Extracellular vesicles such as exosomes convey biological messages between cells, either by surface-to-surface interaction or by shuttling of bioactive molecules to a recipient cell's cytoplasm. Here we show that exosomes released by mast cells harbour both active and latent transforming growth factor beta-1 (TGF beta-1) on their surfaces. The latent form of TGF beta-1 is associated with the exosomes via heparinase-II and pH-sensitive elements. These vesicles traffic to the endocytic compartment of recipient human mesenchymal stem cells (MSCs) within 60 min of exposure. Further, the exosomes-associated TGF beta-1 is retained within the endosomal compartments at the time of signalling, which results in prolonged cellular signalling compared to free-TGF beta-1. These exosomes induce a migratory phenotype in primary MSCs involving SMAD-dependent pathways. Our results show that mast cell-derived exosomes are decorated with latent TGF beta-1 and are retained in recipient MSC endosomes, influencing recipient cell migratory phenotype. We conclude that exosomes can convey signalling within endosomes by delivering bioactive surface ligands to this intracellular compartment.

Protein-based encapsulation systems have a wide spectrum of applications in targeted delivery of cargo molecules and for chemical transformations in confined spaces. By engineering affinity between cargo and container proteins it has been possible to enable the efficient and specific encapsulation of target molecules. Missing in current approaches is the ability to turn off the interaction after encapsulation to enable the cargo to freely diffuse in the lumen of the container. Separation between cargo and container is desirable in drug delivery applications and in the use of capsids as catalytic nanoparticles. We describe an encapsulation system based on the hepatitisB virus capsid in which an engineered high-affinity interaction between cargo and capsid proteins can be modulated by Ca2+. Cargo proteins are loaded into capsids in the presence of Ca2+, while ligand removal triggers unbinding inside the container. We observe that confinement leads to hindered rotation of cargo inside the capsid. Application of the designed container for catalysis was also demonstrated by encapsulation of an enzyme with beta-glucosidase activity.

Cellulose nanofibers (CNFs) have recently attracted a lot of attention in sensing because of their multifunctional character and properties such as renewability, nontoxicity, biodegradability, printability, and optical transparency in addition to unique physicochemical, barrier, and mechanical properties. However, the focus has exclusively been devoted toward developing two-dimensional sensing platforms in the form of nanopaper or nanocellulose-based hydrogels. To improve the flexibility and sensing performance in situ, for example, to detect biomarkers in vivo for early disease diagnostics, more advanced CNF-based structures are needed. Here, we developed porous and hollow, yet robust, CNF-based microcapsules using only the primary plant cell wall components, CNF, pectin, and xyloglucan, to assemble the capsule wall. The fluorescein isothiocyanate-labeled dextrans with M-w of 70 and 2000 kDa could enter the hollow capsules at a rate of 0.13 +/- 0.04 and 0.014 +/- 0.009 s(-1), respectively. This property is very attractive because it minimizes the influence of mass transport through the capsule wall on the response time. As a proof of concept, glucose oxidase (GOx) enzyme was loaded (and cross-linked) in the microcapsule interior with an encapsulation efficiency of 68 +/- 2%. The GOx-loaded microcapsules were immobilized on a variety of surfaces (here, inside a flow channel, on a carbon-coated sensor or a graphite rod) and glucose concentrations up to 10 mM could successfully be measured. The present concept offers new opportunities in the development of simple, more efficient, and disposable nanocellulose-based analytical devices for several sensing applications including environmental monitoring, healthcare, and diagnostics.

Inverse Fluorescence Correlation Spectroscopy (iFCS) is a variant of FCS where unlabeled particles in solution, or domains in membranes, displace their surrounding, signal-generating molecules and thereby generate fluctuations. iFCS has to date been applied to unlabeled as well as labeled particles and protein molecules, using fluorescence as well as Raman scattering as a signal source, in diffraction-limited detection volumes as well as in nano-wells, and on fixed surfaces as well as in lipid bilayers. This review describes these applications and discusses the potentials and pitfalls when using iFCS.

The occurrence of autoantibodies is a hallmark of rheumatoid arthritis, specifically those autoantibodies targeting proteins containing the arginine-derived amino acid citrulline. There is strong evidence showing that the occurrence of anticitrullinated protein/peptide antibodies (ACPA) are involved in disease progression, and ACPA was recently shown to induce pain in animals. Here, we explore a novel concept useful for research, diagnostics, and possibly therapy of autoimmune diseases, namely, to directly target and neutralize autoantibodies using peptide binders. A high-affinity peptide-based scavenger of ACPA was developed by grafting a citrullinated epitope derived from human fibrinogen into a naturally occurring stable peptide scaffold. The best scavenger comprises the truncated epitope α-fibrinogen, [Cit573]fib(566-580), grafted into the scaffold sunflower trypsin inhibitor-1, SFTI-1. The final peptide demonstrates low nanomolar apparent affinity and superior stability.

