A long noncoding RNA controls muscle differentiation by functioning as a competing endogenous RNA.

Recently, a new regulatory circuitry has been identified in which RNAs can crosstalk with each other by competing for shared microRNAs. Such competing endogenous RNAs (ceRNAs) regulate the distribution of miRNA molecules on their targets and thereby impose an additional level of post-transcriptional regulation. Here we identify a muscle-specific long noncoding RNA, linc-MD1, which governs the time of muscle differentiation by acting as a ceRNA in mouse and human myoblasts. Downregulation or overexpression of linc-MD1 correlate with retardation or anticipation of the muscle differentiation program, respectively. We show that linc-MD1 "sponges" miR-133 and miR-133 [corrected] to regulate the expression of MAML1 and MEF2C, transcription factors that activate muscle-specific gene expression. Finally, we demonstrate that linc-MD1 exerts the same control over differentiation timing in human myoblasts, and that its levels are strongly reduced in Duchenne muscle cells. We conclude that the ceRNA network plays an important role in muscle differentiation.

The emerging landscape of single-molecule protein sequencing technologies.

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.

Reversible Cavitation-Induced Junctional Opening in an Artificial Endothelial Layer.

Targeting pharmaceuticals through the endothelial barrier is crucial for drug delivery. In this context, cavitation-assisted permeation shows promise for effective and reversible opening of intercellular junctions. A vessel-on-a-chip is exploited to investigate and quantify the effect of ultrasound-excited microbubbles-stable cavitation-on endothelial integrity. In the vessel-on-a-chip, the endothelial cells form a complete lumen under physiological shear stress, resulting in intercellular junctions that exhibit barrier functionality. Immunofluorescence microscopy is exploited to monitor vascular integrity following vascular endothelial cadherin staining. It is shown that microbubbles amplify the ultrasound effect, leading to the formation of interendothelial gaps that cause barrier permeabilization. The total gap area significantly increases with pressure amplitude compared to the control. Gap opening is fully reversible with gap area distribution returning to the control levels 45 min after insonication. The proposed integrated platform allows for precise and repeatable in vitro measurements of cavitation-enhanced endothelium permeability and shows potential for validating irradiation protocols for in vivo applications.

Amyloid Growth, Inhibition, and Real-Time Enzymatic Degradation Revealed with Single Conical Nanopore.

Amyloid fibrils are involved in several neurodegenerative diseases. However, because of their polymorphism and low concentration, they are challenging to assess in real-time with conventional techniques. Here, we present a new approach for the characterization of the intermediates: protofibrils and "end-off" aggregates which are produced during the amyloid formation. To do so, we have fashioned conical track-etched nanopores that are functionalized to prevent the fouling. Using these nanopores, we have followed the kinetic of amyloid growth to discriminate the different intermediates protofibrils and "end-off. Then, the nanopore was used to characterize the effect of promoter and inhibitor of the fibrillation process. Finally, we have followed in real-time the degradation of amyloid with peptase. Compare with the SiN nanopore, the track-etched one features exceptionally high success rate via functionalization and detection in "one-pot". Our results demonstrate the potential for a conical nanopore to be used as a routine technique for the characterization of the amyloid growth and/or degradation.

Charge polarization, local electroneutrality breakdown and eddy formation due to electroosmosis in varying-section channels.

We characterize the dynamics of an electrolyte embedded in a varying-section channel under the action of a constant external electrostatic field. By means of molecular dynamics simulations we determine the stationary density, charge and velocity profiles of the electrolyte. Our results show that when the Debye length is comparable to the width of the channel bottlenecks a concentration polarization along with two eddies sets inside the channel. Interestingly, upon increasing the external field, local electroneutrality breaks down and charge polarization sets leading to the onset of net dipolar field. This novel scenario, that cannot be captured by the standard approaches based on local electroneutrality, opens the route for the realization of novel micro and nano-fluidic devices.

Flagellated microswimmers: Hydrodynamics in thin liquid films.

The hydrodynamics of a flagellated microswimmer moving in thin films is discussed. The fully resolved hydrodynamics is exploited by solving the Stokes equations for the actual geometry of the swimmer. Two different interfaces are used to confine the swimmer: a bottom solid wall and a top air-liquid interface, as appropriate for a thin film. The swimmer follows curved clockwise trajectories that can converge towards an asymptotically stable circular path or can result in a collision with one of the two interfaces. A bias towards the air-liquid interface emerges. Slight changes in the swimmer geometry and film thickness strongly affect the resulting dynamics suggesting that a very reach phenomenology occurs in the presence of confinement. Under specific conditions, the swimmer follows a "crown-like" path. Implications for the motion of bacteria close to an air bubble moving in a microchannel are discussed.

Analysis of Pore Formation and Protein Translocation Using Large Biological Nanopores.

