Exosomes are extracellular vesicles of endosomal origin that are released by practically all cell types across metazoans. Exosomes are active vehicles of intercellular communication and can transfer lipids, RNAs, and proteins between different cells, tissues, or organs. Here, we describe a mechanism whereby proteins containing a KFERQ motif pentapeptide are loaded into a subpopulation of exosomes in a process that is dependent on the membrane protein LAMP2A. Moreover, we demonstrate that this mechanism is independent of the ESCRT machinery but dependent on HSC70, CD63, Alix, Syntenin-1, Rab31, and ceramides. We show that the master regulator of hypoxia HIF1A is loaded into exosomes by this mechanism to transport hypoxia signaling to normoxic cells. In addition, by tagging fluorescent proteins with KFERQ-like sequences, we were able to follow the interorgan transfer of exosomes. Our findings open new avenues for exosome engineering by allowing the loading of bioactive proteins by tagging them with KFERQ-like motifs.

The structural and molecular mechanism of double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) solvation in ten room-temperature ionic liquids (RTILs) is disclosed herein. We show that the Drew Dickerson B DNA structure solvated in neat RTILs based on imidazolium, oxazolium, pyrrolidinium, pyrimidinium and quaternary ammonium cations, combined with hexafluorophosphate and tetrafluorobromide anions (Figure 1), is able to maintain the native dsDNA B form conformation. We show that cations are preferentially located near the DNA main chain, establishing strong electrostatic interactions with the phosphate groups. On the other hand, anions are predominantly involved in hydrogen-bond interactions with cytosine, adenine and guanine bases. Furthermore, cations are also able to establish hydrogen-bonding and edge-toface NH···p interactions with the DNA bases. We also show that ssDNA bases in RTILs are preferentially solvated by the anions via hydrogen bonding with the fluor atoms. Our study provides the first fundamental structural insights of DNA in RTILs that can support the future development of novel RTILs, specific for nucleic acids solutes.RTILs are becoming ubiquitous in many chemical and biochemical processes, [1] and are replacing organic solvents traditionally used in non-aqueous enzymology [1c] and separation processes.[1a] Recently, the use of RTILs with DNA has also been investigated.[2] DNA separation using a capillary coated with imidazolium-based RTILs was reported, [2c] as well as the use of RTILs to extract trace amounts of DNA from aqueous solutions.[2e] Other applications of DNA in RTILs have also been reported, such as, long-term storage [2d] and DNA nanopore translocation.[2a]The molecular mechanism of interaction between RTILs and DNA is still not clearly understood. Fundamental chemical and physical information about the interaction between a few RTILs and DNA have been reported.[3] The 31P NMR and IR spectra indicate that the molecular interaction between dsDNA and 1-butyl-3-methylimidazolium ([BMIM] + ) chloride is accomplished via strong electrostatic interactions between the cation head group of [BMIM] + with the phosphate groups of DNA, [2f, 3] and hydrophobic association between the aliphatic chain of [BMIM] + with the DNA bases.[3] Furthermore, DNA in [BMIM]Cl maintains its B form, has shown by the similar circular dichroism (CD) spectra of DNA in this RTIL and in tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl). The same behaviour was also observed using choline-based RTILs.[2d] So far, we have some direct and indirect evidence of the interaction of the cation RTILs with DNA and no clear role for the anionic species in the solvation mechanism of DNA.Our modeling and molecular dynamics simulation studies show that the DNA structure is highly stabilized by the RTILs, in agreement with the Vijayaraghavan et al. evidence of the long-term DNA stabilization by [BMIM]Cl.[2d] The root mean square deviation (RMSD) of the DNA structure solvated in neat RTILs is low (Table 1) i...

We aim to characterize the pathways required for autofluorescent granule (AFG) formation by RPE cells using cultured monolayers. METHODS.We fed RPE monolayers in culture with a single pulse of photoreceptor outer segments (POS). After 24 hours the cells started accumulating AFGs that were comparable to lipofuscin in vivo. Using this model, we used a variety of light and electron microscopical techniques, flow cytometry and Western blot to analyze the formation of AFGs. We also generated a mutant RPE line lacking cathepsin D by gene editing. RESULTS.AFGs seem to derive from incompletely digested POS-containing phagosomes and after 3 days are surrounded by a single membrane positive for lysosome markers. We show by various methods that lysosome-phagosome fusion is required for AFG formation, and that impairment of lysosomal pH or catalytic activity, particularly cathepsin D activity, enhances AF accumulation. CONCLUSIONS.We conclude that lysosomal dysfunction results in incomplete POS degradation and enhanced AFG accumulation.

The nutritional requirements of novel microalgal strains are key for their effective cultivation and metabolite content. Therefore, the optimization of heterotrophic and photoautotrophic culture media is crucial for novel Chlorococcum amblystomatis growth. Heterotrophic and photoautotrophic biomass samples were characterized to identify the differences between their heterotrophic and photoautotrophic biomass composition and their biotechnological potential. Media optimization through surface response methodology led to 44.9 and 51.2% increments in C. amblystomatis-specific growth rates under heterotrophic and photoautotrophic growth, respectively. This microalga registered high protein content (61.49–73.45% dry weight), with the highest value being observed in the optimized photoautotrophic growth medium. The lipid fraction mainly constituted polyunsaturated fatty acids, ranging from 44.47 to 51.41% for total fatty acids (TFA) in cells under heterotrophy. However, these contents became significantly higher (70.46–72.82% TFA) in cultures cultivated under photoautotrophy. An interesting carotenoids content was achieved in the cultures grown in optimized photoautotrophic medium: 5.84 mg·g−1 β-carotene, 5.27 mg·g−1 lutein, 3.66 mg·g−1 neoxanthin, and 0.75 mg·g−1 violaxanthin. Therefore, C. amblystomatis demonstrated an interesting growth performance and nutritional profile for food supplements and feed products that might contribute to meeting the world’s nutritional demand.

Microalgae attract interest worldwide due to their potential for several applications. Scenedesmus is one of the first in vitro cultured algae due to their rapid growth and handling easiness. Within this genus, cells exhibit a highly resistant wall and propagate both auto- and heterotrophically. The main goal of the present work is to find scalable ways to produce a highly concentrated biomass of Scenedesmus rubescens in heterotrophic conditions. Scenedesmus rubescens growth was improved at the lab-scale by 3.2-fold (from 4.1 to 13 g/L of dry weight) through medium optimization by response surface methodology. Afterwards, scale-up was evaluated in 7 L stirred-tank reactor under fed-batch operation. Then, the optimized medium resulted in an overall productivity of 8.63 g/L/day and a maximum biomass concentration of 69.5 g/L. S. rubescens protein content achieved approximately 31% of dry weight, similar to the protein content of Chlorella vulgaris in heterotrophy.