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Biological and Medical Applications of Materials and Interfaces

Quantitative Detection of Biological Nanovesicles in Drops of Saliva Using Microcantilevers
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ACS Applied Materials & Interfaces

Cite this: ACS Appl. Mater. Interfaces 2024, 16, 1, 44–53
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https://doi.org/10.1021/acsami.3c12035
Published December 29, 2023

Copyright © 2023 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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Extracellular nanovesicles (EVs) are lipid-based vesicles secreted by cells and are present in all bodily fluids. They play a central role in communication between distant cells and have been proposed as potential indicators for the early detection of a wide range of diseases, including different types of cancer. However, reliable quantification of a specific subpopulation of EVs remains challenging. The process is typically lengthy and costly and requires purification of relatively large quantities of biopsy samples. Here, we show that microcantilevers operated with sufficiently small vibration amplitudes can successfully quantify a specific subpopulation of EVs directly from a drop (0.1 mL) of unprocessed saliva in less than 20 min. Being a complex fluid, saliva is highly non-Newtonian, normally precluding mechanical sensing. With a combination of standard rheology and microrheology, we demonstrate that the non-Newtonian properties are scale-dependent, enabling microcantilever measurements with a sensitivity identical to that in pure water when operating at the nanoscale. We also address the problem of unwanted sensor biofouling by using a zwitterionic coating, allowing efficient quantification of EVs at concentrations down to 0.1 μg/mL, based on immunorecognition of the EVs’ surface proteins. We benchmark the technique on model EVs and illustrate its potential by quantifying populations of natural EVs commonly present in human saliva. The method effectively bypasses the difficulty of targeted detection in non-Newtonian fluids and could be used for various applications, from the detection of EVs and viruses in bodily fluids to the detection of molecular clusters or nanoparticles in other complex fluids.

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Introduction

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The ability to achieve quantitative detection of specific molecules, chemical markers, or nanoscale assemblies in bodily fluids is at the heart of medical laboratory diagnostics. (1) Examples range from early detection of toxins, (2) microbiological agents, (3) or tumoral biomarkers, (4) to the routine monitoring of patients with chronic diseases such as diabetes, (5) thrombophilia, (6) or myelodysplastic syndromes. (7) Typical diagnostic bioassays comprise enzyme immunohistochemistry, (8) liquid chromatography, (6) protein and genetic electrophoresis, (9) and blood cultures. (10) While these methods offer good levels of sensitivity and specificity, they suffer from several drawbacks. First, they tend to be time-consuming and require purification and preparation steps, which can potentially alter the antigen under investigation and affect the detection itself. (9) Second, they are often expensive due to their complexity and the need for reagents or advanced experimental setup. Finally, they tend to require relatively large volumes of bodily fluids. Part of the problem comes from the complexity of bodily fluids, which contain a wide size and compositional range of molecules, proteins, biopolymers, and cells, often at concentrations significantly larger than that of the desired detection target. Additionally, most bodily fluids are highly non-Newtonian, (11) and their inherent compositional heterogeneity renders physical detection methods such as mechanical resonators (12) or nanofluidics-based approaches (13) challenging. As a result, detection methods operating directly into raw bodily fluids are sparse, especially when aiming to detect and quantify a specific target within that fluid. Being able to operate with small quantities of unprocessed bodily fluids could prove a game changer for detection and medical prognosis, potentially cutting costs and diagnosis time as well as offering measurements better reflecting the natural environment of the desired target.
Here, we show that quantitative measurements can be achieved in single drops of saliva by combining immunorecognition with mechanical detection optimized to operate on the correct scale. Saliva is arguably one of the most challenging bodily fluids to operate in given the presence of large biopolymers forming gel–liquid structures, but this can be overcome by accordingly adapting the mechanical sensing. The choice of saliva is also motivated by its potential for noninvasive and real-time diagnostics of infective and neoplastic diseases. (14,15) To illustrate the capabilities of our method, we target the model and native extracellular nanovesicles (EVs). EVs are small (30–300 nm) phospholipid-based vesicles present in most bodily fluids including blood, saliva, and urine. (16,17) They are secreted by cells into the surrounding connective matrix and are naturally used as vehicles to cargo small molecules, proteins, and nucleic acids between distant cells and throughout the body. (16) EVs play a key role as autocrine and paracrine signals (16) regulating multiple cellular functions from growth and apoptosis (18) to gene expression and antigen presentation. (19) They have been suggested as biomarkers for early detection and monitoring of various diseases including cancer, (20,21) diabetes and metabolic conditions, (5,22) neurodegenerative pathology, and viral or microbiological infections. (23) Several pathologies promote the release of specific EVs with a unique combination of antigens in terms of both type and concentration. (17) However, routine use of EVs in diagnostics is currently still limited by the costs and slowness previously highlighted. Additionally, existing characterization methods tend to focus on genetics and proteomics and require relatively expensive purification and concentration of milliliters of bodily fluids (24,25) with no accepted standards.
Using vibrating microcantilevers suitably functionalized, we are able to bypass these issues and quantify specific EV populations directly into saliva. Vibrating microcantilevers have long been used as biosensors, (26,27) including proposed approaches for cancer detection, (12) but operating in liquids tends to limit the sensitivity of the technique, (28) and most applications rely on sensing in vacuum, air, or purified solutions. (26) More recent developments in the field of nanomechanical systems have focused on optomechanical resonators (29) or sophisticated bespoke systems (30) to achieve high levels of sensitivity. However, operating directly in complex biological fluids remains a significant challenge, limiting applicability, use, and direct diagnostics. Here, we show that saliva exhibits scale-dependent viscoelasticity, a property that we exploit to operate in raw saliva with a sensitivity comparable to that achieved in pure water. The proposed approach can be upscaled, parallelized, and in principle applied for the detection of a wide range of targets directly in complex fluids.

Experimental Section

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Ultrapure water was purchased from Water AnalaR NORMAPUR, VWR International Ltd., Leicestershire, UK. For all of the experiments using saliva, fresh samples were obtained from healthy volunteers (two of the authors) and used on the same day. The saliva samples were collected in the morning between 7:00 am–9:00 am after overnight fasting, directly into a sterile glass vial, and used without any further processing or purification. The samples not immediately used were kept at 5 °C (measurements conducted later in the day) and warmed to the measuring temperature (25 °C) immediately before use, taking care to keep the sample homogeneous. For the experiments using model EVs, the desired quantity of EVs was added to the sample, which was then homogenized in a mild sonication bath (see details in the Model EVs section hereafter).

Shear Rheometry

Shear rheometry was performed using a commercial Advanced Rheometer model AR 2000 (TA Instrument, New Castle, DE, USA), equipped with 8 mm parallel plates and an environmental test chamber under nitrogen gas. The fluids were compressed between the parallel plates under atmospheric pressure until a gap of approximately 1 mm thickness and a small normal force was registered by the rheometer. To determine the full rheological response, oscillatory tests were performed at angular frequencies between 0.1 and 600 rad/s, and with strain amplitudes of 1%, after examination of the dynamic strain sweep as a function of frequency. The temperature was kept constant at 25 °C.

Microrheology

Microrheology measurements were performed using a Malvern Zetasizer NanoZSP (Malvern Panalytical, Worcestershire, UK). The tracer particles were silica nanospheres with nominal diameters of 50, 100, and 300 nm monodispersed in water with a concentration of 10 mg/mL (nanoComposix, San Diego, CA, USA). The silica particles were then diluted in water or saliva to a final concentration of 0.1 mg/mL and tip-sonicated for 45 s to remove any aggregates. Before microrheological measurements were conducted, standard dynamic light scattering (DLS) ensured a monodisperse distribution of the particles.

Lipid-Coating of the Tracers

Lipid-coating of the tracers was performed by incubating small unilamellar lipid vesicles (SUVs) of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) into a PBS solution (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4 at pH 7.4) containing the dissolved silica tracers. To ensure full coating, we estimated the total surface area of the tracers in solution and used a 10-fold excess of fluid lipid vesicles (in terms of total bilayer area) adsorbing onto the silica particles. The particles were then diluted in PBS to the desired concentrations for the experiment. DOPC was purchased in liquid form, dissolved in chloroform (Avanti Polar Lipids, AL, USA), and used without any further purification. After chloroform evaporation in a vacuum overnight, lipids were resuspended in a PBS solution at a final concentration of 10 mg/mL. PBS solution was produced using preprepared tablets (Sigma-Aldrich, St Louis, MO, USA). SUVs of diameter ∼100 nm were obtained by bath-sonicating the lipid solution at 25 °C for 10 min to produce a uniformly clear solution, followed by extrusion through a 100 nm filter (WhatMan, Sigma-Aldrich) with at least 31 passes, and then used immediately.

Model EVs

Model EVs were prepared with a biotinylated lipid mixture. The lipid mixture comprised 99.5% dipalmitoylphosphatidylcholine (DPPC) and 0.5% biotinylated-DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine). Both lipids were purchased from Avanti Polar Lipids, AL, USA, and mixed to the desired ratio in chloroform. The chloroform was then evaporated in a vacuum overnight. After resuspending the lipids in PBS and bath-sonicating them at 60 °C for 15 min, SUVs were then prepared by extrusion through a 100 nm filter. The desired proportion of model EVs was then added to the saliva samples and bath-sonicated for 5 min before use.

Cantilever Functionalization

Cantilever functionalization was performed using the same lipid mixture used for the model EVs (99.5% DPPC + 0.5% biotinylated DPPE). Before functionalization, the cantilevers underwent thorough cleaning procedures to ensure the removal of any potential contaminants. (31−33) The cantilevers were immersed in a bath of ultrapure water, followed by propan-2-ol (Merck Millipore, Billerica, MA, USA), and finally ultrapure water, for 60 min at each step. The propan-2-ol was used as purchased without further purification.
The cantilevers were then exposed to low-pressure air plasma, at a pressure of 1 mbar and power of 300 W (VacuLAB Plasma Treater, Tantec) for 30 s. Plasma-oxidation increased the hydrophilicity of the cantilevers and removed unwanted carbon contaminants. A drop of SUVs (150 μL) with a concentration of 1 mg/mL was drop-cast on the cantilever. After a 20 min incubation, the cantilevers were gently rinsed with freshly prepared PBS and left soaking in clean PBS for 1 h to ensure removal of any excess nonadsorbed vesicles. After this, the cantilevers were rinsed again with PBS.
The cantilevers were then further functionalized using streptavidin (Thermofisher, UK), which acted as a bridge between the two biotinylated lipid bilayers forming the EVs’ surface and the cantilever coating. The streptavidin functionalization was performed by first soaking the probes in a solution containing 0.1 mg/mL streptavidin in PBS. After incubation for 1.30 h, the cantilevers were gently rinsed with PBS and then left soaking in clean PBS for 1 h.
The cantilevers used in the detection of naturally occurring EVs needed a further functionalization step. This involved the binding of anti-CD9 and anti-CD81 monoclonal antibodies to the streptavidin functionalized cantilevers. The antibodies were obtained by recombinant DNA technique, with a human species reactivity and purchased from ABCAM, UK. The binding of the antibodies to the streptavidin was performed by biotinylation conjugation using the specific Biotin Conjugation Kit (Fast, Type A) Lightning-Link (ABCAM, UK). The cantilevers used for the negative controls were functionalized with streptavidin (Results section: Testing the Method with Model EVs) and pure DPPC (Results section: Detecting Specific Natural EV Subpopulations in Human Saliva). The design of suitable controls is further discussed in the Supporting Information.

Atomic Force Microscopy (AFM)

The experiments on the dynamic response of vibrating microcantilevers in water and saliva were conducted using a commercial Cypher ES AFM (Oxford Instruments, CA, USA) instrument equipped with temperature control. We used two types of commercial cantilevers with each cantilever calibrated using its thermal spectrum. (34) Initial experiments (Figure 1) were conducted with OMCL-RC800PSA silicon oxide cantilevers (Olympus, Japan) that exhibit a stiffness of 0.1–0.2 N m–1 and a resonance frequency of 20 ± 5 kHz in air. All the quantitative measurements were conducted with stiffer and shorter AC55-TS silicon oxide cantilevers (Olympus, Japan), which exhibit a typical flexural stiffness of 50 ± 5 nN/nm and a resonance frequency of 1600 ± 300 kHz in air. High-quality V1 muscovite mica discs (SPI supplies, West Chester, PA, USA) acted as a substrate where to deposit the fluid of interest; 150 μL of the fluid of interest was deposited on the mica substrate. All the experiments were performed at 25.0 °C to ensure thermal stability. (35,36) Thermal equilibrium is achieved ensuring that the cooling/heating rate of the temperature control system within the AFM is constant for at least 20 min. After thermal equilibrium was achieved, the cantilever was then fully immersed in the fluid, with its motion driven by photothermal excitation.

Figure 1

Figure 1. Probing saliva’s viscosity at different length scales. (a) Macroscopic standard shear rheological measurements highlight the non-Newtonian behavior of raw saliva (red) in comparison with pure water (blue). (b) The nanoscale viscoelastic behavior of raw saliva is also probed using a vibrating microcantilever operated with an AFM. The viscosity of the liquid surrounding the cantilever can be quantified from the frequency shift of the vibration resonances, (41) with the method applied here to comparatively probe the viscosity of water and of raw saliva. No shift is observed between water and saliva (dashed lines), and the derived viscosities for saliva are identical to water within error (inset in b) (see Experimental Section and Figure S1 for more details). The microcantilever measurements were performed using a commercial microcantilever (Olympus, OMCL-RC800 PSA) at 25.0 ± 0.1 °C, with the probe fully immersed in liquid.

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Results

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Scale-Dependence of Saliva’s Viscoelasticity

Saliva, like most bodily fluids, is a complex fluid and exhibits a nonlinear viscoelastic behavior upon applied mechanical strain. (37) Although the specific properties of saliva are person-, time-, and condition-dependent, (38,39) typical rheological measurements reveal viscosities 1 order of magnitude larger than for water in identical conditions (Figure 1). This is due to the presence of numerous large biopolymers which underpin saliva’s non-Newtonian behavior, (39) even preventing flow through a 200 nm filter. (40) At the macroscopic scale, the viscoelastic differences between pure water and saliva are obvious at all accessible shear rates (Figure 1a) and consistent with previous studies. (39) At the nanoscale, viscomechanical sensing can be performed with vibrating microresonators. (29) Considering the rheological findings, mechanical sensing in saliva could be expected to induce a significant reduction in the frequency and amplitude of any vibrating resonators compared to pure water. Interestingly, this is not necessarily the case, as illustrated here using a vibrating commercial microcantilever immersed into either water or saliva and operated with an AFM (Figure 1b). We could not observe any significant variation between the measurements obtained in pure water and saliva for oscillation amplitudes smaller than ∼20 nm: the amplitude and frequency of the microcantilever’s resonances remain broadly unchanged (Figures 1b and S1 for more details). This suggests that the cantilever experiences a nearly identical environment between water and saliva. To further quantify this observation, we use the resonance frequencies of the vibrating microcantilever to determine the viscosity of saliva as experienced at the probed vibration frequencies. (41) A constant viscosity value identical to that of pure water within error was found at both 32 and 98 kHz (second and third resonances, inset Figure 1b, see also Supporting Information section 1 for further details).
A careful comparison of all the resonances in water and in saliva (Figures 1b and S1) indicates that the same conclusion holds, regardless of the frequency probed: no systematic frequency shifts to lower values are observed from water to saliva as would have been expected for a higher viscosity liquid. (41) To rationalize this apparently counterintuitive finding, it is necessary to consider the structure of saliva across scales. Saliva can be understood as a (bio)polymeric mesh, filled primarily with water. (42) Proteins and small biological objects such as EVs are dissolved into the water and fill the mesh structure where they can diffuse freely within gaps. The mesh itself is not a static cross-linked structure, but any structural rearrangement of the polymeric network is significantly slower than that of the water it contains, hence conferring saliva its non-Newtonian macroscopic behavior. Because standard rheological measurements are macroscopic, they are dominated by the viscoelastic behavior of the polymeric mesh under strain, with its pronounced viscous and elastic responses (see Supporting Information Figure S2). In contrast, microcantilevers operate with nanoscale oscillation amplitudes comparable in size to the gaps naturally existing in the polymeric mesh. If the oscillation amplitude A of the vibrating cantilever is smaller than the average gap size S of the mesh, the cantilever primarily experiences the viscous water diffusing within the mesh, with limited impact of the polymers on the measurements.
It is important to keep in mind that, while useful, the idea of saliva as a mesh is a simplification of reality, and no single value of S exists. Instead, S can be understood as an effective size marking the transition from a polymer-dominated (at larger scale) to a water-dominated viscoelastic behavior at smaller scale. This scale-dependent viscoelasticity has previously been reported (43) in complex fluids and is likely common given their hierarchical structure. Here, we exploit this scale-dependence to enhance the detection capability of microcantilevers operating directly into saliva: if the oscillation amplitudes are small enough, the sensing microcantilever effectively operates in a simple aqueous solution, where a significantly better signal-to-noise ratio can be achieved.
To achieve enhanced microcantilever detection in saliva, we first set out to objectively identify the value of S. The fact that saliva cannot flow through a ∼200 nm filter while water can easily pass through it (13,40) suggests that S < 200 nm. This is consistent with the fact that no significant differences between water and saliva could be observed for immersed microcantilevers vibrating with amplitudes below ∼20 nm (Figure 1b), suggesting S to be in the 20–200 nm range. To independently quantify S, we used microrheology (44) with tracers ranging from 70 to 370 nm (see Supporting Information section 3 and Table S1). If the tracers are able to diffuse freely within the mesh (Figure 2a), their mean square displacement (MSD) is expected to follow standard Brownian diffusion and grow linearly over time. (44,45) In contrast, if the tracers’ diffusion is hampered by the mesh (Figure 2b,c), a subdiffusive (45) behavior is expected whereby the MSD is proportional to the time at a power α < 1. It is therefore convenient to track the anomalous diffusion exponent α for each tracer in order to distinguish “free” (α = 1) from mesh-hindered (α < 1) diffusion. An example of microrheological measurements is shown in Figure 2d,e, carried out with silica spherical nanoparticles as tracers. The average diameter of the particles is 73 ± 6 nm (50 nm nominal, see Table S1), and the use of silica tracers is motivated by the fact that the surface of the microcantilevers is primarily silica.

Figure 2

Figure 2. Passive microrheology of raw saliva with silica tracers. A cartoon representation of the system (a–c) illustrates the fact that smaller tracers (a) can diffuse more easily through the mesh formed by saliva compared to larger tracers (b). However, this assumes a limited interaction of the tracer particles with the mesh. Otherwise, interactions with the mesh reduce mobility (c) and affect smaller particles relatively more due to their larger surface to volume ratio. (d) Example of a measurements with a 73 ± 6 nm tracer (without coating) showing the MSD <r2> as a function of time t on a log–log plot. In pure water, <r2> ∝ t indicating normal Brownian diffusion. In saliva, <r2> ∝ tα with α < 1, the so-called anomalous diffusion exponent indicating subdiffusion. (e) The evolution of α at different time scales highlighting differences for water (α ≈ 1, blue curve) and saliva (α < 1) with and without a zwitterionic antifouling coating on the tracer (red and black curves, respectively). Over a short observational time scale (<10 μs), the tracers are able to freely diffuse unhindered. Over longer time scales or for larger tracers, the impact of interactions with saliva components tends to hamper the diffusion. Measurements >0.5 ms are less reliable, being close to the tracking limit of the equipment. Here, this is visible in α becoming lower for coated than uncoated tracers, despite a significantly larger MSD. (f) Time evolution of α for tracers of different sizes. Comparison of α vs tracer size at selected times (10 μs, 25 μs, 50 μs, 0.1 ms, 0.25 ms, and 0.5 ms, vertical dashed lines) suggests consistent unhindered diffusion for tracers <25 nm (inset). All the particles’ diameters are measured by DLS using the same setup as for the microrheology (see Figure S3 and Table S1 within section 3 of the Supporting Information).

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As immediately obvious from the measurements, when operating in saliva, it is crucial for the tracers to be coated with an antifouling layer so as to prevent nonspecific binding to saliva’s constituents. In principle, nonspecific binding can occur with salivary proteins (e.g., mucin fibers, lactoferrin, IgA), ions (calcium, phosphate, carbonate, and thiocyanate ions), (1) and the biopolymeric mesh itself, resulting in a lower mobility of the tracers (Figure 2c). The issue of biofouling is common to most measurements in biologically active environments, (13) often deteriorating the accuracy and precision of the measurements over time. (46) Here, it must be addressed since the microcantilever-based detection strategy implicitly assumes that saliva’s biopolymers do not bind to the cantilever but rather move around it if occasionally disturbed. If the polymers attach to the cantilever, the latter becomes part of the mesh and hence primarily measures the mesh’s viscoelastic behavior, something we aim to prevent. To tackle this issue, we coat the tracers with a self-assembled zwitterionic lipid bilayer. The zwitterionic nature of the lipid headgroups significantly reduces unwanted interactions and enhances the tracers’ diffusion, as evidenced by the clear increase in α (black to red, Figure 2d,e). Coating with a zwitterionic bilayer is a simple and effective antifouling strategy and is systematically used hereafter. Practically, the coating also increases the measured diameter of the tracers by ∼8 nm, consistent with the size of two bilayers (Supplementary section 3 and Table S1).
Focusing on the evolution of the anomalous diffusion coefficient for different size tracers (Figure 2f), it is immediately clear that the larger the size of the tracer, the smaller the value of α. In other words, larger tracers are more hindered by the polymeric mesh, resulting in an accentuated subdiffusive behavior regardless of time. By plotting the α value as a function of the tracers’ size at set times, it is possible to infer the tracer size S which would satisfy α = 1 (Figure 2f inset). We find S = 25 ± 10 nm regardless of the time considered. This provides an objective estimate for S, indicating that smaller objects can, on average, diffuse freely through saliva as if it were a purely Newtonian fluid.

Testing the Method with Model EVs

Based on the microrheology results, we use microcantilevers coated with an antifouling zwitterionic layer and oscillating with an amplitude smaller than S = 25 nm. In practice, the smaller the oscillation amplitude, the better, providing a sufficient signal-to-noise ratio. This is typically achieved using microcantilevers as small and stiff as possible, thereby ensuring a comparatively high resonance frequency and quality factor (see Supporting Information section 4) and hence sensitivity. Here, we use Olympus AC55 cantilevers (see Experimental Section) which offer some of the highest resonance frequencies and quality factors among commercially available levers.
To validate the proposed approach, we conducted a set of experiments aiming to quantify the amount of synthetic model EVs dissolved into raw saliva. The interest of using model EVs is twofold: first, since the saliva sample is prepared with a known concentration of model EVs, it allows for independent determination of the setup sensitivity. The concentration of a specific native EV’s subpopulations in saliva varies between individuals (47) and experiments, (47,48) making any independent measurements highly challenging. Here, the model EVs occur precisely as one of these subpopulations but with a unique protein marker and a specific concentration. Second, a comparison of the known EV concentrations with the measured quantities allows for calibration of the setup. To best mimic natural EVs in size and composition, we create 100 nm gel-phase phospholipid (DPPC) vesicles, with 0.5% of the lipids exposing a tether biotin acting as a specific EV marker (Figure 3a). The model EVs, dissolved in a standard phosphate buffer saline (PBS) solution, are then mixed with the raw saliva to achieve the desired final concentration, but always ensuring that the EV solution represents only 5% of the total saliva volume to minimally affect saliva’s properties (Figure 3b).

Figure 3

Figure 3. Characterization and testing of the proposed approach for targeted detection of specific EV subpopulations directly in raw saliva. The testing is carried out with model EVs composed of DPPC with 0.5% biotinylated lipids acting as surface markers (a) and dissolved into saliva (5% of the total volume from a phosphate buffer saline solution (b)). The cantilever is coated with a DPPC bilayer containing 0.5% biotinylated headgroups (c), preventing biofouling while ensuring specific binding of the model EVs after further streptavidin functionalization (d). From the changes in the cantilever’s vibration amplitude, phase, and frequency, the total mass of the target EVs binding to the cantilever can be precisely quantified despite the saliva background (e). Experimental data are fitted globally with a double exponential function, imposing the same two time scales (τ1 and τ2) for all the experiments. The most important differences occur within the first 5–10 min (τ1 = 346 ± 56 s), with only the slower evolution (τ2 = 5781 ± 778 s) present at 0.1 μg/mL and in pure saliva. (f) The maximum mass uptake (added mass at time = ∞) derived from the fitting also exhibits a double exponential increase against the EV concentration in saliva. All of the measurements are conducted at 25.0 ± 0.1 °C. The error in concentration (f) is assumed to be 10%, likely an overestimate.

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The microcantilevers are functionalized with the same gel-phase phospholipid bilayer containing 0.5% of biotinylated lipid headgroups. In this configuration, 99.5% of the zwitterionic headgroups act as a relatively robust antifouling layer with the membrane in gel phase; the biotinylated headgroups can specifically bind the model EVs after further functionalization of the microcantilever with streptavidin (Figure 3c,d). An AFM is used to track any changes in the cantilever resonance over time, allowing for quantification of the mass uptake associated with specific EV binding to the cantilever (see Supporting Information sections 4–5 and Figures S4–S5 for more details).
The results show a clear sensitivity to the model EVs binding to the cantilever (Figure 3e), with meaningful measurements achieved at concentrations down to 0.3 μg/mL in a single drop (100 μL) of saliva. The sensitivity threshold appears to be around 0.3 μg/mL, where the readout becomes close to the control. While the natural concentration of native EVs in saliva is not known, various studies estimate a range 1 or 2 orders of magnitude greater than the present sensitivity achieved. (49,50) Interestingly, a rapid uptake is visible over the first 5–10 min, followed by a slower uptake also present in the control experiment (pure saliva). This suggests that a quantitative readout is possible in less than 30 min despite the small sample volume and the absence of any sample preparation or conditioning of the sample. This compares favorably to the standard EV characterization methods based on affinity columns, (51) provided no quantification of the EVs’ encapsulated cargo is needed.
A consistent analysis of the results was achieved by globally fitting all the experimental results with a double exponential and imposing the same two time scales for all the experiments (Figure 3f). The evolution of the mass uptake with time, m(t), is fitted with the following equation:
m(t)=M−m1e−t/τ1−m2e−t/τ2
(1)
where M is the maximum mass uptake in each experiment, and m1 and m2 are the concentration-dependent fitting coefficients associated with the global time scales τ1 and τ2. The use of a double exponential model to describe an adsorption process evolving over two distinct time scales is usually referred to as the Largitte double step kinetics model. (52) The initial rapid uptake is not visible for the control and the lowest EV concentration (0.1 μg/mL), and the associated coefficient m1 are hence set to zero. When plotting M against the EV concentration C present in saliva at the start of the experiment (Figure 3f), a two-regime behavior emerges. Near the detection threshold, M increases rapidly with C, likely limited only by diffusion of the target EVs to the surface of the sensing cantilever. As more and more EVs get tethered to the surface, the binding rate decreases due to the fact that diffusing EVs need to find an uncovered region of the cantilever to bind. In this interpretation, this second regime dominates at larger C where it reduces the dependence of M over C, as visible in Figure 3e. Significantly, since the evolution of M with C is determined by the ability of EVs to bind to the microcantilever, Figure 3f effectively acts as a calibration curve for microcantilevers with the specific surface geometry used here. Additionally, since the technique requires only a drop of fluid, it can easily be multiplexed to simultaneously quantify multiple EV targets and improve accuracy.

Detecting Specific Natural EV Subpopulations in Human Saliva

The results presented in Figure 3 validate the possibility of EV detection directly in bodily fluids using vibrating microcantilevers. However, in the absence of a precise reference or accepted standard for the EV populations naturally present inside bodily fluids, it is not yet obvious whether the method can achieve meaningful measurements of natural EV subpopulations. To test this, cantilevers identical to those used in Figure 3 are functionalized with antibodies able to selectively bind common natural markers. We selected members of the tetraspanin family (CD9 and CD81) as markers. (53,54) CD9 and CD81 are cell surface glycoproteins which mediate a wide range of cell functions from B–T cell interactions to platelet activation (53) and aggregation. Naturally occurring EVs with these markers are present in every bodily fluid of a healthy human being, (53) but variations in their concentration could indicate neoplastic evolution (54) or other diseases. (55) Even if the range of concentration in healthy subjects remains to be determined with currently no accepted standard, the associated EVs have been suggested for early cancer diagnostics. (54,56) Here, they are used as a generic test for the setup’s capabilities.
Figure 4 shows the results for the detection of natural EVs exhibiting CD9 or CD81 in the saliva of two different healthy individuals. The functionalization process is similar to that used for model EVs but with an additional step wherein a biotinylated version of the desired antibody is tethered to the exposed streptavidin receptor of the cantilever. Informed by the calibration experiment (Figure 3), the measurements are conducted over only 60 min and analyzed using the same double exponential fitting with the values of τ1 and τ2 imposed as the values found in Figure 3 (τ1 = 346 s, τ2 = 5781 s) except for the baseline fitted with a single exponential. The results show a clear difference in mass uptake between the baseline (black) and the cantilever functionalized with the tetraspanin antibodies (blue and red). The data are well fitted by the double exponential function with the imposed time scale, confirming the generality of the model in this configuration. Interestingly, the results highlight interindividual differences in the total content and ratio of EVs expressing CD9 and CD81 antigens. This further shows the potential of the proposed method to contribute to novel diagnostics and developing personalized medicine. Using the results from Figure 3f as a calibration, we estimate that the concentrations of the EVs expressing CD9 and CD81 antigens are respectively 10.3 ± 0.9 and 1.5 ± 0.2 μg/mL for individual 1 and 8.2 ± 1.1 and 36.1 ± 9.0 μg/mL for individual 2. We emphasize that these values cannot be independently verified in this study and were derived on the implicit assumptions that the setup behaves similarly as with model EVs, with a similar affinity for the microcantilever and without any uspecific fusion of the EVs with the cantilever’s antifouling lipid layer. (57) These assumptions are not obvious and will require further work to confirm their reliability and benchmark the technique. (58) Additionally, complications with the current multistep functionalization make it difficult to achieve consistent control measurements (see Supporting Information section 6 and Figures S6–7). Nevertheless, the present results show that the setup has the potential to detect and quantify specific subpopulations of EVs naturally occurring in human saliva.