Elevated basal insulin secretion under fasting conditions together with insufficient stimulated insulin release is an important hallmark of type 2 diabetes, but the mechanisms controlling basal insulin secretion remain unclear. Membrane rafts exist in pancreatic islet cells and spatially organize membrane ion channels and proteins controlling exocytosis, which may contribute to the regulation of insulin secretion. Membrane rafts (cholesterol and sphingolipid containing microdomains) were dramatically reduced in human type 2 diabetic and diabetic Goto-Kakizaki (GK) rat islets when compared with healthy islets. Oxidation of membrane cholesterol markedly reduced microdomain staining intensity in healthy human islets, but was without effect in type 2 diabetic islets. Intriguingly, oxidation of cholesterol affected glucose-stimulated insulin secretion only modestly, whereas basal insulin release was elevated. This was accompanied by increased intracellular Ca2+ spike frequency and Ca2+ influx and explained by enhanced single Ca2+ channel activity. These results suggest that the reduced presence of membrane rafts could contribute to the elevated basal insulin secretion seen in type 2 diabetes.

Nanoparticles (NPs) and molecules can be analyzed by inverse fluorescence correlation spectroscopy (iFCS) as they pass through an open detection volume, displacing fractions of the fluorescence-emitting solution in which they are dissolved. iFCS does not require the NPs or molecules to be labeled. However, fluorophores in m-mm concentrations are needed for the solution signal. Here, we instead use coherent anti-Stokes Raman scattering (CARS) from plain water molecules as the signal from the solution. By this fully label-free approach, termed inverse CARS-based correlation spectroscopy (iCARS-CS), NPs that are a few tenths of nm in diameter and at pM concentrations can be analyzed, and their absolute volumes/concentrations can be determined. Likewise, lipid vesicles can be analyzed as they diffuse/flow through the detection volume by using CARS fluctuations from the surrounding water molecules. iCARS-CS could likely offer a broadly applicable, label-free characterization technique of, for example, NPs, small lipid exosomes, or microparticles in biomolecular diagnostics and screening, and can also utilize CARS signals from biologically relevant media other than water.

Lysosomotropic detergents (LDs) selectively rupture lysosomal membranes through mechanisms that have yet to be characterized. A consensus view, currently, holds that LDs, which are weakly basic, diffuse across cellular membranes as monomers in an uncharged state, and via protonation in the acidic lysosomal compartment, they become trapped, accumulate, and subsequently solubilize the membrane and induce lysosomal membrane permeabilization. Here we demonstrate that the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH) spontaneously assembles into vesicles at, and above, cytosolic pH, and that the vesicles disassemble as the pH reaches 6.4 or lower. The aggregation commences at concentrations below the range of those used in cell studies. Assembly and disassembly of the vesicles was studied via dynamic light scattering, zeta potential measurements, cryo-TEM, and fluorescence correlation spectroscopy and was found to be reversible via control of the pH. Aggregation of MSDH into closed vesicles under cytosolic conditions is at variance with the commonly held view of LD behavior, and we propose that endocytotic pathways should be considered as possible routes of LD entry into lysosomes. We further demonstrate that MSDH vesicles can be loaded with fluorophores via a solution transition from low to high pH, for subsequent release when the pH is lowered again. The ability to encapsulate molecular cargo into MSDH vesicles together with its ability to disaggregate at low pH and to permeabilize the lysosomal membrane presents an intriguing possibility to use MSDH as a delivery system.

Oligomers formed by the amyloid beta-peptide (A beta) are pathogens in Alzheimers disease. Increased knowledge on the oligomerization process is crucial for understanding the disease and for finding treatments. Ideally, A beta oligomerization should be studied in solution and at physiologically relevant concentrations, but most popular techniques of today are not capable of such analyses. We demonstrate here that the combination of FOrster Resonance Energy Transfer and Fluorescence Correlation Spectroscopy (FRET-FCS) has a unique ability to detect small subpopulations of FRET-active molecules and oligomers. FRET-FCS could readily detect a FRET-active oligonucleotide present at levels as low as 0.5% compared to FRET-inactive dye molecules. In contrast, three established fluorescence fluctuation techniques (FCS, FCCS, and PCH) required fractions between 7 and 11%. When applied to the analysis of A beta, FRET-FCS detected oligomers consisting of less than 10 A beta molecules, which coexisted with the monomers at fractions as low as 2 +/- 2%. Thus, we demonstrate for the first time direct detection of small fractions of A beta oligomers in solution at physiological concentrations. This ability of FRET-FCS could be an indispensable tool for studying biological oligomerization processes, in general, and for finding therapeutically useful oligomerization inhibitors.

We report on label-free volume, concentration, and mobility analysis of single protein molecules and nanoparticles during their diffusion through a subattoliter detection volume, confined by a 100 nm aperture in a thin gold film. A high concentration of small fluorescent molecules renders the aqueous solution in the aperture brightly fluorescent. Nonfluorescent analytes diffusing into the aperture displace the fluorescent molecules in the solution, leading to a decrease of the detected fluorescence signal, while analytes diffusing out of the aperture return the fluorescence level. The resulting fluorescence fluctuations provide direct information on the volume, concentration, and mobility of the nonfluorescent analytes through fluctuation analysis in both time and amplitude.