RESUMEN
Milk extracellular vesicles (EV) have gained extensive attention as promising diagnostic and therapeutic tools. Pre-analytical raw milk storage at low temperatures is an ordinary and usually necessary step after sample collection. It is known that direct freezing of unprocessed whole milk contaminates the native pool of milk EV with other cell structures. However, less evidence is available regarding prolonged cooling at 4°C. The current study assessed whether pre-analytical storage of bovine raw milk for several days affected EV isolation and further analysis. To confirm the independence from the health status of the mammary gland, we analyzed milk samples stored at 4°C for 1, 2, 3, and 7 d past collection, respectively, from 2 quarters of the same cow with different somatic cell counts (SCC). Seven days of refrigeration did not change the milk EV size, concentration, or morphology. We did not detect any changes in the EV cargo regarding the amount of protein and RNA, nor in the specific EV markers TSG101, CD9, and CD81 in milk from quarters with high and low SCC. Overall, we observed fewer CD81 and CD9 markers in quarters with high SCC. Moreover, we found no reduction in the mastitis-related miRNA bta-miR-223-3p, suggesting that refrigeration for several days up to 1 wk is a possible storage option compatible with further EV analyses. The findings of this study enhance the confidence that milk EV are highly stable in the raw milk matrix.
Asunto(s)
Enfermedades de los Bovinos , Vesículas Extracelulares , Mastitis Bovina , Femenino , Bovinos , Animales , Leche/química , Recuento de Células/veterinaria , Congelación , Refrigeración/veterinaria , Vesículas Extracelulares/metabolismo , Mastitis Bovina/metabolismoRESUMEN
OBJECTIVE: To compare the transcriptome of human cumulus cells (CCs) from oocytes with different outcomes (pregnancy yes/no, live birth [LB] yes/no), to identify noninvasive biomarkers for oocyte selection as well as new therapeutic targets to increase LB rates from assisted reproductive technologies (ART). DESIGN: Retrospective observational study. SETTINGS: This study was conducted at a University Hospital in Switzerland. PATIENTS: Subfertile couples undergoing controlled ovarian superstimulation and intracytoplasmic sperm injection with subsequent unbiopsied embryo transfer below the female age of 43 years. INTERVENTION(S): RNA sequencing of CCs from oocytes results in a pregnancy, no pregnancy, LB, or no LB. MAIN OUTCOME MEASURES: Differential gene expression (DEG) between CCs of oocytes results in "no pregnancy" vs. "pregnancy" and "pregnancy only" vs. "live birth." RESULTS: Although RNA sequencing did not reveal DEGs when comparing the transcriptomic profiles of the groups "no pregnancy" with "pregnancy," we identified 139 DEGs by comparing "pregnancy only" with "live birth," of which 28 belonged to clusters relevant to successful ART outcomes (i.e., CTGF, SERPINE2, PCK1, HHIP, HS3ST, and BIRC5). A functional enrichment analysis revealed that the transcriptome of CCs associated with LB depicts pathways of extracellular matrix, inflammatory cascades leading to ovulation, cell patterning, proliferation, and differentiation, and silencing pathways leading to apoptosis. CONCLUSION: We identified a CCs transcriptomic profile associated with LB after embryo transfer that, after further validation, could serve to predict successful ART outcomes. The definition of relevant pathways of CCs related to oocyte competency contributes to a broader understanding of the cumulus oocyte complex and helps identify further therapeutic targets for improving ART success.
Asunto(s)
Nacimiento Vivo , Inyecciones de Esperma Intracitoplasmáticas , Adulto , Femenino , Humanos , Masculino , Embarazo , Células del Cúmulo/metabolismo , Perfilación de la Expresión Génica , Oocitos/metabolismo , Semen , Serpina E2/metabolismo , Transcriptoma , Estudios RetrospectivosRESUMEN
Extracellular vesicles (EVs) and their microRNA (miRNA) cargo have been proposed as possible mammary gland health biomarkers in cattle. However, throughout the day, the biologically active milk components, such as miRNAs, may change due to the dynamic nature of milk. The current study aimed to evaluate the circadian fluctuation of milk EVs miRNA cargo to assess the feasibility of milk EVs as future biomarkers for mammary gland health management. Milk from four healthy dairy cows was collected for four consecutive days in the two daily milking sessions in the morning and the evening. The isolated EVs were heterogeneous, intact, and carried the EV protein markers CD9, CD81, and TSG101, as shown by transmission electron microscopy and western blot. The miRNA sequencing results demonstrate that the abundance of miRNA cargo in milk EVs remained stable, unlike other milk components, such as somatic cells, that changed during milking sessions. These findings indicated that the miRNA cargo within milk EVs remains stable irrespective of the time of day, suggesting their potential utility as diagnostic markers for mammary gland health.