Figure 4

Figure 4. Detection of two natural EV subpopulations expressing CD9 and CD81 antigens on their surface, measured in saliva. The experiments are performed on a drop of unprocessed saliva samples from two healthy individuals. The cantilevers are functionalized as described in Figure 3 but with antibodies (Ab) to the targeted tetraspanin (see Experimental Section). The control probes (black) are coated only with the antifouling DPPC bilayer. As for model EVs, the mass uptake is rapid over the first 5–10 min and can be suitably analyzed imposing the same time scales as in Figure 3. The control is analyzed with a single exponential yielding in both cases a characteristic time scale of ∼550 s (see Supporting Information sections 6 for details). Experiments with anti-CD9 and anti-CD81 Abs were repeated twice for each sample with the average shown in the figure.

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Discussion

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Complex fluids are ubiquitous in nature from bodily fluids to water waste, oil reservoirs, lubricants, and food products. They play a key role in science and technology and are at the forefront of active research. (59) Most complex fluids are composed of a mixture of macromolecules dissolved in a Newtonian fluid, with the dynamics of these macromolecules conferring the resulting solution its non-Newtonian behavior. Here, we show that saliva exhibits a scale-dependent viscoelastic behavior due to the finite size of the biopolymers forming a macromolecular mesh-like structure. At the nanometer level, saliva can be considered as a Newtonian fluid, whereas the non-Newtonian behavior emerges at a scale characteristic of the mesh. While we only investigate saliva in this study, the observed scale-dependent viscoelasticity is likely to be valid for many complex fluids that exhibit a similar structure over scales, but the relevant scales are likely to be fluid specific. For saliva alone, autoimmune pathologies altering the content of mucin fibers and antibodies in saliva and other bodily fluids (1) could influence the diffusion length scale of tracers. This is also the case for any other complex fluids whose specific composition is expected to impact the diffusion length scale and, consequently, the mobility of sensing microcantilevers. Thus, the design of any detection methods for complex fluids should include a “calibration step” assessing this characteristic length scale.
We show that the scale-dependence can be exploited for conducting mechanical sensing directly in the complex fluid while retaining the signal-to-noise ratio normally only possible in simple Newtonian fluids. Calibration and testing of the setup with model lipid vesicles dissolved in raw saliva show a sensitivity in the picogram range, with the intrinsic detection noise level of the setup below this range. Based on the size and composition of the model EVs, we estimate their mass to be on the order of 0.001 pg (∼1 femtogram/model EV), suggesting an effective EV detection sensitivity in the range of 500–1000 EVs. The mass of natural EVs is likely significantly higher when taking into account the nucleic and proteic compounds, thereby increasing the sensitivity per EV. Additionally, the system used here solely relies on commercial equipment and could be significantly improved. First, the sensing cantilevers could be replaced by bespoke cantilevers with a geometry optimized for maximizing the sensing surface while retaining the ability to operate with small amplitudes and high frequencies. Second, the sensing could be developed with self-actuating microresonators, bypassing the need for the expensive laser system of the AFM. Finally, the process is suitable for parallelizing with multiple sensors operating over the same drop-size sample. This would open the possibility for simultaneous repeats and averaging as well as complementary detection of multiple targets, thereby improving the statistical accuracy and predictive power of the detection.
Detection of native EVs expressing the tetraspanin CD9 and CD81 antigens suggests that the setup is able to directly pick these subpopulations from saliva samples of healthy individuals. Here, tetraspanins are used as a test owing to their ubiquity in bodily fluids EVs. Several studies suggest that CD9 and CD81 expression may have a clinical significance in neoplastic diseases, but these are usually not seen as specific enough to represent a clear diagnostic tool. (60−65) In fact, the results shown in Figure 4 indicate significant variations in the concentration detected between healthy individuals (see also Supporting Information section 7 and Figure S8 for more details). Variations may also occur over time for the same healthy individual, something we did not explore. Further work is needed to assess the suitability of the method on more specific targets, something potentially more challenging to achieve if the associated EV subpopulation is significantly smaller. Several antigens have already been identified for immunocapture of EVs from patients with different diseases including cancer, and the method could make a significant difference in routine testing and early detection, especially considering the relatively rapid readout (<20 min). However, while it is generally well established that EVs carry information about diseases such as lung, (8) esophageal, (66) pancreatic, (67) and breast (68) cancers, there is no robust consensus on the most reliable markers with multiple candidates reported. It is therefore necessary to comparatively test several candidate markers on biopsies from healthy, precancerous, and malignant cancer patients. If successful, this would fully validate the technology and open the door for testing other potential diseases with this method.

Conclusion

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In this study, we show that the viscoelastic properties of saliva are scale-dependent, with the liquid behaving as a Newtonian fluid at the nanoscale. This is due to the comparatively large scale of the dissolved biopolymers and cell materials that create a slow-evolving mesh through which water and small molecules can move freely. Using microrheology, we demonstrate that tracers smaller than ∼25 nm can diffuse freely provided they do not interact with the biomesh. We exploit this finding to achieve quantitative mechanically based detection of model lipid nanovesicles with a specific biomarker directly inside drops of unprocessed saliva. We illustrate the potential of the technique to detect specific subpopulations of EV based on proteic markers. More work is needed to independently benchmark the technique and confirm the detection of specific EVs. The fact that the detection method is mechanical could also be further exploited to sense pathological variations in the EVs’ mechanical properties, (69) for example using higher vibration eigenmodes of the lever. (12,27) Finally, although the focus of the present study is on EV quantification motivated by cancer detection, the method can be in principle applied to the detection of any kind of nano-objects in suitable complex fluids, from viruses to nanoparticles and toxins. It could be particularly useful where samples are limited in quantity or where a relatively rapid answer is needed, for example, the analysis of toxicity changes and pollutant level after each treatment step in wastewater recovery.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsami.3c12035.

  • Example thermal spectra of AFM cantilevers in air, water, and saliva; discussion of storage and loss moduli from standard rheological measurements; characterization of the microrheology tracers; details about the acquisition and analysis of the mass uptake data; characterization of the cantilevers’ functionalization; disucssion of the baseline noise and negative controls; Figures S1–S8 and Table S1 (PDF)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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Acknowledgments

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This work was funded by the Institute of Advanced Studies and the Biophysical Science Institute (Durham University) and a Northern Accelerator Proof of Concept (grant NACCF - 224). A.E. is supported by an Australian Research Council (ARC) Discovery Early Career Research Award (DECRA) (DE220100511). We are grateful to Professor Richard Thompson for his help conducting the shear rheometer experiments.

