Yuji O. Kamatari

Bovine leukemia virus (BLV) is an oncogenic retrovirus responsible for enzootic bovine leukosis (EBL) in cattle. Previous studies indicated that increased levels of the miRNA hsa-miR-424-5p in milk small extracellular vesicles (sEVs) may serve as an EBL biomarker in cattle; however, its alteration across the clinical stages of BLV infection to the onset of EBL remains unclear. This study evaluated the prognostic potential of hsa-miR-424-5p in milk sEVs across distinct clinical stages of BLV infection to EBL onset. qPCR analysis showed that the abundance of hsa-miR-424-5p was lower in milk sEVs from BLV-infected cattle with low and high proviral load (LPVL and HPVL) compared to BLV-uninfected healthy cattle (Control), but was not significant (p > 0.05). In contrast, the abundance of hsa-miR-424-5p in milk sEVs was significantly higher in EBL cattle than in the Control and HPVL cattle (p < 0.05). Spearman’s correlation analysis revealed a significant inverse association between hsa-miR-424-5p and the percentage of lactate dehydrogenase 2 + 3 (r = −0.28, p < 0.001), whereas no significant correlations were observed with other parameters. hsa-miR-424-5p showed the highest discriminative performance in distinguishing BLV-infected cattle from EBL cattle (area under the curve [AUC] = 0.71), followed by Control vs. BLV (AUC = 0.63), indicating statistically meaningful biomarker utility across disease stages. These findings suggest that the amount of hsa-miR-424-5p in milk sEVs increases during the progression from the late stage of BLV infection to EBL onset, demonstrating that hsa-miR-424-5p may serve as a part of the biomarker panel for monitoring BLV progression and predicting EBL onset.

The dynamic balance between formation and disaggregation of amyloid fibrils is associated with many neurodegenerative diseases. Multiple chaperones interact with and disaggregate amyloid fibrils, which impacts amyloid propagation and cellular phenotypes. However, it remains poorly understood whether and how site-specific binding of chaperones to amyloids facilitates the concerted disaggregation process and modulates physiological consequences in vivo. Here, we identified binding sites of Ssa1, Sis1, and Hsp104 chaperones for Sup35, the protein determinant of yeast prion [ PSI ⁺ ] yeast. Our biophysical and genetic analyses with various Sup35 deletion mutants and amyloid conformations revealed that the Ssa1-binding to the region outside amyloid core plays a key role in facilitating disaggregation and propagation of yeast prions both in vitro and in vivo. Furthermore, we developed a reconstitution system, including the Ssa1-binding tag and the HAP/Caseinolytic protease P (ClpP) hybrid chaperones, and found that this reconstitution system successfully degraded distinct prion strain conformations. Together, these results show that the properly positioned, exposed Ssa1-binding region in amyloid fibrils influences the efficiency of amyloid disaggregation and propagation, and eventually prion strain phenotypes. More broadly, our findings provide molecular foundations for previous, puzzling observations of prion propagation in vivo, and offer insights into elimination of amyloid deposits in cells.

Structural features of N,N-dimethylaniline derivatives as potent ferroptosis inhibitors:lysosomal localization and stabilization of ferrous ion complexation.

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 5 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.

Enzootic bovine leukosis (EBL) is a B-cell lymphosarcoma caused by the bovine leukemia virus (BLV). While most infected cattle show no clinical signs, approximately 30% of infected cattle develop persistent lymphocytosis (PL), and a small percentage may develop EBL. Currently, there is no method for predicting the possibility of EBL onset. In this study, we analyzed the microRNAs (miRNAs) encapsulated in small extracellular vesicles (sEVs) in the blood to explore the biomarkers of EBL. To identify candidate biomarkers, blood samples were collected from three BLV-uninfected and three EBL cattle. Total RNA was extracted from filtered serum and used for microarray analysis. Due to their association with cancer in human orthologs, we selected three miRNAs as candidate biomarkers, bta-miR-17-5p, bta-miR-24-3p, and bta-miR-210, which were more than twice as abundant in EBL cattle than in BLV-uninfected cattle. Quantitative real-time polymerase chain reaction (qPCR) using serum RNAs from six cattle used for the microarray analysis was carried out for the detection of the three selected miRNAs. Additionally, bta-miR-92a, whose ortholog has been associated with cancer in humans, was also examined by qPCR. bta-miR-17-5p, bta-miR-24-3p, and bta-miR-92a, were successfully detected, but bta-miR-210 was not. To further evaluate the utility of these three miRNAs as biomarkers, new blood samples were collected from 31 BLV-uninfected and 30 EBL cattle. The levels of bta-miR-17-5p, bta-miR-24-3p, and bta-miR-92a, were significantly higher in EBL cattle than in BLV-uninfected cattle. These results suggest that increased levels of bta-miR-17-5p, bta-miR-24-3p, and bta-miR-92a in the blood could be used as biomarkers for EBL. This study may contribute to the control of BLV infections and develop a prediction method of EBL onset.

