Characterization of nucleic acids from extracellular vesicle-enriched human sweat.

BACKGROUND: The human sweat is a mixture of secretions from three types of glands: eccrine, apocrine, and sebaceous. Eccrine glands open directly on the skin surface and produce high amounts of water-based fluid in response to heat, emotion, and physical activity, whereas the other glands produce oily fluids and waxy sebum. While most body fluids have been shown to contain nucleic acids, both as ribonucleoprotein complexes and associated with extracellular vesicles (EVs), these have not been investigated in sweat. In this study we aimed to explore and characterize the nucleic acids associated with sweat particles. RESULTS: We used next generation sequencing (NGS) to characterize DNA and RNA in pooled and individual samples of EV-enriched sweat collected from volunteers performing rigorous exercise. In all sequenced samples, we identified DNA originating from all human chromosomes, but only the mitochondrial chromosome was highly represented with 100% coverage. Most of the DNA mapped to unannotated regions of the human genome with some regions highly represented in all samples. Approximately 5 % of the reads were found to map to other genomes: including bacteria (83%), archaea (3%), and virus (13%), identified bacteria species were consistent with those commonly colonizing the human upper body and arm skin. Small RNA-seq from EV-enriched pooled sweat RNA resulted in 74% of the trimmed reads mapped to the human genome, with 29% corresponding to unannotated regions. Over 70% of the RNA reads mapping to an annotated region were tRNA, while misc. RNA (18,5%), protein coding RNA (5%) and miRNA (1,85%) were much less represented. RNA-seq from individually processed EV-enriched sweat collection generally resulted in fewer percentage of reads mapping to the human genome (7-45%), with 50-60% of those reads mapping to unannotated region of the genome and 30-55% being tRNAs, and lower percentage of reads being rRNA, LincRNA, misc. RNA, and protein coding RNA. CONCLUSIONS: Our data demonstrates that sweat, as all other body fluids, contains a wealth of nucleic acids, including DNA and RNA of human and microbial origin, opening a possibility to investigate sweat as a source for biomarkers for specific health parameters.

Dietary supplement of conjugated linoleic acids or polyunsaturated fatty acids suppressed the mobilization of body fat reserves in dairy cows at early lactation through different pathways.

To investigate the metabolic changes in the adipose tissue (AT) of dairy cows under milk fat depression (MFD), 30 cows were randomly allocated to a control diet, a conjugated linoleic acid (CLA)-supplemented diet, or a high-starch diet supplemented with a mixture of sunflower and fish oil (2:1; as HSO diet) from 1 to 112 d in milk. Performance of animals, milk yield, milk composition, energy balance, and blood metabolites were measured during lactation. Quantitative PCR analyses were conducted on the AT samples collected at wk 3 and 15 of lactation. The CLA and HSO diets considerably depressed milk fat yield and milk fat content at both wk 3 and 15 in the absence of significant changes in milk protein and lactose contents. In addition, the HSO diet lowered milk yield at wk 15 and decreased dry matter intake of cows from wk 3 to 15. Compared with the control, both CLA and HSO groups showed reduced body weight loss, improved energy balance, and decreased plasma concentrations of nonesterified fatty acids and ß-hydroxybutyrate at early lactation. The gene expression analyses reflected suppressed lipolysis in AT of the CLA and HSO groups compared with the control at wk 3, as suggested by the downregulation of hormone-sensitive lipase and fatty acid binding protein 4 and the upregulation of perilipin 2. In addition, the HSO diet promoted lipogenesis in AT at wk 15 through the upregulation of 1-acylglycerol-3-phosphate O-acyltransferase 2, mitochondrial glycerol-3-phosphate acyltransferase, perilipin 2, and peroxisome proliferator-activated receptor γ. The CLA diet likely regulated insulin sensitivity in AT as it upregulated the transcription of various genes involved in insulin signaling, inflammatory responses, and ceramide metabolism, including protein kinase B2, nuclear factor κ B1, toll-like receptor 4, caveolin 1, serine palmitoyltransferase long chain base subunit 1, and N-acylsphingosine amidohydrolase 1. In contrast, the HSO diet resulted in little or no change in the pathways relevant to insulin sensitivity. In conclusion, the CLA and HSO diets induced a shift in energy partitioning toward AT instead of mammary gland during lactation through the regulation of different pathways.

Ruminal Infusions of Cobalt EDTA Modify Milk Fatty Acid Composition via Decreases in Fatty Acid Desaturation and Altered Gene Expression in the Mammary Gland of Lactating Cows.