This paper describes the analysis of pore formation and detection of a single protein molecule using a large nanopore among five different pore-forming proteins. We demonstrate that the identification of appropriate pores for nanopore sensing can be achieved by classifying the channel current signals and performing noise analysis. Through these analyses, we selected a perforin nanopore from the membrane attack complex/perforin superfamily and attempted to use it to detect the granzyme B protein, a serine protease. As a result, we found that granzyme B might pass through the perforin nanopore if it adopts an unfolded structure. Our proposed analytical approach should be useful for exploring several types of nanopore as large biological nanopores other than α-hemolysin.

Protein Nanopore-Based Discrimination between Selected Neutral Amino Acids from Polypeptides.

Nanopore probing of biological polymers has the potential to achieve single-molecule sequencing at low cost, high throughput, portability, and minimal sample preparation and apparatus. In this article, we explore the possibility of discrimination between neutral amino acid residues from the primary structure of 30 amino acids long, engineered peptides, through the analysis of single-molecule ionic current fluctuations accompanying their slowed-down translocation across the wild type α-hemolysin (α-HL) nanopore, and molecular dynamics simulations. We found that the transient presence inside the α-HL of alanine or tryptophan residues from the primary sequence of engineered peptides results in distinct features of the ionic current fluctuation pattern associated with the peptide reversibly blocking the nanopore. We propose that α-HL sensitivity to the molecular exclusion at the most constricted region mediates ionic current blockade events correlated with the volumes that are occluded by at least three alanine or tryptophan residues, and provides the specificity needed to discriminate between groups of neutral amino acids. Further, we find that the pattern of current fluctuations depends on the orientation of the threaded amino acid residues, suggestive of a conformational anisotropy of the ensemble of conformations of the peptide on the restricted nanopore region, related to its relative axial orientation inside the nanopore.

Driven diffusion against electrostatic or effective energy barrier across α-hemolysin.

We analyze the translocation of a charged particle across an α-Hemolysin (αHL) pore in the framework of a driven diffusion over an extended energy barrier generated by the electrical charges of the αHL. A one-dimensional electrostatic potential is extracted from the full 3D solution of the Poisson's equation. We characterize the particle transport under the action of a constant forcing by studying the statistics of the translocation time. We derive an analytical expression of translocation time average that compares well with the results from Brownian dynamic simulations of driven particles over the electrostatic potential. Moreover, we show that the translocation time distributions can be perfectly described by a simple theory which replaces the true barrier by an equivalent structureless square barrier. Remarkably, our approach maintains its accuracy also for low-applied voltage regimes where the usual inverse-Gaussian approximation fails. Finally, we discuss how the comparison between the simulated time distributions and their theoretical prediction results to be greatly simplified when using the notion of the empirical Laplace transform technique.

Controlling Electroosmosis in Nanopores Without Altering the Nanopore Sensing Region.

Nanopores are powerful tools for single-molecule sensing of biomolecules and nanoparticles. The signal coming from the molecule to be analyzed strongly depends on its interaction with the narrower section of the nanopore (constriction) that may be tailored to increase sensing accuracy. Modifications of nanopore constriction have also been commonly used to induce electroosmosis, that favors the capture of molecules in the nanopore under a voltage bias and independently of their charge. However, engineering nanopores for increasing both electroosmosis and sensing accuracy is challenging. Here it is shown that large electroosmotic flows can be achieved without altering the nanopore constriction. Using continuum electrohydrodynamic simulations, it is found that an external charged ring generates strong electroosmosis in cylindrical nanopores. Similarly, for conical nanopores it is shown that moving charges away from the cone tip still results in an electroosmotic flow (EOF), whose intensity reduces increasing the diameter of the nanopore section where charges are placed. This paradigm is applied to engineered biological nanopores showing, via atomistic simulations and experiments, that mutations outside the constriction induce a relatively intense electroosmosis. This strategy provides much more flexibility in nanopore design since electroosmosis can be controlled independently from the constriction, which can be optimized to improve sensing accuracy.

Geometry as a catalyst: how vapor cavities nucleate from defects.

The onset of cavitation is strongly enhanced by the presence of rough surfaces or impurities in the liquid. Despite decades of research, the way the geometry of these defects promote the nucleation of bubbles and its effect on the kinetics of the process remains largely unclear. We present here a comprehensive explanation of the catalytic action that roughness elements exert on the nucleation process for both pure vapor cavities and gas ones. This approach highlights that nucleation may follow nontrivial paths connected with a sharp decrease of the free energy barriers as compared to flat surfaces. Furthermore, we demonstrate the existence of intermediate metastable states that break the nucleation process in multiple steps; these states correspond to what is commonly known as cavitation nuclei. A single dimensionless parameter, the nucleation number, is found to control this rich phenomenology. The devised theory allows one to quantify the effect of the geometry and hydrophobicity of surface asperities on nucleation. Within the same framework, it is possible to treat both vapor cavitation, which is relevant, e.g., for organic liquids, and gas-promoted cavitation, which is commonly encountered in water. The theory is shown to be valid from the nano- to the macroscale.

