Characterization of exosomes from body fluids of dairy cows.

Exosomes are a specific subpopulation of extracellular vesicles that are widely released by cells of different origins with divergent functions that make their way into body fluids that can be conveniently sampled. In the current study, we isolated and evaluated exosomes from concurrently collected samples of milk, plasma, saliva, and urine from a group of 6 pregnant Holstein-Friesian dairy cows (aged 7 mo, 174 to 203 d of gestation). The cows had BCS of 3.5 to 5.25 (on a scale of 1 to 10), and the milk production for the season to the time of sampling ranged between 5,118 and 6,959 kg. The low levels of extracellular vesicles in saliva and urine (more than 86% fewer compared to the extracellular vesicles in milk and plasma) precluded further detailed evaluation since utility for diagnostics was deemed unlikely. In exosomes isolated from milk and plasma, size distribution, morphology, and the presence of exosome markers was confirmed by nanoparticle tracking analysis, electron microscopy, and Western blot. In addition, a targeted proteomic approach using the quadrupole ion trap mass spectrometer was also used in the study to screen for the exosome marker (e.g., Tumor susceptibility gene 101). Following confirmation of the presence of exosomes, the proteomic profiles of milk and plasma exosomes were evaluated using information-dependent acquisition-mediated liquid chromatography-tandem mass spectrometry (LC-MS/MS). The milk exosomes contain proteins that differed greatly from the plasma exosomes, with only 8 similar proteins harbored in both the milk and plasma exosomes. The milk and plasma exosomes were found to contain proteins (e.g., immunoglobulin J chain and α2 macroglobulin) associated with specific biological processes and molecular functions. Hence, the fluid of origin required for exosome analysis will be dependent on the specific information needed. In conclusion, isolated exosomes from milk and plasma samples collected at the same time point from the same dairy cows encapsulated different profiles of proteins associated with different biological processes and molecular functions.

Cryptococcus neoformans ADS lyase is an enzyme essential for virulence whose crystal structure reveals features exploitable in antifungal drug design.

There is significant clinical need for new antifungal agents to manage infections with pathogenic species such as Cryptococcus neoformans Because the purine biosynthesis pathway is essential for many metabolic processes, such as synthesis of DNA and RNA and energy generation, it may represent a potential target for developing new antifungals. Within this pathway, the bifunctional enzyme adenylosuccinate (ADS) lyase plays a role in the formation of the key intermediates inosine monophosphate and AMP involved in the synthesis of ATP and GTP, prompting us to investigate ADS lyase in C. neoformans. Here, we report that ADE13 encodes ADS lyase in C. neoformans. We found that an ade13Δ mutant is an adenine auxotroph and is unable to successfully cause infections in a murine model of virulence. Plate assays revealed that production of a number of virulence factors essential for dissemination and survival of C. neoformans in a host environment was compromised even with the addition of exogenous adenine. Purified recombinant C. neoformans ADS lyase shows catalytic activity similar to its human counterpart, and its crystal structure, the first fungal ADS lyase structure determined, shows a high degree of structural similarity to that of human ADS lyase. Two potentially important amino acid differences are identified in the C. neoformans crystal structure, in particular a threonine residue that may serve as an additional point of binding for a fungal enzyme-specific inhibitor. Besides serving as an antimicrobial target, C. neoformans ADS lyase inhibitors may also serve as potential therapeutics for metabolic disease; rather than disrupt ADS lyase, compounds that improve the stability the enzyme may be used to treat ADS lyase deficiency disease.

GMP Synthase Is Required for Virulence Factor Production and Infection by Cryptococcus neoformans.

Over the last four decades the HIV pandemic and advances in medical treatments that also cause immunosuppression have produced an ever-growing cohort of individuals susceptible to opportunistic pathogens. Of these, AIDS patients are particularly vulnerable to infection by the encapsulated yeast Cryptococcus neoformans Most commonly found in the environment in purine-rich bird guano, C. neoformans experiences a drastic change in nutrient availability during host infection, ultimately disseminating to colonize the purine-poor central nervous system. Investigating the consequences of this challenge, we have characterized C. neoformans GMP synthase, the second enzyme in the guanylate branch of de novo purine biosynthesis. We show that in the absence of GMP synthase, C. neoformans becomes a guanine auxotroph, the production of key virulence factors is compromised, and the ability to infect nematodes and mice is abolished. Activity assays performed using recombinant protein unveiled differences in substrate binding between the C. neoformans and human enzymes, with structural insights into these kinetic differences acquired via homology modeling. Collectively, these data highlight the potential of GMP synthase to be exploited in the development of new therapeutic agents for the treatment of disseminated, life-threatening fungal infections.

