SARS-CoV-2 Infection Alters the Phenotype and Gene Expression of Adipocytes.

Epidemiological evidence emphasizes that excess fat mass is associated with an increased risk of severe COVID-19 disease. Nevertheless, the intricate interplay between SARS-CoV-2 and adipocytes remains poorly understood. It is crucial to decipher the progression of COVID-19 both in the acute phase and on long-term outcomes. In this study, an in vitro model using the human SGBS cell line (Simpson-Golabi-Behmel syndrome) was developed to investigate the infectivity of SARS-CoV-2 in adipocytes, and the effects of virus exposure on adipocyte function. Our results show that SGBS adipocytes expressing ACE2 are susceptible to SARS-CoV-2 infection, as evidenced by the release of the viral genome into the medium, detection of the nucleocapsid in cell lysates, and positive immunostaining for the spike protein. Infected adipocytes show remarkable changes compared to uninfected controls: increased surface area of lipid droplets, upregulated expression of genes of inflammation (Haptoglobin, MCP-1, IL-6, PAI-1), increased oxidative stress (MnSOD), and a concomitant reduction of transcripts related to adipocyte function (leptin, fatty acid synthase, perilipin). Moreover, exogenous expression of spike protein in SGBS adipocytes also led to an increase in lipid droplet size. In conclusion using the human SGBS cell line, we detected SARS-CoV-2 infectivity in adipocytes, revealing substantial morphological and functional changes in infected cells.

Aggregation and Gelation Behavior of Stereocomplexed Four-Arm PLA-PEG Copolymers Containing Neutral or Cationic Linkers.

Poly(lactide) (PLA) and poly(ethylene glycol) (PEG)-based hydrogels were prepared by mixing phosphate buffer saline (PBS, pH 7.4) solutions of four-arm (PEG-PLA)2-R-(PLA-PEG)2 enantiomerically pure copolymers having the opposite chirality of the poly(lactide) blocks. Dynamic Light Scattering, rheology measurements, and fluorescence spectroscopy suggested that, depending on the nature of the linker R, the gelation process followed rather different mechanisms. In all cases, mixing of equimolar amounts of the enantiomeric copolymers led to micellar aggregates with a stereocomplexed PLA core and a hydrophilic PEG corona. Yet, when R was an aliphatic heptamethylene unit, temperature-dependent reversible gelation was mainly induced by entanglements of PEG chains at concentrations higher than 5 wt.%. When R was a linker containing cationic amine groups, thermo-irreversible hydrogels were promptly generated at concentrations higher than 20 wt.%. In the latter case, stereocomplexation of the PLA blocks randomly distributed in micellar aggregates is proposed as the major determinant of the gelation process.

An Efficient Aequorea victoria Green Fluorescent Protein for Stimulated Emission Depletion Super-Resolution Microscopy.

In spite of their value as genetically encodable reporters for imaging in living systems, fluorescent proteins have been used sporadically for stimulated emission depletion (STED) super-resolution imaging, owing to their moderate photophysical resistance, which does not enable reaching resolutions as high as for synthetic dyes. By a rational approach combining steady-state and ultrafast spectroscopy with gated STED imaging in living and fixed cells, we here demonstrate that F99S/M153T/V163A GFP (c3GFP) represents an efficient genetic reporter for STED, on account of no excited state absorption at depletion wavelengths <600 nm and a long emission lifetime. This makes c3GFP a valuable alternative to more common, but less photostable, EGFP and YFP/Citrine mutants for STED imaging studies targeting the green-yellow region of the optical spectrum.

Extremely Low Forces Induce Extreme Axon Growth.

