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.
In recent years, there has been an increase in infectious diseases in marine mammals, including brucellosis, infections of morbillivirus, herpesvirus, and poxvirus. Several serological diagnostic ...methods, including enzyme-linked immunosorbent assays, immunofluorescence assays (ELISA), and western blotting, have been used to detect antibodies against pathogens in marine mammals. However, options for commercial secondary antibodies used to detect antibodies in marine mammals are limited; therefore, the use of proteins A, G, or chimeric protein AG may provide a suitable alternative. This study aimed to assess the use of proteins A, G, and chimeric protein AG to detect marine mammal immunoglobulins. Currently, there are no comparative studies on the use of proteins A, G, and chimeric protein AG for the detection of immunoglobulins in marine mammals. In this study, we used ten pinnipeds’ species (Baikal seal, California sea lion, harbor seal, northern fur seal, ringed seal, South American fur seal, South American sea lion, spotted seal, Steller sea lion, and walrus) and five cetacean species (beluga whale, bottlenose dolphin, harbor porpoise, killer whale, and Pacific white-sided dolphin) and compare binding ability to proteins A, G, or chimeric protein AG by ELISA. The results revealed that the immunoglobulins from pinniped and cetacean species reacted more strongly to protein A than protein G. In addition, the immunoglobulins of pinnipeds and cetaceans showed a strong binding ability to chimeric protein AG. These results suggest that proteins A, G, and chimeric protein AG would be used to help further develop serological assays.
The visible light promoted cross-dehydrogenative coupling reaction has emerged as an excellent strategy for the direct formation of C-C/C-heteroatom bonds from simple compounds. The use of renewable ...energy resources without the need for prefunctionalization of the reactant synergistically promote the synthetic pathway towards green synthesis. Although the introduction of the terminology "cross-dehydrogenative coupling (CDC)" was done by Li's group in 2004, visible light promoted CDC has attracted tremendous attention from synthetic chemists since the first report of Ir-photocatalysis by Stephenson
et al.
in 2010. The efficiency of different transition-metal salts (Ir-, Ru-, Rh-, Cu-, Pt-, Co-,
etc.
), organic molecules (eosin Y, eosin B, rose bengal, rhodamine, methylene blue, acridines,
etc.
), I
2
, and heterogeneous catalysts as photocatalysts in this transformation has been extensively investigated during this period. A number of methodologies have been also developed under visible light irradiation even in the absence of any photocatalysts. In this review, all the visible light promoted cross-dehydrogenative coupling methodologies developed over the last decade have been disclosed. Furthermore, the applicability and the mechanistic pathways of the methodologies have been also discussed.
In this review, all the visible light promoted cross-dehydrogenative coupling methodologies that have been developed over the last decade are disclosed.
Milk small extracellular vesicles (sEV) contain proteins that provide potential information of host physiology and immunology. Bovine leukemia virus (BLV) is an oncogenic virus that causes ...progressive B-cell lymphosarcoma in cattle. In this study, we aimed to explore the proteomic profile of milk sEV from BLV-infected cattle compared with those from uninfected cattle. Milk sEV were isolated from three BLV-infected and three uninfected cattle. Proteomic analysis was performed by using a comprehensive nanoLC-MS/MS method. Furthermore, gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were used to evaluate the candidates for uniquely or differentially expressed proteins in milk sEV from BLV-infected cattle. Proteomic analysis revealed a total of 1330 common proteins in milk sEV among BLV-infected cattle, whereas 118 proteins were uniquely expressed compared with those from uninfected cattle. Twenty-six proteins in milk sEV were differentially expressed proteins more than two-fold significant difference (p < 0.05) in BLV-infected cattle. GO and KEGG analyses indicated that the candidates for uniquely or differentially expressed proteins in milk sEV had been involved in diverse biological activities including metabolic processes, cellular processes, respond to stimulus, binding, catalytic activities, cancer pathways, focal adhesion, and so on. Taken together, the present findings provided a novel insight into the proteomes of milk sEV from BLV-infected cattle.