Review: Eicosanoids in preterm labor and delivery: Potential roles of exosomes in eicosanoid functions.

Preterm delivery is a major obstetric health problem contributing to poor neonatal outcome including low birth weight, respiratory distress syndrome, gastrointestinal, immunologic, central nervous system, hearing, and vision problems. Worldwide, approximately 15 million babies are born prematurely each year. The critical question which remains is how to identify women destined to deliver preterm from those who will achieve a term delivery. Prostaglandins, in all mammals, are important in the parturient process. Increased intrauterine prostaglandin production is associated with labor and in fact prostaglandin E2 (PGE2) or analogs are widely used clinically for cervical ripening and labor induction. Measurements of circulating eicosanoids have been problematic because of the rapid and major clearance by the lungs and then kidneys resulting in very low concentrations in plasma. Moreover, since eicosanoids are produced by all mammalian tissues, the sources of the measured eicosanoids are unknown. Our understanding of how cells communicate has undergone a paradigm shift with the recognition of the role of exosomes in intercellular signaling. Recent publications have identified enzymes and products of arachidonic acid metabolism (eicosanoids) within exosomes. This review will explore the potential roles of exosomes in eicosanoid functions that are critical in preterm labor and delivery.

Disruption of de Novo Adenosine Triphosphate (ATP) Biosynthesis Abolishes Virulence in Cryptococcus neoformans.

Opportunistic fungal pathogens such as Cryptococcus neoformans are a growing cause of morbidity and mortality among immunocompromised populations worldwide. To address the current paucity of antifungal therapeutic agents, further research into fungal-specific drug targets is required. Adenylosuccinate synthetase (AdSS) is a crucial enzyme in the adeosine triphosphate (ATP) biosynthetic pathway, catalyzing the formation of adenylosuccinate from inosine monophosphate and aspartate. We have investigated the potential of this enzyme as an antifungal drug target, finding that loss of function results in adenine auxotrophy in C. neoformans, as well as complete loss of virulence in a murine model. Cryptococcal AdSS was expressed and purified in Escherichia coli and the enzyme's crystal structure determined, the first example of a structure of this enzyme from fungi. Together with enzyme kinetic studies, this structural information enabled comparison of the fungal enzyme with the human orthologue and revealed species-specific differences potentially exploitable via rational drug design. These results validate AdSS as a promising antifungal drug target and lay a foundation for future in silico and in vitro screens for novel antifungal compounds.

Plasma exosome profiles from dairy cows with divergent fertility phenotypes.

Cell-to-cell communication in physiological and pathological conditions may be influenced by neighboring cells, distant tissues, or local environmental factors. Exosomes are specific subsets of extracellular vesicles that internalize and deliver their content to near and distant sites. Exosomes may play a role in the maternal-embryo crosstalk vital for the recognition and maintenance of a pregnancy; however, their role in dairy cow reproduction has not been established. This study aimed to characterize the exosome profile in the plasma of 2 strains of dairy cow with divergent fertility phenotypes. Plasma was obtained and characterized on the basis of genetic ancestry as fertile (FERT; <23% North American genetics, New Zealand Holstein-Friesian strain, n=8) or subfertile (SUBFERT; >92% North American genetics, North American Holstein-Friesian strain, n=8). Exosomes were isolated by differential and buoyant density centrifugation and characterized by size distribution (nanoparticle tracking analysis, NanoSight NS500, NanoSight Ltd., Amesbury, UK), the presence of CD63 (Western blot), and their morphology (electron microscopy). The total number of exosomes was determined by quantifying the immunoreactive CD63 (ExoELISA kit, System Biosciences), and the protein content established by mass spectrometry. Enriched exosome fractions were identified as cup-shape vesicles with diameters around 100 nm and positive for the CD63 marker. The concentration of exosomes was 50% greater in FERT cows. Mass spectrometry identified 104 and 117 proteins in FERT and SUBFERT cows, of which 23 and 36 were unique, respectively. Gene ontology analysis revealed enrichment for proteins involved in immunomodulatory processes and cell-to-cell communication. Although the role of exosomes in dairy cow reproduction remains to be elucidated, their quantification and content in models with divergent fertility phenotypes could provide novel information to support both physiological and genetic approaches to improving dairy cow fertility.