This study aimed to identify a suitable RNA extraction kit and stable internal control microRNA (miRNA) in bovine milk small extracellular vesicles (sEVs) for a quantitative polymerase chain reaction (qPCR) analysis. Two RNA extraction kits, miRNeasy Micro Kit, and Maxwell RSC miRNA Tissue Kit, were compared and evaluated using bovine milk sEVs via qPCR analysis. Five miRNAs, bta-miR-29a, bta-miR-200a, bta-miR-26b, hsa-miR-27b-3p, and hsa-miR-30b-5p, were selected by microarray analyses, and their cycle threshold (Ct) values were further evaluated mathematically using geNorm, NormFinder, BestKeeper, and ∆Ct algorithms. The results revealed that both the miRNeasy Micro Kit and Maxwell RSC miRNA Tissue Kit are useful for the efficient recovery of RNA from bovine milk sEVs. According to the final stability ranking analyzed by RefFinder, hsa-miR-27b-3p and bta-miR-29a can be used as suitable internal control miRNAs in bovine milk sEVs. The study also indicated that using a suitable internal control miRNA may improve the reliability and accuracy of the qPCR analysis for normalization in bovine milk sEVs. To the best of our knowledge, this is the first study to uncover the suitable internal control miRNAs in bovine milk sEVs.

Canine degenerative myelopathy (DM) is a human amyotrophic lateral sclerosis (ALS)-like neurodegenerative disease. It is a unique, naturally occurring animal model of human ALS. Canine DM is associated with the aggregation of canine superoxide dismutase 1 (cSOD1), which is similar to human ALS. Almost 100% of cases in dogs are familial, and the E40K mutation in cSOD1 is a major causative mutation of DM. Therefore, it is important to understand the molecular mechanisms underlying cSOD1(E40K) aggregation. To address this, we first analyzed the structural model of wild type cSOD1. Interactions were evident between amino acid E40 and K91. Therefore, the mutation at residue E40 causes loss of the interaction and may destabilize the native structure of cSOD1. Differential scanning fluorimetry revealed that the E40K mutant was less stable than the wild type. Moreover, stability could be recovered by the E40K and K91E double mutation. Acceleration of amyloid fibril formation in vitro and aggregate formation in cells of cSOD1(E40K) was also suppressed by the introduction of this double mutation in thioflavin T fluorescence assay results and in transfectant cells, respectively. These results clearly show the importance of the interaction between amino acid residues E40 and K91 in cSOD1 for the stability of the native structure and aggregation.

Enzootic bovine leukosis (EBL) is a B-cell lymphosarcoma caused by the bovine leukemia virus (BLV). Most BLV-infected cattle show no clinical signs and only some develop EBL. The pathogenesis of EBL remains unclear and there are no methods for predicting EBL before its onset. Previously, it was reported that miRNA profiles in milk small extracellular vesicles (sEVs) were affected in cattle in the late stage of BLV infection. It raised a possibility that miRNA profile in milk sEVs from EBL cattle could be also affected. To characterize the difference in milk of EBL cattle and healthy cattle, we examined the miRNA profiles in milk sEVs from four EBL and BLV-uninfected cattle each using microarray analysis. Among the detected miRNAs, three miRNAs—bta-miR-1246, hsa-miR-1290, and hsa-miR-424-5p—which were detectable using quantitative real-time PCR (qPCR) and are associated with cancers in humans—were selected as biomarker candidates for EBL. To evaluate the utility of these miRNAs as biomarkers for EBL, their levels were measured using milk that was freshly collected from 13 EBL and seven BLV-uninfected cattle. bta-miR-1246 and hsa-miR-424-5p, but not hsa-miR-1290, were detected using qPCR and their levels in milk sEVs from EBL cattle were significantly higher than those in BLV-uninfected cattle. bta-miR-1246 and hsa-miR-424-5p in sEVs may promote metastasis by targeting tumor suppressor genes, resulting in increased amounts in milk sEVs in EBL cattle. These results suggest that bta-miR-1246 and hsa-miR-424-5p levels in milk sEVs could serve as biomarkers for EBL.