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Probiotics are increasingly administered to premature infants to prevent necrotizing enterocolitis and neonatal sepsis. However, their effects on gut microbiome assembly and immunity are poorly understood. Using a randomized intervention trial in extremely premature infants, we tested the effects of a probiotic product containing four strains of Bifidobacterium species autochthonous to the infant gut and one Lacticaseibacillus strain on the compositional and functional trajectory of microbiome. Daily administration of the mixture accelerated the transition into a mature, term-like microbiome with higher stability and species interconnectivity. Besides infant age, Bifidobacterium strains and stool metabolites were the best predictors of microbiome maturation, and structural equation modeling confirmed probiotics as a major determinant for the trajectory of microbiome assembly. Bifidobacterium-driven microbiome maturation was also linked to an anti-inflammatory intestinal immune milieu. This demonstrates that Bifidobacterium strains are ecosystem engineers that lead to an acceleration of microbiome maturation and immunological consequences in extremely premature infants.

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Development of elastin-like polypeptide (ELP) biomaterials is widespread, but information critical for clinical deployment is limited, with biocompatibility studies focused on a narrow cross-section of ELP sequences. Macrophages can impair biomaterial systems by degrading or isolating the biomaterial and by activating additional immune functions. Their phagocytic response will reveal early immune biocompatibility of ELP nanoparticles (NPs). This study examines that response, induced by the adsorbed protein corona, as a function of ELP guest amino acid, chain length and NP diameter. The breadth of proteins adsorbed to ELP NPs varied, with valine-containing ELP NPs adsorbing fewer types of proteins than leucine-containing constructs. Particle diameter was also a factor, with smaller leucine-containing ELP NPs adsorbing the broadest range of proteins. Macrophage viability was unaffected by the ELP NPs, and their phagocytic capabilities were unimpeded except when incubated with a 500 nm valine-containing 40-mer. This NP significantly decreased the phagocytic capacity of macrophages relative to the control and to a corresponding 500 nm leucine-containing 40-mer. NP size and the proportion of opsonin to dysopsonin proteins likely influenced this outcome. These results suggest that certain combinations of ELP sequence and particle size can result in an adsorbed protein corona, which may hinder macrophage function.

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Stress is broadly defined as the non-specific biological response to changes in homeostatic demands and is mediated by the evolutionarily conserved neuroendocrine networks of the hypothalamus-pituitary-adrenal (HPA) axis and the sympathetic nervous system. Activation of these networks results in transient release of glucocorticoids (cortisol) and catecholamines (epinephrine) into circulation, as well as activation of sympathetic fibers innervating end organs. These interventions thus regulate numerous physiological processes, including energy metabolism, cardiovascular physiology, and immunity, thereby adapting to cope with the perceived stressors. The developmental trajectory of the stress-axis is influenced by a number of factors, including the gut microbiome, which is the community of microbes that colonizes the gastrointestinal tract immediately following birth. The gut microbiome communicates with the brain through the production of metabolites and microbially derived signals, which are essential to human stress response network development. Ecological perturbations to the gut microbiome during early life may result in the alteration of signals implicated in developmental programming during this critical window, predisposing individuals to numerous diseases later in life. The vulnerability of stress response networks to maladaptive development has been exemplified through animal models determining a causal role for gut microbial ecosystems in HPA axis activity, stress reactivity, and brain development. In this review, we explore the evolutionary significance of the stress-axis system for health maintenance and review recent findings that connect early-life microbiome disturbances to alterations in the development of stress response networks.

