Investigation of on-line electrokinetic enrichment strategies for capillary electrophoresis of extracellular vesicles.

This work explores strategies for electrokinetic preconcentration of extracellular vesicles (EVs) that are potential source of biomarkers for different diseases. The first approach that led to successful preconcentration of EVs is based on large volume sample stacking (LVSS), allowing an enrichment factor of 7 for CE of EVs with long-end injection (using a capillary with an effective length of 50 cm). Attempts were also made to perform multiple cycles of LVSS, field amplified sample stacking (FASS) and field amplified sample injection (FASI), to improve EVs preconcentration performance. The focus was then put on development of capillary isotachophoresis under high ionic strengths (IS) for electrokinetic enrichment of slow migrating EVs having heterogeneous mobilities. This approach relies on the use of extremely high concentrations of the terminating electrolyte (TE) to slow down the mobility of TE co-ions, rendering them slower than those of EVs. The limit of detection for intact EVs using the developed ITP-UV method reached 8.3 × 108 EVs/mL, allowing an enrichment of 25 folds and a linear calibration up to 4 × 1010 EVs/mL. The ITP-UV and ITP-LIF approaches were applied to provide the electrokinetic signature of EVs of bovine milk and human plasma as well as to visualize more specifically intravesicular fluorescently labelled EVs. The investigation of these strategies shredded light into the challenges still encountered with electrokinetic preconcentration and separation of heterogeneous EVs sub-populations which are discussed herein based on our results and other attempts reported in the literature.

Udder, Claw, and Reproductive Health in Genomic Selection of the Czech Holstein.

The aim of this study was to construct an enhanced selection index using the genomic and economic parameters of new health disorders and current production and functional traits. Genomic evaluation for the incidence of clinical mastitis (CM), three claw disease traits, retained placenta (RET), metritis (MET), and cystic ovaries (CYS) was performed using linear animal models based on producer-recorded data. Good correlations among the health disorders were found, and their heritability estimates did not exceed 7%. Economic weights (EWs) for the health disorders were EUR -132.10 for CM, EUR -128.87 for overall claw diseases, EUR -52.10 for RET, EUR -80.48 for MET, and EUR -16.16 for CYS. These EWs indicate changes in the present value of the annual profit per cow when increasing the incidence of the traits by one case per cow year. Selection using the enhanced index resulted in favourable responses for most of the new health disorders (e.g., -0.001 and -0.006 cases of RET and MET per cow year, respectively), and also in the current breeding objective traits (+49 kg of milk, -0.02% of calf losses). An index contribution of 7% for the new health disorders was assessed as acceptable for the breeders.

An Assessment of the Genetic Parameters of Boars' Reproductive Traits.

The aim of this study was to estimate genetic parameters for the reproductive traits of boars based on single-nucleotide polymorphism data. A total of 109,836 semen samples from 2249 boars were collected between 2010 and 2022. Five basic traits were assessed: sperm volume, sperm concentration, motility, number of abnormal sperm, and, for the first time for the local population, libido. In addition, two derived traits were assessed: total sperm count and number of functional sperm. Genetic parameters were estimated using the single-step genomic best linear unbiased prediction method (ssGBLUP). Dam and sire breeds were evaluated separately. The five basic traits were evaluated using five-trait models, while the two derived traits were evaluated using single-trait models. The heritability coefficients had lower values for all sperm quality traits with both methods. For the basic traits, the heritability ranged from 0.099 to 0.342. The greatest difference between dam and sire breeds was observed for the heritability of the sperm concentration trait (0.099 and 0.271, respectively). The heritability of the libido trait was twice as high for boars of sire breeds as it was for boars of dam breeds. The genetic parameters estimated with ssGBLUP can be used in routine genetic evaluations to improve the pig breeding process.

Comparison of goat and cow milk-derived extracellular vesicle miRNomes.

miRNAs present in milk are mainly found in extracellular vesicles (EVs), which are nanosized membrane vesicles released by most of the cell types to ensure intercellular communication. The majority of the studies performed so far on these vesicles have been conducted on human and cow's milk and focused on their miRNA content. The objectives of this study were to profile the miRNA content of purified EVs from five healthy goats and to compare their miRNome to those obtained from five healthy cows, at an early stage of lactation. EV populations were morphologically characterized using Transmission Electron Microscopy and Nanoparticle Tracking Analysis. The presence of EV protein markers checked by Western blotting and the absence of contamination of preparations by milk proteins. The size distribution and concentration of bovine and goat milk-derived EVs were similar. RNA-sequencing were performed, and all sequences were mapped to the cow genome identifying a total of 295 miRNAs. This study reports for the first-time a goat miRNome from milk EVs and its validation using cow miRNomes.

