Ultrasonication of Milk Decreases the Content of Exosomes and MicroRNAs in an Exosome-Defined Rodent Diet.

BACKGROUND: Bovine milk exosomes (BMEs) harbor regulatory proteins, lipids, and microRNAs. Consumption of an exosome- and RNA-depleted (ERD) diet elicited phenotypes compared with controls fed an exosome- and RNA-sufficient (ERS) diet in mice. All other ingredients were identical in the diets. ERD and ERS diets were prepared by substituting ultrasonicated and nonultrasonicated milk, respectively, for casein in the AIN-93G formulation. OBJECTIVES: The objective of this study was to assess the effect of ultrasonication of milk on exosome content and bioavailability, and cargo content. METHODS: Bovine milk was ultrasonicated and exosomes were isolated by ultracentrifugation [ultrasonicated exosomes (USEs)]; controls were not ultrasonicated [nonultrasonicated exosomes (NSEs)]. Exosome count, size, and morphology were assessed using a nanoparticle tracker and electron microscopy. RNAs, lipids, and proteins were analyzed by RNA sequencing and MS. Intestinal transport, bioavailability, and distribution were measured by using fluorophore-labeled USEs and NSEs in Caco-2 cells, FHs 74 Int cells, and C57BL/6J mice (n = 3; age: 6-8 wk). RESULTS: The exosome count was 76% ± 22% lower in USEs than in NSEs (P < 0.05). Ultrasonication caused a degradation of ≤100% of microRNAs. USEs and NSEs contained 145 and 332 unique lipid signatures, respectively (P < 0.05). We detected a total of 525 and 484 proteins in USEs and NSEs, respectively. The uptake of USEs decreased by 46% ± 30% and 40% ± 27% compared with NSEs in Caco-2 and FHs 74 Int cells, respectively (P < 0.05). The hepatic accumulation of USEs was 48% ± 28% lower than the accumulation of NSEs in mice (P < 0.05). CONCLUSIONS: Ultrasonication of milk depletes bioavailable BMEs in studies of Caco-2 cells, FHs 74 Int cells, and C57BL/6J mice and causes a near-complete degradation of microRNA cargos.

Isolation of extracellular vesicles from byproducts of cheesemaking by tangential flow filtration yields heterogeneous fractions of nanoparticles.

Extracellular vesicles (EV) in milk, particularly exosomes, have attracted considerable attention as bioactive food compounds and for their use in drug delivery. The utility of small EV in milk (sMEV) as an animal feed additive and in drug delivery would be enhanced by cost-effective large-scale protocols for the enrichment of sMEV from byproducts in dairy plants. Here, we tested the hypothesis that sMEV may be enriched from byproducts of cheesemaking by tangential flow filtration (EV-FF) and that the sMEV have properties similar to sMEV prepared by ultracentrifugation (sMEV-UC). Three fractions of EV were purified from the whey fraction of cottage cheese making by using EV-FF that passed through a membrane with a 50-kDa cutoff (50 penetrate; 50P), and subfractions of 50P that were retained (100 retentate; 100R) or passed through (100 penetrate; 100P) a membrane with a 100-kDa cutoff; sMEV-UC controls were prepared by serial ultracentrifugation. The abundance of sMEV (<200 nm) was less than 0.3% in EV-FF compared with sMEV-UC (1012/mL of milk). Despite the low EV count, the protein content (mg/mL) of 100R (63 ± 0.02; ± standard deviation) was higher than that of 50P (0.75 ± 0.10), 100P (0.65 ± 0.40), and sMEV-UC (27 ± 0.02). There were 17, 14, 35, and 75 distinct proteins detected by nontargeted mass spectrometry analysis in 50P, 100R, 100P, and sMEV-UC, respectively. Exosome markers CD9, CD63, CD81, HSP-70, PDCD6IP, and TSG101 were detected in control sMEV-UC but not in EV-FF by using targeted mass spectrometry and immunoblot analyses. Negative exosome markers, APOB, ß-integrin, and histone H3 were below the limit of detection in EV-FF and control sMEV-UC analyzed by immunoblotting. The abundance of the major milk fat globule protein butyrophilin showed the following pattern: 100R ≫ 100P = 50P > sMEV-UC. More than 100 mature microRNA were detected in sMEV-UC by using sequencing analysis, compared with 36 to 60 microRNA in EV-FF. Only 100R and sMEV-UC yielded mRNA in quantities and qualities sufficient for sequencing analysis; an average of 276,000 and 838,000 reads were mapped to approximately 14,600 and 18,500 genes in 100R and sMEV-UC, respectively. In principal component analysis, microRNA, mRNA, and protein in EV-FF preparations clustered separately from control sMEV-UC. We conclude that under the conditions used here, flow filtration yields a heterogeneous population of milk EV.

Desulfuromonas carbonis sp. nov., an Fe(III)-, S0- and Mn(IV)-reducing bacterium isolated from an active coalbed methane gas well.

