Physical exercise stimulates organs, mainly the skeletal muscle, to release a broad range of molecules, recently dubbed exerkines. Among them, RNAs, such as miRNAs, piRNAs, and tRNAs loaded in extracellular vesicles (EVs) have the potential to play a significant role in the way muscle and other organs communicate to translate exercise into health. Low, moderate and high intensity treadmill protocols were applied to rat groups, aiming to investigate the impact of exercise on serum EVs and their associated small RNA molecules. Transmission electron microscopy, resistive pulse sensing, and western blotting were used to investigate EVs morphology, size distribution, concentration and EVs marker proteins. Small RNA libraries from EVs RNA were sequenced. Exercise did not change EVs size, while increased EVs concentration. Twelve miRNAs were found differentially expressed after exercise: rno-miR-128-3p, 103-3p, 330-5p, 148a-3p, 191a-5p, 10b-5p, 93-5p, 25-3p, 142-5p, 3068-3p, 142-3p, and 410-3p. No piRNA was found differentially expressed, and one tRNA, trna8336, was found down-regulated after exercise. The differentially expressed miRNAs were predicted to target genes involved in the MAPK pathway. A single bout of exercise impacts EVs and their small RNA load, reinforcing the need for a more detailed investigation into EVs and their load as mediators of health-promoting exercise.

Circulating miRNAs are potential biomarkers that can be important molecules driving cell-to-cell communication. To investigate circulating muscle-specific miRNAs in recreational athletes. Three miRNAs from whole plasma before and after a half-marathon were analyzed by qPCR. MiR-1, -133a, and -206 significantly increased after the race. Increased levels of miRNAs after exercise point to potential biomarkers and to the possibility of being functional players following endurance training. These miRNAs are potential biomarkers of muscle damage or adaptation to exercise.

Extracellular vesicles (EVs) have recently emerged as intercellular conveyors of biological information and disease biomarkers. Identification and characterization of RNA species in single EVs are currently challenging. Molecular beacons (MBs) represent an attractive means for detecting specific RNA molecules. Coupling the MBs to cell-penetrating peptides (CPPs) provides a fast, effective, and membrane-type agnostic means to deliver MBs across the plasma membrane and into the cytosol. Here, we generated RBCs-derived EVs by complement activation and tested the ability of MBs coupled with CPP to detect miRNAs from RBC-EVs. Our results showed that RBC and RBC-EVs miRNA-451a can be detected using MB-CPP, and the respective fluorescence levels can be measured by nano-flow cytometry. MB-based detection of RNA via nano-flow cytometry creates a powerful new analytical framework in which a simple addition of a reagent allows profiling of specific RNA species present within certain EV subsets.

Cryptococcus spp. are human pathogens that cause 181,000 deaths per year. In this work, we systematically investigated the virulence attributes of Cryptococcus spp. clinical isolates and correlated them with patient data to better understand cryptococcosis. We collected 66 C. neoformans and 19 C. gattii clinical isolates and analyzed multiple virulence phenotypes and host–pathogen interaction outcomes. C. neoformans isolates tended to melanize faster and more intensely and produce thinner capsules in comparison with C. gattii. We also observed correlations that match previous studies, such as that between secreted laccase and disease outcome in patients. We measured Cryptococcus colony melanization kinetics, which followed a sigmoidal curve for most isolates, and showed that faster melanization correlated positively with LC3-associated phagocytosis evasion, virulence in Galleria mellonella and worse prognosis in humans. These results suggest that the speed of melanization, more than the total amount of melanin Cryptococcus spp. produces, is crucial for virulence.

Extracellular vesicles (EVs) are membrane‐bound structures released by cells and tissues into biofluids, involved in cell‐cell communication. In humans, circulating red blood cells (RBCs), represent the most common cell‐type in the body, generating daily large numbers of microvesicles. In vitro, RBC vesiculation can be mimicked by stimulating RBCs with calcium ionophores, such as ionomycin and A23187. The fate of microvesicles released during in vivo aging of RBCs and their interactions with circulating cells is hitherto unknown. Using SEC plus DEG isolation methods, we have found that human RBCs generate microvesicles with two distinct sizes, densities and protein composition, identified by flow cytometry, and MRPS, and further validated by immune TEM. Furthermore, proteomic analysis revealed that RBC‐derived microvesicles (RBC‐MVs) are enriched in proteins with important functions in ion channel regulation, calcium homeostasis and vesicular transport, such as of sorcin, stomatin, annexin A7 and RAB proteins. Cryo‐electron microscopy identified two separate pathways of RBC‐MV‐neutrophil interaction, direct fusion with the plasma membrane and internalization, respectively. Functionally, RBC‐MVs decrease neutrophil ability to phagocytose Escherichia coli but do not affect their survival at 24 h. This work brings new insights regarding the complexity of the RBC‐MVs biogenesis, as well as their possible role in circulation.