BACKGROUND: Intravenous or ruminal infusion of lithium salt of cobalt EDTA (Co-EDTA) or cobalt-acetate alters milk fat composition in cattle, but the mechanisms involved are not known. OBJECTIVE: The present study evaluated the effect of ruminal Co-EDTA infusion on milk FA composition, mammary lipid metabolism, and mammary lipogenic gene expression. METHODS: For the experiment, 4 cows in midlactation and fitted with rumen cannulae were used in a 4 × 4 Latin square with 28-d periods. Co-EDTA was administered in the rumen to supply 0, 1.5, 3.0, or 4.5 g Co/d over an 18-d interval with a 10-d washout between experimental periods. Milk production was recorded daily, and milk FA composition was determined on alternate days. Mammary tissue was biopsied on day 16, and arteriovenous differences of circulating lipid fractions and FA uptake across the mammary gland were measured on day 18. RESULTS: Co-EDTA had no effect on intake, proportions of rumen volatile FA, or milk production but caused dose-dependent changes in milk FA composition. Alterations in milk fat composition were evident within 3 d of infusion and characterized by linear or quadratic decreases (P < 0.05) in FAs containing a cis-9 double bond, an increase in 4:0 and 16:0, and linear decreases in milk 8:0, 10:0, 12:0, and 14:0 concentrations. Co-EDTA progressively decreased (P < 0.05) the stearoyl-CoA desaturase (SCD)-catalyzed desaturation of FAs in the mammary gland by up to 72% but had no effect on mammary SCD1 mRNA or SCD protein abundance. Changes in milk FA composition were accompanied by altered expression of specific genes involved in de novo FA and triacylglycerol synthesis. CONCLUSION: Ruminal infusion of Co-EDTA alters milk FA composition in cattle via a mechanism that involves decreases in the desaturation of FAs synthesized de novo or extracted from blood and alterations in mammary lipogenic gene expression, without affecting milk fat yield.

Comparative analysis of the effects of different purification methods on the yield and purity of cow milk extracellular vesicles.

Isolation of extracellular vesicles (EV) has been developing rapidly in parallel with the interest in EVs. However, commonly utilized protocols may not suit more challenging sample matrixes and could potentially yield suboptimal results. Knowing and assessing the pitfalls of isolation procedure to be used, should be involved to some extent for EV analytics. EVs in cow milk are of great interest due to their abundancy and large-scale availability as well as their cross-species bioavailability and possible use as drug carriers. However, the characteristics of milk EVs overlap with those of other milk components. This makes it difficult to isolate and study EVs individually. There exists also a lack of consensus for isolation methods. In this study, we demonstrated the differences between various differential centrifugation-based approaches for isolation of large quantities of EVs from cow milk. Samples were further purified with gradient centrifugation and size exclusion chromatography (SEC) and differences were analyzed. Quality measurements were conducted on multiple independent platforms. Particle analysis, electron microscopy and RNA analysis were used, to comprehensively characterize the isolated samples and to identify the limitations and possible sources of contamination in the EV isolation protocols. Vesicle concentration to protein ratio and RNA to protein ratios were observed to increase as samples were purified, suggesting co-isolation with major milk proteins in direct differential centrifugation protocols. We demonstrated a novel size assessment of vesicles using a particle mobility analyzer that matched the sizing using electron microscopy in contrast to commonly utilized nanoparticle tracking analysis. Based on the standards of the International Society for Extracellular Vesicles and the quick checklist of EV-Track.org for EV isolation, we emphasize the need for complete characterization and validation of the isolation protocol with all EV-related work to ensure the accuracy of results and allow further analytics and experiments.

Enrichment of bovine milk-derived extracellular vesicles using surface-functionalized cellulose nanofibers.

The isolation of extracellular vesicles (EVs) from milk, a complex mixture of colloidal structures having a comparable size to EVs, is challenging. Although ultracentrifugation (UC) has been widely used for EV isolation, this has significant limitations, including a long processing time at high g-force conditions and large sample volume requirements. We introduced a new approach based on nature nanoentities cellulose nanofibers (CNFs) and short time and low g-force centrifugation to isolate EVs from various milk fractions. The flexible and entangled network of CNFs forms nanoporous, which entraps the EVs. Further, positively charged CNFs interact with anionic EVs through an electrostatic attraction, promoting their isolation with efficiency comparable with UC. The functionality and toxicity of isolated milk EVs were tested in Caco2 cells. Overall, the newly developed approach provides straightforward isolation and biocompatibility and preserves the natural properties of the isolated EVs, enabling further applications.

Identification of extracellular nanoparticle subsets by nuclear magnetic resonance.