Stretch-growth has been defined as a process that extends axons via the application of mechanical forces. In the present article, we used a protocol based on magnetic nanoparticles (NPs) for labeling the entire axon tract of hippocampal neurons, and an external magnetic field gradient to generate a dragging force. We found that the application of forces below 10 pN induces growth at a rate of 0.66 ± 0.02 µm h-1 pN-1 Calcium imaging confirmed the strong increase in elongation rate, in comparison with the condition of tip-growth. Enhanced growth in stretched axons was also accompanied by endoplasmic reticulum (ER) accumulation and, accordingly, it was blocked by an inhibition of translation. Stretch-growth was also found to stimulate axonal branching, glutamatergic synaptic transmission, and neuronal excitability. Moreover, stretched axons showed increased microtubule (MT) density and MT assembly was key to sustaining stretch-growth, suggesting a possible role of tensile forces in MT translocation/assembly. Additionally, our data showed that stretched axons do not respond to BDNF signaling, suggesting interference between the two pathways. As these extremely low mechanical forces are physiologically relevant, stretch-growth could be an important endogenous mechanism of axon growth, with a potential for designing novel strategies for axonal regrowth.SIGNIFICANCE STATEMENT Axon growth involves motion, and motion is driven by forces. The growth cone (GC) itself can generate very low intracellular forces by inducing a drastic cytoskeleton remodeling, in response to signaling molecules. Here, we investigated the key role of intracellular force as an endogenous regulator of axon outgrowth, which it has been neglected for decades because of the lack of methodologies to investigate the topic. Our results indicate a critical role of force in promoting axon growth by facilitating microtubule (MT) polymerization.

New Coumarin Dipicolinate Europium Complexes with a Rich Chemical Speciation and Tunable Luminescence.

Europium (III) luminescent chelates possess intrinsic photophysical properties that are extremely useful in a wide range of applications. The lack of examples of coumarin-based lanthanide complexes is mainly due to poor photo-sensitization attempts. However, with the appeal of using such a versatile scaffold as antenna, especially in the development of responsive molecular probes, it is worth the effort to research new structural motifs. In this work, we present a series of two new tris coumarin-dipicolinate europium (III) complexes, specifically tailored to be either a mono or a dual emitter, tuning their properties with a simple chemical modification. We also encountered a rich chemical speciation in solution, studied in detail by means of paramagnetic NMR and emission spectroscopy.

New 1,3-Disubstituted Benzo[h]Isoquinoline Cyclen-Based Ligand Platform: Synthesis, Eu3+ Multiphoton Sensitization and Imaging Applications.

The development of lanthanide-based luminescent probes with a long emission lifetime has the potential to revolutionize imaging-based diagnostic techniques. By a rational design strategy taking advantage of computational predictions, a novel, water-soluble Eu3+ complex from a cyclen-based ligand bearing 1,3-disubstituted benzo[h]isoquinoline arms was realized. The ligand has been obtained overcoming the lack of reactivity of position 3 of the isoquinoline moiety. Notably, steric hindrance of the heteroaromatic chromophore allowed selective and stoichiometry-controlled insertion of two or three antennas on the cyclen platform without any protection strategy. The complex bears a fourth heptanoic arm for easy conjugation to biomolecules. This new chromophore allowed the sensitization of the metal center either with one or two photons excitation. The suitability as a luminescent bioprobe was validated by imaging BMI1 oncomarker in lung carcinoma cells following an established immunofluorescence approach. The use of a conventional epifluorescence microscope equipped with a linear structured illumination module disclosed a simple and inexpensive way to image confocally Ln-bioprobes by single photon excitation in the 350-400 nm window, where ordinary confocal systems have no excitation sources.

Lipid-Conjugated Rigidochromic Probe Discloses Membrane Alteration in Model Cells of Krabbe Disease.

