Effects of a mannan-rich yeast cell wall-derived preparation on cecal concentrations and tissue prevalence of Salmonella Enteritidis in layer chickens.

Salmonella Enteritidis (SE) has been the most common Salmonella serotype associated with foodborne infections in the last several years. Dietary applications of yeast-based preparations in feed have shown to reduce Salmonella colonization in chickens augmenting SE control strategies. This study was conducted to evaluate the effects of a mannan-rich yeast cell wall-derived preparation (Actigen®) administered in feed at a rate of 400 g/ton on SE colonization in the cecum and internal organs of commercial layer chickens. Sixteen week-old layer pullets were orally challenged with a selected nalidixic acid resistant SE strain at a dose of 1.7×10^9 colony forming units (CFU) per bird. SE colonization was assessed by evaluating isolation rates from ovary and pooled liver/spleen samples as well as enumeration of SE in cecal pouches one week post-challenge. Recovery rates of SE from the ovaries of directly challenged birds receiving Actigen® were significantly lower (P <0.02) than those in directly challenged birds fed an unsupplemented control diet. Recovery rates of SE from pooled liver/spleen samples were not significantly different between Actigen®-treated pullets and controls (P = 0.22). Using direct plate count methods, cecal SE concentrations were 1 log10 lower (P <0.001) in challenged pullets in the Actigen®-supplemented group than in the challenged controls. The SE concentration distributions in the ceca were similar in groups testing positive and groups testing negative for SE in the ovaries and liver/spleens tissues. As a result, SE concentrations in the ceca could not be directly related to the occurrence or prevalence of SE in these tissues. In conclusion, Actigen® supplementation appears to decrease the prevalence of SE in ovarian tissue and concentrations of SE in cecal contents and may be useful as a tool for reducing the risk of eggshell contamination and transovarian transmission of SE in eggs.

Can dietary zinc diminish the impact of necrotic enteritis on growth performance of broiler chickens by modulating the intestinal immune-system and microbiota?

The objective of this study was to compare the effects of inorganic and proteinate Zn in chickens challenged with coccidia and Clostridium perfringens. A 3 × 2 factorial design was used, with 3 dietary formulations (0 or 90 mg/kg supplemental Zn from ZnSO4 or Zn proteinate, with or without challenge). On day 14, challenged birds were orally gavaged with approx. 5,000 Eimeria maxima sporulated oocysts, and on day 19 to 21 with C. perfringens (108 CFU/D). Productive performance was assessed at day 21 and 28. At 21 D, necrotic enteritis (NE) lesion severity, intestinal permeability, gene expression, and ileal and cecal microbiota were evaluated. An interaction of Zn source by challenge was observed for lesion score and mortality, wherein Zn supplementation decreased the degree of NE lesions (P = 0.02) and mortality due to NE (P = 0.008). In the jejunum, an interaction of Zn source by challenge was observed for the expression of IL-8 (P = 0.001) and INF-γ (P = 0.03), wherein the NE challenge upregulated their expression, but not in the Zn proteinate supplemented birds. Zn proteinate supplementation downregulated iNOS vs. ZnSO4 supplemented birds (P = 0.0003), and supplemental Zn downregulated TLR-2 (P = 0.05) and ZnT5 (P = 0.04), regardless of the source. In the ileal microbiota, Zn proteinate supplementation decreased the frequency of Lactobacillus (P = 0.01), and the challenge increased Enterobacteriaceae (P = 0.01). Dietary Zn decreased NE lesion severity and mortality due to NE; Zn proteinate led to lower expression of IL-8 and INF-γ in challenged birds which may be an indicative of a lessened inflammatory response.

Zinc source modulates intestinal inflammation and intestinal integrity of broiler chickens challenged with coccidia and Clostridium perfringens.