Fast, simple and calibration-free size characterization and quality control of extracellular vesicles using capillary Taylor dispersion analysis.

This study reports the development of a Taylor Dispersion Analysis (TDA) method for the size characterization of Extracellular Vesicles (EVs), which are highly heterogeneous nanoscale cell-derived vesicles (30-1000 nm). Here, we showed that TDA, conducted in uncoated fused silica capillaries (50 µm i.d.) using a conventional Capillary Electrophoresis instrument, is able to provide absolute sizing (requiring no calibration) of bovine milk-derived EVs in a small sample volume (∼ 7 nL) and over their entire size range, even the smallest ones (< 70 nm) not accessible via other techniques that provide nanoparticle sizing in suspension. TDA size measurements were repeatable (RSD < 10%) and the average EV sizes were found in the range of 120-210 nm, in very good agreement with those measured with Nanoparticle Tracking Analysis, commonly used for EV characterization. TDA allowed quantitative estimation of EVs for concentrations ≥ 2 × 1011 EVs/mL. Furthermore, TDA was able to detect minor changes in EV size (i.e. by ∼25 nm upon interaction with specific anti-CD9 antibodies of ∼150 kDa), and to highlight the impact of extraction methods (i.e. milk pretreatment: freezing, acid precipitation or centrifugation; the type of size-exclusion chromatography column) and of fluorescent labeling (i.e. intravesicular or surface labeling) on the isolated EV population size. In parallel to EV sizing, TDA allowed to detect molecular contaminants (average sizes ∼1-13 nm) present within the sample, rendering this method a valuable tool to assess the quality and quantity of EV isolates.

Comparison of interferometric light microscopy with nanoparticle tracking analysis for the study of extracellular vesicles and bacteriophages.

Research on extracellular vesicles (EVs) and bacteriophages (phages) has been steadily expanding over the past decades as many of their roles in medicine, biology, and ecosystems have been unveiled. Such interest has brought about the need for new tools to quantify and determine the sizes of these biological nanoparticles. A new device based on interferometric light microscopy (ILM), the Videodrop, was recently developed for this purpose. Here, we compared this new device to two nanoparticle tracking analysis (NTA) devices, the NanoSight and the ZetaView, for the analysis of EVs and phages. We used EVs isolated from bacteria, fecal samples, bovine milk and human cells, and phages of various sizes and shape, ranging from 30 to 120 nm of diameter. While NTA instruments correctly enumerated most phages, the Videodrop detected only the largest one, indicating a lower sensitivity threshold compared to the NTA devices. Nevertheless, the performance of the Videodrop compared favourably to that of the NTA devices for the determination of the concentration of eukaryotic EV samples. The NanoSight instrument provided the most precise size distributions but the Videodrop was by far the most time-saving device, making it worthy of consideration for studies conducted on a large number of samples composed of nanoparticles larger than 90 nm.

The effect of obesity on uterine receptivity is mediated by endometrial extracellular vesicles that control human endometrial stromal cell decidualization and trophoblast invasion.

The objectives of the present study were to determine whether obesity impacts human decidualization and the endometrial control of trophoblast invasion (both of which are required for embryo implantation) and evaluate the potential involvement of endometrial extracellular vesicles (EVs) in the regulation of these physiological processes. Using primary human cell cultures, we first demonstrated that obesity is associated with significantly lower in vitro decidualization of endometrial stromal cells (ESCs). We then showed that a trophoblastic cell line's invasive ability was greater in the presence of conditioned media from cultures of ESCs from obese women. The results of functional assays indicated that supplementation of the culture medium with EVs from nonobese women can rescue (at least in part) the defect in in vitro decidualization described in ESCs from obese women. Furthermore, exposure to endometrial EVs from obese women (vs. nonobese women) was associated with significantly greater invasive activity by HTR-8/SVneo cells. Using mass-spectrometry-based quantitative proteomics, we found that EVs isolated from uterine supernatants of biopsies from obese women (vs. nonobese women) presented a molecular signature focused on cell remodelling and angiogenesis. The proteomics analysis revealed two differentially expressed proteins (fibronectin and angiotensin-converting enzyme) that might be involved specifically in the rescue of the decidualization capacity in ESCs from obese women; both of these proteins are abundantly present in endometrial EVs from nonobese women, and both are involved in the decidualization process. In conclusion, our results provided new insights into the endometrial EVs' pivotal role in the poor uterine receptivity observed in obese women.

Top-Down proteomics based on LC-MS combined with cDNA sequencing to characterize multiple proteoforms of Amiata donkey milk proteins.