The plasma membrane of cells has a complex architecture based on the bidimensional liquid-crystalline bilayer arrangement of phospho- and sphingolipids, which in turn embeds several proteins and is connected to the cytoskeleton. Several studies highlight the spatial membrane organization into more ordered (Lo or lipid raft) and more disordered (Ld) domains. We here report on a fluorescent analog of the green fluorescent protein chromophore that, when conjugated to a phospholipid, enables the quantification of the Lo and Ld domains in living cells on account of its large fluorescence lifetime variation in the two phases. The domain composition is straightforwardly obtained by the phasor approach to confocal fluorescence lifetime imaging, a graphical method that does not require global fitting of the fluorescence decay in every spatial position of the sample. Our imaging strategy was applied to recover the domain composition in human oligodendrocytes at rest and under treatment with galactosylsphingosine (psychosine). Exogenous psychosine administration recapitulates many of the molecular fingerprints of a severe neurological disease, globoid cell leukodystrophy, better known as Krabbe disease. We found out that psychosine progressively destabilizes plasma membrane, as witnessed by a shrinking of the Lo fraction. The unchanged levels of galactosyl ceramidase, i.e., the enzyme lacking in Krabbe disease, upon psychosine treatment suggest that psychosine alters the plasma membrane structure by direct physical effect, as also recently demonstrated in model membranes.

Simultaneous Detection of Local Polarizability and Viscosity by a Single Fluorescent Probe in Cells.

Many intracellular reactions are dependent on the dielectric ("polarity") and viscosity properties of their milieu. Fluorescence imaging offers a convenient strategy to report on such environmental properties. Yet, concomitant and independent monitoring of polarity and viscosity in cells at submicron scale is currently hampered by the lack of fluorescence probes characterized by unmixed responses to both parameters. Here, the peculiar photophysics of a green fluorescent protein chromophore analog is exploited for quantifying and imaging polarity and viscosity independently in living cells. We show that the polarity and viscosity profile around a novel hybrid drug-delivery peptide changes dramatically upon cell internalization via endosomes, shedding light on the spatiotemporal features of the release mechanism. Accordingly, our fluorescent probe opens the way to monitor the environmental effects on several processes relevant to cell biochemistry and nanomedicine.

Temperature and pressure effects on GFP mutants: explaining spectral changes by molecular dynamics simulations and TD-DFT calculations.

By combining spectroscopic measurements under high pressure with molecular dynamics simulations and quantum mechanics calculations we investigate how sub-angstrom structural perturbations are able to tune protein function. We monitored the variations in fluorescence output of two green fluorescent protein mutants (termed Mut2 and Mut2Y, the latter containing the key T203Y mutation) subjected to pressures up to 600 MPa, at various temperatures in the 280-320 K range. By performing 150 ns molecular dynamics simulations of the protein structures at various pressures, we evidenced subtle changes in conformation and dynamics around the light-absorbing chromophore. Such changes explain the measured spectral tuning in the case of the sizable 120 cm(-1) red-shift observed for pressurized Mut2Y, but absent in Mut2. Previous work [Barstow et al., Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 13362] on pressure effects on GFP also involved a T203Y mutant. On the basis of cryocooling X-ray crystallography, the pressure-induced fluorescence blue shift at low temperature (77 K) was attributed to key changes in relative conformation of the chromophore and Tyr203 phenol ring. At room temperature, however, a red shift was observed at high pressure, analogous to the one we observe in Mut2Y. Our investigation of structural variations in compressed Mut2Y also explains their result, bridging the gap between low-temperature and room-temperature high-pressure effects.

The effects of ferulic acid on ß-amyloid fibrillar structures investigated through experimental and computational techniques.

BACKGROUND: Current research has indicated that small natural compounds could interfere with ß-amyloid fibril growth and have the ability to disassemble preformed folded structures. Ferulic acid (FA), which possesses both hydrophilic and hydrophobic moieties and binds to peptides/proteins, is a potential candidate against amyloidogenesis. The molecular mechanisms connected to this action have not been elucidated in detail yet. METHODS: Here the effects of FA on preformed fibrils are investigated by means of a concerted experimental-computational approach. Spectroscopic techniques, such as FTIR, fluorescence, size exclusion chromatography and confocal microscopy in combination with molecular dynamics simulations are used to identify those features which play a key role in the destabilization of the aggregates. RESULTS: Experimental findings highlight that FA has disruptive effects on the fibrils. The computational analysis suggests that dissociation of peptides from the amyloid superstructures could take place along the fibril axis and be primarily determined by the cooperative rupture of the backbone hydrogen bonds and of the Asp-Lys salt bridges. CONCLUSION: FA clusters could induce a sort of stabilization and tightening of the fibril structure in the short term and its disruption in the long term, inhibiting further fibril re-assembly through FA screening effects. GENERAL SIGNIFICANCE: The combination of experimental and computational techniques could be successfully used to identify the disrupting action of FA on preformed Aß fibrils in water solution.