References

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    Seminars in Cancer Biology (2018), 52 (Part_1), 26-38CODEN: SECBE7; ISSN:1044-579X. (Elsevier Ltd.)
    Most of the cancer deaths could be avoided by early detection of the tumor when it is confined to its primary site and it has not metastasized. To this aim, one of the most promising strategies is the discovery and detection of protein biomarkers shed by the young tumor to the bloodstream. Proteomic technologies, mainly mass spectrometry and multiplexed immunoassays, have rapidly developed during last years with improved limits of detection and multiplexing capability. Unfortunately, these developments together major investments and large international efforts have not resulted into new useful protein biomarkers. Here, we analyze the potential and limitations of current proteomic technologies for detecting protein biomarkers released into circulation by the tumor. We find that these technologies can hardly probe the deepest region of the plasma proteome, at concns. below the pg/mL level, where protein biomarkers for early cancer detection may exist. This clearly indicates the need of incorporating novel ultrasensitive techniques to the proteomic tool-box that can cover the inaccessible regions of the plasma proteome. We here propose biol. detectors based on nanomech. systems for discovery and detection of cancer protein biomarkers in plasma. We review the modes of operation of these devices, putting our focus on recent developments on nanomech. sandwich immunoassays and nanomech. spectrometry. The first technique enables reproducible immunodetection of proteins at concns. well below the pg/mL level, with a limit of detection on the verge of 10 ag/mL. This technol. can potentially detect low abundance tumor-assocd. proteins in plasma at the very early stages of the tumor. The second technique enables the identification of individual intact proteins by two phys. coordinates, the mass and stiffness, instead of the mass-to-charge ratio of the protein constituents. This technol. enormously simplifies the identification of proteins and it can provide useful information on interactions and posttranslational modifications, that otherwise is lost in mass spectrometry.
  13. 13
    Helton, K. L.; Nelson, K. E.; Fu, E.; Yager, P. Conditioning Saliva for Use in a Microfluidic Biosensor. Lab on Chip 2008, 8 (11), 1847– 1851,  DOI: 10.1039/b811150b
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    Conditioning saliva for use in a microfluidic biosensor
    Helton, Kristen L.; Nelson, Kjell E.; Fu, Elain; Yager, Paul
    Lab on a Chip (2008), 8 (11), 1847-1851CODEN: LCAHAM; ISSN:1473-0197. (Royal Society of Chemistry)
    This report details an approach to saliva conditioning for compatibility of raw patient samples with microfluidic immunoassay components, principally biosensor surfaces susceptible to fouling. Stimulated whole human saliva spiked with a small mol. analyte (phenytoin, 252 Da) was first depleted of cells, debris and high mol. wt. glycoproteins (mucins) using membrane filtration. This process significantly reduced but did not eliminate fouling of biosensor surfaces exposed to the sample. An H-filter, which separates solutes from mixed samples based on their diffusion in laminar flow, was used to ext. the analyte from the remaining large mol. wt. species in the filtered saliva sample. Patient samples treated in this way retained 23% of the analyte with 97% and 92% redn. in glycoproteins and proteins, resp., and resulted in 3.6 times less surface fouling than either untreated or filtered saliva alone. These sample conditioning steps will enable the use of fouling-sensitive detection techniques in future studies using clin. saliva samples.
  14. 14
    Defina, S. M.; Wang, J.; Yang, L.; Zhou, H.; Adams, J.; Cushing, W.; Tuohy, B.; Hui, P.; Liu, C.; Pham, K. SaliVISION: A Rapid Saliva-Based COVID-19 Screening and Diagnostic Test with High Sensitivity and Specificity. Sci. Rep. 2022, 12 (1), 5729,  DOI: 10.1038/s41598-022-09718-4
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    14
    SaliVISION: a rapid saliva-based COVID-19 screening and diagnostic test with high sensitivity and specificity
    DeFina, Samuel M.; Wang, Jianhui; Yang, Lei; Zhou, Han; Adams, Jennifer; Cushing, William; Tuohy, Beth; Hui, Pei; Liu, Chen; Pham, Kien
    Scientific Reports (2022), 12 (1), 5729CODEN: SRCEC3; ISSN:2045-2322. (Nature Portfolio)
    The Coronavirus disease 2019 (COVID-19) pandemic-caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)- has posed a global threat and presented with it a multitude of economic and public-health challenges. Establishing a reliable means of readily available, rapid diagnostic testing is of paramount importance in halting the spread of COVID-19, as governments continue to ease lockdown restrictions. The current std. for lab. testing utilizes reverse transcription quant. polymerase chain reaction (RT-qPCR); however, this method presents clear limitations in requiring a longer run-time as well as reduced on-site testing capability. Therefore, we investigated the feasibility of a reverse transcription looped-mediated isothermal amplification (RT-LAMP)-based model of rapid COVID-19 diagnostic testing which allows for less invasive sample collection, named SaliVISION. This novel, two-step, RT-LAMP assay utilizes a customized multiplex primer set specifically targeting SARS-CoV-2 and a visual report system that is ready to interpret within 40 min from the start of sample processing and does not require a BSL-2 level testing environment or special lab. equipment. When compared to the SalivaDirect and Thermo Fisher Scientific TaqPath RT-qPCR testing platforms, the resp. sensitivities of the SaliVISION assay are 94.29% and 98.28% while assay specificity was 100% when compared to either testing platform. Our data illustrate a robust, rapid diagnostic assay in our novel RT-LAMP test design, with potential for greater testing throughput than is currently available through lab. testing and increased on-site testing capability.
  15. 15
    Errazquin, R.; Carrasco, E.; Del Marro, S.; Suñol, A.; Peral, J.; Ortiz, J.; Rubio, J. C.; Segrelles, C.; Dueñas, M.; Garrido-Aranda, A.; Alvarez, M.; Belendez, C.; Balmaña, J.; Garcia-Escudero, R. Early Diagnosis of Oral Cancer and Lesions in Fanconi Anemia Patients: A Prospective and Longitudinal Study Using Saliva and Plasma. Cancers 2023, 15 (6), 1871,  DOI: 10.3390/cancers15061871
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    15
    Early Diagnosis of Oral Cancer and Lesions in Fanconi Anemia Patients: A Prospective and Longitudinal Study Using Saliva and Plasma
    Errazquin, Ricardo; Carrasco, Estela; Del Marro, Sonia; Sunol, Anna; Peral, Jorge; Ortiz, Jessica; Rubio, Juan Carlos; Segrelles, Carmen; Duenas, Marta; Garrido-Aranda, Alicia; Alvarez, Martina; Belendez, Cristina; Balmana, Judith; Garcia-Escudero, Ramon
    Cancers (2023), 15 (6), 1871CODEN: CANCCT; ISSN:2072-6694. (MDPI AG)
    Simple Summary: Patients with Fanconi anemia (FA) have a very high risk of developing oral lesions and squamous carcinomas at early ages. As treatment strategies in this setting are very limited, new early diagnosis methods are urgently needed. We performed a pilot, prospective clin. study in which saliva and plasma samples were analyzed with the deep sequencing of cancer genes. The patients included had apparently normal oral mucosa when recruited. Mutations were detected in the liq. biopsies with allele frequencies of down to 0.07%. We found that patients with mutations displayed a higher risk of developing lesions/carcinomas after mutation detection. We propose that this non-invasive, highly sensitive technol. could allow for the better management of these pathologies in FA individuals. Abstr.: Fanconi anemia (FA) patients display an exacerbated risk of oral squamous cell carcinoma (OSCC) and oral potentially malignant lesions (OPMLs) at early ages. As patients have defects in their DNA repair mechanisms, std.-of-care treatments for OSCC such as radiotherapy and chemotherapy, give rise to severe toxicities. New methods for early diagnosis are urgently needed to allow for treatment in early disease stages and achieve better clin. outcomes. We conducted a prospective, longitudinal study wherein liq. biopsies from sixteen patients with no clin. diagnoses of OPML and/or OSCC were analyzed for the presence of mutations in cancer genes. The DNA from saliva and plasma were sequentially collected and deep-sequenced, and the clin. evaluation followed over a median time of approx. 2 years. In 9/16 FA patients, we detected mutations in cancer genes (mainly TP53) with minor allele frequencies (MAF) of down to 0.07%. Importantly, all patients that had mutations and clin. follow-up data after mutation detection (n = 6) developed oral precursor lesions or OSCC. The lead-time between mutation detection and tumor diagnosis ranged from 23 to 630 days. Strikingly, FA patients without mutations displayed a significantly lower risk of developing precursor lesions or OSCCs. Therefore, our diagnostic approach could help to stratify FA patients into risk groups, which would allow for closer surveillance for OSCCs or precursor lesions.
  16. 16
    Deshmukh, S. K.; Khan, M. A.; Singh, S.; Singh, A. P. Extracellular Nanovesicles: From Intercellular Messengers to Efficient Drug Delivery Systems. ACS Omega 2021, 6 (3), 1773– 1779,  DOI: 10.1021/acsomega.0c05539
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    Extracellular Nanovesicles: From Intercellular Messengers to Efficient Drug Delivery Systems
    Deshmukh, Sachin Kumar; Khan, Mohammad Aslam; Singh, Seema; Singh, Ajay Pratap
    ACS Omega (2021), 6 (3), 1773-1779CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)
    A review. Nanosized extracellular vesicles (nEV) are released by all the eukaryotic cells into the extracellular spaces. They serve as crucial mediators of intercellular communication, and their presence has been detected in a variety of body fluids. nEV carry nucleic acids, lipids, proteins, and metabolites from the donor cells and transfer them to the recipient cells in the vicinity or distant locations to cause changes in their biol. phenotypes. This very property of nEV makes them a suitable carrier of the drugs for therapeutic applications. The use of nEV as a drug delivery system offers several advantages over synthetic nanoparticles, including biocompatibility, natural targeting ability, and long-term safety. Further, nEV can be isolated from various biol. sources, quickly loaded with the drug of choice, and modified to further enhance their utility as targeted drug delivery vehicles. Here we review these aspects of nEV and discuss the parameters that should be kept in mind while choosing the nEV source, drug loading method, and surface modification strategies. We also discuss the challenges assocd. with the nEV-based drug delivery platforms that must be overcome before realizing their full potential in clin. applications.
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    Fais, S.; O’Driscoll, L.; Borras, F. E.; Buzas, E.; Camussi, G.; Cappello, F.; Carvalho, J.; Cordeiro da Silva, A.; Del Portillo, H.; El Andaloussi, S.; Ficko Trček, T.; Furlan, R.; Hendrix, A.; Gursel, I.; Kralj-Iglic, V.; Kaeffer, B.; Kosanovic, M.; Lekka, M. E.; Lipps, G.; Logozzi, M.; Marcilla, A.; Sammar, M.; Llorente, A.; Nazarenko, I.; Oliveira, C.; Pocsfalvi, G.; Rajendran, L.; Raposo, G.; Rohde, E.; Siljander, P.; van Niel, G.; Vasconcelos, M. H.; Yáñez-Mó, M.; Yliperttula, M. L.; Zarovni, N.; Zavec, A. B.; Giebel, B. Evidence-Based Clinical Use of Nanoscale Extracellular Vesicles in Nanomedicine. ACS Nano 2016, 10 (4), 3886– 3899,  DOI: 10.1021/acsnano.5b08015
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    Evidence-Based Clinical Use of Nanoscale Extracellular Vesicles in Nanomedicine
    Fais, Stefano; O'Driscoll, Lorraine; Borras, Francesc E.; Buzas, Edit; Camussi, Giovanni; Cappello, Francesco; Carvalho, Joana; Cordeiro da Silva, Anabela; Del Portillo, Hernando; El Andaloussi, Samir; Ficko Trcek, Tanja; Furlan, Roberto; Hendrix, An; Gursel, Ihsan; Kralj-Iglic, Veronika; Kaeffer, Bertrand; Kosanovic, Maja; Lekka, Marilena E.; Lipps, Georg; Logozzi, Mariantonia; Marcilla, Antonio; Sammar, Marei; Llorente, Alicia; Nazarenko, Irina; Oliveira, Carla; Pocsfalvi, Gabriella; Rajendran, Lawrence; Raposo, Graca; Rohde, Eva; Siljander, Pia; van Niel, Guillaume; Vasconcelos, M. Helena; Yanez-Mo, Maria; Yliperttula, Marjo L.; Zarovni, Natasa; Zavec, Apolonija Bedina; Giebel, Bernd
    ACS Nano (2016), 10 (4), 3886-3899CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    Recent research has demonstrated that all body fluids assessed contain substantial amts. of vesicles that range in size from 30 to 1000 nm and that are surrounded by phospholipid membranes contg. different membrane microdomains such as lipid rafts and caveolae. The most prominent representatives of these so-called extracellular vesicles (EVs) are nanosized exosomes (70-150 nm), which are derivs. of the endosomal system, and microvesicles (100-1000 nm), which are produced by outward budding of the plasma membrane. Nanosized EVs are released by almost all cell types and mediate targeted intercellular communication under physiol. and pathophysiol. conditions. Contg. cell-type-specific signatures, EVs have been proposed as biomarkers in a variety of diseases. Furthermore, according to their phys. functions, EVs of selected cell types have been used as therapeutic agents in immune therapy, vaccination trials, regenerative medicine, and drug delivery. Undoubtedly, the rapidly emerging field of basic and applied EV research will significantly influence the biomedicinal landscape in the future. In this Perspective, we, a network of European scientists from clin., academic, and industry settings collaborating through the H2020 European Cooperation in Science and Technol. (COST) program European Network on Microvesicles and Exosomes in Health and Disease (ME-HAD), demonstrate the high potential of nanosized EVs for both diagnostic and therapeutic (i.e., theranostic) areas of nanomedicine.
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    Sun, W.; Ren, Y.; Lu, Z.; Zhao, X. The Potential Roles of Exosomes in Pancreatic Cancer Initiation and Metastasis. Molecular Cancer 2020, 19 (1), 135,  DOI: 10.1186/s12943-020-01255-w
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    The potential roles of exosomes in pancreatic cancer initiation and metastasis
    Sun Wei; Ren Ying; Lu Zaiming; Zhao Xiangxuan
    Molecular cancer (2020), 19 (1), 135 ISSN:.
    Pancreatic cancer (PaCa) is an insidious and highly metastatic malignancy, with a 5-year survival rate of less than 5%. So far, the pathogenesis and progression mechanisms of PaCa have been poorly characterized. Exosomes correspond to a class of extracellular nanovesicles, produced by a broad range of human somatic and cancerous cells. These particular nanovesicles are mainly composed by proteins, genetic substances and lipids, which mediate signal transduction and material transport. A large number of studies have indicated that exosomes may play decisive roles in the occurrence and metastatic progression of PaCa. This article summarizes the specific functions of exosomes and their underlying molecular mechanisms in mediating the initiation and metastatic capability of PaCa.
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    Bobrie, A.; Colombo, M.; Raposo, G.; Théry, C. Exosome Secretion: Molecular Mechanisms and Roles in Immune Responses. Traffic 2011, 12 (12), 1659– 1668,  DOI: 10.1111/j.1600-0854.2011.01225.x
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    Exosome secretion: molecular mechanisms and roles in immune responses
    Bobrie, Angelique; Colombo, Marina; Raposo, Graca; Thery, Clotilde
    Traffic (Oxford, United Kingdom) (2011), 12 (12), 1659-1668CODEN: TRAFFA; ISSN:1398-9219. (Wiley-Blackwell)
    A review. Exosomes are small membrane vesicles, secreted by most cell types from multivesicular endosomes, and thought to play important roles in intercellular communications. Initially described in 1983, as specifically secreted by reticulocytes, exosomes became of interest for immunologists in 1996, when they were proposed to play a role in antigen presentation. More recently, the finding that exosomes carry genetic materials, mRNA and miRNA, has been a major breakthrough in the field, unveiling their capacity to vehicle genetic messages. It is now clear that not only immune cells but probably all cell types are able to secrete exosomes: their range of possible functions expands well beyond immunol. to neurobiol., stem cell and tumor biol., and their use in clin. applications as biomarkers or as therapeutic tools is an extensive area of research. Despite intensive efforts to understand their functions, two issues remain to be solved in the future: (i) what are the physiol. function(s) of exosomes in vivo and (ii) what are the relative contributions of exosomes and of other secreted membrane vesicles in these proposed functions. Here, we will focus on the current ideas on exosomes and immune responses, but also on their mechanisms of secretion and the use of this knowledge to elucidate the latter issue.
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    Huang, T.; Deng, C. X. Current Progresses of Exosomes as Cancer Diagnostic and Prognostic Biomarkers. Int. J. Biol. Sci. 2019, 15 (1), 1,  DOI: 10.7150/ijbs.27796
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    Current progresses of exosomes as cancer diagnostic and prognostic biomarkers
    Huang, Tao; Deng, Chu-Xia
    International Journal of Biological Sciences (2019), 15 (1), 1-11CODEN: IJBSB9; ISSN:1449-2288. (Ivyspring International Publisher)
    A review. Cancer related exosomes are nano-size membrane vesicles that play important roles in tumor microenvironment. Emerging evidence indicates that exosomes can load unique cargoes, including proteins and nucleic acids that reflect the condition of tumor. Therefore, exosomes are being used as diagnostic and prognostic biomarkers for various cancers. In this review, we describe the current progresses of cancer related exosomes, including their biogenesis, mol. contents, biol. functions, sources where they are derived from, and methods for their detection. We will also discuss the current exosomal biomarkers and the utilization of them for early diagnosis and prognostics in cancer.
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    Li, W.; Li, C.; Zhou, T.; Liu, X.; Liu, X.; Li, X.; Chen, D. Role of Exosomal Proteins in Cancer Diagnosis. Mol. Cancer 2017, 16 (1), 145,  DOI: 10.1186/s12943-017-0706-8
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    Role of exosomal proteins in cancer diagnosis
    Li, Weihua; Li, Chuanyun; Zhou, Tong; Liu, Xiuhong; Liu, Xiaoni; Li, Xiuhui; Chen, Dexi
    Molecular Cancer (2017), 16 (), 145/1-145/12CODEN: MCOACG; ISSN:1476-4598. (BioMed Central Ltd.)
    Exosomes are emerging as a new type of cancer biomarkers. Exosome is a bilayered nano-sized vesicle secreted by various living cells in all body fluids. Based on the expanding albeit incomplete knowledge of their biogenesis, secretion by cells and cancer cell-specific mol. and genetic contents, exosomes are viewed as promising, clin.-relevant surrogates of cancer progression and response to therapy. Preliminary proteomic, genetic and functional profiling of cancer cell-derived or cancer plasma-derived exosomes confirms their unique characteristics. Alterations in protein or nucleic acid profiles of exosomes in plasma correlate with pathol. processes of many diseases including cancer. However, previous studies on exosome application in cancer diagnosis and treatment mainly focussed on miRNAs. With the development of rapid large-scale prodn., purifn., extn. and screening of exosomal contents, exosomal protein application can be explored for early stage cancer diagnosis, monitoring and prognosis evaluation. Here, we summarized the recent developments in application of exosomal proteins for cancer diagnosis.
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    Zhou, F.; Huang, L.; Qu, S. L.; Chao, R.; Yang, C.; Jiang, Z. S.; Zhang, C. The Emerging Roles of Extracellular Vesicles in Diabetes and Diabetic Complications. Clin. Chim. Acta 2019, 497, 130– 136,  DOI: 10.1016/j.cca.2019.07.032
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    The emerging roles of extracellular vesicles in diabetes and diabetic complications
    Zhou, Fan; Huang, Liang; Qu, Shun-Lin; Chao, Ru; Yang, Chen; Jiang, Zhi-Sheng; Zhang, Chi
    Clinica Chimica Acta (2019), 497 (), 130-136CODEN: CCATAR; ISSN:0009-8981. (Elsevier B.V.)
    A review. Diabetes and diabetic vascular complications are now the leading cause of death in the world. The effects of traditional medical treatment are usually limited and accompanied by many side effects, such as hypoglycemia, obesity, liver and kidney damage, and gastrointestinal adverse reactions. Thus, it is urgent to explore some new strategies for the treatment of patients with diabetes. Recently, extracellular vesicles have received increased attention because of their emerging roles of cell-to-cell communication under physiol. and pathol. conditions. In addn., because of their abundant existence in almost all body fluids, as well as their plentiful cargos of bioactive proteins and miRNAs they carry, extracellular vesicles have a strong potential for therapeutic and diagnostic applications in many metabolic diseases, such as obesity and insulin resistance. Here, with the aim of providing the basis for the development of new treatments for diabetes, we review current understanding of extracellular vesicles and the crit. roles it has played in the onset and progression of diabetes and diabetic complications.
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    Vella, L. J.; Hill, A. F.; Cheng, L. Focus on Extracellular Vesicles: Exosomes and Their Role in Protein Trafficking and Biomarker Potential in Alzheimer’s and Parkinson’s Disease. Int. J. Mol. Sci. 2016 2016, 17 (2), 173,  DOI: 10.3390/ijms17020173
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    Cvjetkovic, A.; Lötvall, J.; Lässer, C. The Influence of Rotor Type and Centrifugation Time on the Yield and Purity of Extracellular Vesicles. J. Extracell. Ves. 2014, 3 (1), 23111,  DOI: 10.3402/jev.v3.23111
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    There is no corresponding record for this reference.
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    Minciacchi, V. R.; Freeman, M. R.; Di Vizio, D. Extracellular Vesicles in Cancer: Exosomes, Microvesicles and the Emerging Role of Large Oncosomes. Seminars Cell Develop. Biol. 2015, 40, 41– 51,  DOI: 10.1016/j.semcdb.2015.02.010
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    Extracellular Vesicles in Cancer: Exosomes, Microvesicles and the Emerging Role of Large Oncosomes
    Minciacchi, Valentina R.; Freeman, Michael R.; Di Vizio, Dolores
    Seminars in Cell & Developmental Biology (2015), 40 (), 41-51CODEN: SCDBFX; ISSN:1084-9521. (Elsevier Ltd.)
    Since their first description, extracellular vesicles (EVs) have been the topic of avid study in a variety of physiol. contexts and are now thought to play an important role in cancer. The state of knowledge on biogenesis, mol. content and horizontal communication of diverse types of cancer EVs has expanded considerably in recent years. As a consequence, a plethora of information about EV compn. and mol. function has emerged, along with the notion that cancer cells rely on these particles to invade tissues and propagate oncogenic signals at distance. The no. of in vivo studies, designed to achieve a deeper understanding of the extent to which EV biol. can be applied to clin. relevant settings, is rapidly growing. This review summarizes recent studies on cancer-derived EV functions, with an overview about biogenesis and mol. cargo of exosomes, microvesicles and large oncosomes. We also discuss current challenges and emerging technologies that might improve EV detection in various biol. systems. Further studies on the functional role of EVs in specific steps of cancer formation and progression will expand our understanding of the diversity of paracrine signaling mechanisms in malignant growth.
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    McKendry, R.; Zhang, J.; Arntz, Y.; Strunz, T.; Hegner, M.; Lang, H. P.; Baller, M. K.; Certa, U.; Meyer, E.; Güntherodt, H. J.; Gerber, C. Multiple Label-Free Biodetection and Quantitative DNA-Binding Assays on a Nanomechanical Cantilever Array. Proc. Natl. Acad. Sci. U. S. A. 2002, 99 (15), 9783– 9788,  DOI: 10.1073/pnas.152330199
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    26
    Multiple label-free biodetection and quantitative DNA-binding assays on a nanomechanical cantilever array
    McKendry, Rachel; Zhang, Jiayun; Arntz, Youri; Strunz, Torsten; Hegner, Martin; Lang, Hans Peter; Baller, Marko K.; Certa, Ulrich; Meyer, Ernst; Guntherodt, Hans-Joachim; Gerber, Christoph
    Proceedings of the National Academy of Sciences of the United States of America (2002), 99 (15), 9783-9788CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    We report a microarray of cantilevers to detect multiple unlabeled biomols. simultaneously at nanomolar concns. within minutes. Ligand-receptor binding interactions such as DNA hybridization or protein recognition occurring on microfabricated silicon cantilevers generate nanomech. bending, which is detected optically in situ. Differential measurements including ref. cantilevers on an array of eight sensors can sequence-specifically detect unlabeled DNA targets in 80-fold excess of nonmatching DNA as a background and discriminate 3' and 5' overhangs. Our expts. suggest that the nanomech. motion originates from predominantly steric hindrance effects and depends on the concn. of DNA mols. in soln. We show that cantilever arrays can be used to investigate the thermodn. of biomol. interactions mech., and we have found that the specificity of the reaction on a cantilever is consistent with soln. data. Hence cantilever arrays permit multiple binding assays in parallel and can detect femtomoles of DNA on the cantilever at a DNA concn. in soln. of 75 nM.
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    De Pastina, A.; Padovani, F.; Brunetti, G.; Rotella, C.; Niosi, F.; Usov, V.; Hegner, M. Multimodal Real-Time Frequency Tracking of Cantilever Arrays in Liquid Environment for Biodetection: Comprehensive Setup and Performance Analysis. Rev. Sci. Instrum. 2021, 92 (6), 065001  DOI: 10.1063/5.0047631
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    27
    Multimodal real-time frequency tracking of cantilever arrays in liquid environment for biodetection: Comprehensive setup and performance analysis
    De Pastina, Annalisa; Padovani, Francesco; Brunetti, Giulio; Rotella, Chiara; Niosi, Fabio; Usov, Victor; Hegner, Martin
    Review of Scientific Instruments (2021), 92 (6), 065001CODEN: RSINAK; ISSN:0034-6748. (American Institute of Physics)
    We present a nanomech. platform for real-time quant. label-free detection of target biomols. in a liq. environment with mass sensitivity down to few pg. Newly fabricated arrays of up to 18 cantilevers are integrated in a micromachined fluidic chamber, connected to software-controlled fluidic pumps for automated sample injections. We discuss two functionalization approaches to independently sensitize the interface of different cantilevers. A custom piezo-stack actuator and optical readout system enable the measurement of resonance frequencies up to 2 MHz. We implement a new measurement strategy based on a phase-locked loop (PLL), built via inhouse developed software. The PLL allows us to track, within the same expt., the evolution of resonance frequency over time of up to four modes for all the cantilevers in the array. With respect to the previous measurement technique, based on std. frequency sweep, the PLL enhances the estd. detection limit of the device by a factor of 7 (down to 2 pg in 5 min integration time) and the time resoln. by more than threefold (below 15 s), being on par with com. gold-std. techniques. The detection limit and noise of the new setup are investigated via Allan deviation and std. deviation anal., considering different resonance modes and interface chemistries. As a proof-of-concept, we show the immobilization and label-free in situ detection of live bacterial cells (E. coli), demonstrating qual. and quant. agreement in the mech. response of three different resonance modes. (c) 2021 American Institute of Physics.
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    Burg, T. P.; Godin, M.; Knudsen, S. M.; Shen, W.; Carlson, G.; Foster, J. S.; Babcock, K.; Manalis, S. R. Weighing of Biomolecules, Single Cells and Single Nanoparticles in Fluid. Nature 2007, 446 (7139), 1066– 1069,  DOI: 10.1038/nature05741
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    Weighing of biomolecules, single cells and single nanoparticles in fluid
    Burg, Thomas P.; Godin, Michel; Knudsen, Scott M.; Shen, Wenjiang; Carlson, Greg; Foster, John S.; Babcock, Ken; Manalis, Scott R.
    Nature (London, United Kingdom) (2007), 446 (7139), 1066-1069CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Nanomech. resonators enable the measurement of mass with extraordinary sensitivity. Previously, samples as light as 7 zeptograms (1 zg = 10-21 g) have been weighed in vacuum, and proton-level resoln. seems to be within reach. Resolving small mass changes requires the resonator to be light and to ring at a very pure tone - i.e., with a high quality factor. In soln., viscosity severely degrades both of these characteristics, thus preventing many applications in nanotechnol. and the life sciences where fluid is required. Although the resonant structure can be designed to minimize viscous loss, resoln. is still substantially degraded when compared to measurements made in air or vacuum. An entirely different approach eliminates viscous damping by placing the soln. inside a hollow resonator that is surrounded by vacuum. Here the authors demonstrate that suspended microchannel resonators can weigh single nanoparticles, single bacterial cells and sub-monolayers of adsorbed proteins in water with sub-femtogram resoln. (1 Hz bandwidth). Central to these results is the authors' observation that viscous loss due to the fluid is negligible compared to the intrinsic damping of the authors' silicon crystal resonator. The combination of the low resonator mass (100 ng) and high quality factor (15,000) enables an improvement in mass resoln. of six orders of magnitude over a high-end com. quartz crystal microbalance. This gives access to intriguing applications, such as mass-based flow cytometry, the direct detection of pathogens, or the nonoptical sizing and mass d. measurement of colloidal particles.
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    Gil-Santos, E.; Ruz, J. J.; Malvar, O.; Favero, I.; Lemaître, A.; Kosaka, P. M.; García-López, S.; Calleja, M.; Tamayo, J. Optomechanical Detection of Vibration Modes of a Single Bacterium. Nat. Nanotechnol. 2020, 15 (6), 469– 474,  DOI: 10.1038/s41565-020-0672-y
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    Optomechanical detection of vibration modes of a single bacterium
    Gil-Santos, Eduardo; Ruz, Jose J.; Malvar, Oscar; Favero, Ivan; Lemaitre, Aristide; Kosaka, Priscila. M.; Garcia-Lopez, Sergio; Calleja, Montserrat; Tamayo, Javier
    Nature Nanotechnology (2020), 15 (6), 469-474CODEN: NNAABX; ISSN:1748-3387. (Nature Research)
    Low-frequency vibration modes of biol. particles, such as proteins, viruses and bacteria, involve coherent collective vibrations at frequencies in the terahertz and gigahertz domains. These vibration modes carry information on their structure and mech. properties, which are good indicators of their biol. state. A particular regime in the physics of coupled mech. resonators was harnessed to directly measure these low-frequency mech. resonances of a single bacterium. The bacterium was deposited on the surface of an ultrahigh frequency optomech. disk resonator in ambient conditions. The vibration modes of the disk and bacterium hybridize when their assocd. frequencies are similar. A general theor. framework was developed to describe this coupling, which allows retrieving the eigenfrequencies and mech. loss of the bacterium low-frequency vibration modes (quality factor). The effect of hydration on these vibrational modes was analyzed. Ultrahigh frequency optomech. resonators can be used for vibrational spectrometry with the unique capability to obtain information on single biol. entities.
  30. 30
    Dominguez-Medina, S.; Fostner, S.; Defoort, M.; Sansa, M.; Stark, A.-K.; Halim, M. A.; Vernhes, E.; Gely, M.; Jourdan, G.; Alava, T.; Boulanger, P.; Masselon, C.; Hentz, S. Neutral Mass Spectrometry of Virus Capsids above 100 Megadaltons with Nanomechanical Resonators. Science 2018, 362 (6417), 918– 922,  DOI: 10.1126/science.aat6457
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    Neutral mass spectrometry of virus capsids above 100 megadaltons with nanomechanical resonators
    Dominguez-Medina, Sergio; Fostner, Shawn; Defoort, Martial; Sansa, Marc; Stark, Ann-Kathrin; Halim, Mohammad Abdul; Vernhes, Emeline; Gely, Marc; Jourdan, Guillaume; Alava, Thomas; Boulanger, Pascale; Masselon, Christophe; Hentz, Sebastien
    Science (Washington, DC, United States) (2018), 362 (6417), 918-922CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Measurement of the mass of particles in the mega- to gigadalton range is challenging with conventional mass spectrometry. Although this mass range appears optimal for nanomech. resonators, nanomech. mass spectrometers often suffer from prohibitive sample loss, extended anal. time, or inadequate resoln. We report on a system architecture combining nebulization of the analytes from soln., their efficient transfer and focusing without relying on electromagnetic fields, and the mass measurements of individual particles using nanomech. resonator arrays. This system detd. the mass distribution of ∼30-megadalton polystyrene nanoparticles with high detection efficiency and effectively performed mol. mass measurements of empty or DNA-filled bacteriophage T5 capsids with masses up to 105 megadaltons using less than 1 pmol of sample and with an instrument resoln. above 100.
  31. 31
    Miller, E. J.; Trewby, W.; Farokh Payam, A.; Piantanida, L.; Cafolla, C.; Voïtchovsky, K. Sub-Nanometer Resolution Imaging with Amplitude-Modulation Atomic Force Microscopy in Liquid. J. Visualiz. Experiments 2016, (118), e54924,  DOI: 10.3791/54924
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    Cafolla, C.; Voitchovsky, K. Lubricating Properties of Single Metal Ions at Interfaces. Nanoscale 2018, 10 (25), 11831– 11840,  DOI: 10.1039/C8NR02859A
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    Lubricating properties of single metal ions at interfaces
    Cafolla, Clodomiro; Voitchovsky, Kislon
    Nanoscale (2018), 10 (25), 11831-11840CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
    The behavior of ionic solns. confined in nanoscale gaps is central to countless processes, from biomol. function to electrochem., energy storage and lubrication. However, no clear link exists between the mol.-level behavior of the liq. and macroscopic observations. The problem mainly comes from the difficulty to interrogate a small no. of liq. mols. Here, we use at. force microscopy to investigate the viscoelastic behavior of pure water and ionic solns. down to the single ion level. The results show a glassy-like behavior for pure water, with single metal ions acting as lubricants by reducing the elasticity of the nano-confined soln. and the magnitude of the hydrodynamic friction. At small ionic concn. (<20 mM) the results can be quant. explained by the ions moving via a thermally-activated process resisted by the ion's hydration water (Prandtl-Tomlinson model). The model breaks down at higher salt concns. due to ion-ion interaction effects that can no longer be neglected. The correlations are confirmed by direct sub-nanometer imaging of the interface at equil. The results provide a mol.-level basis for explaining the tribol. properties of aq. solns. and suggest that ion-ion interactions create mesoscale effects that prevent a direct link between nanoscale and macroscopic measurements.
  33. 33
    Cafolla, C.; Foster, W.; Voitchovsky, K. Lubricated Friction around Nanodefects. Sci. Adv. 2020, 6 (14), eaaz3673,  DOI: 10.1126/sciadv.aaz3673
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    Butt, H.-J.; Jaschke, M. Calculation of Thermal Noise in Atomic Force Microscopy. Nanotechnology 1995, 6 (1), 1– 7,  DOI: 10.1088/0957-4484/6/1/001
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    Cafolla, C.; Voitchovsky, K. Real-Time Tracking of Ionic Nano-Domains under Shear Flow. Sci. Rep. 2021, 11 (1), 19540,  DOI: 10.1038/s41598-021-98137-y
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    Real-time tracking of ionic nano-domains under shear flow
    Cafolla, Clodomiro; Voitchovsky, Kislon
    Scientific Reports (2021), 11 (1), 19540CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
    The behavior of ions at solid-liq. interfaces underpins countless phenomena, from the conduction of nervous impulses to charge transfer in solar cells. In most cases, ions do not operate as isolated entities, but in conjunction with neighboring ions and the surrounding soln. In aq. solns., recent studies suggest the existence of group dynamics through water-mediated clusters but results allowing direct tracking of ionic domains with at. precision are scarce. Here, we use high-speed at. force microscopy to track the evolution of Rb+, K+, Na+ and Ca2+ nano-domains contg. 20 to 120 ions adsorbed at the surface of mica in aq. soln. The interface is exposed to a shear flow able to influence the lateral motion of single ions and clusters. The results show that, when in groups, metal ions tend to move with a relatively slow dynamics, as can be expected from a correlated group motion, with an av. residence timescale of ∼ 1-2 s for individual ions at a given at. site. The av. group velocity of the clusters depends on the ions charge d. and can be explained by the ion's hydration state. The lateral shear flow of the fluid is insufficient to desorb ions, but indirectly influences the diffusion dynamics by acting on ions in close vicinity to the surface. The results provide insights into the dynamics of ion clusters when adsorbed onto an immersed solid under shear flow.
  36. 36
    Trewby, W.; Faraudo, J.; Voitchovsky, K. Long-Lived Ionic Nano-Domains Can Modulate the Stiffness of Soft Interfaces. Nanoscale 2019, 11, 4376– 4384,  DOI: 10.1039/C8NR06339G
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    Long-lived ionic nano-domains can modulate the stiffness of soft interfaces
    Trewby, William; Faraudo, Jordi; Voitchovsky, Kislon
    Nanoscale (2019), 11 (10), 4376-4384CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
    Metal ions underpin countless processes at bio-interfaces, including maintaining electroneutrality, modifying mech. properties and driving bioenergetic activity. These processes are typically described by ions behaving as independently diffusing point charges. Here we show that Na+ and K+ ions instead spontaneously form correlated nanoscale networks that evolve over seconds at the interface with an anionic bilayer in soln. Combining single-ion level at. force microscopy and mol. dynamic simulations we investigate the configuration and dynamics of Na+, K+, and Rb+ at the lipid surface. We identify two distinct ionic states: the well-known direct electrostatic interaction with lipid headgroups and a water-mediated interaction that can drive the formation of remarkably long-lived ionic networks which evolve over many seconds. We show that this second state induces ionic network formation via correlative ion-ion interactions that generate an effective energy well of -0.4kBT/ion. These networks locally reduce the stiffness of the membrane, providing a spontaneous mechanism for tuning its mech. properties with nanoscale precision. The ubiquity of water-mediated interactions suggest that our results have far-reaching implications for controlling the properties of soft interfaces.
  37. 37
    Granick, S.; Zhu, Y.; Lee, H. Slippery Questions about Complex Fluids Flowing Past Solids. Nat. Mater. 2003 2:4 2003, 2 (4), 221– 227,  DOI: 10.1038/nmat854
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    Mate, M.; Carpick, R. W. Tribology on the Small Scale: A Modern Textbook on Friction, Lubrication, and Wear, 2nd ed; Oxford University Press, 2019.
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    Inoue, H.; Ono, K.; Masuda, W.; Inagaki, T.; Yokota, M.; Inenaga, K. Rheological Properties of Human Saliva and Salivary Mucins. J. Oral Biosci. 2008, 50 (2), 134– 141,  DOI: 10.1016/S1349-0079(08)80027-8
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    Rheological properties of human saliva and salivary mucins
    Inoue, Hiroko; Ono, Kentaro; Masuda, Wataru; Inagaki, Tomohiro; Yokota, Makoto; Inenaga, Kiyotoshi
    Journal of Oral Biosciences (2008), 50 (2), 134-141CODEN: JOBOA8; ISSN:1349-0079. (Japanese Association for Oral Biology)
    It is unclear which factors in saliva contribute to its rheol. properties such as viscosity and spinnbarkeit. In this study, we investigated relationships between the rheol. properties (viscosity and spinnbarkeit) and two salivary mucin (MUC5B and MUC7) levels in saliva. Unstimulated (UWS) and chewing-stimulated (CWS) whole saliva were collected from healthy young adults, and the viscosities and spinnbarkeits were measured. The image densities of MUC5B and MUC7 bands in saliva were measured by Stains-All staining after electrophoresis. Both the viscosity and spinnbarkeit of UWS were significantly higher than those of CWS. The viscosities were correlated pos. with the spinnbarkeit in UWS, but not in CWS. Image-d. anal. of mucins demonstrated that the viscosities increased with the levels of MUC5B and the spinnbarkeit with the levels of MUC7. These results suggest that MUC5B contributes to viscosity and MUC7 to spinnbarkeit.
  40. 40
    Fors, R.; Persson, M. Nickel in Dental Plaque and Saliva in Patients with and without Orthodontic Appliances. Eur. J. Orthodont. 2005, 28, 292– 297,  DOI: 10.1093/ejo/cji091
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    Payam, A. F.; Trewby, W.; Voïtchovsky, K. Simultaneous Viscosity and Density Measurement of Small Volumes of Liquids Using a Vibrating Microcantilever. Analyst 2017, 142 (9), 1492– 1498,  DOI: 10.1039/C6AN02674E
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    41
    Simultaneous viscosity and density measurement of small volumes of liquids using a vibrating microcantilever
    Payam, A. F.; Trewby, W.; Voitchovsky, K.
    Analyst (Cambridge, United Kingdom) (2017), 142 (9), 1492-1498CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