Two dietary sources of zinc (ZnSO4 or organic Zn) were tested in chickens challenged with coccidiosis (Co) or coccidiosis plus Clostridium perfringens (CoCPF). On day 14, the chickens were orally gavaged with ∼5,000 Eimeria maxima sporulated oocysts. On day 19, 20, and 21 chickens challenged with C. perfringens were given a broth culture containing 108 cfu of this bacterium. Productive performance parameters were determined at d 14, 21, and 28. On day 21, necrotic enteritis (NE) lesions were scored, and intestinal permeability was evaluated. Jejunum and cecal tonsils were collected for morphology and gene expression analysis. On day 21, organic Zn improved BW gain by 18.6% (P = 0.07), and FCR by 12% (P = 0.09) in CoCPF challenged chickens vs. birds fed ZnSO4. From 1 to 28, organic Zn increased BW gain (P = 0.02), and improved FCR (P = 0.03) vs. birds fed ZnSO4. At 21 d, NE lesions were only observed in CoCPF birds (P < 0.001), and mortality due to NE was only observed when CoCPF birds were fed ZnSO4 (P = 0.001). Organic Zn fed birds had increased villus height in the jejunum (P = 0.005) and decreased intestinal permeability (P = 0.01) vs. ZnSO4. In the jejunum, organic Zn fed birds showed a downregulation of expression of IL-8 (P = 0.02), and upregulation of IL-10 (P = 0.05) in CoCPF birds vs. ZnSO4- CoCPF birds. As main effect, birds supplemented with organic Zn had higher mRNA expression of TLR-2 (P = 0.02) and IgA (P = 0.01). In the cecal tonsils, organic Zn fed birds showed upregulation of iNOS (P = 0.008) in CoCPF birds vs. ZnSO4-CoCPF birds. Organic Zn supplementation reduced intestinal permeability and attenuated intestinal inflammation of broilers co-challenged with coccidia and C. perfringens.

Effect of manganese preconditioning and replacing inorganic manganese with organic manganese on performance of male broiler chicks.

The effects of manganese (Mn) preconditioning, 96 h post-hatch followed by the replacement of inorganic Mn with different levels of organic Mn (5 to 21 D), on growth, tissue excreta Mn content, gene expression, and enzyme activity were evaluated. A total of 420 day-old male Cobb 500 broilers were divided into 2 groups. One group was fed a corn-soybean meal basal diet containing 17 mg of Mn/kg (preconditioning diet, MnPD); the second group was fed the non-preconditioning diet (NPCD), which was the MnPD supplemented with 60 mg of Mn/kg from manganese sulfate (MnSO4). On day 5, each group was divided into 5 subgroups and were randomly assigned to dietary treatments consisting of MnPD alone or MnPD supplemented with 12 or 60 mg Mn/kg Mn as MnSO4 or Mn proteinate (6 replicate cages of 6 birds). Broiler chicks that were fed the MnPD had lower (P ≤ 0.05) body weight gain (BWG) and G:F ratio when compared to those that were fed the NPCD for 4 D. Birds that were fed MnPD (1 to 4 D) and switched to MnPD supplemented with 60 mg/kg Mn (5 to 21 D) had lower (P ≤ 0.05) BWG compared to those that were fed NPCD (1 to 4 D) and switched to MnPD supplemented with 60 mg/kg Mn for 21 D. Excreta, tibia ash, liver, and heart Mn levels were increased (P ≤ 0.05) by supplemental Mn. The expression of jejunum divalent metal transporter-1 mRNA levels, as well as activities of plasma total super oxide dismutase and liver alanine transaminase, was not affected by MnPD or Mn source and levels. These results confirmed that feeding marginally deficient Mn diets to broiler chicks post-hatch does affect growth rate and tissue Mn concentration.

Zinc source influences the gene expression of zinc transporters in jejunum and cecal tonsils during broiler challenge with Eimeria maxima and Clostridium perfringens.

The objective of this experiment was to study the effects of dietary zinc (Zn) source on gene expression of Zn transporters (metallothionein [MT], ZIP 3, 5, 8, 9, 10, 11, 13, and 14, and ZnT 1, 4, 5, 6, 7, 9, and 10) in the jejunum and cecal tonsils of broilers challenged with coccidia or coccidia plus Clostridium perfringens. A 2 × 2 factorial design was used with 2 Zn sources (90 mg Zn/kg from either ZnSO4 or an organic Zn, Bioplex® Zn) and challenged with approximately 5,000 oocysts of Eimeria maxima at 14 d of age with or without C. perfringens (108 CFU/bird) at 18, 19, and 20 d of age (8 pens per treatment and 8 birds per pen) after which 1 bird/pen was sampled at 21 d of age. In the jejunum, co-infection resulted in higher ZnT 5 and 6 gene expression, while organic Zn fed birds had lower ZIP 5 and 11, and higher ZnT1. Additionally, an interaction of challenge by Zn source was noted wherein ZnT10 was unaffected by the C. perfringens in the organic Zn treatment but was 2.7-fold lower in the co-infected ZnSO4 fed birds. S100A9 gene expression, a biomarker of inflammatory response in necrotic enteritis, increased 2 and 2.8-fold in the cecal tonsils and jejunum with the co-infection, respectively. Supplementation with organic Zn lowered S100A9 by 1.9 and 4.4-fold in the cecal tonsils and jejunum, respectively, when birds were supplemented with ZnSO4. Notably, MT, ZIP 3, 8, 9, 10, 13, or 14, and ZnT 4, 7, and 9 were unaffected by Zn source and/or method of challenge. An interaction of challenge by Zn source was also noted for serum Zn concentration, which was reduced when birds were challenged with C. perfringens and fed ZnSO4 but no difference between challenge method when birds were fed organic Zn. Based on the expression of ZnT and ZIP genes, more Zn trafficking due to treatment occured in the jejunum than cecal tonsils, but further studies are needed to ascertain how Zn source regulates intracellular free Zn concentrations and whole-body Zn status during an enteric challenge.

