Particle size distribution of bovine milk-derived exosomes isolated via ExoQuick precipitation.

ExoQuick (A) and Differential Ultracentrifugation (B) as determined by Nanoparticle Tracking Analysis (NTA). Group (G)1: whole milk frozen immediately upon collection and processed post-thaw. G2: Whole milk processed to remove fat globules and cream prior to ultracold storage. G3: Whole milk processed to remove fat globules, cream, milk cells, and casein proteins prior to ultracold storage. Mean particle size is based on Stokes-Einstein equation with a 1:700 dilution in 1X-filtered PBS, 2 independent runs with 3 technical replicates of 30 s capture/run. (TIF)</p

RNA yield [ng/μL], purity and quality of human milk-derived exosome pellets and supernatants isolated via ExoQuick (EQ) precipitation and differential ultracentrifugation methods (DUC).

RNA was extracted using four protocols, 1) QIAzol + miRNeasy MiniKit (Q), 2) TRIzol LS (TLS), 3) TRIzol + RNA Clean and Concentrator Kit (Tri+RCC), and 4) TRIzol LS + RNA Clean and Concentrator Kit (TLS+RCC). RNA concentration [ng/μL] (A), RNA purity—absorbance at 260nm/280nm (B), and absorbance at 260nm/230nm (C), and 1% TAE agarose gel electrophoresis (D) of the RNA samples. Data are mean ± SEM with n = 3 independent trials/group. @ Main effect of RNA extraction protocol (p ≤ 0.05). * Significant difference in RNA concentration and purity between the pellets and supernatants (p ≤ 0.05).</p

Morphology of bovine milk-derived exosomes isolated via the ExoQuick (EQ) method and visualized by Transmission Electron Microscopy (TEM).

Group (G)1: whole milk frozen immediately upon collection and processed post-thaw. G2: Whole milk processed to remove fat globules and cream prior to ultracold storage. G3: Whole milk processed to remove fat globules, cream, milk cells, and casein proteins prior to ultracold storage. Scale bars: 200 nm—500 nm.</p

Data_Sheet_1_Effects of Supplemental Leucine on Growth, Nutrient Use, and Muscle and Visceral Tissue Mass in Holstein Bull Calves Fed Milk Replacer.docx

To determine the effects of leucine supplementation on body weight (BW), tissue mass, nutrient digestibility, the concentration of serum amino acids (AAs) and metabolites, and protein abundance of elongation initiation factor 4E (eIF4E) in skeletal muscle, 23 Holstein bull calves (43. 3 ± 1.16 kg; 11.3 ± 0.57 days of age) fed milk replacer at 2.5% of body weight (BW; dry matter basis) were used in a randomized complete block design. Leucine was supplemented at 0, 0.4, 0.6, or 0.8 g Leu/kg BW per day for 28 d. Data were analyzed using the MIXED procedure of SAS. Leucine supplementation did not affect calf BW (P = 0.73), and digestibility of nitrogen (P = 0.21), organic matter (P = 0.28), and dry matter (P = 0.28). Masses proportional to BW of the pancreas (P = 0.04), omasum (P < 0.01), and spleen (P = 0.01) were quadratically affected by treatment where tissue mass decreased at 0.4 g Leu/kg BW and increased at 0.6 and 0.8 g Leu/kg BW. Semitendinosus mass proportional to BW tended (P = 0.07) to be quadratically affected, as tissue mass increased at 0.4 g Leu/kg BW, and decreased at 0.6 and 0.8 g Leu/kg BW. Serum Leu concentration increased linearly (P = 0.002; day × time × treatment) across days and after feedings with increased supplemental Leu. Increasing supplemental Leu linearly decreased serum Ala (P < 0.01), Arg (P = 0.04), Ile (P = 0.02), Met (P < 0.01), and Pro (P = 0.05) concentrations, and quadratically affected serum Glu (P = 0.04) and Lys (P = 0.03) concentrations where serum Glu and Lys concentrations were decreased at 0.4 g Leu/kg BW and increased at 0.6 and 0.8 g Leu/kg BW. There was no effect of treatment on protein abundance of eIF4E in semitendinosus or longissimus dorsi. These data indicate that supplemental Leu did not influence ADG and nitrogen retention in calves fed milk replacer. However, changes in serum AA concentrations and tissue masses proportional to BW suggest that supplementation of Leu at lower levels could increase the use of AA for non-visceral tissue growth.</p

CD9 expression in bovine milk-derived exosomes isolated with differential ultracentrifugation protocol as determined by western immunoblotting.

CD9 is presented as a positive, exosome marker and is approximately 28 kDa in size. Lane 1: 1kB Pink Plus pre-stained protein ladder (range 10.5–175 kDa). Lane 2: Total soluble protein from human microglia (ATCC: HMC3 Cell line), used as the cellular control. Group 1: Frozen whole milk prior to processing. Group 2: Frozen milk without fat globules and cream. Group 3: Frozen whey fraction without fat globules, cream and casein proteins. (PDF)</p

Calnexin expression in bovine milk-derived exosomes isolated with differential ultracentrifugation protocol as determined by western immunoblotting.

Calnexin is presented as a negative, cellular control and is approximately 68 kDa in size. Lane 1: 1kB Pink Plus pre-stained protein ladder (range 10.5–175 kDa). Lane 2: Total soluble protein from human microglia (ATCC: HMC3 Cell line), used as the cellular control. Group 1: Frozen whole milk prior to processing. Group 2: Frozen milk without fat globules and cream. Group 3: Frozen whey fraction without fat globules, cream, and casein proteins. (PDF)</p

Four RNA extraction protocols.

1) QIAzol + miRNeasy MiniKit (Q), 2) TRIzol LS (TLS), 3) TRIzol + RNA Clean and Concentrator Kit (Tri+RCC), and 4) TRIzol LS + RNA Clean and Concentrator Kit (TLS+RCC) used for the isolation of total RNA from bovine and human milk exosomes.</p

Calnexin expression in human milk-derived exosomes as determined by western immunoblotting.

Calnexin is presented as a negative, cellular control and is approximately 68 kDa in size. Lane 1: 1kB Pink Plus pre-stained protein ladder (range 10.5–175 kDa). Lane 2: Total soluble protein from human microglia (ATCC: HMC3 Cell line), used as the cellular control. Lane 3–4: Milk exosomes isolated with ExoQuick protocol. Lane 5–6: milk exosomes isolated with the ultracentrifugation protocol. (PDF)</p

Pre-processing of bovine colostrum prior to long-term storage at -80°C.

Group (G)1: whole milk frozen immediately upon collection and processed post-thaw. G2: Whole milk processed to remove fat globules and cream prior to ultracold storage. G3: Whole milk processed to remove fat globules, cream, milk cells, and casein proteins prior to ultracold storage.</p

Morphology of human milk-derived exosomes visualized by Transmission Electron Microscopy (TEM) with negative staining (uranyl acetate).

Human milk-derived exosomes were isolated via ExoQuick (EQ) precipitation and differential ultracentrifugation (DUC) methods. Scale bars: 200 nm.</p