An in-depth molecular characterization of the main milk proteins, caseins (CNs) and whey proteins, from Amiata donkey combining top-down proteomic analysis (LC-MS) and cDNA sequencing revealed multiple proteoforms arising from complex splicing patterns, including cryptic splice site usage and exon skipping events. Post-translational modifications, in particular phosphorylation, increased the variety and complexity of proteoforms. αs2-CN perfectly exemplifies such a complexity. With 2 functional genes, CSN1S2 I and CSN1S2 II, made of 20 and 16 exons respectively, nearly 30 different molecules of this CN were detected in the milk of one Amiata donkey. A cryptic splice site usage, leading to a singular shift of the open reading frame and generating two αs2-CN I isoforms with different C-terminal sequences, was brought to light. Twenty different αs1-CN molecules with different phosphorylation levels ranging between 4 and 9P were identified in a single milk sample, most of them resulting from exon skipping events and cryptic splice site usage. Novel genetic polymorphisms were detected for CNs (ß- and αs-CN) as well as for whey proteins (lysozyme C and ß-LG I). The probable new ß-LG I variant, with a significantly higher mass than known variants, appears to display an N-terminal extension possibly related to the signal peptide sequence. This represents the most comprehensive report to date detailing the complexity of donkey milk protein micro-heterogeneity, a prerequisite for discovering new elements to objectify the original properties of donkey's milk.

Development of a microfluidic droplet platform with an antibody-free magnetic-bead-based strategy for high through-put and efficient EVs isolation.

In this study, we present a novel microfluidic droplet-based strategy for high performance isolation of extracellular vesicles (EVs). For EVs capture and release, a magnetic bead-based approach without having recourse to any antibody was optimized in batch and then adapted to the microfluidic droplet system. This antibody-free capture approach relies on the presence of a water-excluding polymer, polyethylene glycol (PEG), to precipitate EVs on the surface of negatively charged magnetic beads. We significantly improved the reproducibility of EV recovery and avoided positive false bias by including a washing step and optimizing the protocol. Well-characterized EV standards derived from pre-purified bovine milk were used for EVs isolation performance evaluation. An EVs recovery of up to 25% estimated with nanoparticle tracking analysis (NTA) was achieved for this batchwise PEG-based approach. The confirmation of isolated EVs identity was also made with our recently developed method using capillary electrophoresis (CE) coupled with laser-induced fluorescent (LIF) detection. In parallel, a purpose-made droplet platform working with magnetic tweezers was developed for translation of this PEG-based method into a droplet microfluidic protocol to further improve the performance in terms of EVs capture efficiency and high throughput. The droplet-based protocol offers a significant improvement of recovery rate (up to 50%) while reducing sample and reagent volumes (by more than 10 folds) and operation time (by 3 folds) compared to the batch-wise mode.

Assessment of the Genetic Diversity of a Local Pig Breed Using Pedigree and SNP Data.

Herein, the genetic diversity of the local Prestice Black-Pied pig breed was assessed by the simultaneous analysis of the pedigree and single nucleotide polymorphism (SNP) data. The information about sire line, dam, date of birth, sex, breeding line, and herd for 1971 individuals was considered in the pedigree analysis. The SNP analysis (n = 181) was performed using the Illumina PorcineSNP60 BeadChip kit. The quality of pedigree and SNPs and the inbreeding coefficients (F) and effective population size (Ne) were evaluated. The correlations between inbreeding based on the runs of homozygosity (FROH) and pedigree (FPED) were also calculated. The average FPED for all animals was 3.44%, while the FROH varied from 10.81% for a minimum size of 1 Mbp to 3.98% for a minimum size of 16 Mbp. The average minor allele frequency was 0.28 ± 0.11. The observed and expected within breed heterozygosities were 0.38 ± 0.13 and 0.37 ± 0.12, respectively. The Ne, obtained using both the data sources, reached values around 50 animals. Moderate correlation coefficients (0.49-0.54) were observed between FPED and FROH. It is necessary to make decisions that stabilize the inbreeding rate in the long-term using optimal contribution selection based on the available SNP data.

Electrokinetic characterization of extracellular vesicles with capillary electrophoresis: A new tool for their identification and quantification.