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Nanoparticles (NPs) that are exposed to blood are coated with an assortment of proteins that establish their biological identity by forming the interface between the NP and the cells and tissues of the body. The biological relevance of this protein corona is often overlooked during toxicological assessments of NPs. However, accurate interpretation of biological outcomes following exposure to NPs, including activation of coagulation, opsonization of pathogens, and cellular phagocytosis, must take this adsorbed proteome into account. In this study, we examined protein coronas on the surface of five poly(acrylic acid) (PAA) metal-oxide NPs (TiO2, CeO2, Fe2O3, ZnO, and PAA-capsules) following exposure to human plasma for key markers of various host response pathways, including humoral immunity and coagulation. We also evaluated the impacts of pre-exposing serum proteins to PAA-NPs on the opsonization and phagocytosis of bacteria by two immune cell lines. Results demonstrated that each PAA-NP type adsorbed a unique profile of blood proteins and that protein-coated PAA-NPs significantly inhibited human plasma coagulation with PAA-zinc oxide NPs and their associated proteome fully abrogating clotting. Protein-coated PAA-NPs also resulted in a 50% increase in phagocytic activity of RBL-2H3 cells and a 12.5% increase in phagocytic activity in the RAW 264.7 cell line. We also identified numerous structural, coagulation, and immune-activating proteins in the adsorbed protein corona, which resulted in altered biological function. Overall, our findings demonstrate that the formation of protein coronas on the surface of NPs plays an important role in directing the biological outcomes of opsonization, cell phagocytosis, and blood coagulation.

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The ever-growing production of engineered nanoparticles (NPs) for use in many agricultural, commercial, consumer, and industrial applications will lead to their accidental or intentional release into the environment. Potential routes of environmental exposure include manufacturing or transport spills, disposal of NP-containing products down the drain and/or in landfills, as well as direct usage on agricultural land. Therefore, NPs will inevitably contaminate aquatic environments and interact with resident organisms. However, there is limited information regarding the mechanisms that regulate NP transport into fish from the environment. Thus, our primary objective was to elucidate the mechanism(s) underlying cellular uptake and intracellular fate of 3-9nm poly (acrylic acid) NPs loaded with the fluorescent dye Nile red using a rainbow trout (Oncorhynchus mykiss) gill epithelial cell line (RTgill-W1). In vitro measurements with NP-treated RTgill-W1 cells were carried out using a combination of laser scanning confocal microscopy, flow cytometry, fluorescent biomarkers (transferrin, cholera toxin B subunit, and dextran), endocytosis inhibitors (chlorpromazine, genistein, and wortmannin), and stains (4', 6-diamidino-2-phenylindole, Hoechst 33342, CellMask Deep Red, and LysoTracker Yellow). Clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis and macropinocytosis pathways were active in RTgill-W1 cells, and these pathways were exploited by the non-cytotoxic NPs to enter these cells. We have demonstrated that NP uptake by RTgill-W1 cells was impeded when clathrin-coated pit formation was blocked by chlorpromazine. Furthermore, colocalization analysis revealed a moderate positive relationship between NPs and LysoTracker Yellow-positive lysosomal compartments indicating that CME was the dominant operative mechanism involved in NP internalization by RTgill-W1 cells. Overall, our results clearly show that fish gill epithelial cells internalized NPs via energy-dependent endocytotic processes. This study enhances our understanding of complex NP-cell interactions and the results obtained in vitro imply a potential risk to aquatic organisms.

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In this study, the effects of rosette nanotube (RNT) exposure on immune cell viability and function were investigated in vitro using the rat basophilic leukemia (RBL)-2H3 cell line. RBL-2H3 viability was decreased in a dose- and time-dependent manner after lysine-functionalized RNT (K-RNT) exposure. In addition, K-RNTs had a significant effect on RBL-2H3 degranulation. When K-RNT exposure was concurrent with IgE sensitization, 50 and 100 mg l(-1) K-RNTs elicited a heightened degranulatory response compared with IgE alone. Exposure to 50 and 100 mg l(-1) K-RNTs also caused degranulation in RBL-2H3 cells not sensitized with IgE (0 ng ml(-1) IgE). Furthermore, in cells preexposed to K-RNTs for 2 h and subsequently washed, sensitized, and stimulated with IgE, a potentiated degranulatory response was observed. Using confocal laser scanning microscopy and a fluorescein isothiocyanate (FITC)-functionalized RNT construct (termed FITC(1)/TBL(19)-RNT), we demonstrated a strong and direct affiliation between RNTs and RBL-2H3 cell membranes. We also demonstrated cellular internalization of RNTs after 2 h of exposure. Together, these data demonstrate that RNTs may affiliate with the cellular membrane of RBL-2H3 cells and can be internalized. These interactions can affect viability and alter the ability of these cells to elicit IgE-FcεR mediated degranulation.