    Many industrial and technol. applications require precise detn. of the viscosity and d. of liqs. Such measurements can be time consuming and often require sampling substantial amts. of the liq. These problems can partly be overcome with the use of microcantilevers but most existing methods depend on the specific geometry and properties of the cantilever, which renders simple, accurate measurement difficult. Here we present a new approach able to simultaneously quantify both the d. and the viscosity of microliters of liqs. The method, based solely on the measurement of two characteristic frequencies of an immersed microcantilever, is completely independent of the choice of a cantilever. We derive anal. expressions for the liq.'s d. and viscosity and validate our approach with several simple liqs. and different cantilevers. Application of our model to non-Newtonian fluids shows that the calcd. viscosities are remarkably robust when compared to measurements obtained from a std. rheometer. However, the results become increasingly dependent on the cantilever geometry as the frequency-dependent nature of the liq.'s viscosity becomes more significant.
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    Carpenter, G. H. The Secretion, Components, and Properties of Saliva. Annu. Rev. Food Sci. Technol. 2013, 4 (1), 267– 276,  DOI: 10.1146/annurev-food-030212-182700
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    The secretion, components, and properties of saliva
    Carpenter, Guy H.
    Annual Review of Food Science and Technology (2013), 4 (), 267-276CODEN: ARFSBV; ISSN:1941-1413. (Annual Reviews Inc.)
    A review. Saliva has one of the most difficult roles to perform in the body. It must facilitate the taste and detection of foods nutritious to the body but also defend the mucosa from infection by the ever-present microbiota present in the mouth. It achieves these roles by having a complex compn. and versatile phys. properties. The protein and ion components make a soln. that is 99% water into a viscoelastic soln. capable of many roles, such as acting as a lubricant and an antimicrobial, preventing the dissoln. of teeth, aiding digestion, and facilitating taste. This review describes the neural regulation of salivary secretion in terms of fluid, protein, and ion secretion. It then describes some of the components and phys. properties of saliva and attempts to relate them to the functions that saliva must perform.
  43. 43
    Kalwarczyk, T.; Sozanski, K.; Ochab-Marcinek, A.; Szymanski, J.; Tabaka, M.; Hou, S.; Holyst, R. Motion of Nanoprobes in Complex Liquids within the Framework of the Length-Scale Dependent Viscosity Model. Adv. Coll. Interface Sci. 2015, 223, 55– 63,  DOI: 10.1016/j.cis.2015.06.007
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    Motion of nanoprobes in complex liquids within the framework of the length-scale dependent viscosity model
    Kalwarczyk, Tomasz; Sozanski, Krzysztof; Ochab-Marcinek, Anna; Szymanski, Jedrzej; Tabaka, Marcin; Hou, Sen; Holyst, Robert
    Advances in Colloid and Interface Science (2015), 223 (), 55-63CODEN: ACISB9; ISSN:0001-8686. (Elsevier B.V.)
    This paper deals with the recent phenomenol. model of the motion of nanoscopic objects (colloidal particles, proteins, nanoparticles, mols.) in complex liqs. We analyzed motion in polymer, micellar, colloidal and protein solns. and the cytoplasm of living cells using the length-scale dependent viscosity model. Viscosity monotonically approaches macroscopic viscosity as the size of the object increases and thus gives a single, coherent picture of motion at the nano and macro scale. The model includes interparticle interactions (solvent-solute), temp. and the internal structure of a complex liq. The depletion layer ubiquitously occurring in complex liqs. is also incorporated into the model. We also discuss the biol. aspects of crowding in terms of the length-scale dependent viscosity model.
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    Furst, E. M.; Squires, T. M. Microrheology; Oxford University Press, 2017.
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    Mao, Y.; Nielsen, P.; Ali, J. Passive and Active Microrheology for Biomedical Systems. Front. Bioengin. Biotechnol. 2022, 10, 916354,  DOI: 10.3389/fbioe.2022.916354
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    Sabaté del Río, J.; Henry, O. Y. F.; Jolly, P.; Ingber, D. E. An Antifouling Coating That Enables Affinity-Based Electrochemical Biosensing in Complex Biological Fluids. Nat. Nanotechnol. 2019, 14 (12), 1143– 1149,  DOI: 10.1038/s41565-019-0566-z
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    An antifouling coating that enables affinity-based electrochemical biosensing in complex biological fluids
    Sabate del Rio, Jonathan; Henry, Olivier Y. F.; Jolly, Pawan; Ingber, Donald E.
    Nature Nanotechnology (2019), 14 (12), 1143-1149CODEN: NNAABX; ISSN:1748-3387. (Nature Research)
    Affinity-based electrochem. detection in complex biol. fluids could enable multiplexed point-of-care diagnostics for home healthcare; however, commercialization of point-of-care devices has been limited by the rapid loss of sensitivity caused by electrode surface inactivation and biofouling. Here, we describe a simple and robust antifouling coating for electrodes consisting of a three-dimensional porous matrix of cross-linked bovine serum albumin supported by a network of conductive nanomaterials composed of either gold nanowires, gold nanoparticles or carbon nanotubes. These nanocomposites prevent non-specific interactions while enhancing electron transfer to the electrode surface, preserving 88% of the original signal after 1 mo of exposure to unprocessed human plasma, and functionalization with specific antibodies enables quantification of anti-interleukin 6 in plasma with high sensitivity. The easy prepn., stability and simplicity of this nanocomposite allow the generation of electrochem. biosensors that can operate in complex biol. fluids such as blood plasma or serum.
  47. 47
    Nair, S.; Tang, K. D.; Kenny, L.; Punyadeera, C. Salivary Exosomes as Potential Biomarkers in Cancer. Oral Oncol. 2018, 84, 31– 40,  DOI: 10.1016/j.oraloncology.2018.07.001
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    Salivary exosomes as potential biomarkers in cancer
    Nair, Soumyalekshmi; Tang, Kai Dun; Kenny, Liz; Punyadeera, Chamindie
    Oral Oncology (2018), 84 (), 31-40CODEN: EJCCER; ISSN:1368-8375. (Elsevier Ltd.)
    Over the past decade, there has been emerging research in the field of extracellular vesicles, esp. those originating from endosomes, referred to as 'exosomes. Exosomes are membrane-bound nanovesicles secreted by most cell types upon fusion of multivesicular bodies (MVBs) to the cell plasma membrane. These vesicles are present in almost all body fluids such as blood, urine, saliva, breast milk, cerebrospinal and peritoneal fluids. Exosomes participate in intercellular communication by transferring the biol. active mols. like proteins, nucleic acids, and lipids to neighboring cells. Exosomes are enriched in the tumor microenvironment and growing evidence demonstrates that exosomes mediate cancer progression and metastasis. Given the important biol. role played by these nanovesicles in cancer pathogenesis, these can be used as ideal non-invasive biomarkers in detecting and monitoring tumors as well as therapeutic targets. The scope of the current review is to provide an overview of exosomes with a special focus on salivary exosomes as potential biomarkers in head and neck cancers.
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    Martins, T. S.; Vaz, M.; Henriques, A. G. A Review on Comparative Studies Addressing Exosome Isolation Methods from Body Fluids. Analyt. Bioanalyt. Chem. 2023, 415 (7), 1239– 1263,  DOI: 10.1007/s00216-022-04174-5
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    Sjoqvist, S.; Otake, K. Saliva and Saliva Extracellular Vesicles for Biomarker Candidate Identification─Assay Development and Pilot Study in Amyotrophic Lateral Sclerosis. Int. J. Mol. Sci. 2023, 24 (6), 5237,  DOI: 10.3390/ijms24065237
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    49
    Saliva and Saliva Extracellular Vesicles for Biomarker Candidate Identification-Assay Development and Pilot Study in Amyotrophic Lateral Sclerosis
    Sjoqvist, Sebastian; Otake, Kentaro
    International Journal of Molecular Sciences (2023), 24 (6), 5237CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
    Saliva is gaining increasing attention as a source of biomarkers due to non-invasive and undemanding collection access. Extracellular vesicles (EVs) are nano-sized, cell-released particles that contain mol. information about their parent cells. In this study, we developed methods for saliva biomarker candidate identification using EV-isolation and proteomic evaluation. We used pooled saliva samples for assay development. EVs were isolated using membrane affinity-based methods followed by their characterization using nanoparticle tracking anal. and transmission electron microscopy. Subsequently, both saliva and saliva-EVs were successfully analyzed using proximity extension assay and label-free quant. proteomics. Saliva-EVs had a higher purity than plasma-EVs, based on the expression of EV-proteins and albumin. The developed methods could be used for the anal. of individual saliva samples from amyotrophic lateral sclerosis (ALS) patients and controls (n = 10 each). The starting vol. ranged from 2.1 to 4.9 mL and the amt. of total isolated EV-proteins ranged from 5.1 to 42.6μg. Although no proteins were significantly differentially expressed between the two groups, there was a trend for a downregulation of ZNF428 in ALS-saliva-EVs and an upregulation of IGLL1 in ALS saliva. In conclusion, we have developed a robust workflow for saliva and saliva-EV anal. and demonstrated its tech. feasibility for biomarker discovery.
  50. 50
    Aqrawi, L. A.; Galtung, H. K.; Vestad, B.; Øvstebø, R.; Thiede, B.; Rusthen, S.; Young, A.; Guerreiro, E. M.; Utheim, T. P.; Chen, X.; Utheim, Ø. A.; Palm, Ø.; Jensen, J. L. Identification of Potential Saliva and Tear Biomarkers in Primary Sjögren’s Syndrome, Utilising the Extraction of Extracellular Vesicles and Proteomics Analysis. Arthritis Res. Therapy 2017, 19 (1), 14,  DOI: 10.1186/s13075-017-1228-x
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    50
    Identification of potential saliva and tear biomarkers in primary Sjogren's syndrome, utilising the extraction of extracellular vesicles and proteomics analysis
    Aqrawi, Lara A.; Galtung, Hilde Kanli; Vestad, Beate; Oevsteboe, Reidun; Thiede, Bernd; Rusthen, Shermin; Young, Alix; Guerreiro, Eduarda M.; Utheim, Tor Paaske; Chen, Xiangjun; Utheim, Oeygunn Aass; Palm, Oeyvind; Jensen, Janicke Liaaen
    Arthritis Research & Therapy (2017), 19 (), 14/1-14/15CODEN: ARTRCV; ISSN:1478-6362. (BioMed Central Ltd.)
    Background: There is a long-lasting need for non-invasive, more accurate diagnostic techniques when evaluating primary Sj0gren's syndrome (pSS) patients. Incorporation of addnl. diagnostics involving screening for disease-specific biomarkers in biol. fluid is a promising concept that requires further investigation. In the current study we aimed to explore novel disease biomarkers in saliva and tears from pSS patients. Methods: Liq. chromatog.-mass spectrometry (LC-MS) was performed on stimulated whole saliva and tears from 27 pSS patients and 32 healthy controls, and salivary and tear proteomic biomarker profiles were generated. LC-MS was also combined with size exclusion chromatog. to isolate extracellular vesicles (EVs) from both fluids. Nanoparticle tracking anal. was conducted on joint fractions from the saliva and tears to det. size distribution and concn. of EVs. Further EV characterization was performed by immunoaffinity capture of CD9-pos. EVs using magnetic beads, detected by flow cytometry. The LC-MS data were analyzed for quant. differences between patient and control groups using Scaffold, and the proteins were further analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID), for gene ontol. overrepresentation, and the Search Tool for the Retrieval of Interacting Genes/Proteins for protein-protein interaction network anal. Results: Upregulation of proteins involved in innate immunity (LCN2), cell signalling (CALM) and wound repair (GRN and CALML5) were detected in saliva in pSS. Saliva EVs also displayed biomarkers crit. for activation of the innate immune system (SIRPA and LSP1) and adipocyte differentiation (APMAP). Tear anal. indicated overexpression of proteins involved in TNF-α signalling (CPNE1) and B cell survival (PRDX3). Moreover, neutrophil gelatinase-assocd. lipocalin was upregulated in saliva and tears in pSS. Consistently, DAVID anal. demonstrated pathways of the adaptive immune response in saliva, of cellular component assembly for saliva EVs, and of metab. and protein folding in tears in pSS patients. Conclusions: LC-MS of saliva and tears from pSS patients, solely and in combination with size-exclusion chromatog. allowed screening for possible novel biomarkers encompassing both salivary and lacrimal disease target organs. This approach could provide addnl. diagnostic accuracy in pSS, and could possibly also be applied for staging and monitoring the disease.
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    Contreras, H.; Alarcón-Zapata, P.; Nova-Lamperti, E.; Ormazabal, V.; Varas-Godoy, M.; Salomon, C.; Zuniga, F. A. Comparative Study of Size Exclusion Chromatography for Isolation of Small Extracellular Vesicle from Cell-Conditioned Media, Plasma, Urine, and Saliva. Front. Nanotechnol. 2023, 5, 1146772,  DOI: 10.3389/fnano.2023.1146772
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    Largitte, L.; Pasquier, R. New Models for Kinetics and Equilibrium Homogeneous Adsorption. Chem. Eng. Res. Des. 2016, 112, 289– 297,  DOI: 10.1016/j.cherd.2016.06.021
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    New models for kinetics and equilibrium homogeneous adsorption
    Largitte, L.; Pasquier, R.
    Chemical Engineering Research and Design (2016), 112 (), 289-297CODEN: CERDEE; ISSN:1744-3563. (Elsevier B.V.)
    8 New models, named resp. LK 1, LK 2, LK 3, LEq 1, LEq 2, LEq 3, LEq 4 and LEq 5 are suggested to describe the kinetics and the equil. of a homogeneous adsorption. The models, more general than the existing ones, are very useful to describe the different kinetics and isotherms exptl. behavior of many adsorbents. In these new methods, the adsorption is decompd. in 1 or 2 steps, each step following an n order kinetics. The models have scientific, logical and chem. understanding meaning. Some (LK 1, LK 3, LEq 2, LEq 4) can predict the max. adsorption capacity Qmax. LEq 1 and LEq 2 models permit to make the link between the kinetics and the equil. results and det. the order of reaction for both the adsorbent and the adsorbate, then, increasing the knowledge of the sorption parameters.The kinetics and the equil. of the adsorption of lead by a com. activated carbon are studied and these new models are used to fit the exptl. data. The kinetics and the equil. parameters are detd. The fitting of the kinetics results are compared to those obtained with other existing models, idem for the equil. results. Regression results show that the new equil. models better fit the exptl. data than the existing ones for this sorption process.
  53. 53
    Umeda, R.; Satouh, Y.; Takemoto, M.; Nakada-Nakura, Y.; Liu, K.; Yokoyama, T.; Shirouzu, M.; Iwata, S.; Nomura, N.; Sato, K.; Ikawa, M.; Nishizawa, T.; Nureki, O. Structural Insights into Tetraspanin CD9 Function. Nat. Commun. 2020, 11 (1), 1606,  DOI: 10.1038/s41467-020-15459-7
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    Structural insights into tetraspanin CD9 function
    Umeda, Rie; Satouh, Yuhkoh; Takemoto, Mizuki; Nakada-Nakura, Yoshiko; Liu, Kehong; Yokoyama, Takeshi; Shirouzu, Mikako; Iwata, So; Nomura, Norimichi; Sato, Ken; Ikawa, Masahito; Nishizawa, Tomohiro; Nureki, Osamu
    Nature Communications (2020), 11 (1), 1606CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Abstr.: Tetraspanins play crit. roles in various physiol. processes, ranging from cell adhesion to virus infection. The members of the tetraspanin family have four membrane-spanning domains and short and large extracellular loops, and assoc. with a broad range of other functional proteins to exert cellular functions. Here we report the crystal structure of CD9 and the cryo-electron microscopic structure of CD9 in complex with its single membrane-spanning partner protein, EWI-2. The reversed cone-like mol. shape of CD9 generates membrane curvature in the cryst. lipid layers, which explains the CD9 localization in regions with high membrane curvature and its implications in membrane remodeling. The mol. interaction between CD9 and EWI-2 is mainly mediated through the small residues in the transmembrane region and protein/lipid interactions, whereas the fertilization assay revealed the crit. involvement of the LEL region in the sperm-egg fusion, indicating the different dependency of each binding domain for other partner proteins.
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    Vences-Catalán, F.; Rajapaksa, R.; Kuo, C. C.; Miller, C. L.; Lee, A.; Ramani, V. C.; Jeffrey, S. S.; Levy, R.; Levy, S. Targeting the Tetraspanin CD81 Reduces Cancer Invasion and Metastasis. Proc. Natl. Acad. Sci. U. S. A. 2021, 118 (24), e2018961118,  DOI: 10.1073/pnas.2018961118
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    54
    Targeting the tetraspanin CD81 reduces cancer invasion and metastasis
    Vences-Catalan, Felipe; Rajapaksa, Ranjani; Kuo, Chiung-Chi; Miller, Caitlyn L.; Lee, Anderson; Ramani, Vishnu C.; Jeffrey, Stefanie S.; Levy, Ronald; Levy, Shoshana
    Proceedings of the National Academy of Sciences of the United States of America (2021), 118 (24), e2018961118CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Tetraspanins are an evolutionary conserved family of proteins involved in multiple aspects of cell physiol., including proliferation, migration and invasion, protein trafficking, and signal transduction; yet their detailed mechanism of action is unknown. Tetraspanins have no known natural ligands, but their engagement by antibodies has begun to reveal their role in cell biol. Studies of tetraspanin knockout mice and of germline mutations in humans have highlighted their role under normal and pathol. conditions. Previously, we have shown that mice deficient in the tetraspanin CD81 developed fewer breast cancer metastases compared to their wild-type (WT) counterparts. Here, we show that a unique anti-human CD81 antibody (5A6) effectively halts invasion of triple-neg. breast cancer (TNBC) cell lines. We demonstrate that 5A6 induces CD81 clustering at the cell membrane and we implicate JAM-A protein in the ability of this antibody to inhibit tumor cell invasion and migration. Furthermore, in a series of in vivo studies we demonstrate that this antibody inhibits metastases in xenograft models, as well as in syngeneic mice bearing a mouse tumor into which we knocked in the human CD81 epitope recognized by the 5A6 antibody.
  55. 55
    Picca, A.; Guerra, F.; Calvani, R.; Marini, F.; Biancolillo, A.; Landi, G.; Beli, R.; Landi, F.; Bernabei, R.; Bentivoglio, A. R.; Lo Monaco, M. R.; Bucci, C.; Marzetti, E. Mitochondrial Signatures in Circulating Extracellular Vesicles of Older Adults with Parkinson’s Disease: Results from the EXosomes in ParkiNson’s Disease (EXPAND) Study. J. Clin. Med. 2020, 9 (2), 504,  DOI: 10.3390/jcm9020504
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    Mitochondrial signatures in circulating extracellular vesicles of older adults with parkinson's disease: results from the exosomes in parkinson's disease (EXPAND) study
    Picca, Anna; Guerra, Flora; Calvani, Riccardo; Marini, Federico; Biancolillo, Alessandra; Landi, Giovanni; Beli, Raffaella; Landi, Francesco; Bernabei, Roberto; Bentivoglio, Anna Rita; Lo Monaco, Maria Rita; Bucci, Cecilia; Marzetti, Emanuele
    Journal of Clinical Medicine (2020), 9 (2), 504CODEN: JCMOHK; ISSN:2077-0383. (MDPI AG)
    Systemic inflammation and mitochondrial dysfunction are involved in neurodegeneration in Parkinson's disease (PD). Extracellular vesicle (EV) trafficking may link inflammation and mitochondrial dysfunction. In the present study, circulating small EVs (sEVs) from 16 older adults with PD and 12 non-PD controls were purified and characterized. A panel of serum inflammatory biomols. was measured by multiplex immunoassay. Protein levels of three tetraspanins (CD9, CD63, and CD81) and selected mitochondrial markers (ATP 5A (ATP5A), mitochondrial cytochrome C oxidase subunit I (MTCOI), NAD reduced form (NADH):ubiquinone oxidoreductase subunit B8 (NDUFB8), NADH:ubiquinone oxidoreductase subunit S3 (NDUFS3), succinate dehydrogenase complex iron sulfur subunit B (SDHB), and ubiquinol-cytochrome C reductase core protein 2 (UQCRC2)) were quantified in purified sEVs by immunoblotting. Relative to controls, PD participants showed a greater amt. of circulating sEVs. Levels of CD9 and CD63 were lower in the sEV fraction of PD participants, whereas those of CD81 were similar between groups. Lower levels of ATP5A, NDUFS3, and SDHB were detected in sEVs from PD participants. No signal was retrieved for UQCRC2, MTCOI, or NDUFB8 in either participant group. To identify a mol. signature in circulating sEVs in relationship to systemic inflammation, a low level-fused (multi-platform) partial least squares discriminant anal. was applied. The model correctly classified 94.2% ± 6.1% PD participants and 66.7% ± 5.4% controls, and identified seven biomols. as relevant (CD9, NDUFS3, C-reactive protein, fibroblast growth factor 21, interleukin 9, macrophage inflammatory protein 1β, and tumor necrosis factor alpha). In conclusion, a mitochondrial signature was identified in circulating sEVs from older adults with PD, in assocn. with a specific inflammatory profile.
  56. 56
    P.C., S.; Shetty, S.; Nalilu, S.; Shetty, P.; Patil, P. Tetraspanin CD9: A Friend or Foe of Head and Neck Cancer. Oncol. Rep. 2022, 47 (5), 88,  DOI: 10.3892/or.2022.8299
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    Paba, C.; Dorigo, V.; Senigagliesi, B.; Tormena, N.; Parisse, P.; Voïtchovsky, K.; Casalis, L. Lipid Bilayer Fluidity and Degree of Order Regulates Small EVs Adsorption on Model Cell Membrane. J. Colloid Interface Sci. 2023, 652, 1937– 1943,  DOI: 10.1016/j.jcis.2023.08.117
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    Lipid bilayer fluidity and degree of order regulates small EVs adsorption on model cell membrane
    Paba, Carolina; Dorigo, Virginia; Senigagliesi, Beatrice; Tormena, Nicolo; Parisse, Pietro; Voitchovsky, Kislon; Casalis, Loredana
    Journal of Colloid and Interface Science (2023), 652 (Part_B), 1937-1943CODEN: JCISA5; ISSN:0021-9797. (Elsevier B.V.)
    Small extracellular vesicles (sEVs) are known to play an important role in the communication between distant cells and to deliver biol. information throughout the body. To date, many studies have focused on the role of sEVs characteristics such as cell origin, surface compn., and mol. cargo on the resulting uptake by the recipient cell. Yet, a full understanding of the sEV fusion process with recipient cells and in particular the role of cell membrane phys. properties on the uptake are still lacking. Here we explore this problem using sEVs from a cellular model of triple-neg. breast cancer fusing to a range of synthetic planar lipid bilayers both with and without cholesterol, and designed to mimic the formation of 'raft'-like nanodomains in cell membranes. Using time-resolved Atomic Force Microscopy we were able to track the sEVs interaction with the different model membranes, showing the process to be strongly dependent on the local membrane fluidity. The strongest interaction and fusion is obsd. over the less fluid regions, with sEVs even able to disrupt ordered domains at sufficiently high cholesterol concn. Our findings suggest the biophys. characteristics of recipient cell membranes to be crucial for sEVs uptake regulation.
  58. 58
    Théry, C.; Witwer, K. W.; Aikawa, E.; Alcaraz, M. J.; Anderson, J. D.; Andriantsitohaina, R.; Antoniou, A.; Arab, T.; Archer, F.; Atkin-Smith, G. K.; Ayre, D. C.; Bach, J.-M.; Bachurski, D.; Baharvand, H.; Balaj, L.; Baldacchino, S.; Bauer, N. N.; Baxter, A. A.; Bebawy, M.; Beckham, C.; Bedina Zavec, A.; Benmoussa, A.; Berardi, A. C.; Bergese, P.; Bielska, E.; Blenkiron, C.; Bobis-Wozowicz, S.; Boilard, E.; Boireau, W.; Bongiovanni, A.; Borràs, F. E.; Bosch, S.; Boulanger, C. M.; Breakefield, X.; Breglio, A. M.; Brennan, M. Á.; Brigstock, D. R.; Brisson, A.; Broekman, M. L.; Bromberg, J. F.; Bryl-Górecka, P.; Buch, S.; Buck, A. H.; Burger, D.; Busatto, S.; Buschmann, D.; Bussolati, B.; Buzás, E. I.; Byrd, J. B.; Camussi, G.; Carter, D. R.; Caruso, S.; Chamley, L. W.; Chang, Y.-T.; Chen, C.; Chen, S.; Cheng, L.; Chin, A. R.; Clayton, A.; Clerici, S. P.; Cocks, A.; Cocucci, E.; Coffey, R. J.; Cordeiro-da-Silva, A.; Couch, Y.; Coumans, F. A.; Coyle, B.; Crescitelli, R.; Criado, M. F.; D’Souza-Schorey, C.; Das, S.; Datta Chaudhuri, A.; de Candia, P.; De Santana, E. F.; De Wever, O.; del Portillo, H. A.; Demaret, T.; Deville, S.; Devitt, A.; Dhondt, B.; Di Vizio, D.; Dieterich, L. C.; Dolo, V.; Dominguez Rubio, A. P.; Dominici, M.; Dourado, M. R.; Driedonks, T. A.; Duarte, F. V.; Duncan, H. M.; Eichenberger, R. M.; Ekström, K.; EL Andaloussi, S.; Elie-Caille, C.; Erdbrügger, U.; Falcón-Pérez, J. M.; Fatima, F.; Fish, J. E.; Flores-Bellver, M.; Försönits, A.; Frelet-Barrand, A.; Fricke, F.; Fuhrmann, G.; Gabrielsson, S.; Gámez-Valero, A.; Gardiner, C.; Gärtner, K.; Gaudin, R.; Gho, Y. S.; Giebel, B.; Gilbert, C.; Gimona, M.; Giusti, I.; Goberdhan, D. C.; Görgens, A.; Gorski, S. M.; Greening, D. W.; Gross, J. C.; Gualerzi, A.; Gupta, G. N.; Gustafson, D.; Handberg, A.; Haraszti, R. A.; Harrison, P.; Hegyesi, H.; Hendrix, A.; Hill, A. F.; Hochberg, F. H.; Hoffmann, K. F.; Holder, B.; Holthofer, H.; Hosseinkhani, B.; Hu, G.; Huang, Y.; Huber, V.; Hunt, S.; Ibrahim, A. G.-E.; Ikezu, T.; Inal, J. M.; Isin, M.; Ivanova, A.; Jackson, H. K.; Jacobsen, S.; Jay, S. M.; Jayachandran, M.; Jenster, G.; Jiang, L.; Johnson, S. M.; Jones, J. C.; Jong, A.; Jovanovic-Talisman, T.; Jung, S.; Kalluri, R.; Kano, S.; Kaur, S.; Kawamura, Y.; Keller, E. T.; Khamari, D.; Khomyakova, E.; Khvorova, A.; Kierulf, P.; Kim, K. P.; Kislinger, T.; Klingeborn, M.; Klinke, D. J., II; Kornek, M.; Kosanović, M. M.; Kovács, Á. F.; Krämer-Albers, E.-M.; Krasemann, S.; Krause, M.; Kurochkin, I. V.; Kusuma, G. D.; Kuypers, S.; Laitinen, S.; Langevin, S. M.; Languino, L. R.; Lannigan, J.; Lässer, C.; Laurent, L. C.; Lavieu, G.; Lázaro-Ibáñez, E.; Le Lay, S.; Lee, M.-S.; Lee, Y. X. F.; Lemos, D. S.; Lenassi, M.; Leszczynska, A.; Li, I. T.; Liao, K.; Libregts, S. F.; Ligeti, E.; Lim, R.; Lim, S. K.; Line̅, A.; Linnemannstöns, K.; Llorente, A.; Lombard, C. A.; Lorenowicz, M. J.; Lörincz, Á. M.; Lötvall, J.; Lovett, J.; Lowry, M. C.; Loyer, X.; Lu, Q.; Lukomska, B.; Lunavat, T. R.; Maas, S. L.; Malhi, H.; Marcilla, A.; Mariani, J.; Mariscal, J.; Martens-Uzunova, E. S.; Martin-Jaular, L.; Martinez, M. C.; Martins, V. R.; Mathieu, M.; Mathivanan, S.; Maugeri, M.; McGinnis, L. K.; McVey, M. J.; Meckes, D. G., Jr; Meehan, K. L.; Mertens, I.; Minciacchi, V. R.; Möller, A.; Møller Jo̷rgensen, M.; Morales-Kastresana, A.; Morhayim, J.; Mullier, F.; Muraca, M.; Musante, L.; Mussack, V.; Muth, D. C.; Myburgh, K. H.; Najrana, T.; Nawaz, M.; Nazarenko, I.; Nejsum, P.; Neri, C.; Neri, T.; Nieuwland, R.; Nimrichter, L.; Nolan, J. P.; Nolte-’t Hoen, E. N.; Noren Hooten, N.; O’Driscoll, L.; O’Grady, T.; O’Loghlen, A.; Ochiya, T.; Olivier, M.; Ortiz, A.; Ortiz, L. A.; Osteikoetxea, X.; Østergaard, O.; Ostrowski, M.; Park, J.; Pegtel, D. M.; Peinado, H.; Perut, F.; Pfaffl, M. W.; Phinney, D. G.; Pieters, B. C.; Pink, R. C.; Pisetsky, D. S.; Pogge von Strandmann, E.; Polakovicova, I.; Poon, I. K.; Powell, B. H.; Prada, I.; Pulliam, L.; Quesenberry, P.; Radeghieri, A.; Raffai, R. L.; Raimondo, S.; Rak, J.; Ramirez, M. I.; Raposo, G.; Rayyan, M. S.; Regev-Rudzki, N.; Ricklefs, F. L.; Robbins, P. D.; Roberts, D. D.; Rodrigues, S. C.; Rohde, E.; Rome, S.; Rouschop, K. M.; Rughetti, A.; Russell, A. E.; Saá, P.; Sahoo, S.; Salas-Huenuleo, E.; Sánchez, C.; Saugstad, J. A.; Saul, M. J.; Schiffelers, R. M.; Schneider, R.; Schøyen, T. H.; Scott, A.; Shahaj, E.; Sharma, S.; Shatnyeva, O.; Shekari, F.; Shelke, G. V.; Shetty, A. K.; Shiba, K.; Siljander, P. R.-M.; Silva, A. M.; Skowronek, A.; Snyder, O. L., II; Soares, R. P.; Sódar, B. W.; Soekmadji, C.; Sotillo, J.; Stahl, P. D.; Stoorvogel, W.; Stott, S. L.; Strasser, E. F.; Swift, S.; Tahara, H.; Tewari, M.; Timms, K.; Tiwari, S.; Tixeira, R.; Tkach, M.; Toh, W. S.; Tomasini, R.; Torrecilhas, A. C.; Tosar, J. P.; Toxavidis, V.; Urbanelli, L.; Vader, P.; van Balkom, B. W.; van der Grein, S. G.; Van Deun, J.; van Herwijnen, M. J.; Van Keuren-Jensen, K.; van Niel, G.; van Royen, M. E.; van Wijnen, A. J.; Vasconcelos, M. H.; Vechetti, I. J., Jr; Veit, T. D.; Vella, L. J.; Velot, É.; Verweij, F. J.; Vestad, B.; Viñas, J. L.; Visnovitz, T.; Vukman, K. V.; Wahlgren, J.; Watson, D. C.; Wauben, M. H.; Weaver, A.; Webber, J. P.; Weber, V.; Wehman, A. M.; Weiss, D. J.; Welsh, J. A.; Wendt, S.; Wheelock, A. M.; Wiener, Z.; Witte, L.; Wolfram, J.; Xagorari, A.; Xander, P.; Xu, J.; Yan, X.; Yáñez-Mó, M.; Yin, H.; Yuana, Y.; Zappulli, V.; Zarubova, J.; Žėkas, V.; Zhang, J.; Zhao, Z.; Zheng, L.; Zheutlin, A. R.; Zickler, A. M.; Zimmermann, P.; Zivkovic, A. M.; Zocco, D.; Zuba-Surma, E. K. Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018): A Position Statement of the International Society for Extracellular Vesicles and Update of the MISEV2014 Guidelines. J. Extracell. Ves. 2018, 7 (1), 1535750,  DOI: 10.1080/20013078.2018.1535750
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    Jawerth, L.; Fischer-Friedrich, E.; Saha, S.; Wang, J.; Franzmann, T.; Zhang, X.; Sachweh, J.; Ruer, M.; Ijavi, M.; Saha, S.; Mahamid, J.; Hyman, A. A.; Jülicher, F. Protein Condensates as Aging Maxwell Fluids. Science 2020, 370 (6522), 1317– 1323,  DOI: 10.1126/science.aaw4951
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    Protein condensates as aging Maxwell fluids
    Jawerth, Louise; Fischer-Friedrich, Elisabeth; Saha, Suropriya; Wang, Jie; Franzmann, Titus; Zhang, Xiaojie; Sachweh, Jenny; Ruer, Martine; Ijavi, Mahdiye; Saha, Shambaditya; Mahamid, Julia; Hyman, Anthony A.; Juelicher, Frank
    Science (Washington, DC, United States) (2020), 370 (6522), 1317-1323CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
    Protein condensates are complex fluids that can change their material properties with time. However, an appropriate rheol. description of these fluids remains missing. We characterize the time-dependent material properties of in vitro protein condensates using laser tweezer-based active and microbead-based passive rheol. For different proteins, the condensates behave at all ages as viscoelastic Maxwell fluids. Their viscosity strongly increases with age while their elastic modulus varies weakly. No significant differences in structure were seen by electron microscopy at early and late ages. We conclude that protein condensates can be soft glassy materials that we call Maxwell glasses with age-dependent material properties. We discuss possible advantages of glassy behavior for modulation of cellular biochem.
  60. 60
    Murayama, Y.; Oritani, K.; Tsutsui, S. Novel CD9-Targeted Therapies in Gastric Cancer. World J. Gastroenterol. 2015, 21 (11), 3206– 3213,  DOI: 10.3748/wjg.v21.i11.3206
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    Novel CD9-targeted therapies in gastric cancer
    Murayama, Yoko; Oritani, Kenji; Tsutsui, Shusaku
    World Journal of Gastroenterology (2015), 21 (11), 3206-3213CODEN: WJGAF2; ISSN:2219-2840. (Baishideng Publishing Group Inc.)
    There are 33 human tetraspanin proteins, emerging as key players in malignancy, the immune system, fertilization, cellular signaling, adhesion, morphol., motility, proliferation, and tumor invasion. CD9, a member of the tetraspanin family, assocs. with and influences a variety of cell-surface mols. Through these interactions, CD9 modifies multiple cellular events, including adhesion, migration, proliferation, and survival. CD9 is therefore considered to play a role in several stages during cancer development. Reduced CD9 expression is generally related to venous vessel invasion and metastasis as well as poor prognosis. We found that treatment of mice bearing human gastric cancer cells with anti-CD9 antibody successfully inhibited tumor progression via antiproliferative, proapoptotic, and antiangiogenic effects, strongly indicating that CD9 is a possible therapeutic target in patients with gastric cancer. Here, we describe the possibility of CD9 manipulation as a novel therapeutic strategy in gastric cancer, which still shows poor prognosis.
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    Lorico, A.; Lorico-Rappa, M.; Karbanová, J.; Corbeil, D.; Pizzorno, G. CD9, a Tetraspanin Target for Cancer Therapy?. Experiment. Biol. Med. 2021, 246 (9), 1121– 1138,  DOI: 10.1177/1535370220981855
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    CD9, a tetraspanin target for cancer therapy?
    Lorico, Aurelio; Lorico-Rappa, Marco; Karbanova, Jana; Corbeil, Denis; Pizzorno, Giuseppe
    Experimental Biology and Medicine (London, United Kingdom) (2021), 246 (9), 1121-1138CODEN: EBMMBE; ISSN:1535-3699. (Sage Publications Ltd.)
    In the present minireview, we intend to provide a brief history of the field of CD9 involvement in oncogenesis and in the metastatic process of cancer, considering its potential value as a tumor-assocd. antigenic target. Over the years, CD9 has been identified as a favorable prognostic marker or predictor of metastatic potential depending on the cancer type. To understand its implications in cancer beside its use as an antigenic biomarker, it is essential to know its physiol. functions, including its mol. partners in a given cell system. Moreover, the discovery that CD9 is one of the most specific and broadly expressed markers of extracellular membrane vesicles, nanometer-sized entities that are released into extracellular space and various physiol. body fluids and play a role in intercellular communication under physiol. and pathol. conditions, notably the establishment of cancer metastases, has added a new dimension to our knowledge of CD9 function in cancer. Here, we will discuss these issues as well as the possible cancer therapeutic implications of CD9, their limitations, and pitfalls.
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    Nigri, J.; Leca, J.; Tubiana, S. S.; Finetti, P.; Guillaumond, F.; Martinez, S.; Lac, S.; Iovanna, J. L.; Audebert, S.; Camoin, L.; Vasseur, S.; Bertucci, F.; Tomasini, R. CD9 Mediates the Uptake of Extracellular Vesicles from Cancer-Associated Fibroblasts That Promote Pancreatic Cancer Cell Aggressiveness. Science Signaling 2022, 15 (745), eabg8191,  DOI: 10.1126/scisignal.abg8191
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    Koh, H. M.; Jang, B. G.; Lee, D. H.; Hyun, C. L. Increased CD9 Expression Predicts Favorable Prognosis in Human Cancers: A Systematic Review and Meta-Analysis. Cancer Cell Int. 2021, 21 (1), 472,  DOI: 10.1186/s12935-021-02152-y
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    Increased CD9 expression predicts favorable prognosis in human cancers: a systematic review and meta-analysis
    Koh, Hyun Min; Jang, Bo Gun; Lee, Dong Hui; Hyun, Chang Lim
    Cancer Cell International (2021), 21 (1), 472CODEN: CCIACC; ISSN:1475-2867. (BioMed Central Ltd.)
    Meta-anal. of CD9 is implicated in cancer progression and metastasis by its role in suppressing cancer cell proliferation and survival. However, the prognostic and clinicopathol. significance of CD9 expression is controversial. Therefore, the current meta-anal. was conducted to det. the prognostic and clinicopathol. significance of CD9 expression in cancer patients. Eligible studies were selected through database search of PubMed, Embase and Cochrane library up to Apr. 5 2020. The necessary data were extd. from the included studies. Pooled hazard ratio (HR) and odds ratio (OR) with 95% confidence interval (CI) were calcd. to evaluate the prognostic and clinicopathol. significance of CD9 expression in cancer patients. A total of 17 studies consisting of 3456 cancer patients were included in this meta-anal. An increased CD9 expression was significantly assocd. with a more favorable overall survival (OS) (HR 0.47, 95% CI 0.31-0.73, p = 0.001) and disease-free survival (DFS) (HR 0.48, 95% CI 0.30-0.79, p = 0.003). In subgroup anal. of cancer type, an increased CD9 expression was assocd. with increased OS in breast cancer and digestive system cancer, and with increased DFS in head and neck cancer and leukemia/lymphoma. Addnl., an increased CD9 expression significantly correlated with lower overall stage (OR 0.45, 95% CI 0.29-0.72, p = 0.001). An increased CD9 expression was assocd. with favorable survival in cancer patients suggesting that CD9 expression could be a valuable survival factor in cancer patients.
  64. 64
    Salvi, S.; Bandini, E.; Carloni, S.; Casadio, V.; Battistelli, M.; Salucci, S.; Erani, I.; Scarpi, E.; Gunelli, R.; Cicchetti, G.; Guescini, M.; Bonafè, M.; Fabbri, F. Detection and Investigation of Extracellular Vesicles in Serum and Urine Supernatant of Prostate Cancer Patients. Diagnostics 2021, 11 (3), 466,  DOI: 10.3390/diagnostics11030466
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    Detection and investigation of extracellular vesicles in serum and urine supernatant of prostate cancer patients
    Salvi, Samanta; Bandini, Erika; Carloni, Silvia; Casadio, Valentina; Battistelli, Michela; Salucci, Sara; Erani, Ilaria; Scarpi, Emanuela; Gunelli, Roberta; Cicchetti, Giacomo; Guescini, Michele; Bonafe, Massimiliano; Fabbri, Francesco
    Diagnostics (2021), 11 (3), 466CODEN: DIAGC9; ISSN:2075-4418. (MDPI AG)
    Prostate Cancer (PCa) is one of the most frequently identified urol. cancers. PCa patients are often over-diagnosed due to still not highly specific diagnostic methods. The need for more accurate diagnostic tools to prevent overestimated diagnosis and unnecessary treatment of patients with non-malignant conditions is clear, and new markers and methods are strongly desirable. Extracellular vesicles (EVs) hold great promises as liq. biopsy-based markers. Despite the biol. and tech. issues present in their detection and study, these particles can be found highly abundantly in the biofluid and encompass a wealth of macromols. that have been reported to be related to many physiol. and pathol. processes, including cancer onset, metastasis spreading, and treatment resistance. The present study aims to perform a tech. feasibility study to develop a new workflow for investigating EVs from several biol. sources. Serum and urinary supernatant EVs of PCa, benign prostatic hyperplasia (BPH) patients, and healthy donors were isolated and investigated by a fast, easily performable, and cost-effective cytofluorimetric approach for a multiplex detection of 37 EV-antigens. We also obsd. significant alterations in serum and urinary supernatant EVs potentially related to BPH and PCa, suggesting a potential clin. application of this workflow.
  65. 65
    Paolino, G.; Huber, V.; Camerini, S.; Casella, M.; Macone, A.; Bertuccini, L.; Iosi, F.; Moliterni, E.; Cecchetti, S.; Ruspantini, I.; Chiarotti, F.; Vergani, E.; Lalli, L.; Raggi, C.; Di Biase, A.; Calvieri, S.; Mercuri, S. R.; Lugini, L.; Federici, C. The Fatty Acid and Protein Profiles of Circulating Cd81-Positive Small Extracellular Vesicles Are Associated with Disease Stage in Melanoma Patients. Cancers 2021, 13 (16), 4157,  DOI: 10.3390/cancers13164157
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    65
    The Fatty Acid and Protein Profiles of Circulating CD81-Positive Small Extracellular Vesicles Are Associated with Disease Stage in Melanoma Patients
    Paolino, Giovanni; Huber, Veronica; Camerini, Serena; Casella, Marialuisa; Macone, Alberto; Bertuccini, Lucia; Iosi, Francesca; Moliterni, Elisa; Cecchetti, Serena; Ruspantini, Irene; Chiarotti, Flavia; Vergani, Elisabetta; Lalli, Luca; Raggi, Carla; Di Biase, Antonella; Calvieri, Stefano; Mercuri, Santo Raffaele; Lugini, Luana; Federici, Cristina
    Cancers (2021), 13 (16), 4157CODEN: CANCCT; ISSN:2072-6694. (MDPI AG)
    Simple Summary: Early detection of cutaneous melanoma is the key to increasing survival and proper therapeutic adjustment, esp. in stages II-IV. We investigated whether the fatty acid (FA) and protein compns. of small extracellular vesicles (sEV) expressing CD81, derived from the plasma of stage 0-I, II and III-IV melanoma patients, could reflect disease stage. Results showed a higher content of FA and differences in C18:0/C18:1 ratio, a marker of cell membrane fluidity, that distinguished patients' CD81sEV from those of healthy donors (HD). By proteomic anal. (identifier PXD024434) we identified significant increases in CD14, PON1, PON3 and APOA5 in stage II CD81sEV compared to HD. In stage III-IV, CD81sEV' RAP1B expression was decreased. These stage-related signatures may support the potential of sEV to provide information for early diagnosis, prediction of metastatic behavior, treatment and follow-up of melanoma patients. Abstr.: The early detection of cutaneous melanoma, a potentially lethal cancer with rising incidence, is fundamental to increasing survival and therapeutic adjustment. In stages II-IV esp., addnl. indications for adjuvant therapy purposes after resection and for treatment of metastatic patients are urgently needed. We investigated whether the fatty acid (FA) and protein compns. of small extracellular vesicles (sEV) derived from the plasma of stage 0-I, II and III-IV melanoma patients (n = 38) could reflect disease stage. The subpopulation of sEV expressing CD81 EV marker (CD81sEV) was captured by an ad hoc immune affinity technique from plasma depleted of large EV. Biol. macromols. were investigated by gas chromatog. and mass spectrometry in CD81sEV. A higher content of FA was detectable in patients with respect to healthy donors (HD). Moreover, a higher C18:0/C18:1 ratio, as a marker of cell membrane fluidity, distinguished early (stage 0-I) from late (III-IV) stages' CD81sEV. Proteomics detected increases in CD14, PON1, PON3 and APOA5 exclusively in stage II CD81sEV, and RAP1B was decreased in stage III-IV CD81sEV, in comparison to HD. Our results suggest that stage dependent alterations in CD81sEV' FA and protein compn. may occur early after disease onset, strengthening the potential of circulating sEV as a source of discriminatory information for early diagnosis, prediction of metastatic behavior and following up of melanoma patients.
  66. 66
    You, B.; Shan, Y.; Bao, L.; Chen, J.; Yang, L.; Zhang, Q.; Zhang, W.; Zhang, Z.; Zhang, J.; Shi, S.; You, Y. The Biology and Function of Extracellular Vesicles in Nasopharyngeal Carcinoma. Int. J. Oncol. 2017, 52 (1), 38– 46,  DOI: 10.3892/ijo.2017.4202
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    Nuzhat, Z.; Kinhal, V.; Sharma, S.; Rice, G. E.; Joshi, V.; Salomon, C. Tumour-Derived Exosomes as a Signature of Pancreatic Cancer - Liquid Biopsies as Indicators of Tumour Progression. Oncotarget 2017, 8 (10), 17279,  DOI: 10.18632/oncotarget.13973
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    Tumour-derived exosomes as a signature of pancreatic cancer - liquid biopsies as indicators of tumour progression
    Nuzhat Zarin; Kinhal Vyjayanthi; Sharma Shayna; Rice Gregory E; Salomon Carlos; Rice Gregory E; Salomon Carlos; Joshi Virendra
    Oncotarget (2017), 8 (10), 17279-17291 ISSN:.
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  • Abstract

