Storage of Extracellular Vesicles in Human Milk, and MicroRNA Profiles in Human Milk Exosomes and Infant Formulas.

The objectives of this study were to lay the methodological groundwork for field studies of microRNA analysis in exosomes from small sample volumes of human milk, and assess exosome and microRNA content in infant formulas. When human milk was stored at 4°C for 4 weeks, the count of exosome-sized vesicles decreased progressively to 49% ±â€Š13% of that in fresh milk. Exosomes were purified from 1 mL of fresh human milk and their microRNA content was assessed by microRNA-sequencing analysis and compared with that in infant formulas. We identified 221 microRNAs in exosomes from 3 samples of fresh human milk; 84 microRNAs were present in all 3 samples. MicroRNAs were not detectable in infant formulas and their exosome-sized vesicles, which appeared to be casein micelles. We conclude that large-scale studies of microRNAs in human milk exosomes are feasible, and exosomes and microRNAs are not detectable in formulas.

Phenethyl Isothiocyanate Exposure Promotes Oxidative Stress and Suppresses Sp1 Transcription Factor in Cancer Stem Cells.

Aldehyde dehydrogenase 1 (ALDH1) is a cytosolic marker of cancer stem cells (CSCs), which are a sub-population within heterogeneous tumor cells. CSCs associate with therapy-resistance, self-renewal, malignancy, tumor-relapse, and reduced patient-survival window. ALDH1-mediated aldehyde scavenging helps CSCs to survive a higher level of oxidative stress than regular cancer cells. Cruciferous vegetable-derived phenethyl isothiocyanate (PEITC) selectively induces reactive oxygen species (ROS), leading to apoptosis of cancer cells, but not healthy cells. However, this pro-oxidant role of PEITC in CSCs is poorly understood and is investigated here. In a HeLa CSCs model (hCSCs), the sphere-culture and tumorsphere assay showed significantly enriched ALDHhi CSCs from HeLa parental cells (p < 0.05). Aldefluor assay and cell proliferation assay revealed that PEITC treatments resulted in a reduced number of ALDHhi hCSCs in a concentration-dependent manner (p < 0.05). In the ROS assay, PEITC promoted oxidative stress in hCSCs (p ≤ 0.001). Using immunoblotting and flow cytometry techniques, we reported that PEITC suppressed the cancer-associated transcription factor (Sp1) and a downstream multidrug resistance protein (P-glycoprotein) (both, p < 0.05). Furthermore, PEITC-treatment of hCSCs, prior to xenotransplantation in mice, lowered the in vivo tumor-initiating potential of hCSCs. In summary, PEITC treatment suppressed the proliferation of ALDH1 expressing cancer stem cells as well as key factors that are involved with drug-resistance, while promoting oxidative stress and apoptosis in hCSCs.

Milk exosomes are bioavailable and distinct microRNA cargos have unique tissue distribution patterns.

Exosomes participate in cell-to-cell communication, facilitated by the transfer of RNAs, proteins and lipids from donor to recipient cells. Exosomes and their RNA cargos do not exclusively originate from endogenous synthesis but may also be obtained from dietary sources such as the inter-species transfer of exosomes and RNAs in bovine milk to humans. Here, we assessed the bioavailability and distribution of exosomes and their microRNA cargos from bovine, porcine and murine milk within and across species boundaries. Milk exosomes labeled with fluorophores or fluorescent fusion proteins accumulated in liver, spleen and brain following suckling, oral gavage and intravenous administration in mice and pigs. When synthetic, fluorophore-labeled microRNAs were transfected into bovine milk exosomes and administered to mice, distinct species of microRNAs demonstrated unique distribution profiles and accumulated in intestinal mucosa, spleen, liver, heart or brain. Administration of bovine milk exosomes failed to rescue Drosha homozygous knockout mice, presumably due to low bioavailability or lack of essential microRNAs.

Prenatal Exposure to a Maternal High-Fat Diet Affects Histone Modification of Cardiometabolic Genes in Newborn Rats.

Infants born to women with diabetes or obesity are exposed to excess circulating fuels during fetal heart development and are at higher risk of cardiac diseases. We have previously shown that late-gestation diabetes, especially in conjunction with a maternal high-fat (HF) diet, impairs cardiac functions in rat-offspring. This study investigated changes in genome-wide histone modifications in newborn hearts from rat-pups exposed to maternal diabetes and HF-diet. Chromatin-immunoprecipitation-sequencing revealed a differential peak distribution on gene promoters in exposed pups with respect to acetylation of lysines 9 and 14 and to trimethylation of lysines 4 and 27 in histone H3 (all, false discovery rate, FDR < 0.1). In the HF-diet exposed offspring, 54% of the annotated genes showed the gene-activating mark trimethylated lysine 4. Many of these genes (1) are associated with the "metabolic process" in general and particularly with "positive regulation of cholesterol biosynthesis" (FDR = 0.03); (2) overlap with 455 quantitative trait loci for blood pressure, body weight, serum cholesterol (all, FDR < 0.1); and (3) are linked to cardiac disease susceptibility/progression, based on disease ontology analyses and scientific literature. These results indicate that maternal HF-diet changes the cardiac histone signature in offspring suggesting a fuel-mediated epigenetic reprogramming of cardiac tissue in utero.