This work reports on the development of the first capillary electrophoresis methodology for the elucidation of extracellular vesicles' (EVs) electrokinetic distributions. The approach is based on capillary electrophoresis coupled with laser-induced fluorescent (LIF) detection for the identification and quantification of EVs after their isolation. Sensitive detection of these nanometric entities was possible thanks to an 'inorganic-species-free' background electrolyte. This electrolyte was made up of weakly charged molecules at very high concentrations to stabilize EVs, and an intra-membrane labelling approach was used to prevent EV morphology modification. The limit of detection for EVs achieved using the developed CE-LIF method reached 8 × 109 EV/mL, whereas the calibration curve was acquired from 1.22 × 1010 to 1.20 × 1011 EV/mL. The CE-LIF approach was applied to provide the electrokinetic distributions of various EVs of animal and human origins, and visualize different EV subpopulations from our recently developed high-yield EV isolation method.

Extracellular vesicles produced by human and animal Staphylococcus aureus strains share a highly conserved core proteome.

Staphylococcus aureus is an important opportunistic pathogen of humans and animals. It produces extracellular vesicles (EVs) that are involved in cellular communication and enable inter-kingdom crosstalk, the delivery of virulence factors and modulation of the host immune response. The protein content of EVs determines their biological functions. Clarifying which proteins are selected, and how, is of crucial value to understanding the role of EVs in pathogenesis and the development of molecular delivery systems. Here, we postulated that S. aureus EVs share a common proteome containing components involved in cargo sorting. The EV proteomes of five S. aureus strains originating from human, bovine, and ovine hosts were characterised. The clustering of EV proteomes reflected the diversity of the producing strains. A total of 253 proteins were identified, 119 of which composed a core EV proteome with functions in bacterial survival, pathogenesis, and putatively in EV biology. We also identified features in the sequences of EV proteins and the corresponding genes that could account for their packaging into EVs. Our findings corroborate the hypothesis of a selective sorting of proteins into EVs and offer new perspectives concerning the roles of EVs in S. aureus pathogenesis in specific host niches.

An improved LC-MS method to profile molecular diversity and quantify the six main bovine milk proteins, including genetic and splicing variants as well as post-translationally modified isoforms.

Here we describe a method based on Liquid Chromatography coupled with Mass Spectrometry (LC-MS) that provides an accurate determination of the six main bovine milk proteins, including allelic and splicing variants, as well as isoforms resulting from post-translational modifications, with an unprecedented level of resolution. Proteins are identified from observed molecular masses in comparison with theoretical masses of intact proteins indexed in an "in-house" database that includes nearly 3000 entries. Quantification was performed either from UV (214 nm) or mass signals. Thus, up to one hundred molecules, derived from the six major milk proteins, can be identified and quantified from an individual milk sample. This powerful and reliable method, initially developed as an anchoring method to estimate the composition of the six main bovine milk proteins from MIR spectra, is transferable to several mammalian species, including small ruminants, camels, equines, rabbits, etc., for which specific mass databases are available.

Estimation of economic weights for number of teats and sperm quality traits in pigs.

Accurate predictive modelling facilitates efficient and effective trait selection in animal breeding and can decrease costs while maximizing profits when raising economically important animals. The objective of this study was to extend a previously developed bioeconomic model and computer program to calculate the marginal economic values (MEVs) and economic weights (EWs) for direct and maternal pig traits affected by new reproductive traits, namely the number of sow functional teats (NFTs) and boar sperm quality traits (SQTs) that included sperm volume, sperm concentration, motility percentage and percentage of abnormal spermatozoa. The MEV of NFTs represented the cost differences between naturally and artificially reared piglets until weaning and the cost differences between naturally and artificially reared finished animals. The MEVs of SQTs expressed the saved costs for artificial insemination, assuming a decreased price per insemination dose when improving the SQTs. The absolute and relative EWs for the newly defined complex of traits in the breeding objectives for pig breeds involved in the Czech national three-way crossing system (Czech Large White [CLW], Czech Landrace [CL] and Pietrain [PN]) were calculated using gene flow methods. The NFT trait was included only for dam breeds, and the relative EW averaged 3.6% of the total economic importance based on the genetic standard deviations of all 19 simultaneously evaluated traits in CLW and CL breeds. The relative EWs of the four SQTs comprised 2.0% of the total economic importance of the 19 traits in the CLW and CL dam breeds and 8% of the total economic importance of the 18 traits in the PN sire breed. Therefore, inclusion of the NFTs for dam breeds and SQTs for sire breeds in the breeding goal is recommended to aid in obtaining ideal outcomes with optimal economic values.

Host factors determine the evolution of infection with Staphylococcus aureus to gangrenous mastitis in goats.

