extracellular vesicles (eVs) are nano-sized vesicles containing nucleic acid and protein cargo that are released from a multitude of cell types and have gained significant interest as potential diagnostic biomarkers. Human serum is a rich source of readily accessible eVs; however, the separation of eVs from serum proteins and non-eV lipid particles represents a considerable challenge. in this study, we compared the most commonly used isolation techniques, either alone or in combination, for the isolation of EVs from 200 µl of human serum and their separation from non-eV protein and lipid particles present in serum. the size and yield of particles isolated by each method was determined by nanoparticle tracking analysis, with the variation in particle size distribution being used to determine the relative impact of lipoproteins and protein aggregates on the isolated EV population. Purification of eVs from soluble protein was determined by calculating the ratio of eV particle count to protein concentration. finally, lipoprotein particles co-isolated with eVs was determined by Western blot analysis of lipoprotein markers ApoB and Apoe. overall, this study reveals that the choice of eV isolation procedure significantly impacts EV yield from human serum, together with the presence of lipoprotein and protein contaminants. Extracellular vesicles (EVs) were originally identified in reticulocytes as a means of disposing of obsolete membrane proteins such as α4β1 and transferrin receptor during reticulocyte maturation 1-3 , and have since been shown to participate in cell-cell signalling via transfer of proteins, nucleic acids and metabolites 4-6. EVs have been identified in a diverse range of human biofluids including serum, plasma, urine, saliva, breast milk, amniotic fluid, ascites fluid, cerebrospinal fluid and bile 7,8. These EVs are classified into three groups; exosomes, microvesicles and apoptotic bodies depending on their size, biogenesis and method of cellular release. Microvesicles and apoptotic bodies generally range from 100 to 1000 nm and 1-4 µm respectively, and are formed by budding from the plasma membrane 4,9. In contrast, exosomes have a diameter of 30-150 nm and are formed by inward budding of the late endosome lumen to form a multivesicular body (MVB) that is secreted by fusion with the plasma membrane 10. The overlap in exosome and microvesicle size (100-150 nm) and density (1.08-1.19 g/ml) makes it difficult to distinguish the two groups and as a result exosomes are often defined by their content of endosome-associated proteins including tetraspanins CD9, CD63, and CD81. However, since microvesicles from haematopoietic cells are also enriched for endosomal proteins such as CD63 and CD81 11 exosomes and microvesicles <150 nm are collectively referred to as small extracellular vesicles (sEVs) 12. EV secretion has been shown to be elevated in response to inflammation 13 , hypoxia 14,15 and an acidic microenvironment 16,17 and is associated with human diseases such as cancer, where secretion levels have b...

Heusermann et al. use a single-vesicle dye-tracing analysis in live cells showing that exosomes enter cells as intact vesicles, primarily at filopodia-active regions, and sort into endocytic vesicle circuits that are targeted to scan the ER before being directed to lysosomes.

Genetically encoded, ratiometric biosensors based on fluorescence resonance energy transfer (FRET) are powerful tools to study the spatiotemporal dynamics of cell signaling. However, many biosensors lack sensitivity. We present a biosensor library that contains circularly permutated mutants for both the donor and acceptor fluorophores, which alter the orientation of the dipoles and thus better accommodate structural constraints imposed by different signaling molecules while maintaining FRET efficiency. Our strategy improved the brightness and dynamic range of preexisting RhoA and extracellular signal-regulated protein kinase (ERK) biosensors. Using the improved RhoA biosensor, we found micrometer-sized zones of RhoA activity at the tip of F-actin bundles in growth cone filopodia during neurite extension, whereas RhoA was globally activated throughout collapsing growth cones. RhoA was also activated in filopodia and protruding membranes at the leading edge of motile fibroblasts. Using the improved ERK biosensor, we simultaneously measured ERK activation dynamics in multiple cells using low-magnification microscopy and performed in vivo FRET imaging in zebrafish. Thus, we provide a construction toolkit consisting of a vector set, which enables facile generation of sensitive biosensors.

In many species that form socially monogamous pair bonds, a considerable proportion of the offspring is sired by extrapair males. This observation has remained a puzzle for evolutionary biologists: although mating outside the pair bond can obviously increase the offspring production of males, the benefits of such behavior to females are less clear, yet females are known to actively solicit extrapair copulations. For more than two decades adaptionist explanations have dominated the discussions, yet remain controversial, and genetic constraint arguments have been dismissed without much consideration. An intriguing but still untested hypothesis states that extrapair mating behavior by females may be affected by the same genetic variants (alleles) as extrapair mating behavior by males, such that the female behavior could evolve through indirect selection on the male behavior. Here we show that in the socially monogamous zebra finch, individual differences in extrapair mating behavior have a hereditary component. Intriguingly, this genetic basis is shared between the sexes, as shown by a strong genetic correlation between male and female measurements of extrapair mating behavior. Hence, positive selection on males to sire extrapair young will lead to increased extrapair mating by females as a correlated evolutionary response. This behavior leads to a fundamentally different view of female extrapair mating: it may exist even if females obtain no net benefit from it, simply because the corresponding alleles were positively selected in the male ancestors.

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) is known to give reliable results for narrow molecular weight distributions, though in the case of polydisperse polymers the values obtained are not in agreement with those from conventional methods. In this paper we simulate broad polymer distributions with increasing polydispersity by mixing equimolar amounts of either poly(styrene) (PS) or poly(methylmethacrylate) (PMMA) standards in a mass range between 5000 and 100 000 Da with increasing difference in molecular weight. This allows us to investigate the problems of MALDI‐TOF measurements of polydisperse polymers. We found that several reasons are responsible for the false results. High molecular weight polymers require higher laser power for the desorption/ionization process than do low molecular weight polymers. This causes smaller peak areas for the high mass component and can also be responsible for fragmentation of the low molecular weight part of a mixture, if the molecular weights of the polymers are widely distributed. The observed dependence upon laser power is different for PS and PMMA polymers and changes with the matrix used. Furthermore, doubly charged molecular peaks and molecular cluster peaks appear in the spectra and influence the experimentally measured distribution.

In a polymer analogous approach, large dendritic oligophenylenes containing benzene and tetraphenylmethane cores are transformed via oxidative cyclodehydrogenation to novel propeller-shaped molecules with large polycyclic aromatic hydrocarbon units as "blades". Structure analysis is performed by a combination of MALDI-TOF mass spectrometry, UV/vis, fluorescence, and Raman spectroscopy using solid-state sample preparation methods. These methods are also utilized to determine the degree of the cyclodehydrogenation reaction.

Male reproductive success depends on the competitive ability of sperm to fertilize the ova, which should lead to strong selection on sperm characteristics. This raises the question of how heritable variation in sperm traits is maintained. Here we show that in zebra finches (Taeniopygia guttata) nearly half of the variance in sperm morphology is explained by an inversion on the Z chromosome with a 40% allele frequency in the wild. The sperm of males that are heterozygous for the inversion had the longest midpieces and the highest velocity. Furthermore, such males achieved the highest fertility and the highest siring success, both within-pair and extra-pair. Males homozygous for the derived allele show detrimental sperm characteristics and the lowest siring success. Our results suggest heterozygote advantage as the mechanism that maintains the inversion polymorphism and hence variance in sperm design and in fitness.

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