A novel, mesophilic, obligately anaerobic, acetate-oxidizing, dissimilatory iron-, sulfur-, and manganese-reducing bacterium, designated strain ICBM(T), was obtained from an active, coalbed methane gas well in Indiana, USA. Strain ICBM(T) was a Gram-stain-negative, non-spore-forming, rod-shaped, non-motile bacterium that was rich in c-type cytochromes and formed red colonies in solid medium. Strain ICBM(T) conserved energy to support growth from the oxidation of acetate, propionate, pyruvate, malate, fumarate, succinate and dl-lactate, concomitant with dissimilatory iron reduction. Strain ICBM(T) fermented fumarate yielding succinate and acetate. Strain ICBM(T) was able to grow in the temperature range of 10 °C to 37 °C, NaCl concentration range of 0 to 1.2 M, and pH range of 6.5 to 8.0. The physiological characteristics of strain ICBM(T) indicated that it belongs to the Desulfuromonas cluster. The G+C content of its genomic DNA was 61.2 mol%. The predominant cellular fatty acids were C16 : 0 (39.3%), C16 : 1ω7c and/or iso-C15 : 0 2-OH (36.6%). The closest cultured phylogenetic relative of strain ICBM(T) was Desulfuromonas michiganensis BB1(T) with only 95% 16S rRNA gene sequence similarity. This confirmed that strain ICBM(T) is affiliated with the genus Desulfuromonas . On the basis of phenotypic and genotypic differences between strain ICBM(T) and other taxa of the genus Desulfuromonas , strain ICBM(T) represents a novel species for which the name Desulfuromonas carbonis sp. nov. is proposed (type strain ICBM(T) = DSM 29759(T) = JCM 30471(T)). Strain ICBM(T) is the first Fe(III)-, S(0)-, and Mn(IV)-reducing bacterium that was isolated from a coal bed.

Desulfocarbo indianensis gen. nov., sp. nov., a benzoate-oxidizing, sulfate-reducing bacterium isolated from water extracted from a coal bed.

A novel, strictly anaerobic, sulfate-reducing bacterium, designated strain SCBM(T), was isolated from water extracted from a coal bed in Indiana, USA. The isolate was characterized by a polyphasic taxonomic approach that included phenotypic and genotypic characterizations. Cells of strain SCBM(T) were vibrio-shaped, polarly flagellated, Gram-negative, motile, oxidase-negative and weakly catalase-positive. Growth of strain SCBM(T) was observed at NaCl concentrations ranging from 0 to 300 mM. However, no growth was observed when 1 M or more NaCl was present. Growth was observed at 16-37 °C, with optimal growth at 30 °C. The optimum pH for growth was 7, although growth was observed from pH 6.5 to 8. The doubling time under optimal growth conditions (30 °C, pH 7, 2.5 mM benzoate, 14 mM sulfate) was 2.7 days. Bicarbonate, HEPES, PIPES and MES were effective buffers for growth of strain SCBM(T), but citrate inhibited growth. When sulfate was provided as the electron acceptor, strain SCBM(T) grew autotrophically with hydrogen as the electron donor and heterotrophically on benzoate, formate, acetate, pyruvate, butyrate, fumarate, succinate and palmitate. None of the substrates tested supported fermentative growth. Thiosulfate and sulfate were used as electron acceptors coupled to benzoate oxidation, but sulfite, elemental sulfur, DMSO, anthraquinone 2,6-disulfonate, nitrate, nitrite, ferric citrate, hydrous iron oxide and oxygen were not. The G+C content of genomic DNA was 62.5 mol%. The major cellular fatty acids were anteiso-C(15 : 0) and C(18 : 1)ω7c. Phylogenetic analysis based on 16S rRNA gene sequencing placed strain SCBM(T) into a distinct lineage within the class Deltaproteobacteria. The closest, cultivated phylogenetic relative of strain SCBM(T) was Desulfarculus baarsii DSM 2075(T), with only 91.7% 16S rRNA gene sequence identity. On the basis of phenotypic and genotypic analyses, strain SCBM(T) represents a novel genus and species of sulfate-reducing bacteria, for which the name Desulfocarbo indianensis gen. nov., sp. nov. is proposed. The type strain of Desulfocarbo indianensis is SCBM(T) ( = DSM 28127(T) = JCM 19826(T)). Desulfocarbo is the second genus of the order Desulfarculales.

Identification of anti-inflammatory vesicle-like nanoparticles in honey.

Honey has been used as a nutrient, an ointment, and a medicine worldwide for many centuries. Modern research has demonstrated that honey has many medicinal properties, reflected in its anti-microbial, anti-oxidant, and anti-inflammatory bioactivities. Honey is composed of sugars, water and a myriad of minor components, including minerals, vitamins, proteins and polyphenols. Here, we report a new bioactive component‒vesicle-like nanoparticles‒in honey (H-VLNs). These H-VLNs are membrane-bound nano-scale particles that contain lipids, proteins and small-sized RNAs. The presence of plant-originated plasma transmembrane proteins and plasma membrane-associated proteins suggests the potential vesicle-like nature of these particles. H-VLNs impede the formation and activation of the nucleotide-binding domain and leucine-rich repeat related (NLR) family, pyrin domain containing 3 (NLRP3) inflammasome, which is a crucial inflammatory signalling platform in the innate immune system. Intraperitoneal administration of H-VLNs in mice alleviates inflammation and liver damage in the experimentally induced acute liver injury. miR-4057 in H-VLNs was identified in inhibiting NLRP3 inflammasome activation. Together, our studies have identified anti-inflammatory VLNs as a new bioactive agent in honey.

Draft Genome Sequence of Desulfocarbo indianensis SCBM, a New Genus of Sulfate-Reducing Bacteria, Isolated from Water Extracted from an Active Coalbed Methane Gas Well.

We used Illumina MiSeq technology to sequence the whole genome of Desulfocarbo indianensis SCBM, a new genus of sulfate-reducing bacteria isolated from a coal bed in Indiana, USA. This draft genome represents the first sequenced genome of the genus Desulfocarbo and the second known genome of the order Desulfarculales.