Dietary resistant starch type 4-derived butyrate attenuates nuclear factor-kappa-B1 through modulation of histone H3 trimethylation at lysine 27.

Indigestible resistant starches (RS) are substrates for gut-microbial metabolism and have been shown to attenuate intestinal inflammation but the supporting evidence is inconsistent and lacks mechanistic explanation. We have recently reported dietary RS type 4 (RS4) induced improvements in immunometabolic functions in humans and a concomitant increase in butyrogenic gut-bacteria. Since inflammation is a key component in metabolic diseases, here we investigated the effects of RS4-derived butyrate on the epigenetic repression of pro-inflammatory genes in vivo and in vitro. RS4-fed mice, compared to the control-diet group, had higher cecal butyrate and increased tri-methylation of lysine 27 on histone 3 (H3K27me3) in the promoter of nuclear factor-kappa-B1 (NFκB1) in the colon tissue. The H3K27me3-enrichment inversely correlated with the concentration dependent down-regulation of NFκB1 in sodium butyrate treated human colon epithelial cells. Two additional inflammatory genes were attenuated by sodium butyrate, but were not linked with H3K27me3 changes. This exploratory study presents a new opportunity for studying underlying H3K27me3 and other methylation modifying mechanisms linked to RS4 biological activity.

Impact of dietary resistant starch type 4 on human gut microbiota and immunometabolic functions.

Dietary modulation of the gut microbiota impacts human health. Here we investigated the hitherto unknown effects of resistant starch type 4 (RS4) enriched diet on gut microbiota composition and short-chain fatty acid (SCFA) concentrations in parallel with host immunometabolic functions in twenty individuals with signs of metabolic syndrome (MetS). Cholesterols, fasting glucose, glycosylated haemoglobin, and proinflammatory markers in the blood as well as waist circumference and % body fat were lower post intervention in the RS4 group compared with the control group. 16S-rRNA gene sequencing revealed a differential abundance of 71 bacterial operational taxonomic units, including the enrichment of three Bacteroides species and one each of Parabacteroides, Oscillospira, Blautia, Ruminococcus, Eubacterium, and Christensenella species in the RS4 group. Gas chromatography-mass spectrometry revealed higher faecal SCFAs, including butyrate, propionate, valerate, isovalerate, and hexanoate after RS4-intake. Bivariate analyses showed RS4-specific associations of the gut microbiota with the host metabolic functions and SCFA levels. Here we show that dietary RS4 induced changes in the gut microbiota are linked to its biological activity in individuals with signs of MetS. These findings have potential implications for dietary guidelines in metabolic health management.

Expression and distribution of thiol-regulating enzyme glutaredoxin 2 (GRX2) in porcine ocular tissues.

Glutaredoxin2 (Grx2) is a mitochondrial isozyme of the cytosolic glutaredoxin1 (thioltransferase or TTase). Both belong to the large oxidoreductase family and play an important role in maintaining thiol/disulfide redox homeostasis in the cells. Grx2 is recently found in the lens where its activities of disulfide reductase and peroxidase, similar to TTase, can protect the lens against oxidative stress. Since other eye tissues are also highly sensitive to oxidative stress, and TTase's distribution in the eye is known, we focused on this study by investigating the Grx2 distribution in the ocular tissues in comparison to the lens. Fresh porcine eyes were dissected into cornea, iris, ciliary body, the lens, vitreous humor, retina, and optic nerve. Each tissue (pooled from three eyes) was homogenized and processed for mitochondrial isolation. The mitochondrial fraction was analyzed for Grx2 protein using Western blotting with anti-Grx2 antibody, and Grx2 activity using the published procedure. The eye tissues were also measured for Grx2 mRNA expression by RT-PCR with GAPDH as the control. Grx2-rich mouse liver and purified recombinant mouse Grx2 were used as positive controls for the above analyses. It was found that Grx2 was present in all the tested ocular tissues, except vitreous humor. In comparison with the mouse liver, the protein levels of Grx2 in porcine ciliary body and the lens were 27-fold and 0.75-fold, respectively. Comparing to the lens, Grx2 protein was highest in the ciliary body (13.5-fold), followed by retina (9.2-fold), iris and optic nerve (2-fold), and cornea (1.2-fold). Enzyme activity assays showed that the retina had the highest Grx2 specific activity (3.9 mU/mg protein), followed by ciliary body (3.1 mU/mg), the lens (0.58 mU/mg), and optic nerve (0.32 mU/mg). Grx2 gene expression in these ocular tissues was further confirmed by RT-PCR analysis. Grx2 mRNA expression showed the highest in ciliary body, followed by retina, optic nerve, cornea, iris, and the lens. No Grx2 mRNA, protein or enzyme activity could be found in the vitreous humor. The results indicate that Grx2 level was higher in eye tissues rich in vasculature and mitochondria (i.e. ciliary body and retina), corroborating with the levels of mRNA expression and Grx2 activity. The rich presence of Grx2 in these tissues is also consistent with their known sensitivity to oxidative stress.