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Previous reports have shown that nanoparticles (NPs) can both enhance and suppress immune effector functions; however the mechanisms that dictate these responses are still unclear. Here, the effects of polyacrylic acid (PAA) functionalized metal-oxide NP are investigated on RBL-2H3 (representative mammalian granulocyte-like cell line) cell viability, cellular degranulation, immunoglobulin E (IgE) receptor binding, and cell signaling pathways related to immune function. The increasing development of PAA-NPs as pesticide dispersants and as drug carriers in therapeutics necessitates their investigation for safe production. Using two in vitro experimental approaches, this study demonstrates that pre-exposing RBL-2H3 cells, or IgE antibodies, to PAA-NPs (TiO2, CeO2, ZnO, Fe2O3, and PAA-Capsules (NP coating control) over 24 h, significantly decrease the binding capacity of IgE for Fcε receptors, inhibit the phosphorylation of intracellular signaling proteins (e.g., MAPK ERK) that mediate degranulation, and inhibited RBL-2H3 cell degranulation. In addition, and unlike the other NPs tested, PAA-TiO2 significantly reduced RBL-2H3 viability, in a time (4-24 h) and dose-dependent manner (>50 µg mL-1). Together, these data demonstrate that PAA-NPs at sub-lethal doses can interact with cell surface structures, such as receptors, to suppress various stages of the RBL-2H3 degranulatory response to external stimuli, and modify immune cell functions that can impact host-immunity.

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The evaluation of engineered nanomaterial safety has been hindered by conflicting reports demonstrating differential degrees of toxicity with the same nanoparticles. The unique properties of these materials increase the likelihood that they will interfere with analytical techniques, which may contribute to this phenomenon. We tested the potential for: 1) nanoparticle intrinsic fluorescence/absorbance, 2) interactions between nanoparticles and assay components, and 3) the effects of adding both nanoparticles and analytes to an assay, to interfere with the accurate assessment of toxicity. Silicon, cadmium selenide, titanium dioxide, and helical rosette nanotubes each affected at least one of the six assays tested, resulting in either substantial over- or under-estimations of toxicity. Simulation of realistic assay conditions revealed that interference could not be predicted solely by interactions between nanoparticles and assay components. Moreover, the nature and degree of interference cannot be predicted solely based on our current understanding of nanomaterial behaviour. A literature survey indicated that ca. 95% of papers from 2010 using biochemical techniques to assess nanotoxicity did not account for potential interference of nanoparticles, and this number had not substantially improved in 2012. We provide guidance on avoiding and/or controlling for such interference to improve the accuracy of nanotoxicity assessments.

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Nanomaterials in biological solutions are known to interact with proteins and have been documented to affect protein function, such as enzyme activity. Understanding the interactions of nanoparticles with biological components at the molecular level will allow for rational designs of nanomaterials for use in medical technologies. Here we present the first detailed molecular mechanics model of functionalized gold nanoparticle (NP) interacting with an enzyme (L-lactate dehydrogenase (LDH) enzyme). Molecular dynamics (MD) simulations of the response of LDH to the NP binding demonstrate that although atomic motions (dynamics) of the main chain exhibit only a minor response to the binding, the dynamics of side chains are significantly constrained in all four active sites that predict alteration in kinetic properties of the enzyme. It is also demonstrated that the 5 nm gold NPs cause a decrease in the maximal velocity of the enzyme reaction (V(max)) and a trend towards a reduced affinity (increased K(m)) for the ß-NAD binding site, while pyruvate enzyme kinetics (K(m) and V(max)) are not significantly altered in the presence of the gold NPs. These results demonstrate that modeling of NP:protein interactions can be used to understand alterations in protein function.