Biological recognition of graphene nanoflakes.

The systematic study of nanoparticle-biological interactions requires particles to be reproducibly dispersed in relevant fluids along with further development in the identification of biologically relevant structural details at the materials-biology interface. Here, we develop a biocompatible long-term colloidally stable water dispersion of few-layered graphene nanoflakes in the biological exposure medium in which it will be studied. We also report the study of the orientation and functionality of key proteins of interest in the biolayer (corona) that are believed to mediate most of the early biological interactions. The evidence accumulated shows that graphene nanoflakes are rich in effective apolipoprotein A-I presentation, and we are able to map specific functional epitopes located in the C-terminal portion that are known to mediate the binding of high-density lipoprotein to binding sites in receptors that are abundant in the liver. This could suggest a way of connecting the materials' properties to the biological outcomes.

Label-free in-flow detection of receptor recognition motifs on the biomolecular corona of nanoparticles.

Nanomedicine, nanotargeting and nanotherapeutics have in the last few years faced several difficulties in translating the promising results obtained in vitro to an in vivo scenario. The origin of this discrepancy might be found in the lack of a detailed and realistic characterization of the biological surface of nanoparticles. Despite the capability to engineer nanomaterials with a great variety and a precise control of the surface functionalization, the targeting capability is lost when the nanoparticles are embedded in complex biological media, due to the formation of a biological layer (biomolecular corona). This biological layer represents the ultimate nanoparticle surface, likely to interact with the cell machinery. Therefore, in addition to traditional nanoparticle characterization techniques, a more insightful investigation of the biomolecular corona is needed, including the capability to assess the orientation and functionality of specific key molecular features. Here we present a method for the rapid screening of exposed protein recognition motifs on the surface of nanoparticles exploiting quartz crystal microbalance (QCM). We quantify accessible functional epitopes of transferrin-coated nanoparticles and correlate them to differences in nanoparticle size and functionalization. The target recognition occurs label free in flow, thereby pushing our investigation into a more in vivo-like scenario. Our method is applicable to a wide array of nanoparticles and therefore holds the potential to become an advanced technique for the classification of all kinds of nanobioconstructs based on their biological external functionality.

Long-term toxicity of surface-charged polystyrene nanoplastics to marine planktonic species Dunaliella tertiolecta and Artemia franciscana.

Plastic pollution has been globally recognized as a critical issue for marine ecosystems and nanoplastics constitute one of the last unexplored areas to understand the magnitude of this threat. However, current difficulties in sampling and identifying nano-sized debris make hard to assess their occurrence in marine environment. Polystyrene nanoparticles (PS NPs) are largely used as nanoplastics in ecotoxicological studies and although acute exposures have been already investigated, long-term toxicity on marine organisms is unknown. Our study aims at evaluating the effects of 40nm PS anionic carboxylated (PS-COOH) and 50nm cationic amino-modified (PS-NH2) NPs in two planktonic species, the green microalga Dunaliella tertiolecta and the brine shrimp Artemia franciscana, respectively prey and predator. PS NP behaviour in exposure media was determined through DLS, while their toxicity to microalgae and brine shrimps evaluated through 72h growth inhibition test and 14 d long-term toxicity test respectively. Moreover, the expression of target genes (i.e. clap and cstb), having a role in brine shrimp larval growth and molting, was measured in 48h brine shrimp larvae. A different behaviour of the two PS NPs in exposure media as well as diverse toxicity to the two planktonic species was observed. PS-COOH formed micro-scale aggregates (Z-Average>1µm) and did not affect the growth of microalgae up to 50µg/ml or that of brine shrimps up to 10µg/ml. However, these negatively charged NPs were adsorbed on microalgae and accumulated (and excreted) in brine shrimps, suggesting a potential trophic transfer from prey to predator. On the opposite, PS-NH2-formed nano-scale aggregates (Z-Average<200nm), caused inhibition of algal growth (EC50=12.97µg/ml) and mortality in brine shrimps at 14 d (LC50=0.83µg/ml). Moreover, 1µg/ml PS-NH2 significantly induced clap and cstb genes, explaining the physiological alterations (e.g. increase in molting) previously observed in 48h larvae, but also suggesting an apoptotic pathway triggered by cathepsin L-like protease in brine shrimps upon PS-NH2 exposure. These findings provide a first insight into long-term toxicity of nanoplastics to marine plankton, underlining the role of the surface chemistry in determining the behaviour and effects of PS NPs, in terms of adsorption, growth inhibition, accumulation, gene modulation and mortality. The use of long-term end-point has been identified as valuable tool for assessing the impact of nanoplastics on marine planktonic species, being more predictable of real exposure scenarios for risk assessment purposes.