Surface-particle interactions control the escape time of a particle from a nanopore-gated nanocavity system: a coarse grained simulation.

Nanopores and nanocavities are promising single molecule tools for investigating the behavior of individual molecules within confined spaces. For single molecule analysis, the total duration of time the analyte remains within the pore/cavity is highly important. However, this dwell time is ruled by a complex interplay among particle-surface interactions, external forces on the particle and Brownian diffusion, making the prediction of the dwell time challenging. Here, we show how the dwell time of an analyte in a nanocavity that is connected to the external environment by two nanopore gates depends on the sizes of the nanocavity/nanopore, as well as particle-wall interactions. For this purpose, we used a coarse-grained model that allowed us to simulate hundreds of individual analyte trajectories within a nanocavity volume. We found that by increasing the attraction between the particle and the wall, the diffusion process transforms from a usual 3D scenario (repulsive wall) to a 2D motion along the cavity surface (highly attractive wall). This results in a significant reduction of the average dwell time. Additionally, the comparison of our results with existing theories on narrow escape problem allowed us to quantify the reliability of theory derived for ideal conditions to geometries more similar to actual devices.

Translocation of linearized full-length proteins through an engineered nanopore under opposing electrophoretic force.

Nanopores have recently been used to identify and fingerprint proteins. However, because proteins, unlike DNA, do not have a uniform charge, the electrophoretic force cannot in general be used to translocate or linearize them. Here we show that the introduction of sets of charges in the lumen of the CytK nanopore spaced by ~1 nm creates an electroosmotic flow that induces the unidirectional transport of unstructured natural polypeptides against a strong electrophoretic force. Molecular dynamics simulations indicate that this electroosmotic-dominated force has a strength of ~20 pN at -100 mV, which is similar to the electric force on single-stranded DNA. Unfolded polypeptides produce current signatures as they traverse the nanopore, which may be used to identify proteins. This approach can be used to translocate and stretch proteins for enzymatic and non-enzymatic protein identification and sequencing.

Protein Sizing with 15 nm Conical Biological Nanopore YaxAB.

Nanopores are promising single-molecule tools for the electrical identification and sequencing of biomolecules. However, the characterization of proteins, especially in real-time and in complex biological samples, is complicated by the sheer variety of sizes and shapes in the proteome. Here, we introduce a large biological nanopore, YaxAB for folded protein analysis. The 15 nm cis-opening and a 3.5 nm trans-constriction describe a conical shape that allows the characterization of a wide range of proteins. Molecular dynamics showed proteins are captured by the electroosmotic flow, and the overall resistance is largely dominated by the narrow trans constriction region of the nanopore. Conveniently, proteins in the 35-125 kDa range remain trapped within the conical lumen of the nanopore for a time that can be tuned by the external bias. Contrary to cylindrical nanopores, in YaxAB, the current blockade decreases with the size of the trapped protein, as smaller proteins penetrate deeper into the constriction region than larger proteins do. These characteristics are especially useful for characterizing large proteins, as shown for pentameric C-reactive protein (125 kDa), a widely used health indicator, which showed a signal that could be identified in the background of other serum proteins.

Hydrophobically gated memristive nanopores for neuromorphic applications.

Signal transmission in the brain relies on voltage-gated ion channels, which exhibit the electrical behaviour of memristors, resistors with memory. State-of-the-art technologies currently employ semiconductor-based neuromorphic approaches, which have already demonstrated their efficacy in machine learning systems. However, these approaches still cannot match performance achieved by biological neurons in terms of energy efficiency and size. In this study, we utilise molecular dynamics simulations, continuum models, and electrophysiological experiments to propose and realise a bioinspired hydrophobically gated memristive nanopore. Our findings indicate that hydrophobic gating enables memory through an electrowetting mechanism, and we establish simple design rules accordingly. Through the engineering of a biological nanopore, we successfully replicate the characteristic hysteresis cycles of a memristor and construct a synaptic device capable of learning and forgetting. This advancement offers a promising pathway for the realization of nanoscale, cost- and energy-effective, and adaptable bioinspired memristors.

SARS-CoV-2 multi-variant rapid detector based on graphene transistor functionalized with an engineered dimeric ACE2 receptor.