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Corticotropin-releasing factor (CRF), urotensin I (UI) and serotonin (5-HT) are generally recognized as key regulators of the anorexigenic stress response in vertebrates, yet the proximal effects and potential interactions of these central messengers on food intake in salmonids are not known. Moreover, no study to date in fishes has compared the appetite-suppressing effects of CRF and UI using species-specific peptides. Therefore, the objectives of this study were to (1) assess the individual effects of synthesized rainbow trout CRF (rtCRF), rtUI as well as 5-HT on food intake in rainbow trout, and (2) determine whether the CRF and serotonergic systems interact in the regulation of food intake in this species. Intracerebroventricular (icv) injections of rtCRF and rtUI both suppressed food intake in a dose-related manner but rtUI [ED50 = 17.4 ng/g body weight (BW)] was significantly more potent than rtCRF (ED50 = 105.9 ng/g BW). Co-injection of either rtCRF or rtUI with the CRF receptor antagonist α-hCRF(9-41) blocked the reduction in food intake induced by CRF-related peptides. Icv injections of 5-HT also inhibited feeding in a dose-related manner (ED50 = 14.7 ng/g BW) and these effects were blocked by the serotonergic receptor antagonist methysergide. While the anorexigenic effects of 5-HT were reversed by α-hCRF(9-41) co-injection, the appetite-suppressing effects of either rtCRF or rtUI were not affected by methysergide co-injection. These results identify CRF, UI and 5-HT as anorexigenic agents in rainbow trout, and suggest that 5-HT-induced anorexia may be at least partially mediated by CRF- and/or UI-secreting neurons.

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Coated nanoparticles (NPs) will end up in the environment due to their proposed use in agricultural applications and may potentially cause toxic effects due to their unique properties. To determine the effects of coated NPs on zebrafish (Danio rerio) development, we tested aqueous poly(acrylic acid) (PAA)-coated metal-oxide NPs including TiO2, ZnO, Fe2O3, and CeO2, as well as the polymer coating alone (nanocapsule). Zebrafish embryos were exposed to NPs over a 72 h period at 1, 10, 50, 100, 200, 400, 800, 1200, 1600, and 2000 mg/L to measure various end points. We also ran free metal controls. Time-dependent changes in physicochemical properties of NPs were characterized using dynamic light scattering. Dissolution experiments over 72 h showed minimal free metals were present in stock suspensions and released from the NPs. Interestingly, nanocapsules (≥ 800 mg/L) cause inhibition of hatch, and we suggest that a low pH environment may explain this effect. This study has also demonstrated that CeO2 NPs and nanocapsules containing Nile red are able to traverse the chorion. Overall, our findings indicate that each NP type is stable and neither the NP or encapsulating PAA coating causes apparent toxicity to developing zebrafish.

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To assess whether the brain's monoaminergic and/or corticotropin-releasing factor (CRF) systems may be involved in mediating the appetite-suppressing effects of high environmental ammonia levels, we exposed rainbow trout to one of four NH4Cl treatments (0, 500, 750, 1000 micromol l(-1)) for 24 or 96 h and monitored changes in food intake, brain serotonin (5-HT) and dopamine (DA) activity, CRF and urotensin I (UI) mRNA levels, and plasma cortisol levels. Food intake decreased in a dose-dependent manner after 24 h of ammonia exposure and partially recovered in all groups after 96 h. Ammonia also elicited dose-dependent increases in serotonergic activity in the hypothalamus (HYP), telencephalon (TEL) and posterior brain (PB). Whereas the increase in serotonergic activity was timed with the 24 h food intake inhibition, TEL and PB serotonergic activity increased after 96 h. In the PB, exogenous ammonia also elicited dose-dependent increases in dopaminergic activity after both 24 and 96 h of exposure. Transient increases in TEL CRF and UI mRNA levels, HYP UI mRNA levels, and plasma cortisol concentrations were evidence that the hypothalamic-pituitary-interrenal (HPI) stress axis was primarily stimulated in the first 24 h of ammonia exposure when food intake was depressed. Overall, the transient nature of the appetite suppression during chronic ammonia exposure, and the time-dependent changes in brain monoaminergic and CRF systems, implicate 5-HT, DA, CRF and UI as potential mediators of the appetite-suppressing effects of ammonia. Among these anorexigenic signals, our results specifically identify hypothalamic 5-HT as a potentially key neurobiological substrate for the regulation of food intake during exposure to high external ammonia concentrations.

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