Detecting the shape of anisotropic gold nanoparticles in dispersion with single particle extinction and scattering.

The shape and size of nanoparticles are important parameters affecting their biodistribution, bioactivity, and toxicity. The high-throughput characterisation of the nanoparticle shape in dispersion is a fundamental prerequisite for realistic in vitro and in vivo evaluation, however, with routinely available bench-top optical characterisation techniques, it remains a challenging task. Herein, we demonstrate the efficacy of a single particle extinction and scattering (SPES) technique for the in situ detection of the shape of nanoparticles in dispersion, applied to a small library of anisotropic gold particles, with a potential development for in-line detection. The use of SPES paves the way to the routine quantitative analysis of nanoparticles dispersed in biologically relevant fluids, which is of importance for the nanosafety assessment and any in vitro and in vivo administration of nanomaterials.

Towards a classification strategy for complex nanostructures.

The range of possible nanostructures is so large and continuously growing, that collating and unifying the knowledge connected to them, including their biological activity, is a major challenge. Here we discuss a concept that is based on the connection of microscopic features of the nanomaterials to their biological impacts. We also consider what would be necessary to identify the features that control their biological interactions, and make them resemble each other in a biological context.

Phylogenetic and functional characterization of the distal intestinal microbiome of rainbow trout Oncorhynchus mykiss from both farm and aquarium settings.

AIMS: This study focused on comparing the phylogenetic composition and functional potential of the intestinal microbiome of rainbow trout sourced from both farm and aquarium settings. METHODS AND RESULTS: Samples of distal intestinal contents were collected from fish and subjected to high throughput 16S rRNA sequencing, to accurately determine the composition of the intestinal microbiome. The predominant phyla identified from both groups were Tenericutes, Firmicutes, Proteobacteria, Spirochaetae and Bacteroidetes. A novel metagenomic tool, PICRUSt, was used to determine the functional potential of the bacterial communities present in the rainbow trout intestine. Pathways concerning membrane transport activity were dominant in the intestinal microbiome of all fish samples. Furthermore, this analysis revealed that gene pathways relating to metabolism, and in particular amino acid and carbohydrate metabolism, were upregulated in the rainbow trout intestinal microbiome. CONCLUSIONS: The results suggest that the structure of the intestinal microbiome in farmed rainbow trout may be similar regardless of where the fish are located and hence could be shaped by host factors. Differences were, however, noted in the microbial community membership within the intestine of both fish populations, suggesting that more sporadic taxa could be unique to each environment and may have the ability to colonize the rainbow trout gastrointestinal tract. Finally, the functional analysis provides evidence that the microbiome of rainbow trout contains genes that could contribute to the metabolism of dietary ingredients and therefore may actively influence the digestive process in these fish. SIGNIFICANCE AND IMPACT OF THE STUDY: To better understand and exploit the intestinal microbiome and its impact on fish health, it is vital to determine its structure, diversity and potential functional capacity. This study improves our knowledge of these areas and suggests that the intestinal microbiome of rainbow trout may play an important role in the digestive physiology of these fish.

Effect of zinc imprinting and replacing inorganic zinc with organic zinc on early performance of broiler chicks.