Cancer-cell-targeted theranostic cubosomes.

This work was devoted to the development of a new type of lipid-based (cubosome) theranostic nanoparticle able to simultaneously host camptothecin, a potent anticancer drug, and a squarain-based NIR-emitting fluorescent probe. Furthermore, to confer targeting abilities on these nanoparticles, they were dispersed using mixtures of Pluronic F108 and folate-conjugated Pluronic F108 in appropriate ratios. The physicochemical characterization, performed via SAXS, DLS, and cryo-TEM techniques, proved that aqueous dispersions of such cubosomes can be effectively prepared, while the photophysical characterization demonstrated that these nanoparticles may be used for in vivo imaging purposes. The superior ability of these innovative nanoparticles in targeting cancer cells was emphasized by investigating the lipid droplet alterations induced in HeLa cells upon exposure to targeted and nontargeted cubosomes.

Inhibitory effect of the human liver-derived antimicrobial peptide hepcidin 20 on biofilms of polysaccharide intercellular adhesin (PIA)-positive and PIA-negative strains of Staphylococcus epidermidis.

Staphylococcus epidermidis plays a major role in biofilm-related medical device infections. Herein the anti-biofilm activity of the human liver-derived antimicrobial peptide hepcidin 20 (hep20) was evaluated against polysaccharide intercellular adhesin (PIA)-positive and PIA-negative clinical isolates of S. epidermidis. Hep20 markedly inhibited biofilm formation and bacterial cell metabolism of PIA-positive and PIA-negative strains, but the decrease in biofilm biomass only partially correlated with a decrease in viable bacteria. Confocal microscope images revealed that, in the presence of hep20, both PIA-positive and PIA-negative strains formed biofilms with altered architectures and reduced amounts of extracellular matrix. Co-incubation of hep20 with vancomycin produced no synergistic effect, evaluated as number of viable cells, both in preventing biofilm formation and in treating preformed biofilms. In contrast, biofilms obtained in the presence of hep20, and then exposed to vancomycin, displayed an increased susceptibility to vancomycin. These results suggest that hep20 may inhibit the production/accumulation of biofilm extracellular matrix.

Tumor Microenvironment Landscapes Supporting EGFR-mutant NSCLC Are Modulated at the Single-cell Interaction Level by Unesbulin Treatment.

Lung cancer is the leading cause of cancer deaths. Lethal pulmonary adenocarcinomas (ADC) present with frequent mutations in the EGFR. Genetically engineered murine models of lung cancer expedited comprehension of the molecular mechanisms driving tumorigenesis and drug response. Here, we systematically analyzed the evolution of tumor heterogeneity in the context of dynamic interactions occurring with the intermingled tumor microenvironment (TME) by high-resolution transcriptomics. Our effort identified vulnerable tumor-specific epithelial cells, as well as their cross-talk with niche components (endothelial cells, fibroblasts, and tumor-infiltrating immune cells), whose symbiotic interface shapes tumor aggressiveness and is almost completely abolished by treatment with Unesbulin, a tubulin binding agent that reduces B cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) activity. Simultaneous magnetic resonance imaging (MRI) analysis demonstrated decreased tumor growth, setting the stage for future investigations into the potential of novel therapeutic strategies for EGFR-mutant ADCs. SIGNIFICANCE: Targeting the TME is an attractive strategy for treatment of solid tumors. Here we revealed how EGFR-mutant landscapes are affected at the single-cell resolution level during Unesbulin treatment. This novel drug, by targeting cancer cells and their interactions with crucial TME components, could be envisioned for future therapeutic advancements.

