Isolation and Characterization of Chlamydomonas Autophagy-Related Mutants in Nutrient-Deficient Conditions.

Autophagy is a recycling system for amino acids and carbon- and nitrogen (N)-containing compounds. To date, the functional importance of autophagy in microalgae in nutrient-deficient conditions has not been evaluated by using autophagy-defective mutants. Here, we provide evidence which supports the following notions by characterizing an insertional mutant of the autophagy-related gene ATG8, encoding a ubiquitin-like protein necessary for the formation of the autophagosome in the green alga, Chlamydomonas reinhardtii. First, ATG8 is required for maintenance of cell survival and Chl content in N-, sulfur- and phosphate-deficient conditions. Secondly, ATG8 supports the degradation of triacylglycerol and lipid droplets after the resupply of N to cells cultured in N-limiting conditions. Thirdly, ATG8 is also necessary for accumulation of starch in phosphate-deficient conditions. Additionally, autophagy is not essential for maternal inheritance of the organelle genomes in gametogenesis.

Efficient method for isolation of exosomes from raw bovine milk.

OBJECTIVE: This study aimed to establish a rapid and simple method for isolating exosomes from raw bovine milk and to compare the quality of the isolated exosomes with those isolated by a standard method involving ultracentrifugation (UC). METHODS: To remove caseins, which are major milk proteins consisting more than 80% of milk protein (35% in human breast milk) and hamper isolation and purification of exosomes, hydrochloride (HCl) was added to milk for isoelectric precipitation (IP). The effects of acidification on morphological features, particle size distribution, surface charge, and exosome surface proteins were analyzed by electron microscopy, tunable resistive pulse sensing (TRPS), and Western blot (WB) analysis, respectively. RESULTS: Electron microscopy showed that some of the exosomes isolated using IP had rough surfaces; most exosomes were successfully isolated without breakage, and their morphological features were similar to those of exosomes isolated by UC. TRPS showed that their surface charge and peaks (mode) for particle size distribution did not significantly differ between both methods. WB analysis using antibodies against the exosome surface marker proteins - milk fat globule-epidermal growth factor 8 (MFG-E8) and CD63 - revealed that the structures of exosome surface proteins were not affected by adding HCl. CONCLUSIONS: IP can be used to remove caseins to reduce operation time. This method will be useful for efficient isolation and purification of bovine milk exosomes and contribute to progression of research on health management of dairy cattle and drug delivery systems in human medicine, which require large amounts of milk exosomes.

Characterization of mRNA Signature in Milk Small Extracellular Vesicles from Cattle Infected with Bovine Leukemia Virus.

This study aimed to characterize the mRNA signature of milk small extracellular vesicles (sEVs) from BLV-infected cattle. A total of 23 mRNAs, which showed greater abundance in milk sEVs from BLV-infected cattle compared to those from BLV-uninfected (control) cattle, were identified through microarray analyses conducted in our previous study. To assess the significance of these differences in mRNA abundance, milk was collected from six control cattle and twenty-six cattle infected with BLV. The infected cattle were categorized into two distinct groups based on their proviral loads: a group of eight cattle with low proviral loads (LPVL), characterized by <10,000 copies per 105 white blood cells (WBC), and a group of eighteen cattle with high proviral loads (HPVL), marked by ≥10,000 copies per 105 WBC. The qPCR analysis quantified 7 out of 23 mRNAs, including BoLA, CALB1, IL33, ITGB2, MYOF, TGFBR1, and TMEM156, in the milk sEVs from control cattle, LPVL cattle, and HPVL cattle. Significantly, the average relative expression of CALB1 mRNA in milk sEVs was higher in LPVL cattle compared to HPVL cattle and control cattle (p < 0.05), while it was relatively lower in HPVL cattle compared to LPVL cattle and control cattle (p > 0.05). Likewise, the average relative expression of TMEM156 mRNA in milk sEVs was significantly higher in LPVL cattle compared to HPVL cattle (p < 0.05), and relatively lower in HPVL cattle compared to LPVL cattle and control cattle (p > 0.05). The results indicate distinct patterns of CALB1 and TMEM156 mRNA levels in milk sEVs, with higher levels observed in LPVL cattle and lower levels in HPVL cattle. The current study could provide essential information to comprehend the complexities during the progression of BLV infection and direct the exploration of mRNA biomarkers for monitoring the clinical stage of BLV infection.

mRNA Profile in Milk Extracellular Vesicles from Bovine Leukemia Virus-Infected Cattle.

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.

Acidification effects on isolation of extracellular vesicles from bovine milk.

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.

The N-terminal region of serum amyloid A3 protein activates NF-κB and up-regulates MUC2 mucin mRNA expression in mouse colonic epithelial cells.

Serum amyloid A (SAA) is the major acute-phase protein and a precursor of amyloid A (AA) in AA amyloidosis in humans and animals. SAA isoforms have been identified in a wide variety of animals, such as SAA1, SAA2, SAA3, and SAA4 in mouse. Although the biological functions of SAA isoforms are not completely understood, recent studies have suggested that SAA3 plays a role in host defense. Expression of SAA3 is increased on the mouse colon surface in the presence of microbiota in vivo, and it increases mRNA expression of mucin 2 (MUC2) in murine colonic epithelial cells in vitro, which constitutes a protective mucus barrier in the intestinal tract. In this study, to identify responsible regions in SAA3 for MUC2 expression, recombinant murine SAA1 (rSAA1), rSAA3, and rSAA1/3, a chimera protein constructed with mature SAA1 (amino acids 1-36) and SAA3 (amino acids 37-103), and vice versa for rSAA3/1, were added to murine colonic epithelial CMT-93 cells, and the mRNA expressions of MUC2 and cytokines were measured. Inhibition assays with NF-κB inhibitor or TLR4/MD2 inhibitor were also performed. Up-regulation of MUC2 mRNA expression was strongly stimulated by rSAA3 and rSAA3/1, but not by rSAA1 or rSAA1/3. Moreover, NF-κB and TLR4/MD2 inhibitors suppressed the increase of MUC2 mRNA expression. These results suggest that the major responsible region for MUC2 expression exists in amino acids 1-36 of SAA3, and that up-regulations of MUC2 expression by SAA3 and SAA3/1 are involved with activation of NF-κB via the TLR4/MD2 complex.