Globally, Campylobacter spp. are prominent causative agents of food-borne gastroenteritis. These pathogens are commonly detected using conventional culture methods; however, culture methods are ...unable to detect viable but nonculturable (VBNC) bacteria. Currently, the detection rate of Campylobacter spp. on chicken meat does not correlate with the seasonal peak of human campylobacteriosis. We hypothesized that this may be due to the presence of undetectable VBNC Campylobacter spp. Therefore, we previously established a quantitative PCR assay using propidium monoazide (PMA-qPCR), which can detect viable Campylobacter cells. In this study, PMA-qPCR was conducted to detect viable Campylobacter spp. in chicken meat, and the detection rates of PMA-qPCR and the culture method throughout all 4 seasons were compared. A total of 105 chicken meat samples (whole legs, breast fillets, and livers) were screened for the presence of Campylobacter spp. using both PMA-qPCR and the conventional culture method. The detection rates of the 2 methods did not differ significantly; however, the positive and negative samples were not always consistent. Detection rates in March were significantly lower compared to months with the highest detection rates. These results suggest that, to increase the detection rate of Campylobacter spp., the 2 methods should be used in parallel. In this study, PMA-qPCR could not detect VBNC Campylobacter spp. effectively in C. jejuni-spiked chicken meat. Further studies using improved viability-qPCR should be performed to describe the impact of the VBNC state of Campylobacter spp. on the detection of this bacterium in chicken meat.
•Bus travel time distribution type varies with horizon.•A cutoff horizon exists distinguishing the short-and long-term horizon.•Lognormal distribution is found to be more appropriate for short-term ...horizon.•Normal distribution is found to be more suitable for long-term horizon.•Illustrates real-time interval estimate based on horizon dependent distribution type.
Given the increasing interest in real-time bus arrival information, producing reliable estimation is essential to maximize the benefits of real-time systems. The primary objectives of this paper are to analyze the changes of bus travel time characteristics as pseudo horizon varies and how such characteristics can be applied to real-time bus arrival estimation. In this study, “horizon” refers to the distance between a real-time bus location and a bus stop, whereas “pseudo horizon” refers to the distance from a GPS point to an upstream GPS point. In contrast to existing methods that provide point estimates of bus arrival times, this study provides interval estimates that take into account the uncertainty of future bus arrival times given that early and late buses have their own respective ramifications. A methodology is developed to analyze the bus travel time distribution systematically based on different pseudo horizons since such distributions are critical to producing reliable bus arrival information. The analysis of real transit GPS data shows a significant change in bus travel time characteristics around a pseudo horizon range of 8 km. The analysis of changes in probability densities with pseudo horizons shows that bus travel time distribution converges from a rightly skewed distribution to a more symmetrical distribution from a shorter to a longer pseudo horizon. Lognormal and normal distributions are found to be the best models for before and after a cut-off horizon of 7–8 km, respectively. Instead of a single distribution, the outcomes of this study suggest a combination of probability distributions based on the estimation horizon to be used to provide better bus arrival time estimations.
A new copper‐catalyzed oxidative cyclization via CH amination between 2‐aminopyridines and methyl aryl/heteroaryl ketones has been developed under ambient air. Imidazo1,2‐apyridines containing a ...wide range of functional groups have been synthesized from basic and easily available starting materials. This simple, one‐pot reaction protocol is applicable for the direct preparation of zolimidine (a marketed antiulcer drug) on a large scale.
The metal salts ubiquitously present in biological samples cause serious ion suppression, capillary clogging and signal fluctuation in ESI/nESI. Herein, a current-limited high voltage polarity ...reversing approach was applied for the online separation of intrinsic metal ions in biological samples, resulting in the generation of protonated analytes at the nESI tip for mass analysis without interference from salt cations. Stable and durable signals (∼30-60 s) were observed for protonated proteins, peptides and metabolites in complex biological samples, including liquids, solids and viscous samples, even with very high salt concentration (100 mM NaCl), allowing comprehensive tandem MS analysis with on average
ca.
5-times higher analyte signal intensities compared to the conventional nESI analysis. Therefore this approach offers improved performance of nESI/ESI for the sensitive molecular analysis of untreated biological samples, opening new possibilities in various disciplines, including biology, medicine, chemistry, life sciences,
etc.
Current-limited high voltage polarity reversing nanoelectrospray ionization allows online separation of intrinsic metal ions in complex biological samples, resulting in the generation of protonated analytes without interference from salt cations.
Transition metals have been an indispensable component of modern catalysis and among these palladium is the top‐ranked choice of chemists as a catalyst. After the several important developments of ...palladium catalysed C−C cross‐coupling reactions, carbonylative transformations have been realised as an attractive post modification of these reactions. Carbonylative Suzuki‐Miyaura coupling reaction is among such transformations for direct incorporation of CO fragment for the preparation of biaryl or aryl/alkyl ketones via transition‐metal catalysis. Ketone functionality is basically a valuable building block having huge pharmaceutical and agrochemical applications. Moreover, carbonylative Suzuki‐Miyaura coupling is also used in the synthesis of various natural bioactive molecules through the direct joining of molecular fragments via CO bridge. In this review, the past decade developments in the carbonylative Suzuki‐Miyaura coupling reactions are documented in detail with modern approaches in transition‐metal catalysis.
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.
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