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    Figure 1

    Figure 1. Probing saliva’s viscosity at different length scales. (a) Macroscopic standard shear rheological measurements highlight the non-Newtonian behavior of raw saliva (red) in comparison with pure water (blue). (b) The nanoscale viscoelastic behavior of raw saliva is also probed using a vibrating microcantilever operated with an AFM. The viscosity of the liquid surrounding the cantilever can be quantified from the frequency shift of the vibration resonances, (41) with the method applied here to comparatively probe the viscosity of water and of raw saliva. No shift is observed between water and saliva (dashed lines), and the derived viscosities for saliva are identical to water within error (inset in b) (see Experimental Section and Figure S1 for more details). The microcantilever measurements were performed using a commercial microcantilever (Olympus, OMCL-RC800 PSA) at 25.0 ± 0.1 °C, with the probe fully immersed in liquid.

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    Figure 2

    Figure 2. Passive microrheology of raw saliva with silica tracers. A cartoon representation of the system (a–c) illustrates the fact that smaller tracers (a) can diffuse more easily through the mesh formed by saliva compared to larger tracers (b). However, this assumes a limited interaction of the tracer particles with the mesh. Otherwise, interactions with the mesh reduce mobility (c) and affect smaller particles relatively more due to their larger surface to volume ratio. (d) Example of a measurements with a 73 ± 6 nm tracer (without coating) showing the MSD <r2> as a function of time t on a log–log plot. In pure water, <r2> ∝ t indicating normal Brownian diffusion. In saliva, <r2> ∝ tα with α < 1, the so-called anomalous diffusion exponent indicating subdiffusion. (e) The evolution of α at different time scales highlighting differences for water (α ≈ 1, blue curve) and saliva (α < 1) with and without a zwitterionic antifouling coating on the tracer (red and black curves, respectively). Over a short observational time scale (<10 μs), the tracers are able to freely diffuse unhindered. Over longer time scales or for larger tracers, the impact of interactions with saliva components tends to hamper the diffusion. Measurements >0.5 ms are less reliable, being close to the tracking limit of the equipment. Here, this is visible in α becoming lower for coated than uncoated tracers, despite a significantly larger MSD. (f) Time evolution of α for tracers of different sizes. Comparison of α vs tracer size at selected times (10 μs, 25 μs, 50 μs, 0.1 ms, 0.25 ms, and 0.5 ms, vertical dashed lines) suggests consistent unhindered diffusion for tracers <25 nm (inset). All the particles’ diameters are measured by DLS using the same setup as for the microrheology (see Figure S3 and Table S1 within section 3 of the Supporting Information).

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    Figure 3

    Figure 3. Characterization and testing of the proposed approach for targeted detection of specific EV subpopulations directly in raw saliva. The testing is carried out with model EVs composed of DPPC with 0.5% biotinylated lipids acting as surface markers (a) and dissolved into saliva (5% of the total volume from a phosphate buffer saline solution (b)). The cantilever is coated with a DPPC bilayer containing 0.5% biotinylated headgroups (c), preventing biofouling while ensuring specific binding of the model EVs after further streptavidin functionalization (d). From the changes in the cantilever’s vibration amplitude, phase, and frequency, the total mass of the target EVs binding to the cantilever can be precisely quantified despite the saliva background (e). Experimental data are fitted globally with a double exponential function, imposing the same two time scales (τ1 and τ2) for all the experiments. The most important differences occur within the first 5–10 min (τ1 = 346 ± 56 s), with only the slower evolution (τ2 = 5781 ± 778 s) present at 0.1 μg/mL and in pure saliva. (f) The maximum mass uptake (added mass at time = ∞) derived from the fitting also exhibits a double exponential increase against the EV concentration in saliva. All of the measurements are conducted at 25.0 ± 0.1 °C. The error in concentration (f) is assumed to be 10%, likely an overestimate.

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    Figure 4

    Figure 4. Detection of two natural EV subpopulations expressing CD9 and CD81 antigens on their surface, measured in saliva. The experiments are performed on a drop of unprocessed saliva samples from two healthy individuals. The cantilevers are functionalized as described in Figure 3 but with antibodies (Ab) to the targeted tetraspanin (see Experimental Section). The control probes (black) are coated only with the antifouling DPPC bilayer. As for model EVs, the mass uptake is rapid over the first 5–10 min and can be suitably analyzed imposing the same time scales as in Figure 3. The control is analyzed with a single exponential yielding in both cases a characteristic time scale of ∼550 s (see Supporting Information sections 6 for details). Experiments with anti-CD9 and anti-CD81 Abs were repeated twice for each sample with the average shown in the figure.