Exosomes are a subset of secreted lipid envelope-encapsulated extracellular vesicles (EVs) of 50-150 nm diameter that can transfer cargo from donor to acceptor cells. In the current purification protocols of exosomes, many smaller and larger nanoparticles such as lipoproteins, exomers and microvesicles are typically co-isolated as well. Particle size distribution is one important characteristics of EV samples, as it reflects the cellular origin of EVs and the purity of the isolation. However, most of the physicochemical analytical methods today cannot illustrate the smallest exosomes and other small particles like the exomers. Here, we demonstrate that diffusion ordered spectroscopy (DOSY) nuclear magnetic resonance (NMR) method enables the determination of a very broad distribution of extracellular nanoparticles, ranging from 1 to 500 nm. The range covers sizes of all particles included in EV samples after isolation. The method is non-invasive, as it does not require any labelling or other chemical modification. We investigated EVs secreted from milk as well as embryonic kidney and renal carcinoma cells. Western blot analysis and immuno-electron microscopy confirmed expression of exosomal markers such as ALIX, TSG101, CD81, CD9, and CD63 in the EV samples. In addition to the larger particles observed by nanoparticle tracking analysis (NTA) in the range of 70-500 nm, the DOSY distributions include a significant number of smaller particles in the range of 10-70 nm, which are visible also in transmission electron microscopy images but invisible in NTA. Furthermore, we demonstrate that hyperpolarized chemical exchange saturation transfer (Hyper-CEST) with 129Xe NMR indicates also the existence of smaller and larger nanoparticles in the EV samples, providing also additional support for DOSY results. The method implies also that the Xe exchange is significantly faster in the EV pool than in the lipoprotein/exomer pool.

The role of the bovine growth hormone receptor and prolactin receptor genes in milk, fat and protein production in Finnish Ayrshire dairy cattle.

We herein report new evidence that the QTL effect on chromosome 20 in Finnish Ayrshire can be explained by variation in two distinct genes, growth hormone receptor (GHR) and prolactin receptor (PRLR). In a previous study in Holstein-Friesian dairy cattle an F279Y polymorphism in the transmembrane domain of GHR was found to be associated with an effect on milk yield and composition. The result of our multimarker regression analysis suggests that in Finnish Ayrshire two QTL segregate on the chromosomal region including GHR and PRLR. By sequencing the coding sequences of GHR and PRLR and the sequence of three GHR promoters from the pooled samples of individuals of known QTL genotype, we identified two substitutions that were associated with milk production traits: the previously reported F-to-Y substitution in the transmembrane domain of GHR and an S-to-N substitution in the signal peptide of PRLR. The results provide strong evidence that the effect of PRLR S18N polymorphism is distinct from the GHR F279Y effect. In particular, the GHR F279Y has the highest influence on protein percentage and fat percentage while PRLR S18N markedly influences protein and fat yield. Furthermore, an interaction between the two loci is suggested.

Quantitative trait loci for fertility traits in Finnish Ayrshire cattle.

A whole genome scan was carried out to detect quantitative trait loci (QTL) for fertility traits in Finnish Ayrshire cattle. The mapping population consisted of 12 bulls and 493 sons. Estimated breeding values for days open, fertility treatments, maternal calf mortality and paternal non-return rate were used as phenotypic data. In a granddaughter design, 171 markers were typed on all 29 bovine autosomes. Associations between markers and traits were analysed by multiple marker regression. Multi-trait analyses were carried out with a variance component based approach for the chromosomes and trait combinations, which were observed significant in the regression method. Twenty-two chromosome-wise significant QTL were detected. Several of the detected QTL areas were overlapping with milk production QTL previously identified in the same population. Multi-trait QTL analyses were carried out to test if these effects were due to a pleiotropic QTL affecting fertility and milk yield traits or to linked QTL causing the effects. This distinction could only be made with confidence on BTA1 where a QTL affecting milk yield is linked to a pleiotropic QTL affecting days open and fertility treatments.

Birth of correctly genotyped calves after multiplex marker detection from bovine embryo microblade biopsies.

We report a method for multiplex genotyping of bovine embryo microblade biopsies. We have tested the reliability of the method and the viability of the embryos in vitro and in vivo. Two polymorphic gene markers (GHR F279Y and PRLR S18N) associated with milk production traits and one marker for sex diagnosis (ZFX/ZFY) were genotyped simultaneously with a method that combines nested PCR and allelic discrimination. To test the accuracy of genotyping, in the first experiment the genotypes of 134 biopsies from in vitro produced embryos were compared to genotypes determined from the corresponding embryos after biopsy. The method proved to be highly accurate as only in three cases (two for PRLR S18N and one for GHR F279Y) out of 395 genotypes the genotype was in disagreement between the two samples. The viability of similarly biopsied embryos was tested in parallel: after 24-hr culture 94.6% of embryos recovered in vitro. In the second experiment, a total of 150 in vivo-produced embryos were biopsied on Day 7 and genotyped. After the genotyping results were obtained on Day 8, female embryos were selected for transfer. From a total of 57 selected embryos 43 were transferred individually and 14 as pairs. After single embryo transfers, 19 recipients became pregnant and after embryo transfers in pairs one became pregnant. The success of genotyping was tested with the genotypes of donors and bulls and also from the hair samples of born calves. All calves were females and of the same genotypes determined from the biopsy.