Intact microtubules preserve transient receptor potential vanilloid 1 (TRPV1) functionality through receptor binding.

The transient receptor potential cation channel subfamily V member 1 (TRPV1) is a protein currently under scrutiny as a pharmacological target for pain management therapies. Recently, the role of TRPV1-microtubule interaction in transducing nociception stimuli to cells by cytoskeletal rearrangement was proposed. In this work, we investigate TRPV1-microtubule interaction in living cells under the resting or activated state of TRPV1, as well as in presence of structurally intact or depolymerized cytoskeletal microtubules. We combined a toolbox of high resolution/high sensitivity fluorescence imaging techniques (such as FRET, correlation spectroscopy, and fluorescence anisotropy) to monitor TRPV1 aggregation status, membrane mobility, and interaction with microtubules. We found that TRPV1 is a dimeric membrane protein characterized by two populations with different diffusion properties in basal condition. After stimulation with resiniferatoxin, TRPV1 dimers tetramerize. The tetramers and the slower population of TRPV1 dimers bind dynamically to intact microtubules but not to tubulin dimers. Upon microtubule disassembly, the interaction with TRPV1 is lost thereby inducing receptor self-aggregation with partial loss of functionality. Intact microtubules play an essential role in maintaining TRPV1 functionality toward activation stimuli. This previously undisclosed property mirrors the recently reported role of TRPV1 in modulating microtubule assembly/disassembly and suggests the participation of these two players in a feedback cycle linking nociception and cytoskeletal remodeling.

Fluorescent recovery after photobleaching (FRAP) analysis of nuclear export rates identifies intrinsic features of nucleocytoplasmic transport.

A quantitative description of carrier-mediated nuclear export in live cells is presented. To this end, we fused a prototypical leucine-rich nuclear export signal (NES) to GFP as a cargo model and expressed the fluorescent chimera in live CHO-K1 cells. By modeling FRAP data, we calculate the NES affinity for the export machinery and the maximum rate of nuclear export achievable at saturation of endogenous carriers. The measured active-export time through the Nuclear Pore Complex (NPC) is 18 ms, remarkably similar to the previously determined active-import rate. Also, our results reveal that active export/import and active export/passive diffusion fluxes are uncoupled, thus complementing previous reports on active import/passive diffusion uncoupling. These findings suggest differential gating at the NPC level.

Imaging intracellular viscosity by a new molecular rotor suitable for phasor analysis of fluorescence lifetime.

The arsenal of fluorescent probes tailored to functional imaging of cells is rapidly growing and benefits from recent developments in imaging strategies. Here, we present a new molecular rotor, which displays strong absorption in the green region of the spectrum, very little solvatochromism, and strong emission sensitivity to local viscosity. The emission increase is paralleled by an increase in emission lifetime. Owing to its concentration-independent nature, fluorescence lifetime is particularly suitable to image environmental properties, such as viscosity, at the intracellular level. Accordingly, we demonstrate that intracellular viscosity measurements can be efficiently carried out by lifetime imaging with our probe and phasor analysis, an efficient method for measuring lifetime-related properties (e.g., bionalyte concentration or local physicochemical features) in living cells. Notably, we show that it is possible to monitor the partition of our probe into different intracellular regions/organelles and to follow mitochondrial de-energization upon oxidative stress.

On the Advent of Super-Resolution Microscopy in the Realm of Polycomb Proteins.