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  • References

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      BACKGROUND: Epileptic seizures (ES) lead to alterations in the blood laboratory values and reflect changes in different organ systems. Here, we review the diagnostic and prognostic value of various blood laboratory values within the context of epilepsy. METHODS: Narrative review and literature search on PubMed using the term, "seizure" and various laboratory values. RESULTS: Laboratory markers can help clinicians determine whether an unwitnessed event was more likely to be epileptic or non-epileptic. Prolactin testing helps differentiate ES from psychogenic non-epileptic seizures (PNES) in adults and adolescents, and is associated with high specificity and moderate sensitivity. Elevations in the creatine kinase (CK) levels are common after generalized tonic-clonic seizures (GTCS) and display high specificity and moderate sensitivity. Metabolic markers such as ammonia and lactate may have diagnostic potential for postictal blood tests. Analyzing blood postictally is important for identifying the cause of the symptomatic seizures due to endocrine, metabolic, toxic or infectious etiologies. Finally, laboratory analyses are used for identifying patients who are at risk for developing rare, threatening complications such as rhabdomyolysis, acute renal failure (ARF) or cardiomyopathy. CONCLUSIONS: Presently, no postictal laboratory values can definitively prove or rule out the diagnosis of an epileptic seizure. For seizures with unknown causes, simple blood tests can be a valuable aid for quickly defining the etiology, particularly with certain metabolic and toxic encephalopathies. For this reason, CK, electrolytes, creatinine, liver and renal function tests should be measured on at least one occasion. Further research is needed in order to identify new biomarkers that improve the diagnosis and prognosis of seizures and seizure-related complications.
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      Extracellular vesicles (EVs) are submicron-sized lipid envelopes that are produced and released from a parent cell and can be taken up by a recipient cell. EVs are capable of mediating cellular signalling by carrying nucleic acids, proteins, lipids and cellular metabolites between cells and organs. Metabolic dysfunction is associated with changes in plasma concentrations of EVs as well as alterations in their EV cargo. Since EVs can act as messengers between parent and recipient cells, they could be involved in cell-to-cell and organ-to-organ communication in metabolic diseases. Recent literature has shown that EVs are produced by cells within metabolic tissues, such as adipose tissue, pancreas, muscle and liver. These vesicles have therefore been proposed as a novel intercellular communication mode in systemic metabolic regulation. In this review, we will describe and discuss the current literature that investigates the role of adipose-derived EVs in the regulation of obesity-associated metabolic disease. We will particularly focus on the EV-dependent communication between adipocytes, the vasculature and immune cells in type 2 diabetes.
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      OBJECTIVES: Recent studies have suggested that vitamin K may exert significant effects on the central nervous system. The present study investigates the relationship between vitamin K plasmatic levels and cognitive functions in elderly patients on oral anticoagulant therapy (OAT). DESIGN: At the Thrombosis Centre of Haematology, "Sapienza" University of Rome, 85 patients on OAT, aged between 75 and 92, were randomly enrolled in the study. Patients were on OAT with vitamin K antagonists (VKAs). Vitamin K1 concentrations were determined using standardized High-Performance Liquid Chromatography (HPLC). Cognitive functions were assessed using the Milan Overall Dementia Assessment (MODA). RESULTS: MODA scores are positively correlated to vitamin K1 concentration. Patients with vitamin K1 below 0.100 μg/L and between 0.100 and 0.400 μg/L and between 0.100 and 0.400 μg/L and between 0.100 and 0.400 p < 0.001). Even long-term OAT (>10 years) does not affect MODA scores. Education seems to exert a greater role on the cognitive status in comparison with aging. CONCLUSIONS: The study shows a positive association between vitamin K1 concentration and cognitive status in elderly patients (≥75 years) on OAT. The relationship between vitamin K1 concentration and MODA scores is described by a linear model. Cognitive status is not influenced by the duration of OAT but by the years of education.
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      Fattizzo, B.; Levati, G. V.; Giannotta, J. A.; Cassanello, G.; Cro, L. M.; Zaninoni, A.; Barbieri, M.; Croci, G. A.; Revelli, N.; Barcellini, W. Low-Risk Myelodysplastic Syndrome Revisited: Morphological, Autoimmune, and Molecular Features as Predictors of Outcome in a Single Center Experience. Front. Oncol. 2022, 12, 795955,  DOI: 10.3389/fonc.2022.795955
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      Low-risk myelodysplastic syndrome revisited: morphological, autoimmune, and molecular features as predictors of outcome in a single center experience
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      Low-risk myelodysplastic syndromes (LR-MDS) are a very heterogeneous disease, with extremely variable clin. features and outcome. Therapeutic strategies are still limited and mainly consist of erythropoiesis-stimulating agents (ESAs) and transfusion support. The contribution of mol. lesions and of autoimmune phenomena to pathogenesis and clin. course, including leukemic evolution, is a field of open investigation. We analyzed data from a cohort of 226 patients with LR-MDS followed at our center in the last 20 years, focusing on morphol., immunol. (antiplatelets and anti-erythrocyte autoantibodies, anti-erythroblast antibodies), and mol. features. Hypoplastic bone marrow was found in 7% of the cases correlating with younger age, deeper cytopenia, lower dysplasia, and worse response to ESAs. A marker of autoimmunity was obsd. in 46% of the tested cases, who were younger, were less frequent dysplastic changes, and responded better to ESAs and steroids. Finally, 68% of the tested cases displayed at least one somatic mutation, most commonly SF3B1, TET2, ASXL1, and SRSF2, assocd. with older age, presence of neutropenia, and lower response to ESAs. Leukemic evolution (2.2%) was assocd. with presence of somatic mutations, and survival was favorably related to response to ESAs and transfusion independence. Overall, granular evaluation and re-evaluation are pivotal in LR-MDS patients to optimize clin. management.
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      Wu, Y.; Fan, Q.; Chen, Y.; Sun, X.; Shi, G. Production and Selection of Antibody-Antigen Pairs for the Development of Immunoenzyme Assay and Lateral Flow Immunoassay Methods for Carbofuran and Its Analogues. Biosensors 2022, 12 (8), 560,  DOI: 10.3390/bios12080560
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      Production and Selection of Antibody-Antigen Pairs for the Development of Immunoenzyme Assay and Lateral Flow Immunoassay Methods for Carbofuran and Its Analogues
      Wu, Yuxiang; Fan, Qi; Chen, Yinuo; Sun, Xia; Shi, Guoqing
      Biosensors (2022), 12 (8), 560CODEN: BIOSHU; ISSN:2079-6374. (MDPI AG)
      To produce a sensitive monoclonal antibody (mAb) for the simultaneous detection of carbofuran, benfuracarb, carbosulfan and 3-hydroxy-carbofuran, 2,3-dihydro-2,2-dimethyl-7-benzofuranmethanamine (DDB) was conjugated to bovine serum albumin (BSA) to prep. the immunogen DDB-BSA and mice were immunized. Coating antigens were prepd. by conjugating DDB and 5-methoxy-2,3-dihydrobenzofuran-3-acetic acid (MDA) to BSA and ovalbumin (OVA), resp. Furthermore, the effect of different antibody-antigen pairs on the sensitivity of ELISA and LFIA methods for the detection of carbofuran was investigated. After the immunization, a high-affinity mAb 13C8 was obtained. The ability of the coating antigen to compete with carbofuran for binding antibodies was found to be significantly different between ELISA and LFIA methods. With the antibody-antigen pair 13C8-MDA-OVA, the IC50 values of the ELISA and QD-LFIA methods for carbofuran were 0.18 ng/mL and 0.67 ng/mL, resp. The cross-reactivity (CR) values of the two methods for benfuracarb, carbosulfan and 3-hydroxy-carbofuran ranged from 72.0% to 83.7%, while, for other carbamate pesticides, the CR values were less than 1%. The spiked recoveries of carbofuran in vegetables by the QD-LFIA method were 83-111%, with a coeff. of variation below 10%, and the test results of the actual samples were consistent with the HPLC-MS method. Overall, this study provides key materials for the development of immunoassays for carbofuran and its analogs, and the antibody-antigen pair selection strategy established in this study provides useful insights for the development of sensitive immunoassays for other compds.
    9. 9
      Litke, J. L.; Jaffrey, S. R. Highly Efficient Expression of Circular RNA Aptamers in Cells Using Autocatalytic Transcripts. Nat. Biotechnol. 2019, 37 (6), 667– 675,  DOI: 10.1038/s41587-019-0090-6
      9
      Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts
      Litke, Jacob L.; Jaffrey, Samie R.
      Nature Biotechnology (2019), 37 (6), 667-675CODEN: NABIF9; ISSN:1087-0156. (Nature Research)
      RNA aptamers and RNA aptamer-based devices can be genetically encoded and expressed in cells to probe and manipulate cellular function. However, their usefulness in the mammalian cell is limited by low expression and rapid degrdn. Here we describe the Tornado (Twister-optimized RNA for durable overexpression) expression system for achieving rapid RNA circularization, resulting in RNA aptamers with high stability and expression levels. Tornado-expressed transcripts contain an RNA of interest flanked by Twister ribozymes. The ribozymes rapidly undergo autocatalytic cleavage, leaving termini that are ligated by the ubiquitous endogenous RNA ligase RtcB. Using this approach, protein-binding aptamers that otherwise have minimal effects in cells become potent inhibitors of cellular signaling. Addnl., an RNA-based fluorescent metabolite biosensor for S-adenosyl methionine (SAM) that is expressed at low levels when expressed as a linear RNA achieves levels sufficient for detection of intracellular SAM dynamics when expressed as a circular RNA. The Tornado expression system thus markedly enhances the utility of RNA-based approaches in the mammalian cell.
    10. 10
      Ombelet, S.; Barbé, B.; Affolabi, D.; Ronat, J. B.; Lompo, P.; Lunguya, O.; Jacobs, J.; Hardy, L. Best Practices of Blood Cultures in Low- and Middle-Income Countries. Front. Med. 2019, 6, 131,  DOI: 10.3389/fmed.2019.00131
      10
      Best Practices of Blood Cultures in Low- and Middle-Income Countries
      Ombelet Sien; Barbe Barbara; Jacobs Jan; Hardy Liselotte; Ombelet Sien; Jacobs Jan; Affolabi Dissou; Ronat Jean-Baptiste; Lompo Palpouguini; Lunguya Octavie; Lunguya Octavie
      Frontiers in medicine (2019), 6 (), 131 ISSN:2296-858X.
      Bloodstream infections (BSI) have a substantial impact on morbidity and mortality worldwide. Despite scarcity of data from many low- and middle-income countries (LMICs), there is increasing awareness of the importance of BSI in these countries. For example, it is estimated that the global mortality of non-typhoidal Salmonella bloodstream infection in children under 5 already exceeds that of malaria. Reliable and accurate diagnosis of these infections is therefore of utmost importance. Blood cultures are the reference method for diagnosis of BSI. LMICs face many challenges when implementing blood cultures, due to financial, logistical, and infrastructure-related constraints. This review aims to provide an overview of the state-of-the-art of sampling and processing of blood cultures, with emphasis on its use in LMICs. Laboratory processing of blood cultures is relatively straightforward and can be done without the need for expensive and complicated equipment. Automates for incubation and growth monitoring have become the standard in high-income countries (HICs), but they are still too expensive and not sufficiently robust for imminent implementation in most LMICs. Therefore, this review focuses on "manual" methods of blood culture, not involving automated equipment. In manual blood cultures, a bottle consisting of a broth medium supporting bacterial growth is incubated in a normal incubator and inspected daily for signs of growth. The collection of blood for blood culture is a crucial step in the process, as the sensitivity of blood cultures depends on the volume sampled; furthermore, contamination of the blood culture (accidental inoculation of environmental and skin bacteria) can be avoided by appropriate antisepsis. In this review, we give recommendations regarding appropriate blood culture sampling and processing in LMICs. We present feasible methods to detect and speed up growth and discuss some challenges in implementing blood cultures in LMICs, such as the biosafety aspects, supply chain and waste management.
    11. 11
      Marcali, M.; Chen, X.; Aucoin, M. G.; Ren, C. L. Droplet Formation of Biological Non-Newtonian Fluid in T-Junction Generators I. Experimental Investigation. Phys. Rev. E 2022, 105, 025105,  DOI: 10.1103/PhysRevE.105.025105
      11
      Droplet formation of biological non-Newtonian fluid in T-junction generators. I. Experimental investigation
      Marcali, Merve; Chen, Xiaoming; Aucoin, Marc G.; Ren, Carolyn L.
      Physical Review E (2022), 105 (2), 025105CODEN: PREHBM; ISSN:2470-0053. (American Physical Society)
      The extension of microfluidics to many bioassay applications requires the ability to work with non-Newtonian fluids. One case in point is the use of microfluidics with blood having different hematocrit levels. This work is the first part of a two-part study and presents the formation dynamics of blood droplets in a T-junction generator under the squeezing regime. In this regime, droplet formation with Newtonian fluids depends on T-junction geometry; however, we found that in the presence of the non-Newtonian fluid such as red blood cells, the formation depends on not only to the channel geometry, but also the flow rate ratio of fluids, and the viscosity of the phases. In addn., we analyzed the impact of the red blood cell concn. on the formation cycle. In this study, we presented the exptl. data of the blood droplet evolution through the anal. of videos that are captured by a high-speed camera. During this anal., we tracked several parameters such as droplet vol., spacing between droplets, droplet generation frequency, flow conditions, and geometrical designs of the T junction. Our anal. revealed that, unlike other non-Newtonian fluids, where the fourth stage exists (stretching stage), the formation cycle consists of only three stages: lag, filling, and necking stages. Because of the detailed anal. of each stage, a math. model can be generated to predict the final vol. of the blood droplet and can be utilized as a guide in the operation of the microfluidic device for biochem. assay applications; this is the focus of the second part of this study.
    12. 12
      Kosaka, P. M.; Calleja, M.; Tamayo, J. Optomechanical Devices for Deep Plasma Cancer Proteomics. Seminars Cancer Biol. 2018, 52, 26– 38,  DOI: 10.1016/j.semcancer.2017.08.011
      12
      Optomechanical devices for deep plasma cancer proteomics
      Kosaka, Priscila M.; Calleja, Montserrat; Tamayo, Javier
      Seminars in Cancer Biology (2018), 52 (Part_1), 26-38CODEN: SECBE7; ISSN:1044-579X. (Elsevier Ltd.)
      Most of the cancer deaths could be avoided by early detection of the tumor when it is confined to its primary site and it has not metastasized. To this aim, one of the most promising strategies is the discovery and detection of protein biomarkers shed by the young tumor to the bloodstream. Proteomic technologies, mainly mass spectrometry and multiplexed immunoassays, have rapidly developed during last years with improved limits of detection and multiplexing capability. Unfortunately, these developments together major investments and large international efforts have not resulted into new useful protein biomarkers. Here, we analyze the potential and limitations of current proteomic technologies for detecting protein biomarkers released into circulation by the tumor. We find that these technologies can hardly probe the deepest region of the plasma proteome, at concns. below the pg/mL level, where protein biomarkers for early cancer detection may exist. This clearly indicates the need of incorporating novel ultrasensitive techniques to the proteomic tool-box that can cover the inaccessible regions of the plasma proteome. We here propose biol. detectors based on nanomech. systems for discovery and detection of cancer protein biomarkers in plasma. We review the modes of operation of these devices, putting our focus on recent developments on nanomech. sandwich immunoassays and nanomech. spectrometry. The first technique enables reproducible immunodetection of proteins at concns. well below the pg/mL level, with a limit of detection on the verge of 10 ag/mL. This technol. can potentially detect low abundance tumor-assocd. proteins in plasma at the very early stages of the tumor. The second technique enables the identification of individual intact proteins by two phys. coordinates, the mass and stiffness, instead of the mass-to-charge ratio of the protein constituents. This technol. enormously simplifies the identification of proteins and it can provide useful information on interactions and posttranslational modifications, that otherwise is lost in mass spectrometry.
    13. 13
      Helton, K. L.; Nelson, K. E.; Fu, E.; Yager, P. Conditioning Saliva for Use in a Microfluidic Biosensor. Lab on Chip 2008, 8 (11), 1847– 1851,  DOI: 10.1039/b811150b
      13
      Conditioning saliva for use in a microfluidic biosensor
      Helton, Kristen L.; Nelson, Kjell E.; Fu, Elain; Yager, Paul
      Lab on a Chip (2008), 8 (11), 1847-1851CODEN: LCAHAM; ISSN:1473-0197. (Royal Society of Chemistry)
      This report details an approach to saliva conditioning for compatibility of raw patient samples with microfluidic immunoassay components, principally biosensor surfaces susceptible to fouling. Stimulated whole human saliva spiked with a small mol. analyte (phenytoin, 252 Da) was first depleted of cells, debris and high mol. wt. glycoproteins (mucins) using membrane filtration. This process significantly reduced but did not eliminate fouling of biosensor surfaces exposed to the sample. An H-filter, which separates solutes from mixed samples based on their diffusion in laminar flow, was used to ext. the analyte from the remaining large mol. wt. species in the filtered saliva sample. Patient samples treated in this way retained 23% of the analyte with 97% and 92% redn. in glycoproteins and proteins, resp., and resulted in 3.6 times less surface fouling than either untreated or filtered saliva alone. These sample conditioning steps will enable the use of fouling-sensitive detection techniques in future studies using clin. saliva samples.
    14. 14
      Defina, S. M.; Wang, J.; Yang, L.; Zhou, H.; Adams, J.; Cushing, W.; Tuohy, B.; Hui, P.; Liu, C.; Pham, K. SaliVISION: A Rapid Saliva-Based COVID-19 Screening and Diagnostic Test with High Sensitivity and Specificity. Sci. Rep. 2022, 12 (1), 5729,  DOI: 10.1038/s41598-022-09718-4
      14
      SaliVISION: a rapid saliva-based COVID-19 screening and diagnostic test with high sensitivity and specificity
      DeFina, Samuel M.; Wang, Jianhui; Yang, Lei; Zhou, Han; Adams, Jennifer; Cushing, William; Tuohy, Beth; Hui, Pei; Liu, Chen; Pham, Kien
      Scientific Reports (2022), 12 (1), 5729CODEN: SRCEC3; ISSN:2045-2322. (Nature Portfolio)
      The Coronavirus disease 2019 (COVID-19) pandemic-caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)- has posed a global threat and presented with it a multitude of economic and public-health challenges. Establishing a reliable means of readily available, rapid diagnostic testing is of paramount importance in halting the spread of COVID-19, as governments continue to ease lockdown restrictions. The current std. for lab. testing utilizes reverse transcription quant. polymerase chain reaction (RT-qPCR); however, this method presents clear limitations in requiring a longer run-time as well as reduced on-site testing capability. Therefore, we investigated the feasibility of a reverse transcription looped-mediated isothermal amplification (RT-LAMP)-based model of rapid COVID-19 diagnostic testing which allows for less invasive sample collection, named SaliVISION. This novel, two-step, RT-LAMP assay utilizes a customized multiplex primer set specifically targeting SARS-CoV-2 and a visual report system that is ready to interpret within 40 min from the start of sample processing and does not require a BSL-2 level testing environment or special lab. equipment. When compared to the SalivaDirect and Thermo Fisher Scientific TaqPath RT-qPCR testing platforms, the resp. sensitivities of the SaliVISION assay are 94.29% and 98.28% while assay specificity was 100% when compared to either testing platform. Our data illustrate a robust, rapid diagnostic assay in our novel RT-LAMP test design, with potential for greater testing throughput than is currently available through lab. testing and increased on-site testing capability.
    15. 15
      Errazquin, R.; Carrasco, E.; Del Marro, S.; Suñol, A.; Peral, J.; Ortiz, J.; Rubio, J. C.; Segrelles, C.; Dueñas, M.; Garrido-Aranda, A.; Alvarez, M.; Belendez, C.; Balmaña, J.; Garcia-Escudero, R. Early Diagnosis of Oral Cancer and Lesions in Fanconi Anemia Patients: A Prospective and Longitudinal Study Using Saliva and Plasma. Cancers 2023, 15 (6), 1871,  DOI: 10.3390/cancers15061871
      15
      Early Diagnosis of Oral Cancer and Lesions in Fanconi Anemia Patients: A Prospective and Longitudinal Study Using Saliva and Plasma
      Errazquin, Ricardo; Carrasco, Estela; Del Marro, Sonia; Sunol, Anna; Peral, Jorge; Ortiz, Jessica; Rubio, Juan Carlos; Segrelles, Carmen; Duenas, Marta; Garrido-Aranda, Alicia; Alvarez, Martina; Belendez, Cristina; Balmana, Judith; Garcia-Escudero, Ramon
      Cancers (2023), 15 (6), 1871CODEN: CANCCT; ISSN:2072-6694. (MDPI AG)
      Simple Summary: Patients with Fanconi anemia (FA) have a very high risk of developing oral lesions and squamous carcinomas at early ages. As treatment strategies in this setting are very limited, new early diagnosis methods are urgently needed. We performed a pilot, prospective clin. study in which saliva and plasma samples were analyzed with the deep sequencing of cancer genes. The patients included had apparently normal oral mucosa when recruited. Mutations were detected in the liq. biopsies with allele frequencies of down to 0.07%. We found that patients with mutations displayed a higher risk of developing lesions/carcinomas after mutation detection. We propose that this non-invasive, highly sensitive technol. could allow for the better management of these pathologies in FA individuals. Abstr.: Fanconi anemia (FA) patients display an exacerbated risk of oral squamous cell carcinoma (OSCC) and oral potentially malignant lesions (OPMLs) at early ages. As patients have defects in their DNA repair mechanisms, std.-of-care treatments for OSCC such as radiotherapy and chemotherapy, give rise to severe toxicities. New methods for early diagnosis are urgently needed to allow for treatment in early disease stages and achieve better clin. outcomes. We conducted a prospective, longitudinal study wherein liq. biopsies from sixteen patients with no clin. diagnoses of OPML and/or OSCC were analyzed for the presence of mutations in cancer genes. The DNA from saliva and plasma were sequentially collected and deep-sequenced, and the clin. evaluation followed over a median time of approx. 2 years. In 9/16 FA patients, we detected mutations in cancer genes (mainly TP53) with minor allele frequencies (MAF) of down to 0.07%. Importantly, all patients that had mutations and clin. follow-up data after mutation detection (n = 6) developed oral precursor lesions or OSCC. The lead-time between mutation detection and tumor diagnosis ranged from 23 to 630 days. Strikingly, FA patients without mutations displayed a significantly lower risk of developing precursor lesions or OSCCs. Therefore, our diagnostic approach could help to stratify FA patients into risk groups, which would allow for closer surveillance for OSCCs or precursor lesions.
    16. 16
      Deshmukh, S. K.; Khan, M. A.; Singh, S.; Singh, A. P. Extracellular Nanovesicles: From Intercellular Messengers to Efficient Drug Delivery Systems. ACS Omega 2021, 6 (3), 1773– 1779,  DOI: 10.1021/acsomega.0c05539
      16
      Extracellular Nanovesicles: From Intercellular Messengers to Efficient Drug Delivery Systems
      Deshmukh, Sachin Kumar; Khan, Mohammad Aslam; Singh, Seema; Singh, Ajay Pratap
      ACS Omega (2021), 6 (3), 1773-1779CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)
      A review. Nanosized extracellular vesicles (nEV) are released by all the eukaryotic cells into the extracellular spaces. They serve as crucial mediators of intercellular communication, and their presence has been detected in a variety of body fluids. nEV carry nucleic acids, lipids, proteins, and metabolites from the donor cells and transfer them to the recipient cells in the vicinity or distant locations to cause changes in their biol. phenotypes. This very property of nEV makes them a suitable carrier of the drugs for therapeutic applications. The use of nEV as a drug delivery system offers several advantages over synthetic nanoparticles, including biocompatibility, natural targeting ability, and long-term safety. Further, nEV can be isolated from various biol. sources, quickly loaded with the drug of choice, and modified to further enhance their utility as targeted drug delivery vehicles. Here we review these aspects of nEV and discuss the parameters that should be kept in mind while choosing the nEV source, drug loading method, and surface modification strategies. We also discuss the challenges assocd. with the nEV-based drug delivery platforms that must be overcome before realizing their full potential in clin. applications.
    17. 17
      Fais, S.; O’Driscoll, L.; Borras, F. E.; Buzas, E.; Camussi, G.; Cappello, F.; Carvalho, J.; Cordeiro da Silva, A.; Del Portillo, H.; El Andaloussi, S.; Ficko Trček, T.; Furlan, R.; Hendrix, A.; Gursel, I.; Kralj-Iglic, V.; Kaeffer, B.; Kosanovic, M.; Lekka, M. E.; Lipps, G.; Logozzi, M.; Marcilla, A.; Sammar, M.; Llorente, A.; Nazarenko, I.; Oliveira, C.; Pocsfalvi, G.; Rajendran, L.; Raposo, G.; Rohde, E.; Siljander, P.; van Niel, G.; Vasconcelos, M. H.; Yáñez-Mó, M.; Yliperttula, M. L.; Zarovni, N.; Zavec, A. B.; Giebel, B. Evidence-Based Clinical Use of Nanoscale Extracellular Vesicles in Nanomedicine. ACS Nano 2016, 10 (4), 3886– 3899,  DOI: 10.1021/acsnano.5b08015
      17
      Evidence-Based Clinical Use of Nanoscale Extracellular Vesicles in Nanomedicine
      Fais, Stefano; O'Driscoll, Lorraine; Borras, Francesc E.; Buzas, Edit; Camussi, Giovanni; Cappello, Francesco; Carvalho, Joana; Cordeiro da Silva, Anabela; Del Portillo, Hernando; El Andaloussi, Samir; Ficko Trcek, Tanja; Furlan, Roberto; Hendrix, An; Gursel, Ihsan; Kralj-Iglic, Veronika; Kaeffer, Bertrand; Kosanovic, Maja; Lekka, Marilena E.; Lipps, Georg; Logozzi, Mariantonia; Marcilla, Antonio; Sammar, Marei; Llorente, Alicia; Nazarenko, Irina; Oliveira, Carla; Pocsfalvi, Gabriella; Rajendran, Lawrence; Raposo, Graca; Rohde, Eva; Siljander, Pia; van Niel, Guillaume; Vasconcelos, M. Helena; Yanez-Mo, Maria; Yliperttula, Marjo L.; Zarovni, Natasa; Zavec, Apolonija Bedina; Giebel, Bernd
      ACS Nano (2016), 10 (4), 3886-3899CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      Recent research has demonstrated that all body fluids assessed contain substantial amts. of vesicles that range in size from 30 to 1000 nm and that are surrounded by phospholipid membranes contg. different membrane microdomains such as lipid rafts and caveolae. The most prominent representatives of these so-called extracellular vesicles (EVs) are nanosized exosomes (70-150 nm), which are derivs. of the endosomal system, and microvesicles (100-1000 nm), which are produced by outward budding of the plasma membrane. Nanosized EVs are released by almost all cell types and mediate targeted intercellular communication under physiol. and pathophysiol. conditions. Contg. cell-type-specific signatures, EVs have been proposed as biomarkers in a variety of diseases. Furthermore, according to their phys. functions, EVs of selected cell types have been used as therapeutic agents in immune therapy, vaccination trials, regenerative medicine, and drug delivery. Undoubtedly, the rapidly emerging field of basic and applied EV research will significantly influence the biomedicinal landscape in the future. In this Perspective, we, a network of European scientists from clin., academic, and industry settings collaborating through the H2020 European Cooperation in Science and Technol. (COST) program European Network on Microvesicles and Exosomes in Health and Disease (ME-HAD), demonstrate the high potential of nanosized EVs for both diagnostic and therapeutic (i.e., theranostic) areas of nanomedicine.
    18. 18
      Sun, W.; Ren, Y.; Lu, Z.; Zhao, X. The Potential Roles of Exosomes in Pancreatic Cancer Initiation and Metastasis. Molecular Cancer 2020, 19 (1), 135,  DOI: 10.1186/s12943-020-01255-w
      18
      The potential roles of exosomes in pancreatic cancer initiation and metastasis
      Sun Wei; Ren Ying; Lu Zaiming; Zhao Xiangxuan
      Molecular cancer (2020), 19 (1), 135 ISSN:.
      Pancreatic cancer (PaCa) is an insidious and highly metastatic malignancy, with a 5-year survival rate of less than 5%. So far, the pathogenesis and progression mechanisms of PaCa have been poorly characterized. Exosomes correspond to a class of extracellular nanovesicles, produced by a broad range of human somatic and cancerous cells. These particular nanovesicles are mainly composed by proteins, genetic substances and lipids, which mediate signal transduction and material transport. A large number of studies have indicated that exosomes may play decisive roles in the occurrence and metastatic progression of PaCa. This article summarizes the specific functions of exosomes and their underlying molecular mechanisms in mediating the initiation and metastatic capability of PaCa.
    19. 19
      Bobrie, A.; Colombo, M.; Raposo, G.; Théry, C. Exosome Secretion: Molecular Mechanisms and Roles in Immune Responses. Traffic 2011, 12 (12), 1659– 1668,  DOI: 10.1111/j.1600-0854.2011.01225.x
      19
      Exosome secretion: molecular mechanisms and roles in immune responses
      Bobrie, Angelique; Colombo, Marina; Raposo, Graca; Thery, Clotilde
      Traffic (Oxford, United Kingdom) (2011), 12 (12), 1659-1668CODEN: TRAFFA; ISSN:1398-9219. (Wiley-Blackwell)
      A review. Exosomes are small membrane vesicles, secreted by most cell types from multivesicular endosomes, and thought to play important roles in intercellular communications. Initially described in 1983, as specifically secreted by reticulocytes, exosomes became of interest for immunologists in 1996, when they were proposed to play a role in antigen presentation. More recently, the finding that exosomes carry genetic materials, mRNA and miRNA, has been a major breakthrough in the field, unveiling their capacity to vehicle genetic messages. It is now clear that not only immune cells but probably all cell types are able to secrete exosomes: their range of possible functions expands well beyond immunol. to neurobiol., stem cell and tumor biol., and their use in clin. applications as biomarkers or as therapeutic tools is an extensive area of research. Despite intensive efforts to understand their functions, two issues remain to be solved in the future: (i) what are the physiol. function(s) of exosomes in vivo and (ii) what are the relative contributions of exosomes and of other secreted membrane vesicles in these proposed functions. Here, we will focus on the current ideas on exosomes and immune responses, but also on their mechanisms of secretion and the use of this knowledge to elucidate the latter issue.
    20. 20
      Huang, T.; Deng, C. X. Current Progresses of Exosomes as Cancer Diagnostic and Prognostic Biomarkers. Int. J. Biol. Sci. 2019, 15 (1), 1,  DOI: 10.7150/ijbs.27796
      20
      Current progresses of exosomes as cancer diagnostic and prognostic biomarkers
      Huang, Tao; Deng, Chu-Xia
      International Journal of Biological Sciences (2019), 15 (1), 1-11CODEN: IJBSB9; ISSN:1449-2288. (Ivyspring International Publisher)
      A review. Cancer related exosomes are nano-size membrane vesicles that play important roles in tumor microenvironment. Emerging evidence indicates that exosomes can load unique cargoes, including proteins and nucleic acids that reflect the condition of tumor. Therefore, exosomes are being used as diagnostic and prognostic biomarkers for various cancers. In this review, we describe the current progresses of cancer related exosomes, including their biogenesis, mol. contents, biol. functions, sources where they are derived from, and methods for their detection. We will also discuss the current exosomal biomarkers and the utilization of them for early diagnosis and prognostics in cancer.
    21. 21
      Li, W.; Li, C.; Zhou, T.; Liu, X.; Liu, X.; Li, X.; Chen, D. Role of Exosomal Proteins in Cancer Diagnosis. Mol. Cancer 2017, 16 (1), 145,  DOI: 10.1186/s12943-017-0706-8
      21
      Role of exosomal proteins in cancer diagnosis
      Li, Weihua; Li, Chuanyun; Zhou, Tong; Liu, Xiuhong; Liu, Xiaoni; Li, Xiuhui; Chen, Dexi
      Molecular Cancer (2017), 16 (), 145/1-145/12CODEN: MCOACG; ISSN:1476-4598. (BioMed Central Ltd.)
      Exosomes are emerging as a new type of cancer biomarkers. Exosome is a bilayered nano-sized vesicle secreted by various living cells in all body fluids. Based on the expanding albeit incomplete knowledge of their biogenesis, secretion by cells and cancer cell-specific mol. and genetic contents, exosomes are viewed as promising, clin.-relevant surrogates of cancer progression and response to therapy. Preliminary proteomic, genetic and functional profiling of cancer cell-derived or cancer plasma-derived exosomes confirms their unique characteristics. Alterations in protein or nucleic acid profiles of exosomes in plasma correlate with pathol. processes of many diseases including cancer. However, previous studies on exosome application in cancer diagnosis and treatment mainly focussed on miRNAs. With the development of rapid large-scale prodn., purifn., extn. and screening of exosomal contents, exosomal protein application can be explored for early stage cancer diagnosis, monitoring and prognosis evaluation. Here, we summarized the recent developments in application of exosomal proteins for cancer diagnosis.