The goal of this study was to determine the effects of feeding a zinc (Zn) deficient diet to broiler chicks for 96 h post-hatch followed by feeding diets with different Zn sources and supplemental levels (5 to 21 d) on the growth performance, tissue, and excreta Zn content. At the start of the study, four hundred 20-day-old male broiler chicks were divided into two groups. One group was fed a corn soybean meal based diet containing 25 mg of Zn/kg (imprinting diet, ID). The second group was fed the basal diet supplemented with 40 mg of Zn/kg from Zn oxide (ZnO) (non-imprinting diet, NID). Both groups were fed these diets for 96 h. At d 5, chicks from each group were randomly assigned to the dietary treatments consisting of the basal diet alone or the basal diet supplemented with 8 or 40 mg/kg Zn as ZnO or Zn proteinate. Main effects of post-hatch Zn ID were observed on feed intake and G:F. ID decreased (P < 0.05) feed intake and improved (P < 0.05) the gain to feed ratio (G:F) of 14 and 21 d old chicks compared to G:F of chicks fed NID. Additionally, G:F for 14 and 21 d was improved (P < 0.05) by interaction of Zn source × level. Furthermore, at d 21 chicks fed the ID had a lower (P < 0.05) Zn content in the tibia ash and excreta, and a higher (P < 0.05) Zn content in the pancreas tissue compared to chicks fed NID. These results suggest that Zn imprinting can affect body Zn stores and early performance.

Fibre type-dependent response of broiler muscles to dietary antioxidant supplementation for oxidative stability enhancement.

The influence of dietary antioxidants and quality of oil on the oxidative and physico-chemical properties of chicken broiler breast and thigh meat stored was studied in either an oxygen-enriched (HiOx: 80% O2/20% CO2) or an air-permeable polyvinylchloride (PVC) packaging system during retail display at 2-4°C for up to 14 and 7 d, respectively. Broilers were fed on a diet with either a low-oxidised (peroxide value (POV) 23 meq O2/kg) or a high-oxidised (POV 121 meq O2/kg) oil, supplemented with or without an algae/selenium-based antioxidant with organic minerals, for 42 d. Lipid and protein oxidation, myofibrillar protein profile and purge loss were analysed. In both packaging systems, lipid oxidation (thiobarbituric acid-reactive substances [TBARS]) was inhibited by up to 65% and 57% in chicken breast and thigh, respectively, with an antioxidant-supplemented diet compared to those without. In both breast and thigh samples, protein sulfhydryls and water-holding capacity (purge loss) were better protected by the antioxidant dietary treatment, regardless of oil quality. Thigh muscles had up to sevenfold greater TBARS formation and more myosin heavy chain losses compared to breast samples. Antioxidant supplementation was more protective against lipid oxidation and water-holding capacity in the group fed on high-oxidised oil compared to those fed on low-oxidised oil. The results suggest that dietary antioxidants can minimise the negative impact of oxidised oil on broiler meat quality, and this protection was more pronounced for thigh than breast muscle, indicating inherent variations between muscle fibre types.

Characterization of the bionano interface and mapping extrinsic interactions of the corona of nanomaterials.

Nanoparticles in physiological environments are known to selectively adsorb proteins and other biomolecules forming a tightly bound biomolecular 'corona' on their surface. Where the exchange times of the proteins are sufficiently long, it is believed that the protein corona constitutes the particle identity in biological milieu. Here we show that proteins in the corona retain their functional characteristics and can specifically bind to cognate proteins on arrays of thousands of immobilised human proteins. The biological identity of the nanomaterial is seen to be specific to the blood plasma concentration in which they are exposed. We show that the resulting in situ nanoparticle interactome is dependent on the protein concentration in plasma, with the emergence of a small number of dominant protein-protein interactions. These interactions are those driven by proteins that are adsorbed onto the particle surface and whose binding epitopes are subsequently expressed or presented suitably on the particle surface. We suggest that, since specific tailored protein arrays for target systems and organs can be designed, their use may be an important element in an overall study of the biomolecular corona.

Evidence for immunomodulation and apoptotic processes induced by cationic polystyrene nanoparticles in the hemocytes of the marine bivalve Mytilus.