Staphylococcus aureus is the major cause of very severe mastitis of dairy goats. The initial objective of our study was to fine-tune an experimental model of infection of the goat mammary gland with two strains of S. aureus and two lines of goats (low and high somatic cell score lines). Following the challenge, the 10 infected goats divided in two clear-cut severity groups, independently of the S. aureus strain and the goat line. Five goats developed very severe mastitis (of which four were gangrenous) characterized by uncontrolled infection (UI group), whereas the other five kept the infection under control (CI group). The outcome of the infection was determined by 18 h post-infection (hpi), as heralded by the bacterial milk concentration at 18 hpi: more than 107/mL in the UI group, about 106/mL in the CI group. Leukocyte recruitment and composition did not differ between the groups, but the phagocytic killing at 18 hpi efficiency did. Contributing factors involved milk concentrations of α-toxin and LukMF' leukotoxin, but not early expression of the genes encoding the pentraxin PTX3, the cytokines IL-1α and IL-1ß, and the chemokines IL-8 and CCL5. Concentrations of TNF-α, IFN-γ, IL-17A, and IL-22 rose sharply in the milk of UI goats when infection was out of control. The results indicate that defenses mobilized by the mammary gland at an early stage of infection were essential to prevent staphylococci from reaching critical concentrations. Staphylococcal exotoxin production appeared to be a consequent event inducing the evolution to gangrenous mastitis.

Characterisation of adipocyte-derived extracellular vesicle subtypes identifies distinct protein and lipid signatures for large and small extracellular vesicles.

Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells by conveying signalling proteins and genomic material. The level of EVs in plasma is significantly increased in cardiometabolic diseases associated with obesity, suggesting their possible participation in the development of metabolic dysfunction. With regard to the poor definition of adipocyte-derived EVs, the purpose of this study was to characterise both qualitatively and quantitatively EVs subpopulations secreted by fat cells. Adipocyte-derived EVs were isolated by differential centrifugation of conditioned media collected from 3T3-L1 adipocytes cultured for 24 h in serum-free conditions. Based on morphological and biochemical properties, as well as quantification of secreted EVs, we distinguished two subpopulations of adipocyte-derived EVs, namely small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs). Proteomic analyses revealed that lEVs and sEVs exhibit specific protein signatures, allowing us not only to define novel markers of each population, but also to predict their biological functions. Despite similar phospholipid patterns, the comparative lipidomic analysis performed on these EV subclasses revealed a specific cholesterol enrichment of the sEV population, whereas lEVs were characterised by high amounts of externalised phosphatidylserine. Enhanced secretion of lEVs and sEVs is achievable following exposure to different biological stimuli related to the chronic low-grade inflammation state associated with obesity. Finally, we demonstrate the ability of primary murine adipocytes to secrete sEVs and lEVs, which display physical and biological characteristics similar to those described for 3T3-L1. Our study provides additional information and elements to define EV subtypes based on the characterisation of adipocyte-derived EV populations. It also underscores the need to distinguish EV subpopulations, through a combination of multiple approaches and markers, since their specific composition may cause distinct metabolic responses in recipient cells and tissues.

Association of germ-free mice with a simplified human intestinal microbiota results in a shortened intestine.

Genetic, nutritional, and gut microbiota-derived factors have been proposed to play a role in the development of the whole intestine that is around 40% longer in PRM/Alf mice compared with other mouse strains. The PRM/Alf genotype explains 60% of this length difference. The remaining 40% are due to a maternal effect that could depend on the gut microbiota transmitted by the mother to their pups. Germ-free PRM/Alf mice and C3H/He mice were associated with a simplified human microbiota (SIHUMI) to study its impact on gut length. The small intestines of the SIHUMI-associated mice were 16.4% (PRM/Alf) and 9.7% (C3H/He) shorter than those of the corresponding germ-free counterparts. Temporal temperature gradient gel electrophoresis and quantitative real-time PCR revealed differences in microbiota composition between both SIHUMI-associated mouse strains. Anaerostipes caccae was one log lower in PRM/Alf mice than in C3H/He mice. Since polyamines and short-chain fatty acids (SCFAs) are important intestinal growth factors, their concentrations were explored. Cecal concentrations of putrescine, spermine, spermidine, and N-acetylspermine were 1.5-fold, 3.7-fold, 2.2-fold, and 1.4-fold higher, respectively, in the SIHUMI-C3H/He mice compared with the SIHUMI-PRM/Alf mice. In addition, cecal acetate, propionate, and butyrate concentrations in SIHUMI-C3H/He mice were 1.4-fold, 1.1-fold, and 2.1-fold higher, respectively, than in SIHUMI-PRM/Alf mice. These results indicate that polyamines and SCFAs did not promote gut lengthening in any of the two mouse strains. This suggests that as yet unknown factors provided by the SIHUMI prevented gut lengthening in the SIHUMI-associated mice compared with the germfree mice.