    22. 22
      Zhou, F.; Huang, L.; Qu, S. L.; Chao, R.; Yang, C.; Jiang, Z. S.; Zhang, C. The Emerging Roles of Extracellular Vesicles in Diabetes and Diabetic Complications. Clin. Chim. Acta 2019, 497, 130– 136,  DOI: 10.1016/j.cca.2019.07.032
      22
      The emerging roles of extracellular vesicles in diabetes and diabetic complications
      Zhou, Fan; Huang, Liang; Qu, Shun-Lin; Chao, Ru; Yang, Chen; Jiang, Zhi-Sheng; Zhang, Chi
      Clinica Chimica Acta (2019), 497 (), 130-136CODEN: CCATAR; ISSN:0009-8981. (Elsevier B.V.)
      A review. Diabetes and diabetic vascular complications are now the leading cause of death in the world. The effects of traditional medical treatment are usually limited and accompanied by many side effects, such as hypoglycemia, obesity, liver and kidney damage, and gastrointestinal adverse reactions. Thus, it is urgent to explore some new strategies for the treatment of patients with diabetes. Recently, extracellular vesicles have received increased attention because of their emerging roles of cell-to-cell communication under physiol. and pathol. conditions. In addn., because of their abundant existence in almost all body fluids, as well as their plentiful cargos of bioactive proteins and miRNAs they carry, extracellular vesicles have a strong potential for therapeutic and diagnostic applications in many metabolic diseases, such as obesity and insulin resistance. Here, with the aim of providing the basis for the development of new treatments for diabetes, we review current understanding of extracellular vesicles and the crit. roles it has played in the onset and progression of diabetes and diabetic complications.
    23. 23
      Vella, L. J.; Hill, A. F.; Cheng, L. Focus on Extracellular Vesicles: Exosomes and Their Role in Protein Trafficking and Biomarker Potential in Alzheimer’s and Parkinson’s Disease. Int. J. Mol. Sci. 2016 2016, 17 (2), 173,  DOI: 10.3390/ijms17020173
      There is no corresponding record for this reference.
    24. 24
      Cvjetkovic, A.; Lötvall, J.; Lässer, C. The Influence of Rotor Type and Centrifugation Time on the Yield and Purity of Extracellular Vesicles. J. Extracell. Ves. 2014, 3 (1), 23111,  DOI: 10.3402/jev.v3.23111
      There is no corresponding record for this reference.
    25. 25
      Minciacchi, V. R.; Freeman, M. R.; Di Vizio, D. Extracellular Vesicles in Cancer: Exosomes, Microvesicles and the Emerging Role of Large Oncosomes. Seminars Cell Develop. Biol. 2015, 40, 41– 51,  DOI: 10.1016/j.semcdb.2015.02.010
      25
      Extracellular Vesicles in Cancer: Exosomes, Microvesicles and the Emerging Role of Large Oncosomes
      Minciacchi, Valentina R.; Freeman, Michael R.; Di Vizio, Dolores
      Seminars in Cell & Developmental Biology (2015), 40 (), 41-51CODEN: SCDBFX; ISSN:1084-9521. (Elsevier Ltd.)
      Since their first description, extracellular vesicles (EVs) have been the topic of avid study in a variety of physiol. contexts and are now thought to play an important role in cancer. The state of knowledge on biogenesis, mol. content and horizontal communication of diverse types of cancer EVs has expanded considerably in recent years. As a consequence, a plethora of information about EV compn. and mol. function has emerged, along with the notion that cancer cells rely on these particles to invade tissues and propagate oncogenic signals at distance. The no. of in vivo studies, designed to achieve a deeper understanding of the extent to which EV biol. can be applied to clin. relevant settings, is rapidly growing. This review summarizes recent studies on cancer-derived EV functions, with an overview about biogenesis and mol. cargo of exosomes, microvesicles and large oncosomes. We also discuss current challenges and emerging technologies that might improve EV detection in various biol. systems. Further studies on the functional role of EVs in specific steps of cancer formation and progression will expand our understanding of the diversity of paracrine signaling mechanisms in malignant growth.
    26. 26
      McKendry, R.; Zhang, J.; Arntz, Y.; Strunz, T.; Hegner, M.; Lang, H. P.; Baller, M. K.; Certa, U.; Meyer, E.; Güntherodt, H. J.; Gerber, C. Multiple Label-Free Biodetection and Quantitative DNA-Binding Assays on a Nanomechanical Cantilever Array. Proc. Natl. Acad. Sci. U. S. A. 2002, 99 (15), 9783– 9788,  DOI: 10.1073/pnas.152330199
      26
      Multiple label-free biodetection and quantitative DNA-binding assays on a nanomechanical cantilever array
      McKendry, Rachel; Zhang, Jiayun; Arntz, Youri; Strunz, Torsten; Hegner, Martin; Lang, Hans Peter; Baller, Marko K.; Certa, Ulrich; Meyer, Ernst; Guntherodt, Hans-Joachim; Gerber, Christoph
      Proceedings of the National Academy of Sciences of the United States of America (2002), 99 (15), 9783-9788CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      We report a microarray of cantilevers to detect multiple unlabeled biomols. simultaneously at nanomolar concns. within minutes. Ligand-receptor binding interactions such as DNA hybridization or protein recognition occurring on microfabricated silicon cantilevers generate nanomech. bending, which is detected optically in situ. Differential measurements including ref. cantilevers on an array of eight sensors can sequence-specifically detect unlabeled DNA targets in 80-fold excess of nonmatching DNA as a background and discriminate 3' and 5' overhangs. Our expts. suggest that the nanomech. motion originates from predominantly steric hindrance effects and depends on the concn. of DNA mols. in soln. We show that cantilever arrays can be used to investigate the thermodn. of biomol. interactions mech., and we have found that the specificity of the reaction on a cantilever is consistent with soln. data. Hence cantilever arrays permit multiple binding assays in parallel and can detect femtomoles of DNA on the cantilever at a DNA concn. in soln. of 75 nM.
    27. 27
      De Pastina, A.; Padovani, F.; Brunetti, G.; Rotella, C.; Niosi, F.; Usov, V.; Hegner, M. Multimodal Real-Time Frequency Tracking of Cantilever Arrays in Liquid Environment for Biodetection: Comprehensive Setup and Performance Analysis. Rev. Sci. Instrum. 2021, 92 (6), 065001  DOI: 10.1063/5.0047631
      27
      Multimodal real-time frequency tracking of cantilever arrays in liquid environment for biodetection: Comprehensive setup and performance analysis
      De Pastina, Annalisa; Padovani, Francesco; Brunetti, Giulio; Rotella, Chiara; Niosi, Fabio; Usov, Victor; Hegner, Martin
      Review of Scientific Instruments (2021), 92 (6), 065001CODEN: RSINAK; ISSN:0034-6748. (American Institute of Physics)
      We present a nanomech. platform for real-time quant. label-free detection of target biomols. in a liq. environment with mass sensitivity down to few pg. Newly fabricated arrays of up to 18 cantilevers are integrated in a micromachined fluidic chamber, connected to software-controlled fluidic pumps for automated sample injections. We discuss two functionalization approaches to independently sensitize the interface of different cantilevers. A custom piezo-stack actuator and optical readout system enable the measurement of resonance frequencies up to 2 MHz. We implement a new measurement strategy based on a phase-locked loop (PLL), built via inhouse developed software. The PLL allows us to track, within the same expt., the evolution of resonance frequency over time of up to four modes for all the cantilevers in the array. With respect to the previous measurement technique, based on std. frequency sweep, the PLL enhances the estd. detection limit of the device by a factor of 7 (down to 2 pg in 5 min integration time) and the time resoln. by more than threefold (below 15 s), being on par with com. gold-std. techniques. The detection limit and noise of the new setup are investigated via Allan deviation and std. deviation anal., considering different resonance modes and interface chemistries. As a proof-of-concept, we show the immobilization and label-free in situ detection of live bacterial cells (E. coli), demonstrating qual. and quant. agreement in the mech. response of three different resonance modes. (c) 2021 American Institute of Physics.
    28. 28
      Burg, T. P.; Godin, M.; Knudsen, S. M.; Shen, W.; Carlson, G.; Foster, J. S.; Babcock, K.; Manalis, S. R. Weighing of Biomolecules, Single Cells and Single Nanoparticles in Fluid. Nature 2007, 446 (7139), 1066– 1069,  DOI: 10.1038/nature05741
      28
      Weighing of biomolecules, single cells and single nanoparticles in fluid
      Burg, Thomas P.; Godin, Michel; Knudsen, Scott M.; Shen, Wenjiang; Carlson, Greg; Foster, John S.; Babcock, Ken; Manalis, Scott R.
      Nature (London, United Kingdom) (2007), 446 (7139), 1066-1069CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Nanomech. resonators enable the measurement of mass with extraordinary sensitivity. Previously, samples as light as 7 zeptograms (1 zg = 10-21 g) have been weighed in vacuum, and proton-level resoln. seems to be within reach. Resolving small mass changes requires the resonator to be light and to ring at a very pure tone - i.e., with a high quality factor. In soln., viscosity severely degrades both of these characteristics, thus preventing many applications in nanotechnol. and the life sciences where fluid is required. Although the resonant structure can be designed to minimize viscous loss, resoln. is still substantially degraded when compared to measurements made in air or vacuum. An entirely different approach eliminates viscous damping by placing the soln. inside a hollow resonator that is surrounded by vacuum. Here the authors demonstrate that suspended microchannel resonators can weigh single nanoparticles, single bacterial cells and sub-monolayers of adsorbed proteins in water with sub-femtogram resoln. (1 Hz bandwidth). Central to these results is the authors' observation that viscous loss due to the fluid is negligible compared to the intrinsic damping of the authors' silicon crystal resonator. The combination of the low resonator mass (100 ng) and high quality factor (15,000) enables an improvement in mass resoln. of six orders of magnitude over a high-end com. quartz crystal microbalance. This gives access to intriguing applications, such as mass-based flow cytometry, the direct detection of pathogens, or the nonoptical sizing and mass d. measurement of colloidal particles.
    29. 29
      Gil-Santos, E.; Ruz, J. J.; Malvar, O.; Favero, I.; Lemaître, A.; Kosaka, P. M.; García-López, S.; Calleja, M.; Tamayo, J. Optomechanical Detection of Vibration Modes of a Single Bacterium. Nat. Nanotechnol. 2020, 15 (6), 469– 474,  DOI: 10.1038/s41565-020-0672-y
      29
      Optomechanical detection of vibration modes of a single bacterium
      Gil-Santos, Eduardo; Ruz, Jose J.; Malvar, Oscar; Favero, Ivan; Lemaitre, Aristide; Kosaka, Priscila. M.; Garcia-Lopez, Sergio; Calleja, Montserrat; Tamayo, Javier
      Nature Nanotechnology (2020), 15 (6), 469-474CODEN: NNAABX; ISSN:1748-3387. (Nature Research)
      Low-frequency vibration modes of biol. particles, such as proteins, viruses and bacteria, involve coherent collective vibrations at frequencies in the terahertz and gigahertz domains. These vibration modes carry information on their structure and mech. properties, which are good indicators of their biol. state. A particular regime in the physics of coupled mech. resonators was harnessed to directly measure these low-frequency mech. resonances of a single bacterium. The bacterium was deposited on the surface of an ultrahigh frequency optomech. disk resonator in ambient conditions. The vibration modes of the disk and bacterium hybridize when their assocd. frequencies are similar. A general theor. framework was developed to describe this coupling, which allows retrieving the eigenfrequencies and mech. loss of the bacterium low-frequency vibration modes (quality factor). The effect of hydration on these vibrational modes was analyzed. Ultrahigh frequency optomech. resonators can be used for vibrational spectrometry with the unique capability to obtain information on single biol. entities.
    30. 30
      Dominguez-Medina, S.; Fostner, S.; Defoort, M.; Sansa, M.; Stark, A.-K.; Halim, M. A.; Vernhes, E.; Gely, M.; Jourdan, G.; Alava, T.; Boulanger, P.; Masselon, C.; Hentz, S. Neutral Mass Spectrometry of Virus Capsids above 100 Megadaltons with Nanomechanical Resonators. Science 2018, 362 (6417), 918– 922,  DOI: 10.1126/science.aat6457
      30
      Neutral mass spectrometry of virus capsids above 100 megadaltons with nanomechanical resonators
      Dominguez-Medina, Sergio; Fostner, Shawn; Defoort, Martial; Sansa, Marc; Stark, Ann-Kathrin; Halim, Mohammad Abdul; Vernhes, Emeline; Gely, Marc; Jourdan, Guillaume; Alava, Thomas; Boulanger, Pascale; Masselon, Christophe; Hentz, Sebastien
      Science (Washington, DC, United States) (2018), 362 (6417), 918-922CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Measurement of the mass of particles in the mega- to gigadalton range is challenging with conventional mass spectrometry. Although this mass range appears optimal for nanomech. resonators, nanomech. mass spectrometers often suffer from prohibitive sample loss, extended anal. time, or inadequate resoln. We report on a system architecture combining nebulization of the analytes from soln., their efficient transfer and focusing without relying on electromagnetic fields, and the mass measurements of individual particles using nanomech. resonator arrays. This system detd. the mass distribution of ∼30-megadalton polystyrene nanoparticles with high detection efficiency and effectively performed mol. mass measurements of empty or DNA-filled bacteriophage T5 capsids with masses up to 105 megadaltons using less than 1 pmol of sample and with an instrument resoln. above 100.
    31. 31
      Miller, E. J.; Trewby, W.; Farokh Payam, A.; Piantanida, L.; Cafolla, C.; Voïtchovsky, K. Sub-Nanometer Resolution Imaging with Amplitude-Modulation Atomic Force Microscopy in Liquid. J. Visualiz. Experiments 2016, (118), e54924,  DOI: 10.3791/54924
      There is no corresponding record for this reference.
    32. 32
      Cafolla, C.; Voitchovsky, K. Lubricating Properties of Single Metal Ions at Interfaces. Nanoscale 2018, 10 (25), 11831– 11840,  DOI: 10.1039/C8NR02859A
      32
      Lubricating properties of single metal ions at interfaces
      Cafolla, Clodomiro; Voitchovsky, Kislon
      Nanoscale (2018), 10 (25), 11831-11840CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
      The behavior of ionic solns. confined in nanoscale gaps is central to countless processes, from biomol. function to electrochem., energy storage and lubrication. However, no clear link exists between the mol.-level behavior of the liq. and macroscopic observations. The problem mainly comes from the difficulty to interrogate a small no. of liq. mols. Here, we use at. force microscopy to investigate the viscoelastic behavior of pure water and ionic solns. down to the single ion level. The results show a glassy-like behavior for pure water, with single metal ions acting as lubricants by reducing the elasticity of the nano-confined soln. and the magnitude of the hydrodynamic friction. At small ionic concn. (<20 mM) the results can be quant. explained by the ions moving via a thermally-activated process resisted by the ion's hydration water (Prandtl-Tomlinson model). The model breaks down at higher salt concns. due to ion-ion interaction effects that can no longer be neglected. The correlations are confirmed by direct sub-nanometer imaging of the interface at equil. The results provide a mol.-level basis for explaining the tribol. properties of aq. solns. and suggest that ion-ion interactions create mesoscale effects that prevent a direct link between nanoscale and macroscopic measurements.
    33. 33
      Cafolla, C.; Foster, W.; Voitchovsky, K. Lubricated Friction around Nanodefects. Sci. Adv. 2020, 6 (14), eaaz3673,  DOI: 10.1126/sciadv.aaz3673
      There is no corresponding record for this reference.
    34. 34
      Butt, H.-J.; Jaschke, M. Calculation of Thermal Noise in Atomic Force Microscopy. Nanotechnology 1995, 6 (1), 1– 7,  DOI: 10.1088/0957-4484/6/1/001
      There is no corresponding record for this reference.
    35. 35
      Cafolla, C.; Voitchovsky, K. Real-Time Tracking of Ionic Nano-Domains under Shear Flow. Sci. Rep. 2021, 11 (1), 19540,  DOI: 10.1038/s41598-021-98137-y
      35
      Real-time tracking of ionic nano-domains under shear flow
      Cafolla, Clodomiro; Voitchovsky, Kislon
      Scientific Reports (2021), 11 (1), 19540CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
      The behavior of ions at solid-liq. interfaces underpins countless phenomena, from the conduction of nervous impulses to charge transfer in solar cells. In most cases, ions do not operate as isolated entities, but in conjunction with neighboring ions and the surrounding soln. In aq. solns., recent studies suggest the existence of group dynamics through water-mediated clusters but results allowing direct tracking of ionic domains with at. precision are scarce. Here, we use high-speed at. force microscopy to track the evolution of Rb+, K+, Na+ and Ca2+ nano-domains contg. 20 to 120 ions adsorbed at the surface of mica in aq. soln. The interface is exposed to a shear flow able to influence the lateral motion of single ions and clusters. The results show that, when in groups, metal ions tend to move with a relatively slow dynamics, as can be expected from a correlated group motion, with an av. residence timescale of ∼ 1-2 s for individual ions at a given at. site. The av. group velocity of the clusters depends on the ions charge d. and can be explained by the ion's hydration state. The lateral shear flow of the fluid is insufficient to desorb ions, but indirectly influences the diffusion dynamics by acting on ions in close vicinity to the surface. The results provide insights into the dynamics of ion clusters when adsorbed onto an immersed solid under shear flow.
    36. 36
      Trewby, W.; Faraudo, J.; Voitchovsky, K. Long-Lived Ionic Nano-Domains Can Modulate the Stiffness of Soft Interfaces. Nanoscale 2019, 11, 4376– 4384,  DOI: 10.1039/C8NR06339G
      36
      Long-lived ionic nano-domains can modulate the stiffness of soft interfaces
      Trewby, William; Faraudo, Jordi; Voitchovsky, Kislon
      Nanoscale (2019), 11 (10), 4376-4384CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
      Metal ions underpin countless processes at bio-interfaces, including maintaining electroneutrality, modifying mech. properties and driving bioenergetic activity. These processes are typically described by ions behaving as independently diffusing point charges. Here we show that Na+ and K+ ions instead spontaneously form correlated nanoscale networks that evolve over seconds at the interface with an anionic bilayer in soln. Combining single-ion level at. force microscopy and mol. dynamic simulations we investigate the configuration and dynamics of Na+, K+, and Rb+ at the lipid surface. We identify two distinct ionic states: the well-known direct electrostatic interaction with lipid headgroups and a water-mediated interaction that can drive the formation of remarkably long-lived ionic networks which evolve over many seconds. We show that this second state induces ionic network formation via correlative ion-ion interactions that generate an effective energy well of -0.4kBT/ion. These networks locally reduce the stiffness of the membrane, providing a spontaneous mechanism for tuning its mech. properties with nanoscale precision. The ubiquity of water-mediated interactions suggest that our results have far-reaching implications for controlling the properties of soft interfaces.
    37. 37
      Granick, S.; Zhu, Y.; Lee, H. Slippery Questions about Complex Fluids Flowing Past Solids. Nat. Mater. 2003 2:4 2003, 2 (4), 221– 227,  DOI: 10.1038/nmat854
      There is no corresponding record for this reference.
    38. 38
      Mate, M.; Carpick, R. W. Tribology on the Small Scale: A Modern Textbook on Friction, Lubrication, and Wear, 2nd ed; Oxford University Press, 2019.
      There is no corresponding record for this reference.
    39. 39
      Inoue, H.; Ono, K.; Masuda, W.; Inagaki, T.; Yokota, M.; Inenaga, K. Rheological Properties of Human Saliva and Salivary Mucins. J. Oral Biosci. 2008, 50 (2), 134– 141,  DOI: 10.1016/S1349-0079(08)80027-8
      39
      Rheological properties of human saliva and salivary mucins
      Inoue, Hiroko; Ono, Kentaro; Masuda, Wataru; Inagaki, Tomohiro; Yokota, Makoto; Inenaga, Kiyotoshi
      Journal of Oral Biosciences (2008), 50 (2), 134-141CODEN: JOBOA8; ISSN:1349-0079. (Japanese Association for Oral Biology)
      It is unclear which factors in saliva contribute to its rheol. properties such as viscosity and spinnbarkeit. In this study, we investigated relationships between the rheol. properties (viscosity and spinnbarkeit) and two salivary mucin (MUC5B and MUC7) levels in saliva. Unstimulated (UWS) and chewing-stimulated (CWS) whole saliva were collected from healthy young adults, and the viscosities and spinnbarkeits were measured. The image densities of MUC5B and MUC7 bands in saliva were measured by Stains-All staining after electrophoresis. Both the viscosity and spinnbarkeit of UWS were significantly higher than those of CWS. The viscosities were correlated pos. with the spinnbarkeit in UWS, but not in CWS. Image-d. anal. of mucins demonstrated that the viscosities increased with the levels of MUC5B and the spinnbarkeit with the levels of MUC7. These results suggest that MUC5B contributes to viscosity and MUC7 to spinnbarkeit.
    40. 40
      Fors, R.; Persson, M. Nickel in Dental Plaque and Saliva in Patients with and without Orthodontic Appliances. Eur. J. Orthodont. 2005, 28, 292– 297,  DOI: 10.1093/ejo/cji091
      There is no corresponding record for this reference.
    41. 41
      Payam, A. F.; Trewby, W.; Voïtchovsky, K. Simultaneous Viscosity and Density Measurement of Small Volumes of Liquids Using a Vibrating Microcantilever. Analyst 2017, 142 (9), 1492– 1498,  DOI: 10.1039/C6AN02674E
      41
      Simultaneous viscosity and density measurement of small volumes of liquids using a vibrating microcantilever
      Payam, A. F.; Trewby, W.; Voitchovsky, K.
      Analyst (Cambridge, United Kingdom) (2017), 142 (9), 1492-1498CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
      Many industrial and technol. applications require precise detn. of the viscosity and d. of liqs. Such measurements can be time consuming and often require sampling substantial amts. of the liq. These problems can partly be overcome with the use of microcantilevers but most existing methods depend on the specific geometry and properties of the cantilever, which renders simple, accurate measurement difficult. Here we present a new approach able to simultaneously quantify both the d. and the viscosity of microliters of liqs. The method, based solely on the measurement of two characteristic frequencies of an immersed microcantilever, is completely independent of the choice of a cantilever. We derive anal. expressions for the liq.'s d. and viscosity and validate our approach with several simple liqs. and different cantilevers. Application of our model to non-Newtonian fluids shows that the calcd. viscosities are remarkably robust when compared to measurements obtained from a std. rheometer. However, the results become increasingly dependent on the cantilever geometry as the frequency-dependent nature of the liq.'s viscosity becomes more significant.
    42. 42
      Carpenter, G. H. The Secretion, Components, and Properties of Saliva. Annu. Rev. Food Sci. Technol. 2013, 4 (1), 267– 276,  DOI: 10.1146/annurev-food-030212-182700
      42
      The secretion, components, and properties of saliva
      Carpenter, Guy H.
      Annual Review of Food Science and Technology (2013), 4 (), 267-276CODEN: ARFSBV; ISSN:1941-1413. (Annual Reviews Inc.)
      A review. Saliva has one of the most difficult roles to perform in the body. It must facilitate the taste and detection of foods nutritious to the body but also defend the mucosa from infection by the ever-present microbiota present in the mouth. It achieves these roles by having a complex compn. and versatile phys. properties. The protein and ion components make a soln. that is 99% water into a viscoelastic soln. capable of many roles, such as acting as a lubricant and an antimicrobial, preventing the dissoln. of teeth, aiding digestion, and facilitating taste. This review describes the neural regulation of salivary secretion in terms of fluid, protein, and ion secretion. It then describes some of the components and phys. properties of saliva and attempts to relate them to the functions that saliva must perform.
    43. 43
      Kalwarczyk, T.; Sozanski, K.; Ochab-Marcinek, A.; Szymanski, J.; Tabaka, M.; Hou, S.; Holyst, R. Motion of Nanoprobes in Complex Liquids within the Framework of the Length-Scale Dependent Viscosity Model. Adv. Coll. Interface Sci. 2015, 223, 55– 63,  DOI: 10.1016/j.cis.2015.06.007
      43
      Motion of nanoprobes in complex liquids within the framework of the length-scale dependent viscosity model
      Kalwarczyk, Tomasz; Sozanski, Krzysztof; Ochab-Marcinek, Anna; Szymanski, Jedrzej; Tabaka, Marcin; Hou, Sen; Holyst, Robert
      Advances in Colloid and Interface Science (2015), 223 (), 55-63CODEN: ACISB9; ISSN:0001-8686. (Elsevier B.V.)
      This paper deals with the recent phenomenol. model of the motion of nanoscopic objects (colloidal particles, proteins, nanoparticles, mols.) in complex liqs. We analyzed motion in polymer, micellar, colloidal and protein solns. and the cytoplasm of living cells using the length-scale dependent viscosity model. Viscosity monotonically approaches macroscopic viscosity as the size of the object increases and thus gives a single, coherent picture of motion at the nano and macro scale. The model includes interparticle interactions (solvent-solute), temp. and the internal structure of a complex liq. The depletion layer ubiquitously occurring in complex liqs. is also incorporated into the model. We also discuss the biol. aspects of crowding in terms of the length-scale dependent viscosity model.
    44. 44
      Furst, E. M.; Squires, T. M. Microrheology; Oxford University Press, 2017.
      There is no corresponding record for this reference.
    45. 45
      Mao, Y.; Nielsen, P.; Ali, J. Passive and Active Microrheology for Biomedical Systems. Front. Bioengin. Biotechnol. 2022, 10, 916354,  DOI: 10.3389/fbioe.2022.916354
      There is no corresponding record for this reference.
    46. 46
      Sabaté del Río, J.; Henry, O. Y. F.; Jolly, P.; Ingber, D. E. An Antifouling Coating That Enables Affinity-Based Electrochemical Biosensing in Complex Biological Fluids. Nat. Nanotechnol. 2019, 14 (12), 1143– 1149,  DOI: 10.1038/s41565-019-0566-z
      46
      An antifouling coating that enables affinity-based electrochemical biosensing in complex biological fluids
      Sabate del Rio, Jonathan; Henry, Olivier Y. F.; Jolly, Pawan; Ingber, Donald E.
      Nature Nanotechnology (2019), 14 (12), 1143-1149CODEN: NNAABX; ISSN:1748-3387. (Nature Research)
      Affinity-based electrochem. detection in complex biol. fluids could enable multiplexed point-of-care diagnostics for home healthcare; however, commercialization of point-of-care devices has been limited by the rapid loss of sensitivity caused by electrode surface inactivation and biofouling. Here, we describe a simple and robust antifouling coating for electrodes consisting of a three-dimensional porous matrix of cross-linked bovine serum albumin supported by a network of conductive nanomaterials composed of either gold nanowires, gold nanoparticles or carbon nanotubes. These nanocomposites prevent non-specific interactions while enhancing electron transfer to the electrode surface, preserving 88% of the original signal after 1 mo of exposure to unprocessed human plasma, and functionalization with specific antibodies enables quantification of anti-interleukin 6 in plasma with high sensitivity. The easy prepn., stability and simplicity of this nanocomposite allow the generation of electrochem. biosensors that can operate in complex biol. fluids such as blood plasma or serum.
    47. 47
      Nair, S.; Tang, K. D.; Kenny, L.; Punyadeera, C. Salivary Exosomes as Potential Biomarkers in Cancer. Oral Oncol. 2018, 84, 31– 40,  DOI: 10.1016/j.oraloncology.2018.07.001
      47
      Salivary exosomes as potential biomarkers in cancer
      Nair, Soumyalekshmi; Tang, Kai Dun; Kenny, Liz; Punyadeera, Chamindie
      Oral Oncology (2018), 84 (), 31-40CODEN: EJCCER; ISSN:1368-8375. (Elsevier Ltd.)
      Over the past decade, there has been emerging research in the field of extracellular vesicles, esp. those originating from endosomes, referred to as 'exosomes. Exosomes are membrane-bound nanovesicles secreted by most cell types upon fusion of multivesicular bodies (MVBs) to the cell plasma membrane. These vesicles are present in almost all body fluids such as blood, urine, saliva, breast milk, cerebrospinal and peritoneal fluids. Exosomes participate in intercellular communication by transferring the biol. active mols. like proteins, nucleic acids, and lipids to neighboring cells. Exosomes are enriched in the tumor microenvironment and growing evidence demonstrates that exosomes mediate cancer progression and metastasis. Given the important biol. role played by these nanovesicles in cancer pathogenesis, these can be used as ideal non-invasive biomarkers in detecting and monitoring tumors as well as therapeutic targets. The scope of the current review is to provide an overview of exosomes with a special focus on salivary exosomes as potential biomarkers in head and neck cancers.
    48. 48
      Martins, T. S.; Vaz, M.; Henriques, A. G. A Review on Comparative Studies Addressing Exosome Isolation Methods from Body Fluids. Analyt. Bioanalyt. Chem. 2023, 415 (7), 1239– 1263,  DOI: 10.1007/s00216-022-04174-5
      There is no corresponding record for this reference.
    49. 49
      Sjoqvist, S.; Otake, K. Saliva and Saliva Extracellular Vesicles for Biomarker Candidate Identification─Assay Development and Pilot Study in Amyotrophic Lateral Sclerosis. Int. J. Mol. Sci. 2023, 24 (6), 5237,  DOI: 10.3390/ijms24065237
      49
      Saliva and Saliva Extracellular Vesicles for Biomarker Candidate Identification-Assay Development and Pilot Study in Amyotrophic Lateral Sclerosis
      Sjoqvist, Sebastian; Otake, Kentaro
      International Journal of Molecular Sciences (2023), 24 (6), 5237CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
      Saliva is gaining increasing attention as a source of biomarkers due to non-invasive and undemanding collection access. Extracellular vesicles (EVs) are nano-sized, cell-released particles that contain mol. information about their parent cells. In this study, we developed methods for saliva biomarker candidate identification using EV-isolation and proteomic evaluation. We used pooled saliva samples for assay development. EVs were isolated using membrane affinity-based methods followed by their characterization using nanoparticle tracking anal. and transmission electron microscopy. Subsequently, both saliva and saliva-EVs were successfully analyzed using proximity extension assay and label-free quant. proteomics. Saliva-EVs had a higher purity than plasma-EVs, based on the expression of EV-proteins and albumin. The developed methods could be used for the anal. of individual saliva samples from amyotrophic lateral sclerosis (ALS) patients and controls (n = 10 each). The starting vol. ranged from 2.1 to 4.9 mL and the amt. of total isolated EV-proteins ranged from 5.1 to 42.6μg. Although no proteins were significantly differentially expressed between the two groups, there was a trend for a downregulation of ZNF428 in ALS-saliva-EVs and an upregulation of IGLL1 in ALS saliva. In conclusion, we have developed a robust workflow for saliva and saliva-EV anal. and demonstrated its tech. feasibility for biomarker discovery.
    50. 50
      Aqrawi, L. A.; Galtung, H. K.; Vestad, B.; Øvstebø, R.; Thiede, B.; Rusthen, S.; Young, A.; Guerreiro, E. M.; Utheim, T. P.; Chen, X.; Utheim, Ø. A.; Palm, Ø.; Jensen, J. L. Identification of Potential Saliva and Tear Biomarkers in Primary Sjögren’s Syndrome, Utilising the Extraction of Extracellular Vesicles and Proteomics Analysis. Arthritis Res. Therapy 2017, 19 (1), 14,  DOI: 10.1186/s13075-017-1228-x
      50
      Identification of potential saliva and tear biomarkers in primary Sjogren's syndrome, utilising the extraction of extracellular vesicles and proteomics analysis
      Aqrawi, Lara A.; Galtung, Hilde Kanli; Vestad, Beate; Oevsteboe, Reidun; Thiede, Bernd; Rusthen, Shermin; Young, Alix; Guerreiro, Eduarda M.; Utheim, Tor Paaske; Chen, Xiangjun; Utheim, Oeygunn Aass; Palm, Oeyvind; Jensen, Janicke Liaaen
      Arthritis Research & Therapy (2017), 19 (), 14/1-14/15CODEN: ARTRCV; ISSN:1478-6362. (BioMed Central Ltd.)
      Background: There is a long-lasting need for non-invasive, more accurate diagnostic techniques when evaluating primary Sj0gren's syndrome (pSS) patients. Incorporation of addnl. diagnostics involving screening for disease-specific biomarkers in biol. fluid is a promising concept that requires further investigation. In the current study we aimed to explore novel disease biomarkers in saliva and tears from pSS patients. Methods: Liq. chromatog.-mass spectrometry (LC-MS) was performed on stimulated whole saliva and tears from 27 pSS patients and 32 healthy controls, and salivary and tear proteomic biomarker profiles were generated. LC-MS was also combined with size exclusion chromatog. to isolate extracellular vesicles (EVs) from both fluids. Nanoparticle tracking anal. was conducted on joint fractions from the saliva and tears to det. size distribution and concn. of EVs. Further EV characterization was performed by immunoaffinity capture of CD9-pos. EVs using magnetic beads, detected by flow cytometry. The LC-MS data were analyzed for quant. differences between patient and control groups using Scaffold, and the proteins were further analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID), for gene ontol. overrepresentation, and the Search Tool for the Retrieval of Interacting Genes/Proteins for protein-protein interaction network anal. Results: Upregulation of proteins involved in innate immunity (LCN2), cell signalling (CALM) and wound repair (GRN and CALML5) were detected in saliva in pSS. Saliva EVs also displayed biomarkers crit. for activation of the innate immune system (SIRPA and LSP1) and adipocyte differentiation (APMAP). Tear anal. indicated overexpression of proteins involved in TNF-α signalling (CPNE1) and B cell survival (PRDX3). Moreover, neutrophil gelatinase-assocd. lipocalin was upregulated in saliva and tears in pSS. Consistently, DAVID anal. demonstrated pathways of the adaptive immune response in saliva, of cellular component assembly for saliva EVs, and of metab. and protein folding in tears in pSS patients. Conclusions: LC-MS of saliva and tears from pSS patients, solely and in combination with size-exclusion chromatog. allowed screening for possible novel biomarkers encompassing both salivary and lacrimal disease target organs. This approach could provide addnl. diagnostic accuracy in pSS, and could possibly also be applied for staging and monitoring the disease.
