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Povzetek: Aim: Human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) persist in effectively treated HIV-infected individuals, in part due to HIV reservoirs in brain microglia, which express low levels of viral proteins such as Nef. This study aimed to elucidate how microglia release Nef into the extracellular space, where it exerts its biological functions. Methods: Here, we systematically characterized extracellular particles released from immortalized human microglia (h-microglia) expressing Nef alone or after HIV infection. Importantly, we established a novel h-microglia model harboring a stably integrated Nef tagged with green fluorescent protein (Nef.GFP) transgene under an inducible promoter. Extracellular vesicles (EVs) were enriched from culture media and analyzed for morphology, size, concentration and molecular composition, including Nef content, by (super-resolution) fluorescence microscopy, (immunogold) transmission electron microscopy, asymmetric flow field-flow fractionation coupled to a multi-angle light-scattering detector, nanoparticle tracking analysis, and nano-flow cytometry and immunoblotting. Results: Nef.GFP expression increased particle release up to 11.7-fold compared with controls or known stimulants adenosine triphosphate (ATP) and ionomycin. Compared to the latter, the particles were also significantly smaller (root mean square radius, Rrms = 172 nm) and displayed unique protein and density profiles. All data support the EV nature of the released particles. Approximately half of the Nef.GFP-induced EVs contained Nef (45.5% ± 15.8%), with immunogold labeling confirming its intraluminal localization. Notably, infection with HIV isolates NL4-3 and YU-2 likewise produced Nef-positive EVs distinct from virions. Conclusion: Our findings importantly contribute to understanding the source and characteristics of extracellular Nef in the central nervous system of HIV infected individuals and offer new tools to study HIV Nef biology. Nef-laden EVs should be further investigated as potential therapeutic targets in HAND.
Ključne besede: human immunodeficiency virus (HIV), microglia, protein Nef, extracellular vesicles (EVs), HIV-associated neurocognitive disorder (HAND)
Objavljeno v DiRROS: 13.04.2026; Ogledov: 48; Prenosov: 19
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Objavljeno v DiRROS: 20.03.2026; Ogledov: 243; Prenosov: 125
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Povzetek: Potato virus Y (PVY) is one of the top 10 economically most important plant viruses and responsible for major yield losses. We previously suggested the involvement of the N-terminal region of PVY coat protein (CP) in PVY spread. By constructing different N-terminal deletion mutants of the PVY N605 strain, we here show that deletions of 40 or more amino acid residues from the N-terminal region of the CP resulted in the PVY multiplication limited to primary infected cells in Nicotiana clevelandii plants. Deletion of 26 residues profoundly impaired PVY cell-to-cell movement and prevented systemic PVY spread, while deletions of 19-23 residues allowed delayed systemic PVY spread. Introduced point mutations in the identified region prevent (S21G) or delay (G20P) PVY movement. In summary, this work shows the significance of the CP N-terminus for movement of the PVY.
Ključne besede: potato virus Y, potato, coat proteins, viral movement, point mutations
Objavljeno v DiRROS: 18.12.2025; Ogledov: 971; Prenosov: 383
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Povzetek: Extracellular vesicle-bound DNA (evDNA) is an understudied extracellular vesicle (EV) cargo, particularly in cancer-unrelated research. Although evDNA has been detected in urine, little is known about its characteristics, localization, and biomarker potential for kidney pathologies. To address this, we enriched EVs from urine of well-characterized kidney transplant recipients undergoing allograft biopsy, characterized their evDNA and its association to allograft injury. The SEC-based method enriched pure EVs from urine of kidney transplant recipients, regardless of the allograft injury. Urinary evDNA represented up to 29.2 ± 8% (mean ± SD) of cell-free DNA (cfDNA) and correlated with cfDNA in several characteristics but was less fragmented (P < 0.001). Importantly, using DNase treatment and immunogold labelling TEM, we demonstrated that evDNA was bound to the surface of urinary EVs. Normalised evDNA yield (P = 0.042) and evDNA copy number (P = 0.027) significantly differed between patients with normal histology, rejection injury and non-rejection injury, the later groups having significantly larger uEVs (mean diameter, P = 0.045) and more DNA bound per uEV. ddDNA is detectable in uEV samples of kidney allograft recipients, but its quantity is highly variable. In a proof-of-principle study, several evDNA characteristics correlated with clinical and histological parameters (P = 0.040), supporting that the potential of evDNA as a biomarker for kidney allograft injury should be further investigated.
Ključne besede: DNA, donor-derived DNA, extracellular vesicles
Objavljeno v DiRROS: 26.02.2025; Ogledov: 901; Prenosov: 780
Celotno besedilo (7,03 MB)
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Povzetek: The important economic sector of world grapevine production of 74 million tons is threatened by several grapevine yellows diseases associated with the presence of phytoplasmas. There is a lack of availability of well-characterized and certified material suitable for use as reference positive controls in different diagnostic schemes, validation studies, performance studies and proficiency tests, which are all part of the management programs for fast and accurate detection of grapevine yellows phytoplasmas.
Ključne besede: reference material, detection, plant pathogen, digital droplet PCR
Objavljeno v DiRROS: 03.09.2024; Ogledov: 1343; Prenosov: 684
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