Unveiling Cryptosporidium parvum sporozoite-derived extracellular vesicles: profiling, origin, and protein composition.

Cryptosporidium parvum is a common cause of a zoonotic disease and a main cause of diarrhea in newborns. Effective drugs or vaccines are still lacking. Oocyst is the infective form of the parasite; after its ingestion, the oocyst excysts and releases four sporozoites into the host intestine that rapidly attack the enterocytes. The membrane protein CpRom1 is a large rhomboid protease that is expressed by sporozoites and recognized as antigen by the host immune system. In this study, we observed the release of CpRom1 with extracellular vesicles (EVs) that was not previously described. To investigate this phenomenon, we isolated and resolved EVs from the excystation medium by differential ultracentrifugation. Fluorescence flow cytometry and transmission electron microscopy (TEM) experiments identified two types of sporozoite-derived vesicles: large extracellular vesicles (LEVs) and small extracellular vesicles (SEVs). Nanoparticle tracking analysis (NTA) revealed mode diameter of 181 nm for LEVs and 105 nm for SEVs, respectively. Immunodetection experiments proved the presence of CpRom1 and the Golgi protein CpGRASP in LEVs, while immune-electron microscopy trials demonstrated the localization of CpRom1 on the LEVs surface. TEM and scanning electron microscopy (SEM) showed that LEVs were generated by means of the budding of the outer membrane of sporozoites; conversely, the origin of SEVs remained uncertain. Distinct protein compositions were observed between LEVs and SEVs as evidenced by their corresponding electrophoretic profiles. Indeed, a dedicated proteomic analysis identified 5 and 16 proteins unique for LEVs and SEVs, respectively. Overall, 60 proteins were identified in the proteome of both types of vesicles and most of these proteins (48 in number) were already identified in the molecular cargo of extracellular vesicles from other organisms. Noteworthy, we identified 12 proteins unique to Cryptosporidium spp. and this last group included the immunodominant parasite antigen glycoprotein GP60, which is one of the most abundant proteins in both LEVs and SEVs.

EGFR/ErbB Inhibition Promotes OPC Maturation up to Axon Engagement by Co-Regulating PIP2 and MBP.

Remyelination in the adult brain relies on the reactivation of the Neuronal Precursor Cell (NPC) niche and differentiation into Oligodendrocyte Precursor Cells (OPCs) as well as on OPC maturation into myelinating oligodendrocytes (OLs). These two distinct phases in OL development are defined by transcriptional and morphological changes. How this differentiation program is controlled remains unclear. We used two drugs that stimulate myelin basic protein (MBP) expression (Clobetasol and Gefitinib) alone or combined with epidermal growth factor receptor (EGFR) or Retinoid X Receptor gamma (RXRγ) gene silencing to decode the receptor signaling required for OPC differentiation in myelinating OLs. Electrospun polystyrene (PS) microfibers were used as synthetic axons to study drug efficacy on fiber engagement. We show that EGFR inhibition per se stimulates MBP expression and increases Clobetasol efficacy in OPC differentiation. Consistent with this, Clobetasol and Gefitinib co-treatment, by co-regulating RXRγ, MBP and phosphatidylinositol 4,5-bisphosphate (PIP2) levels, maximizes synthetic axon engagement. Conversely, RXRγ gene silencing reduces the ability of the drugs to promote MBP expression. This work provides a view of how EGFR/ErbB inhibition controls OPC differentiation and indicates the combination of Clobetasol and Gefitinib as a potent remyelination-enhancing treatment.

Myiasis of the Tracheostomy Wound Caused by Sarcophaga (Liopygia) argyrostoma (Diptera: Sarcophagidae): Molecular Identification Based on the Mitochondrial Cytochrome c Oxidase I Gene.

Wound myiasis is the infestation of open wounds of mammalian hosts caused by larvae of various species of flies. This kind of myiasis can be a serious problem for immobilized patients with open wounds. Here, we identify a dipteran larva found in the tracheostomy wound of a child affected by a severe spinal muscular atrophy. The collected larva was dissected and microscopically analyzed. DNA was extracted from part of the larva and used for the molecular identification. A 487 bp fragment, including part of 5.8 S, the internal transcribed spacer 2 (ITS2), and part of 28S, was amplified using a novel PCR assay to be cloned and sequenced. The barcode region of cytochrome oxidase I (COI) was also cloned and sequenced after PCR amplification. The larva, designated as SASI1, was identified as a third instar of Sarcophaga sp. The COI sequencing confirmed a low similarity with Sarcophaga ruficornis (F.) (95%), yet COI showed a 100% similarity with Sarcophaga argyrostoma (Robineau-Desvoidy, 1830) species. Therefore, SASI1 was identified as a S. argyrostoma larva on the basis of its COI barcode. This is one of the rare cases of myiasis of tracheostomy wound and the first caused by S. argyrostoma.