Reliable point-of-care (POC) rapid tests are crucial to detect infection and contain the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The emergence of several variants of concern (VOC) can reduce binding affinity to diagnostic antibodies, limiting the efficacy of the currently adopted tests, while showing unaltered or increased affinity for the host receptor, angiotensin converting enzyme 2 (ACE2). We present a graphene field-effect transistor (gFET) biosensor design, which exploits the Spike-ACE2 interaction, the crucial step for SARS-CoV-2 infection. Extensive computational analyses show that a chimeric ACE2-Fragment crystallizable (ACE2-Fc) construct mimics the native receptor dimeric conformation. ACE2-Fc functionalized gFET allows in vitro detection of the trimeric Spike protein, outperforming functionalization with a diagnostic antibody or with the soluble ACE2 portion, resulting in a sensitivity of 20 pg/mL. Our miniaturized POC biosensor successfully detects B.1.610 (pre-VOC), Alpha, Beta, Gamma, Delta, Omicron (i.e., BA.1, BA.2, BA.4, BA.5, BA.2.75 and BQ.1) variants in isolated viruses and patient's clinical nasopharyngeal swabs. The biosensor reached a Limit Of Detection (LOD) of 65 cps/mL in swab specimens of Omicron BA.5. Our approach paves the way for a new and reusable class of highly sensitive, rapid and variant-robust SARS-CoV-2 detection systems.

Metastable wetting on superhydrophobic surfaces: continuum and atomistic views of the Cassie-Baxter-Wenzel transition.

In this Letter, we develop a continuum theory for the Cassie-Baxter-Wenzel (CB-W) transition. The proposed model accounts for the metastabilities in the wetting of rough hydrophobic surfaces, allows us to reconstruct the transition mechanism, and identifies the free energy barriers separating the CB and W states as a function of the liquid pressure. This information is crucial in the context of superhydrophobic surfaces, where there is interest in extending the duration of the metastable superhydrophobic CB state. The model is validated against free energy atomistic simulations.

Cassie-Baxter and Wenzel states on a nanostructured surface: phase diagram, metastabilities, and transition mechanism by atomistic free energy calculations.

In this work, we study the wetting of a surface decorated with one nanogroove by a bulk Lennard-Jones liquid at various temperatures and densities. We used atomistic simulations aimed at computing the free energy of the stable and metastable states of the system, as well as the intermediate states separating them. We found that the usual description in terms of Cassie-Baxter and Wenzel states is insufficient, as the system presents two states of the Cassie-Baxter type. These states are characterized by different curvatures of the meniscus. The measured free energy barrier separating the Cassie-Baxter from the Wenzel state (and vice versa) largely exceeds the thermal energy, attesting the existence of Cassie-Baxter/Wenzel metastabilities. Finally, we found that the Cassie-Baxter/Wenzel transition follows an asymmetric path, with the formation of a liquid finger on one side of the groove and a vapor bubble on the opposite side.

Geometrically Induced Selectivity and Unidirectional Electroosmosis in Uncharged Nanopores.

Selectivity toward positive and negative ions in nanopores is often associated with electroosmotic flow, the control of which is pivotal in several micro-nanofluidic technologies. Selectivity is traditionally understood to be a consequence of surface charges that alter the ion distribution in the pore lumen. Here we present a purely geometrical mechanism to induce ionic selectivity and electroosmotic flow in uncharged nanopores, and we tested it via molecular dynamics simulations. Our approach exploits the accumulation of charges, driven by an external electric field, in a coaxial cavity that decorates the membrane close to the pore entrance. The selectivity was shown to depend on the applied voltage and becomes completely inverted when reversing the voltage. The simultaneous inversion of ionic selectivity and electric field direction causes a unidirectional electroosmotic flow. We developed a quantitatively accurate theoretical model for designing pore geometry to achieve the desired electroosmotic velocity. Finally, we show that unidirectional electroosmosis also occurs in much more complex scenarios, such as a biological pore whose structure presents a coaxial cavity surrounding the pore constriction as well as a complex surface charge pattern. The capability to induce ion selectivity without altering the pore lumen shape or the surface charge may be useful for a more flexible design of selective membranes.

DNA compaction and dynamic observation in a nanopore gated sub-attoliter silicon nanocavity.

Confinement of biopolymers inside volumes with micro- or nanoscale lateral dimensions is ubiquitous in nature. Investigating the behavior of biopolymers in a confined environment is essential to improve our basic understanding in life sciences. In this work, we present a nanopore gated sub-attoliter silicon nanocavity device, which allows DNA compaction similar to that in virus capsids. Single DNA molecules can be electrically driven into the nanocavity, and then get compacted inside the nanocavity under certain conditions. The dynamic fluctuations of the compacted DNA can be monitored via ionic current measurements. The mechanism for the DNA compaction is elucidated by varying the DNA length or concentration, voltage polarity, nanocavity dimensions and ionic strength. Furthermore, Brownian dynamics simulations reveal the dynamic fluctuations of the compacted DNA, which are reflected in the measured ionic current. Our nanocavity device is anticipated to provide a controlled environment in extremely small volumes for investigating the physics of confined biopolymers.