    51. 51
      Contreras, H.; Alarcón-Zapata, P.; Nova-Lamperti, E.; Ormazabal, V.; Varas-Godoy, M.; Salomon, C.; Zuniga, F. A. Comparative Study of Size Exclusion Chromatography for Isolation of Small Extracellular Vesicle from Cell-Conditioned Media, Plasma, Urine, and Saliva. Front. Nanotechnol. 2023, 5, 1146772,  DOI: 10.3389/fnano.2023.1146772
      There is no corresponding record for this reference.
    52. 52
      Largitte, L.; Pasquier, R. New Models for Kinetics and Equilibrium Homogeneous Adsorption. Chem. Eng. Res. Des. 2016, 112, 289– 297,  DOI: 10.1016/j.cherd.2016.06.021
      52
      New models for kinetics and equilibrium homogeneous adsorption
      Largitte, L.; Pasquier, R.
      Chemical Engineering Research and Design (2016), 112 (), 289-297CODEN: CERDEE; ISSN:1744-3563. (Elsevier B.V.)
      8 New models, named resp. LK 1, LK 2, LK 3, LEq 1, LEq 2, LEq 3, LEq 4 and LEq 5 are suggested to describe the kinetics and the equil. of a homogeneous adsorption. The models, more general than the existing ones, are very useful to describe the different kinetics and isotherms exptl. behavior of many adsorbents. In these new methods, the adsorption is decompd. in 1 or 2 steps, each step following an n order kinetics. The models have scientific, logical and chem. understanding meaning. Some (LK 1, LK 3, LEq 2, LEq 4) can predict the max. adsorption capacity Qmax. LEq 1 and LEq 2 models permit to make the link between the kinetics and the equil. results and det. the order of reaction for both the adsorbent and the adsorbate, then, increasing the knowledge of the sorption parameters.The kinetics and the equil. of the adsorption of lead by a com. activated carbon are studied and these new models are used to fit the exptl. data. The kinetics and the equil. parameters are detd. The fitting of the kinetics results are compared to those obtained with other existing models, idem for the equil. results. Regression results show that the new equil. models better fit the exptl. data than the existing ones for this sorption process.
    53. 53
      Umeda, R.; Satouh, Y.; Takemoto, M.; Nakada-Nakura, Y.; Liu, K.; Yokoyama, T.; Shirouzu, M.; Iwata, S.; Nomura, N.; Sato, K.; Ikawa, M.; Nishizawa, T.; Nureki, O. Structural Insights into Tetraspanin CD9 Function. Nat. Commun. 2020, 11 (1), 1606,  DOI: 10.1038/s41467-020-15459-7
      53
      Structural insights into tetraspanin CD9 function
      Umeda, Rie; Satouh, Yuhkoh; Takemoto, Mizuki; Nakada-Nakura, Yoshiko; Liu, Kehong; Yokoyama, Takeshi; Shirouzu, Mikako; Iwata, So; Nomura, Norimichi; Sato, Ken; Ikawa, Masahito; Nishizawa, Tomohiro; Nureki, Osamu
      Nature Communications (2020), 11 (1), 1606CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Abstr.: Tetraspanins play crit. roles in various physiol. processes, ranging from cell adhesion to virus infection. The members of the tetraspanin family have four membrane-spanning domains and short and large extracellular loops, and assoc. with a broad range of other functional proteins to exert cellular functions. Here we report the crystal structure of CD9 and the cryo-electron microscopic structure of CD9 in complex with its single membrane-spanning partner protein, EWI-2. The reversed cone-like mol. shape of CD9 generates membrane curvature in the cryst. lipid layers, which explains the CD9 localization in regions with high membrane curvature and its implications in membrane remodeling. The mol. interaction between CD9 and EWI-2 is mainly mediated through the small residues in the transmembrane region and protein/lipid interactions, whereas the fertilization assay revealed the crit. involvement of the LEL region in the sperm-egg fusion, indicating the different dependency of each binding domain for other partner proteins.
    54. 54
      Vences-Catalán, F.; Rajapaksa, R.; Kuo, C. C.; Miller, C. L.; Lee, A.; Ramani, V. C.; Jeffrey, S. S.; Levy, R.; Levy, S. Targeting the Tetraspanin CD81 Reduces Cancer Invasion and Metastasis. Proc. Natl. Acad. Sci. U. S. A. 2021, 118 (24), e2018961118,  DOI: 10.1073/pnas.2018961118
      54
      Targeting the tetraspanin CD81 reduces cancer invasion and metastasis
      Vences-Catalan, Felipe; Rajapaksa, Ranjani; Kuo, Chiung-Chi; Miller, Caitlyn L.; Lee, Anderson; Ramani, Vishnu C.; Jeffrey, Stefanie S.; Levy, Ronald; Levy, Shoshana
      Proceedings of the National Academy of Sciences of the United States of America (2021), 118 (24), e2018961118CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Tetraspanins are an evolutionary conserved family of proteins involved in multiple aspects of cell physiol., including proliferation, migration and invasion, protein trafficking, and signal transduction; yet their detailed mechanism of action is unknown. Tetraspanins have no known natural ligands, but their engagement by antibodies has begun to reveal their role in cell biol. Studies of tetraspanin knockout mice and of germline mutations in humans have highlighted their role under normal and pathol. conditions. Previously, we have shown that mice deficient in the tetraspanin CD81 developed fewer breast cancer metastases compared to their wild-type (WT) counterparts. Here, we show that a unique anti-human CD81 antibody (5A6) effectively halts invasion of triple-neg. breast cancer (TNBC) cell lines. We demonstrate that 5A6 induces CD81 clustering at the cell membrane and we implicate JAM-A protein in the ability of this antibody to inhibit tumor cell invasion and migration. Furthermore, in a series of in vivo studies we demonstrate that this antibody inhibits metastases in xenograft models, as well as in syngeneic mice bearing a mouse tumor into which we knocked in the human CD81 epitope recognized by the 5A6 antibody.
    55. 55
      Picca, A.; Guerra, F.; Calvani, R.; Marini, F.; Biancolillo, A.; Landi, G.; Beli, R.; Landi, F.; Bernabei, R.; Bentivoglio, A. R.; Lo Monaco, M. R.; Bucci, C.; Marzetti, E. Mitochondrial Signatures in Circulating Extracellular Vesicles of Older Adults with Parkinson’s Disease: Results from the EXosomes in ParkiNson’s Disease (EXPAND) Study. J. Clin. Med. 2020, 9 (2), 504,  DOI: 10.3390/jcm9020504
      55
      Mitochondrial signatures in circulating extracellular vesicles of older adults with parkinson's disease: results from the exosomes in parkinson's disease (EXPAND) study
      Picca, Anna; Guerra, Flora; Calvani, Riccardo; Marini, Federico; Biancolillo, Alessandra; Landi, Giovanni; Beli, Raffaella; Landi, Francesco; Bernabei, Roberto; Bentivoglio, Anna Rita; Lo Monaco, Maria Rita; Bucci, Cecilia; Marzetti, Emanuele
      Journal of Clinical Medicine (2020), 9 (2), 504CODEN: JCMOHK; ISSN:2077-0383. (MDPI AG)
      Systemic inflammation and mitochondrial dysfunction are involved in neurodegeneration in Parkinson's disease (PD). Extracellular vesicle (EV) trafficking may link inflammation and mitochondrial dysfunction. In the present study, circulating small EVs (sEVs) from 16 older adults with PD and 12 non-PD controls were purified and characterized. A panel of serum inflammatory biomols. was measured by multiplex immunoassay. Protein levels of three tetraspanins (CD9, CD63, and CD81) and selected mitochondrial markers (ATP 5A (ATP5A), mitochondrial cytochrome C oxidase subunit I (MTCOI), NAD reduced form (NADH):ubiquinone oxidoreductase subunit B8 (NDUFB8), NADH:ubiquinone oxidoreductase subunit S3 (NDUFS3), succinate dehydrogenase complex iron sulfur subunit B (SDHB), and ubiquinol-cytochrome C reductase core protein 2 (UQCRC2)) were quantified in purified sEVs by immunoblotting. Relative to controls, PD participants showed a greater amt. of circulating sEVs. Levels of CD9 and CD63 were lower in the sEV fraction of PD participants, whereas those of CD81 were similar between groups. Lower levels of ATP5A, NDUFS3, and SDHB were detected in sEVs from PD participants. No signal was retrieved for UQCRC2, MTCOI, or NDUFB8 in either participant group. To identify a mol. signature in circulating sEVs in relationship to systemic inflammation, a low level-fused (multi-platform) partial least squares discriminant anal. was applied. The model correctly classified 94.2% ± 6.1% PD participants and 66.7% ± 5.4% controls, and identified seven biomols. as relevant (CD9, NDUFS3, C-reactive protein, fibroblast growth factor 21, interleukin 9, macrophage inflammatory protein 1β, and tumor necrosis factor alpha). In conclusion, a mitochondrial signature was identified in circulating sEVs from older adults with PD, in assocn. with a specific inflammatory profile.
    56. 56
      P.C., S.; Shetty, S.; Nalilu, S.; Shetty, P.; Patil, P. Tetraspanin CD9: A Friend or Foe of Head and Neck Cancer. Oncol. Rep. 2022, 47 (5), 88,  DOI: 10.3892/or.2022.8299
      There is no corresponding record for this reference.
    57. 57
      Paba, C.; Dorigo, V.; Senigagliesi, B.; Tormena, N.; Parisse, P.; Voïtchovsky, K.; Casalis, L. Lipid Bilayer Fluidity and Degree of Order Regulates Small EVs Adsorption on Model Cell Membrane. J. Colloid Interface Sci. 2023, 652, 1937– 1943,  DOI: 10.1016/j.jcis.2023.08.117
      57
      Lipid bilayer fluidity and degree of order regulates small EVs adsorption on model cell membrane
      Paba, Carolina; Dorigo, Virginia; Senigagliesi, Beatrice; Tormena, Nicolo; Parisse, Pietro; Voitchovsky, Kislon; Casalis, Loredana
      Journal of Colloid and Interface Science (2023), 652 (Part_B), 1937-1943CODEN: JCISA5; ISSN:0021-9797. (Elsevier B.V.)
      Small extracellular vesicles (sEVs) are known to play an important role in the communication between distant cells and to deliver biol. information throughout the body. To date, many studies have focused on the role of sEVs characteristics such as cell origin, surface compn., and mol. cargo on the resulting uptake by the recipient cell. Yet, a full understanding of the sEV fusion process with recipient cells and in particular the role of cell membrane phys. properties on the uptake are still lacking. Here we explore this problem using sEVs from a cellular model of triple-neg. breast cancer fusing to a range of synthetic planar lipid bilayers both with and without cholesterol, and designed to mimic the formation of 'raft'-like nanodomains in cell membranes. Using time-resolved Atomic Force Microscopy we were able to track the sEVs interaction with the different model membranes, showing the process to be strongly dependent on the local membrane fluidity. The strongest interaction and fusion is obsd. over the less fluid regions, with sEVs even able to disrupt ordered domains at sufficiently high cholesterol concn. Our findings suggest the biophys. characteristics of recipient cell membranes to be crucial for sEVs uptake regulation.
    58. 58
      Théry, C.; Witwer, K. W.; Aikawa, E.; Alcaraz, M. J.; Anderson, J. D.; Andriantsitohaina, R.; Antoniou, A.; Arab, T.; Archer, F.; Atkin-Smith, G. K.; Ayre, D. C.; Bach, J.-M.; Bachurski, D.; Baharvand, H.; Balaj, L.; Baldacchino, S.; Bauer, N. N.; Baxter, A. A.; Bebawy, M.; Beckham, C.; Bedina Zavec, A.; Benmoussa, A.; Berardi, A. C.; Bergese, P.; Bielska, E.; Blenkiron, C.; Bobis-Wozowicz, S.; Boilard, E.; Boireau, W.; Bongiovanni, A.; Borràs, F. E.; Bosch, S.; Boulanger, C. M.; Breakefield, X.; Breglio, A. M.; Brennan, M. Á.; Brigstock, D. R.; Brisson, A.; Broekman, M. L.; Bromberg, J. F.; Bryl-Górecka, P.; Buch, S.; Buck, A. H.; Burger, D.; Busatto, S.; Buschmann, D.; Bussolati, B.; Buzás, E. I.; Byrd, J. B.; Camussi, G.; Carter, D. R.; Caruso, S.; Chamley, L. W.; Chang, Y.-T.; Chen, C.; Chen, S.; Cheng, L.; Chin, A. R.; Clayton, A.; Clerici, S. P.; Cocks, A.; Cocucci, E.; Coffey, R. J.; Cordeiro-da-Silva, A.; Couch, Y.; Coumans, F. A.; Coyle, B.; Crescitelli, R.; Criado, M. F.; D’Souza-Schorey, C.; Das, S.; Datta Chaudhuri, A.; de Candia, P.; De Santana, E. F.; De Wever, O.; del Portillo, H. A.; Demaret, T.; Deville, S.; Devitt, A.; Dhondt, B.; Di Vizio, D.; Dieterich, L. C.; Dolo, V.; Dominguez Rubio, A. P.; Dominici, M.; Dourado, M. R.; Driedonks, T. A.; Duarte, F. V.; Duncan, H. M.; Eichenberger, R. M.; Ekström, K.; EL Andaloussi, S.; Elie-Caille, C.; Erdbrügger, U.; Falcón-Pérez, J. M.; Fatima, F.; Fish, J. E.; Flores-Bellver, M.; Försönits, A.; Frelet-Barrand, A.; Fricke, F.; Fuhrmann, G.; Gabrielsson, S.; Gámez-Valero, A.; Gardiner, C.; Gärtner, K.; Gaudin, R.; Gho, Y. S.; Giebel, B.; Gilbert, C.; Gimona, M.; Giusti, I.; Goberdhan, D. C.; Görgens, A.; Gorski, S. M.; Greening, D. W.; Gross, J. C.; Gualerzi, A.; Gupta, G. N.; Gustafson, D.; Handberg, A.; Haraszti, R. A.; Harrison, P.; Hegyesi, H.; Hendrix, A.; Hill, A. F.; Hochberg, F. H.; Hoffmann, K. F.; Holder, B.; Holthofer, H.; Hosseinkhani, B.; Hu, G.; Huang, Y.; Huber, V.; Hunt, S.; Ibrahim, A. G.-E.; Ikezu, T.; Inal, J. M.; Isin, M.; Ivanova, A.; Jackson, H. K.; Jacobsen, S.; Jay, S. M.; Jayachandran, M.; Jenster, G.; Jiang, L.; Johnson, S. M.; Jones, J. C.; Jong, A.; Jovanovic-Talisman, T.; Jung, S.; Kalluri, R.; Kano, S.; Kaur, S.; Kawamura, Y.; Keller, E. T.; Khamari, D.; Khomyakova, E.; Khvorova, A.; Kierulf, P.; Kim, K. P.; Kislinger, T.; Klingeborn, M.; Klinke, D. J., II; Kornek, M.; Kosanović, M. M.; Kovács, Á. F.; Krämer-Albers, E.-M.; Krasemann, S.; Krause, M.; Kurochkin, I. V.; Kusuma, G. D.; Kuypers, S.; Laitinen, S.; Langevin, S. M.; Languino, L. R.; Lannigan, J.; Lässer, C.; Laurent, L. C.; Lavieu, G.; Lázaro-Ibáñez, E.; Le Lay, S.; Lee, M.-S.; Lee, Y. X. F.; Lemos, D. S.; Lenassi, M.; Leszczynska, A.; Li, I. T.; Liao, K.; Libregts, S. F.; Ligeti, E.; Lim, R.; Lim, S. K.; Line̅, A.; Linnemannstöns, K.; Llorente, A.; Lombard, C. A.; Lorenowicz, M. J.; Lörincz, Á. M.; Lötvall, J.; Lovett, J.; Lowry, M. C.; Loyer, X.; Lu, Q.; Lukomska, B.; Lunavat, T. R.; Maas, S. L.; Malhi, H.; Marcilla, A.; Mariani, J.; Mariscal, J.; Martens-Uzunova, E. S.; Martin-Jaular, L.; Martinez, M. C.; Martins, V. R.; Mathieu, M.; Mathivanan, S.; Maugeri, M.; McGinnis, L. K.; McVey, M. J.; Meckes, D. G., Jr; Meehan, K. L.; Mertens, I.; Minciacchi, V. R.; Möller, A.; Møller Jo̷rgensen, M.; Morales-Kastresana, A.; Morhayim, J.; Mullier, F.; Muraca, M.; Musante, L.; Mussack, V.; Muth, D. C.; Myburgh, K. H.; Najrana, T.; Nawaz, M.; Nazarenko, I.; Nejsum, P.; Neri, C.; Neri, T.; Nieuwland, R.; Nimrichter, L.; Nolan, J. P.; Nolte-’t Hoen, E. N.; Noren Hooten, N.; O’Driscoll, L.; O’Grady, T.; O’Loghlen, A.; Ochiya, T.; Olivier, M.; Ortiz, A.; Ortiz, L. A.; Osteikoetxea, X.; Østergaard, O.; Ostrowski, M.; Park, J.; Pegtel, D. M.; Peinado, H.; Perut, F.; Pfaffl, M. W.; Phinney, D. G.; Pieters, B. C.; Pink, R. C.; Pisetsky, D. S.; Pogge von Strandmann, E.; Polakovicova, I.; Poon, I. K.; Powell, B. H.; Prada, I.; Pulliam, L.; Quesenberry, P.; Radeghieri, A.; Raffai, R. L.; Raimondo, S.; Rak, J.; Ramirez, M. I.; Raposo, G.; Rayyan, M. S.; Regev-Rudzki, N.; Ricklefs, F. L.; Robbins, P. D.; Roberts, D. D.; Rodrigues, S. C.; Rohde, E.; Rome, S.; Rouschop, K. M.; Rughetti, A.; Russell, A. E.; Saá, P.; Sahoo, S.; Salas-Huenuleo, E.; Sánchez, C.; Saugstad, J. A.; Saul, M. J.; Schiffelers, R. M.; Schneider, R.; Schøyen, T. H.; Scott, A.; Shahaj, E.; Sharma, S.; Shatnyeva, O.; Shekari, F.; Shelke, G. V.; Shetty, A. K.; Shiba, K.; Siljander, P. R.-M.; Silva, A. M.; Skowronek, A.; Snyder, O. L., II; Soares, R. P.; Sódar, B. W.; Soekmadji, C.; Sotillo, J.; Stahl, P. D.; Stoorvogel, W.; Stott, S. L.; Strasser, E. F.; Swift, S.; Tahara, H.; Tewari, M.; Timms, K.; Tiwari, S.; Tixeira, R.; Tkach, M.; Toh, W. S.; Tomasini, R.; Torrecilhas, A. C.; Tosar, J. P.; Toxavidis, V.; Urbanelli, L.; Vader, P.; van Balkom, B. W.; van der Grein, S. G.; Van Deun, J.; van Herwijnen, M. J.; Van Keuren-Jensen, K.; van Niel, G.; van Royen, M. E.; van Wijnen, A. J.; Vasconcelos, M. H.; Vechetti, I. J., Jr; Veit, T. D.; Vella, L. J.; Velot, É.; Verweij, F. J.; Vestad, B.; Viñas, J. L.; Visnovitz, T.; Vukman, K. V.; Wahlgren, J.; Watson, D. C.; Wauben, M. H.; Weaver, A.; Webber, J. P.; Weber, V.; Wehman, A. M.; Weiss, D. J.; Welsh, J. A.; Wendt, S.; Wheelock, A. M.; Wiener, Z.; Witte, L.; Wolfram, J.; Xagorari, A.; Xander, P.; Xu, J.; Yan, X.; Yáñez-Mó, M.; Yin, H.; Yuana, Y.; Zappulli, V.; Zarubova, J.; Žėkas, V.; Zhang, J.; Zhao, Z.; Zheng, L.; Zheutlin, A. R.; Zickler, A. M.; Zimmermann, P.; Zivkovic, A. M.; Zocco, D.; Zuba-Surma, E. K. Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018): A Position Statement of the International Society for Extracellular Vesicles and Update of the MISEV2014 Guidelines. J. Extracell. Ves. 2018, 7 (1), 1535750,  DOI: 10.1080/20013078.2018.1535750
      There is no corresponding record for this reference.
    59. 59
      Jawerth, L.; Fischer-Friedrich, E.; Saha, S.; Wang, J.; Franzmann, T.; Zhang, X.; Sachweh, J.; Ruer, M.; Ijavi, M.; Saha, S.; Mahamid, J.; Hyman, A. A.; Jülicher, F. Protein Condensates as Aging Maxwell Fluids. Science 2020, 370 (6522), 1317– 1323,  DOI: 10.1126/science.aaw4951
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      Protein condensates as aging Maxwell fluids
      Jawerth, Louise; Fischer-Friedrich, Elisabeth; Saha, Suropriya; Wang, Jie; Franzmann, Titus; Zhang, Xiaojie; Sachweh, Jenny; Ruer, Martine; Ijavi, Mahdiye; Saha, Shambaditya; Mahamid, Julia; Hyman, Anthony A.; Juelicher, Frank
      Science (Washington, DC, United States) (2020), 370 (6522), 1317-1323CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
      Protein condensates are complex fluids that can change their material properties with time. However, an appropriate rheol. description of these fluids remains missing. We characterize the time-dependent material properties of in vitro protein condensates using laser tweezer-based active and microbead-based passive rheol. For different proteins, the condensates behave at all ages as viscoelastic Maxwell fluids. Their viscosity strongly increases with age while their elastic modulus varies weakly. No significant differences in structure were seen by electron microscopy at early and late ages. We conclude that protein condensates can be soft glassy materials that we call Maxwell glasses with age-dependent material properties. We discuss possible advantages of glassy behavior for modulation of cellular biochem.
    60. 60
      Murayama, Y.; Oritani, K.; Tsutsui, S. Novel CD9-Targeted Therapies in Gastric Cancer. World J. Gastroenterol. 2015, 21 (11), 3206– 3213,  DOI: 10.3748/wjg.v21.i11.3206
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      Novel CD9-targeted therapies in gastric cancer
      Murayama, Yoko; Oritani, Kenji; Tsutsui, Shusaku
      World Journal of Gastroenterology (2015), 21 (11), 3206-3213CODEN: WJGAF2; ISSN:2219-2840. (Baishideng Publishing Group Inc.)
      There are 33 human tetraspanin proteins, emerging as key players in malignancy, the immune system, fertilization, cellular signaling, adhesion, morphol., motility, proliferation, and tumor invasion. CD9, a member of the tetraspanin family, assocs. with and influences a variety of cell-surface mols. Through these interactions, CD9 modifies multiple cellular events, including adhesion, migration, proliferation, and survival. CD9 is therefore considered to play a role in several stages during cancer development. Reduced CD9 expression is generally related to venous vessel invasion and metastasis as well as poor prognosis. We found that treatment of mice bearing human gastric cancer cells with anti-CD9 antibody successfully inhibited tumor progression via antiproliferative, proapoptotic, and antiangiogenic effects, strongly indicating that CD9 is a possible therapeutic target in patients with gastric cancer. Here, we describe the possibility of CD9 manipulation as a novel therapeutic strategy in gastric cancer, which still shows poor prognosis.
    61. 61
      Lorico, A.; Lorico-Rappa, M.; Karbanová, J.; Corbeil, D.; Pizzorno, G. CD9, a Tetraspanin Target for Cancer Therapy?. Experiment. Biol. Med. 2021, 246 (9), 1121– 1138,  DOI: 10.1177/1535370220981855
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      CD9, a tetraspanin target for cancer therapy?
      Lorico, Aurelio; Lorico-Rappa, Marco; Karbanova, Jana; Corbeil, Denis; Pizzorno, Giuseppe
      Experimental Biology and Medicine (London, United Kingdom) (2021), 246 (9), 1121-1138CODEN: EBMMBE; ISSN:1535-3699. (Sage Publications Ltd.)
      In the present minireview, we intend to provide a brief history of the field of CD9 involvement in oncogenesis and in the metastatic process of cancer, considering its potential value as a tumor-assocd. antigenic target. Over the years, CD9 has been identified as a favorable prognostic marker or predictor of metastatic potential depending on the cancer type. To understand its implications in cancer beside its use as an antigenic biomarker, it is essential to know its physiol. functions, including its mol. partners in a given cell system. Moreover, the discovery that CD9 is one of the most specific and broadly expressed markers of extracellular membrane vesicles, nanometer-sized entities that are released into extracellular space and various physiol. body fluids and play a role in intercellular communication under physiol. and pathol. conditions, notably the establishment of cancer metastases, has added a new dimension to our knowledge of CD9 function in cancer. Here, we will discuss these issues as well as the possible cancer therapeutic implications of CD9, their limitations, and pitfalls.
    62. 62
      Nigri, J.; Leca, J.; Tubiana, S. S.; Finetti, P.; Guillaumond, F.; Martinez, S.; Lac, S.; Iovanna, J. L.; Audebert, S.; Camoin, L.; Vasseur, S.; Bertucci, F.; Tomasini, R. CD9 Mediates the Uptake of Extracellular Vesicles from Cancer-Associated Fibroblasts That Promote Pancreatic Cancer Cell Aggressiveness. Science Signaling 2022, 15 (745), eabg8191,  DOI: 10.1126/scisignal.abg8191
      There is no corresponding record for this reference.
    63. 63
      Koh, H. M.; Jang, B. G.; Lee, D. H.; Hyun, C. L. Increased CD9 Expression Predicts Favorable Prognosis in Human Cancers: A Systematic Review and Meta-Analysis. Cancer Cell Int. 2021, 21 (1), 472,  DOI: 10.1186/s12935-021-02152-y
      63
      Increased CD9 expression predicts favorable prognosis in human cancers: a systematic review and meta-analysis
      Koh, Hyun Min; Jang, Bo Gun; Lee, Dong Hui; Hyun, Chang Lim
      Cancer Cell International (2021), 21 (1), 472CODEN: CCIACC; ISSN:1475-2867. (BioMed Central Ltd.)
      Meta-anal. of CD9 is implicated in cancer progression and metastasis by its role in suppressing cancer cell proliferation and survival. However, the prognostic and clinicopathol. significance of CD9 expression is controversial. Therefore, the current meta-anal. was conducted to det. the prognostic and clinicopathol. significance of CD9 expression in cancer patients. Eligible studies were selected through database search of PubMed, Embase and Cochrane library up to Apr. 5 2020. The necessary data were extd. from the included studies. Pooled hazard ratio (HR) and odds ratio (OR) with 95% confidence interval (CI) were calcd. to evaluate the prognostic and clinicopathol. significance of CD9 expression in cancer patients. A total of 17 studies consisting of 3456 cancer patients were included in this meta-anal. An increased CD9 expression was significantly assocd. with a more favorable overall survival (OS) (HR 0.47, 95% CI 0.31-0.73, p = 0.001) and disease-free survival (DFS) (HR 0.48, 95% CI 0.30-0.79, p = 0.003). In subgroup anal. of cancer type, an increased CD9 expression was assocd. with increased OS in breast cancer and digestive system cancer, and with increased DFS in head and neck cancer and leukemia/lymphoma. Addnl., an increased CD9 expression significantly correlated with lower overall stage (OR 0.45, 95% CI 0.29-0.72, p = 0.001). An increased CD9 expression was assocd. with favorable survival in cancer patients suggesting that CD9 expression could be a valuable survival factor in cancer patients.
    64. 64
      Salvi, S.; Bandini, E.; Carloni, S.; Casadio, V.; Battistelli, M.; Salucci, S.; Erani, I.; Scarpi, E.; Gunelli, R.; Cicchetti, G.; Guescini, M.; Bonafè, M.; Fabbri, F. Detection and Investigation of Extracellular Vesicles in Serum and Urine Supernatant of Prostate Cancer Patients. Diagnostics 2021, 11 (3), 466,  DOI: 10.3390/diagnostics11030466
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      Detection and investigation of extracellular vesicles in serum and urine supernatant of prostate cancer patients
      Salvi, Samanta; Bandini, Erika; Carloni, Silvia; Casadio, Valentina; Battistelli, Michela; Salucci, Sara; Erani, Ilaria; Scarpi, Emanuela; Gunelli, Roberta; Cicchetti, Giacomo; Guescini, Michele; Bonafe, Massimiliano; Fabbri, Francesco
      Diagnostics (2021), 11 (3), 466CODEN: DIAGC9; ISSN:2075-4418. (MDPI AG)
      Prostate Cancer (PCa) is one of the most frequently identified urol. cancers. PCa patients are often over-diagnosed due to still not highly specific diagnostic methods. The need for more accurate diagnostic tools to prevent overestimated diagnosis and unnecessary treatment of patients with non-malignant conditions is clear, and new markers and methods are strongly desirable. Extracellular vesicles (EVs) hold great promises as liq. biopsy-based markers. Despite the biol. and tech. issues present in their detection and study, these particles can be found highly abundantly in the biofluid and encompass a wealth of macromols. that have been reported to be related to many physiol. and pathol. processes, including cancer onset, metastasis spreading, and treatment resistance. The present study aims to perform a tech. feasibility study to develop a new workflow for investigating EVs from several biol. sources. Serum and urinary supernatant EVs of PCa, benign prostatic hyperplasia (BPH) patients, and healthy donors were isolated and investigated by a fast, easily performable, and cost-effective cytofluorimetric approach for a multiplex detection of 37 EV-antigens. We also obsd. significant alterations in serum and urinary supernatant EVs potentially related to BPH and PCa, suggesting a potential clin. application of this workflow.
    65. 65
      Paolino, G.; Huber, V.; Camerini, S.; Casella, M.; Macone, A.; Bertuccini, L.; Iosi, F.; Moliterni, E.; Cecchetti, S.; Ruspantini, I.; Chiarotti, F.; Vergani, E.; Lalli, L.; Raggi, C.; Di Biase, A.; Calvieri, S.; Mercuri, S. R.; Lugini, L.; Federici, C. The Fatty Acid and Protein Profiles of Circulating Cd81-Positive Small Extracellular Vesicles Are Associated with Disease Stage in Melanoma Patients. Cancers 2021, 13 (16), 4157,  DOI: 10.3390/cancers13164157
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      The Fatty Acid and Protein Profiles of Circulating CD81-Positive Small Extracellular Vesicles Are Associated with Disease Stage in Melanoma Patients
      Paolino, Giovanni; Huber, Veronica; Camerini, Serena; Casella, Marialuisa; Macone, Alberto; Bertuccini, Lucia; Iosi, Francesca; Moliterni, Elisa; Cecchetti, Serena; Ruspantini, Irene; Chiarotti, Flavia; Vergani, Elisabetta; Lalli, Luca; Raggi, Carla; Di Biase, Antonella; Calvieri, Stefano; Mercuri, Santo Raffaele; Lugini, Luana; Federici, Cristina
      Cancers (2021), 13 (16), 4157CODEN: CANCCT; ISSN:2072-6694. (MDPI AG)
      Simple Summary: Early detection of cutaneous melanoma is the key to increasing survival and proper therapeutic adjustment, esp. in stages II-IV. We investigated whether the fatty acid (FA) and protein compns. of small extracellular vesicles (sEV) expressing CD81, derived from the plasma of stage 0-I, II and III-IV melanoma patients, could reflect disease stage. Results showed a higher content of FA and differences in C18:0/C18:1 ratio, a marker of cell membrane fluidity, that distinguished patients' CD81sEV from those of healthy donors (HD). By proteomic anal. (identifier PXD024434) we identified significant increases in CD14, PON1, PON3 and APOA5 in stage II CD81sEV compared to HD. In stage III-IV, CD81sEV' RAP1B expression was decreased. These stage-related signatures may support the potential of sEV to provide information for early diagnosis, prediction of metastatic behavior, treatment and follow-up of melanoma patients. Abstr.: The early detection of cutaneous melanoma, a potentially lethal cancer with rising incidence, is fundamental to increasing survival and therapeutic adjustment. In stages II-IV esp., addnl. indications for adjuvant therapy purposes after resection and for treatment of metastatic patients are urgently needed. We investigated whether the fatty acid (FA) and protein compns. of small extracellular vesicles (sEV) derived from the plasma of stage 0-I, II and III-IV melanoma patients (n = 38) could reflect disease stage. The subpopulation of sEV expressing CD81 EV marker (CD81sEV) was captured by an ad hoc immune affinity technique from plasma depleted of large EV. Biol. macromols. were investigated by gas chromatog. and mass spectrometry in CD81sEV. A higher content of FA was detectable in patients with respect to healthy donors (HD). Moreover, a higher C18:0/C18:1 ratio, as a marker of cell membrane fluidity, distinguished early (stage 0-I) from late (III-IV) stages' CD81sEV. Proteomics detected increases in CD14, PON1, PON3 and APOA5 exclusively in stage II CD81sEV, and RAP1B was decreased in stage III-IV CD81sEV, in comparison to HD. Our results suggest that stage dependent alterations in CD81sEV' FA and protein compn. may occur early after disease onset, strengthening the potential of circulating sEV as a source of discriminatory information for early diagnosis, prediction of metastatic behavior and following up of melanoma patients.
    66. 66
      You, B.; Shan, Y.; Bao, L.; Chen, J.; Yang, L.; Zhang, Q.; Zhang, W.; Zhang, Z.; Zhang, J.; Shi, S.; You, Y. The Biology and Function of Extracellular Vesicles in Nasopharyngeal Carcinoma. Int. J. Oncol. 2017, 52 (1), 38– 46,  DOI: 10.3892/ijo.2017.4202
      There is no corresponding record for this reference.
    67. 67
      Nuzhat, Z.; Kinhal, V.; Sharma, S.; Rice, G. E.; Joshi, V.; Salomon, C. Tumour-Derived Exosomes as a Signature of Pancreatic Cancer - Liquid Biopsies as Indicators of Tumour Progression. Oncotarget 2017, 8 (10), 17279,  DOI: 10.18632/oncotarget.13973
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      Tumour-derived exosomes as a signature of pancreatic cancer - liquid biopsies as indicators of tumour progression
      Nuzhat Zarin; Kinhal Vyjayanthi; Sharma Shayna; Rice Gregory E; Salomon Carlos; Rice Gregory E; Salomon Carlos; Joshi Virendra
      Oncotarget (2017), 8 (10), 17279-17291 ISSN:.
      Pancreatic cancer is the fourth most common cause of death due to cancer in the world. It is known to have a poor prognosis, mostly because early stages of the disease are generally asymptomatic. Progress in pancreatic cancer research has been slow, leaving several fundamental questions pertaining to diagnosis and treatment unanswered. Recent studies highlight the putative utility of tissue-specific vesicles (i.e. extracellular vesicles) in the diagnosis of disease onset and treatment monitoring in pancreatic cancer. Extracellular vesicles are membrane-limited structures derived from the cell membrane. They contain specific molecules including proteins, mRNA, microRNAs and non-coding RNAs that are secreted in the extracellular space. Extracellular vesicles can be classified according to their size and/or origin into microvesicles (~150-1000 nm) and exosomes (~40-120 nm). Microvesicles are released by budding from the plasmatic membrane, whereas exosomes are released via the endocytic pathway by fusion of multivesicular bodies with the plasmatic membrane. This endosomal origin means that exosomes contain an abundance of cell-specific biomolecules which may act as a 'fingerprint' of the cell of origin. In this review, we discuss our current knowledge in the diagnosis and treatment of pancreatic cancer, particularly the potential role of EVs in these facets of disease management. In particular, we suggest that as exosomes contain cellular protein and RNA molecules in a cell type-specific manner, they may provide extensive information about the signature of the tumour and pancreatic cancer progression.
    68. 68
      Sadovska, L.; Egli̅tis, J.; Linë, A. Extracellular Vesicles as Biomarkers and Therapeutic Targets in Breast Cancer. Anticancer Res. 2015, 35 (12), 6379– 6390
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      Extracellular vesicles as biomarkers and therapeutic targets in breast cancer
      Sadovska, Lilite; Eglitis, Janis; Line, Aija
      Anticancer Research (2015), 35 (12), 6379-6390CODEN: ANTRD4; ISSN:0250-7005. (International Institute of Anticancer Research)
      Cancer-derived extracellular vesicles (EVs) contain various cancer-assocd. mols., such as mutated or overexpressed oncoproteins, glycoproteins, mRNAs, various non-coding RNAs and DNA fragments. They have been shown to propagate phenotypic traits, such as drug resistance, increased proliferation rate, invasiveness and stemness across cancer cells and to mediate cancer-induced immunosuppression. Therefore, cancer-derived EVs have gained increasing attention as cancer biomarkers and therapeutic targets. Unlike circulating tumor cells they are highly abundant in biofluids and, on the contrary to single-mol. circulating biomarkers, they protect their mol. cargo against degrdn. and may carry mol. signatures assocd. with specific phenotypes. Herein, we summarize studies investigating EVs as biomarkers in breast cancer and propose scenarios for various clin. applications of EV-based biomarkers in the management of breast cancer. Furthermore, we provide an overview of recent findings regarding the cancer-promoting effects of breast cancer-derived EVs and discuss opportunities for blocking EV-mediated signaling as a therapeutic strategy for breast cancer.
    69. 69
      Whitehead, B.; Wu, L.; Hvam, M. L.; Aslan, H.; Dong, M.; Dyrskjøt, L.; Ostenfeld, M. S.; Moghimi, S. M.; Howard, K. A. Tumour Exosomes Display Differential Mechanical and Complement Activation Properties Dependent on Malignant State: Implications in Endothelial Leakiness. J. Extracell. Ves. 2015, 4 (1), 29685,  DOI: 10.3402/jev.v4.29685
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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsami.3c12035.

    • Example thermal spectra of AFM cantilevers in air, water, and saliva; discussion of storage and loss moduli from standard rheological measurements; characterization of the microrheology tracers; details about the acquisition and analysis of the mass uptake data; characterization of the cantilevers’ functionalization; disucssion of the baseline noise and negative controls; Figures S1–S8 and Table S1 (PDF)


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