Asunto(s)
Vesículas Extracelulares , MicroARNs , Femenino , Animales , Bovinos , MicroARNs/genética , MicroARNs/metabolismo , Leche/metabolismo , Biomarcadores/metabolismo , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Ritmo CircadianoRESUMEN
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces B and T cell responses, contributing to virus neutralization. In a cohort of 2,911 young adults, we identified 65 individuals who had an asymptomatic or mildly symptomatic SARS-CoV-2 infection and characterized their humoral and T cell responses to the Spike (S), Nucleocapsid (N) and Membrane (M) proteins. We found that previous infection induced CD4 T cells that vigorously responded to pools of peptides derived from the S and N proteins. By using statistical and machine learning models, we observed that the T cell response highly correlated with a compound titer of antibodies against the Receptor Binding Domain (RBD), S and N. However, while serum antibodies decayed over time, the cellular phenotype of these individuals remained stable over four months. Our computational analysis demonstrates that in young adults, asymptomatic and paucisymptomatic SARS-CoV-2 infections can induce robust and long-lasting CD4 T cell responses that exhibit slower decays than antibody titers. These observations imply that next-generation COVID-19 vaccines should be designed to induce stronger cellular responses to sustain the generation of potent neutralizing antibodies.
Asunto(s)
COVID-19 , Humanos , Vacunas contra la COVID-19 , SARS-CoV-2 , Anticuerpos Neutralizantes , Aprendizaje AutomáticoRESUMEN
Scientific evidence suggests that not only murine scent communication is regulated by major urinary proteins, but that their expression may also vary in response to metabolism via a yet unknown mechanism. Major urinary proteins are expressed mainly in the liver, showing a sexually dimorphic pattern with substantially higher expression in males. Here, we investigate the metabolic implications of a major urinary protein knockout in twelve-week-old male and female C57BL/6N mice during ad libitum feeding. Despite both sexes of major urinary protein knockout mice displayed numerically increased body weight and visceral adipose tissue proportions compared to sex-matched wildtype mice, the main genotype-specific metabolic differences were observed exclusively in males. Male major urinary protein knockout mice exhibited plasma and hepatic lipid accumulation accompanied by a hepatic transcriptome indicating an activation of lipogenesis. These findings match the higher major urinary protein expression in male compared to female wildtype mice, suggesting a more distinct reduction in energy requirements in male compared to female major urinary protein knockout mice. The observed sex-specific anabolic phenotype confirms a role of major urinary protein in metabolism and, since major urinary proteins are not expressed in humans, suggests the major urinary protein knockout mouse as a potential alternative model for translational metabolism research which needs to be further elucidated.
Asunto(s)
Hígado , Proteínas , Animales , Femenino , Humanos , Metabolismo de los Lípidos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Familia de Multigenes , Proteínas/metabolismoRESUMEN
In dairy cows, Staphylococcus aureus (S. aureus) is among the most prevalent microorganisms worldwide, causing mastitis, an inflammation of the mammary gland. Production of extracellular vesicles (EVs) is a common feature of S. aureus strains, which contributes to its pathogenesis by delivering bacterial effector molecules to host cells. In the current study, we evaluated the differences between five S. aureus mastitis isolates regarding their EV production. We found that different mastitis-related S. aureus strains differ in their behaviour of shedding EVs, with M5512VL producing the largest amount of EVs containing alpha-haemolysin, a strong cytotoxic agent. We stimulated primary cultured bovine mammary epithelial cells (pbMECs) with EVs from the S. aureus strain M5512VL. After 24 h of incubation, we observed a moderate increase in gene expression of tumour necrosis factor-alpha (TNF-α) but, surprisingly, a lack of an associated pronounced pro-inflammatory response. Our results contribute to understanding the damaging nature of S. aureus in its capacity to effectively affect mammary epithelial cells.
RESUMEN
BACKGROUND: Subclinical mastitis, the inflammation of the mammary gland lacking clinical symptoms, is one of the most prevalent and costly diseases in dairy farming worldwide. Milk microRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) have been proposed as potential biomarkers of different mammary gland conditions, including subclinical mastitis. However, little is known about the robustness of EVs analysis regarding sampling time-point and natural infections. To estimate the reliability of EVs measurements in raw bovine milk, we first evaluated changes in EVs size and concentration using Tunable Resistive Pulse Sensing (TRPS) during three consecutive days of sampling. Then, we analysed daily differences in miRNA cargo using small RNA-seq. Finally, we compared milk EVs differences from naturally infected udder quarters with their healthy adjacent quarters and quarters from uninfected udders, respectively. RESULTS: We found that the milk EV miRNA cargo was very stable over the course of three days regardless of the health status of the quarter, and that infected quarters did not induce relevant changes in milk EVs of adjacent healthy quarters. Chronic subclinical mastitis induced changes in milk EV miRNA cargo, but neither in EVs size nor concentration. We observed that the changes in immunoregulatory miRNAs in quarters with chronic subclinical mastitis were cow-individual, however, the most upregulated miRNA was bta-miR-223-3p across all individuals. CONCLUSIONS: Our results showed that the miRNA profile and particle size characteristics remained constant throughout consecutive days, suggesting that miRNAs packed in EVs are physiological state-specific. In addition, infected quarters were solely affected while adjacent healthy quarters remained unaffected. Finally, the cow-individual miRNA changes pointed towards infection-specific alterations.