Phenethyl isothiocyanate upregulates death receptors 4 and 5 and inhibits proliferation in human cancer stem-like cells.

BACKGROUND: The cytokine TRAIL (tumor necrotic factor-related apoptosis-inducing ligand) selectively induces apoptosis in cancer cells, but cancer stem cells (CSCs) that contribute to cancer-recurrence are frequently TRAIL-resistant. Here we examined hitherto unknown effects of the dietary anti-carcinogenic compound phenethyl isothiocyanate (PEITC) on attenuation of proliferation and tumorigenicity and on up regulation of death receptors and apoptosis in human cervical CSC. METHODS: Cancer stem-like cells were enriched from human cervical HeLa cell line by sphere-culture method and were characterized by CSC-specific markers' analyses (flow cytometry) and Hoechst staining. Cell proliferation assays, immunoblotting, and flow cytometry were used to assess anti-proliferative as well as pro-apoptotic effects of PEITC exposure in HeLa CSCs (hCSCs). Xenotransplantation study in a non-obese diabetic, severe combined immunodeficient (NOD/SCID) mouse model, histopathology, and ELISA techniques were further utilized to validate our results in vivo. RESULTS: PEITC attenuated proliferation of CD44(high/+)/CD24(low/-), stem-like, sphere-forming subpopulations of hCSCs in a concentration- and time-dependent manner that was comparable to the CSC antagonist salinomycin. PEITC exposure-associated up-regulation of cPARP (apoptosis-associated cleaved poly [ADP-ribose] polymerase) levels and induction of DR4 and DR5 (death receptor 4 and 5) of TRAIL signaling were observed. Xenotransplantation of hCSCs into mice resulted in greater tumorigenicity than HeLa cells, which was diminished along with serum hVEGF-A (human vascular endothelial growth factor A) levels in the PEITC-pretreated hCSC group. Lung metastasis was observed only in the hCSC-injected group that did not receive PEITC-pretreatment. CONCLUSIONS: The anti-proliferative effects of PEITC in hCSCs may at least partially result from up regulation of DR4 and possibly DR5 of TRAIL-mediated apoptotic pathways. PEITC may offer a novel approach for improving therapeutic outcomes in cancer patients.

Detection of autoreactive CD4 T cells using major histocompatibility complex class II dextramers.

BACKGROUND: Tetramers are useful tools to enumerate the frequencies of antigen-specific T cells. However, unlike CD8 T cells, CD4 T cells - especially self-reactive cells - are challenging to detect with major histocompatibility complex (MHC) class II tetramers because of low frequencies and low affinities of their T cell receptors to MHC-peptide complexes. Here, we report the use of fluorescent multimers, designated MHC dextramers that contain a large number of peptide-MHC complexes per reagent. RESULTS: The utility of MHC dextramers was evaluated in three autoimmune disease models: 1) proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis in SJL/J (H-2s) mice; 2) myelin oligodendrocyte glycoprotein (MOG) 35-55-induced experimental autoimmune encephalomyelitis in C57Bl/6 (H-2b) mice; and 3) cardiac myosin heavy chain (Myhc)-α 334-352-induced experimental autoimmune myocarditis in A/J (H-2a) mice. Flow cytometrically, we demonstrate that IAs/PLP 139-151, IAb/MOG 35-55 and IAk/Myhc-α 334-352 dextramers detect the antigen-sensitized cells with specificity, and with a detection sensitivity significantly higher than that achieved with conventional tetramers. Furthermore, we show that binding of dextramers, but not tetramers, is less dependent on the activation status of cells, permitting enumeration of antigen-specific cells ex vivo. CONCLUSIONS: The data suggest that MHC dextramers are useful tools to track the generation and functionalities of self-reactive CD4 cells in various experimental systems.