Bovine milk extracellular vesicles (EVs) attract research interest as carriers of biologically active cargo including miRNA from donor to recipient cells to facilitate intercellular communication. Since toxicity of edible milk seems to be negligible, milk EVs are applicable to use for therapeutics in human medicine. Casein separation is an important step in obtaining pure EVs from milk, and recent studies reported that adding hydrochloric acid (HCl) and acetic acid (AA) to milk accelerates casein aggregation and precipitation to facilitate EV isolation and purification; however, the effects of acidification on EVs remain unclear. In this study, we evaluated the acidification effects on milk-derived EVs with that by standard ultracentrifugation (UC). We separated casein from milk by either UC method or treatment with HCl or AA, followed by evaluation of EVs in milk serum (whey) by transmission electron microcopy (TEM), spectrophotometry, and tunable resistive pulse sensing analysis to determine EVs morphology, protein concentration, and EVs size and concentration, respectively. Moreover, we used anti-CD9, -CD63, -CD81, -MFG-E8, -HSP70, and -Alix antibodies for the detection of EVs surface and internal marker proteins by western blot (WB). Morphological features of EVs were spherical shape and similar structure was observed in isolated EVs by TEM. However, some of the EVs isolated by HCl and AA had shown rough surface. Although protein concentration was higher in whey obtained by UC, EV concentration was significantly higher in whey following acid treatment. Moreover, although all of the targeted EVs-marker-proteins were detected by WB, HCl- or AA-treatments partially degraded CD9 and CD81. These findings indicated that acid treatment successfully separated casein from milk to allow efficient EV isolation and purification but resulted in partial degradation of EV-surface proteins. Our results suggest that following acid treatment, appropriate EV-surface-marker antibodies should be used for accurate assess the obtained EVs for downstream applications. This study describes the acidification effects on EVs isolated from bovine milk for the first time.

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APOBEC3G (A3G) is a member of the cellular polynucleotide cytidine deaminases, which catalyze the deamination of cytosine (dC) to uracil (dU) in single-stranded DNA. These enzymes potently inhibit the replication of a variety of retroviruses and retrotransposons, including HIV-1. A3G is incorporated into vif-deficient HIV-1 virions and targets viral reverse transcripts, particularly minus-stranded DNA products, in newly infected cells. It is well established that the enzymatic activity of A3G is closely correlated with the potential to greatly inhibit HIV-1 replication in the absence of Vif. However, the details of the underlying molecular mechanisms are not fully understood. One potential mechanism of A3G antiviral activity is that the A3G-dependent deamination may trigger degradation of the dU-containing reverse transcripts by cellular uracil DNA glycosylases (UDGs). More recently, another mechanism has been suggested, in which the virion-incorporated A3G generates lethal levels of the G-to-A hypermutation in the viral DNA genome, thus potentially driving the viruses into “error catastrophe” mode. In this mini review article, we summarize the deaminase-dependent and deaminase-independent molecular mechanisms of A3G and discuss how A3G-mediated deamination is linked to antiviral mechanisms.

Milk extracellular vesicles (EVs) form an excellent source of mRNAs, microRNAs (miRNAs), proteins, and lipids that represent the physiological and pathological status of the host. Recent studies have reported milk EVs as novel biomarkers for many infectious diseases in both humans and animals. For example, miRNAs in milk EVs from cattle were used for early detection of bacterial infection in the mammary gland. Based on these findings, we hypothesized that mRNAs in milk EVs are suitable for gaining a better understanding of the pathogenesis of bovine leukemia virus (BLV) infection and prognosis of the clinical stage in cattle. For that purpose, milk EVs were isolated from BLV-infected and uninfected cattle, and mRNAs were investigated using microarray analysis. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed mainly focusing on the differentially expressed genes (DEGs) in milk EVs from BLV-infected cattle. GO and KEGG analyses suggested the DEGs in milk EVs from BLV-infected cattle had involved in diverse molecular functions, biological processes, and distinct disease-related pathways. The present study suggested that BLV infection causes profound effects on host cellular activity, changing the mRNA expression profile in milk EVs obtained from BLV-infected cattle. Overall, our results suggested that the mRNA profile in milk EVs to be a key factor for monitoring the clinical stage of BLV infection. This is the first report of mRNA profiling of milk EVs obtained from BLV-infected cattle.