The genomes of metazoans are organized at multiple spatial scales, ranging from the double helix of DNA to whole chromosomes. The intermediate genomic scale of kilobases to megabases, which corresponds to the 50-300 nm spatial scale, is particularly interesting, as the 3D arrangement of chromatin is implicated in multiple regulatory mechanisms. In this context, polycomb group (PcG) proteins stand as major epigenetic modulators of chromatin function, acting prevalently as repressors of gene transcription by combining chemical modifications of target histones with physical crosslinking of distal genomic regions and phase separation. The recent development of super-resolution microscopy (SRM) has strongly contributed to improving our comprehension of several aspects of nano-/mesoscale (10-200 nm) chromatin domains. Here, we review the current state-of-the-art SRM applied to PcG proteins, showing that the application of SRM to PcG activity and organization is still quite limited and mainly focused on the 3D assembly of PcG-controlled genomic loci. In this context, SRM approaches have mostly been applied to multilabel fluorescence in situ hybridization (FISH). However, SRM data have complemented the maps obtained from chromosome capture experiments and have opened a new window to observe how 3D chromatin topology is modulated by PcGs.

Quantitative analysis of Tat peptide binding to import carriers reveals unconventional nuclear transport properties.

A detailed study of nuclear import mediated by the HIV-1 Tat peptide (47YGRKKRRQRRR57, TatRRR) is reported. Fluorescence-based measurements, calibration of protein concentrations, and binding assays are exploited to address the physicochemical mechanisms of Tat peptide recognition by the classical importin α (Impα) and importin ß (Impß) receptors both in vitro and in intact cells. We show that TatRRR is an unconventional nuclear localization sequence that binds directly to both Impα and Impß carriers in the absence of competitors (in vitro), whereas this property is silenced in the actual cellular environment. In the latter case, Impα/ß-dependent nuclear import can be successfully restored by replacing the "RRR" stretch with "GGG". We apply a recently developed method to determine quantitatively TatGGG affinity for each receptor. Based on these results, we can rationalize previous controversial reports on the Tat peptide and provide coherent guidelines for the design of novel intracellular targeting sequences.

Fluorescence recovery after photobleaching reveals the biochemistry of nucleocytoplasmic exchange.

Fluorescence recovery after photobleaching (FRAP) can help unveil subtle dynamical and biochemical properties of intracellular components. A peculiar aspect of this method is that it is based on the change of optical properties only, whereas dynamics and biochemistry of the molecules of interest are not perturbed. This makes FRAP particularly suitable for the study of protein translocation, e.g., between nucleus and cytoplasm. Here we present a comprehensive theoretical treatment of FRAP applied to protein nucleocytoplasmic translocation by passive diffusion and/or energy-driven processes across the nuclear envelope. Our mathematical model is validated by experimental FRAP studies with functionalized fluorescent protein chimeras. Using this approach we demonstrate that molecular crowding at the nuclear pore does not hamper passive diffusion and calculate the dimension of the nuclear pore size (5.33 nm). Additionally, our FRAP analysis reveals the biochemical parameters (maximum translocation rate and dissociation constant of the transport complex in cytoplasm) associated with the active import of a prototypical nuclear localization sequence (NLS of SV40) and related mutants. We demonstrate that transportin binding and active import into the nucleus are independent processes that can be separately modulated. The present results are discussed in light of their potential to help in engineering sequences for intracellular targeted delivery of sensors and/or therapeutic compounds. Finally, the limits of validity of our mathematical model are addressed.

Cis-trans photoisomerization properties of GFP chromophore analogs.

The photoswitching behaviour of the green fluorescent protein (GFP) chromophore and its analogs opens up exciting horizons for the engineering and development of molecular devices for high sensitivity in vivo studies. In this work we present the synthesis and photophysical study of four GFP chromophore analogs belonging to butenolide and pyrrolinone classes. These chromophores possess an intriguing photoinduced cis-trans isomerization mechanism. Stereochemical structural assignment was unambiguously performed by 1D Nuclear Overhauser Effect NMR measurements. The spectroscopic properties of both cis and trans isomers were studied, and photoconversion quantum yield for cis-trans isomerization was assessed to be in the 0.1-0.4 range. Finally, the 3J(C,H) coupling constant in the 13C-C=C-H motif was in excellent agreement with theoretical DFT calculations, thus providing a further confirmation of cis-trans photoisomerization of the structurally analog GFP chromophore.