Polymeric nanoparticles can reach the marine environment from different sources as weathering of plastic debris and nanowaste. Nevertheless, few data are available on their fate and impact on marine biota. Polystyrene nanoparticles (PS NPs) can be considered as a model for studying the effects of nanoplastics in marine organisms: recent data on amino-modified PS NPs (PS-NH2) toxicity in sea urchin embryos underlined that marine invertebrates can be biological targets of nanoplastics. Cationic PS NPs have been shown to be toxic to mammalian cells, where they can induce apoptotic processes; however, no information is available on their effects and mechanisms of action in the cells of marine organisms. In this work, the effects of 50 nm PS-NH2 were investigated in the hemocytes of the marine bivalve Mytilus galloprovincialis. Hemocytes were exposed to different concentrations (1, 5, 50 µg/ml) of PS-NH2 suspension in ASW. Clear signs of cytoxicity were evident only at the highest concentrations (50 µg/ml). On the other hand, a dose dependent decrease in phagocytic activity and increase in lysozyme activity were observed. PS-NH2 NPs also stimulated increase in extracellular ROS (reactive oxygen species) and NO (nitric oxide) production, with maximal effects at lower concentrations. Moreover, at the highest concentration tested, PS-NH2 NPs induced apoptotic process, as evaluated by Flow cytometry (Annexin V binding and mitochondrial parameters). The results demonstrate that in marine invertebrates the immune function can represent a significant target for PS-NPs. Moreover, in Mytilus hemocytes, PS-NH2 NPs can act through mechanisms similar to those observed in mammalian cells. Further research is necessary on specific mechanisms of toxicity and cellular uptake of nanoplastics in order to assess their impact on marine biota.

Augmentation of water-holding and textural properties of breast meat from oxidatively stressed broilers by dietary antioxidant regimens.

1. The impact of dietary antioxidants and degree of oil oxidation on textural attributes of chicken broiler breast meat stored in oxygen-enriched, air-permeable polyvinylchloride and skin packaging systems during retail display at 2-4°C for up to 21 d was assessed. 2. Broilers were fed on diets either with a low-oxidised oil (peroxide 23 mEq O2/kg) or with a high-oxidised oil (peroxide 121 mEq O2/kg), with or without an algae-based antioxidant and organic mineral antioxidant supplement for 42 d. 3. Fatty acids and radical scavenging activities of the diets were estimated. Meat colour, pH, myofibrillar protein profile and textural traits were measured. 4. Diets with high-oxidised oil reduced stearic, linoleic and linolenic acid content compared to low-oxidised oil samples, regardless of antioxidant supplementation. Meat colour and pH varied among dietary treatments throughout storage. Meat samples from the antioxidant dietary group, irrespective of oil oxidation level, had lower amounts of purge and cooking losses compared to the unsupplemented diets. For all packaging systems, meat shear force was significantly higher for broilers fed on high-oxidised diets. 5. The results demonstrate that dietary antioxidant supplementation can minimise the negative impact of oxidised oil on the quality of broiler meat packaged in different atmospheric environments.

Accumulation and embryotoxicity of polystyrene nanoparticles at early stage of development of sea urchin embryos Paracentrotus lividus.

Nanoplastic debris, resulted from runoff and weathering breakdown of macro- and microplastics, represents an emerging concern for marine ecosystems. The aim of the present study was to investigate disposition and toxicity of polystyrene nanoparticles (NPs) in early development of sea urchin embryos (Paracentrotus lividus). NPs with two different surface charges where chosen, carboxylated (PS-COOH) and amine (PS-NH2) polystyrene, the latter being a less common variant, known to induce cell death in several in vitro cell systems. NPs stability in natural seawater (NSW) was measured while disposition and embryotoxicity were monitored within 48 h of postfertilization (hpf). Modulation of genes involved in cellular stress response (cas8, 14-3-3ε, p-38 MAPK, Abcb1, Abcc5) was investigated. PS-COOH forms microaggregates (PDI > 0.4) in NSW, whereas PS-NH2 results are better dispersed (89 ± 2 nm) initially, though they also aggregated partially with time. Their respectively anionic and cationic nature was confirmed by ζ-potential measurements. No embryotoxicity was observed for PS-COOH up to 50 µg mL(-1) whereas PS-NH2 caused severe developmental defects (EC50 3.85 µg mL(-1) 24 hpf and EC50 2.61 µg mL(-1) 48 hpf). PS-COOH accumulated inside embryo's digestive tract while PS-NH2 were more dispersed. Abcb1 gene resulted up-regulated at 48 hpf by PS-COOH whereas PS-NH2 induced cas8 gene at 24 hpf, suggesting an apoptotic pathway. In line with the results obtained with the same PS NPs in several human cell lines, also in sea urchin embryos, differences in surface charges and aggregation in seawater strongly affect their embryotoxicity.

Diagnostic nanoparticle targeting of the EGF-receptor in complex biological conditions using single-domain antibodies.

For effective localization of functionalized nanoparticles at diseased tissues such as solid tumours or metastases through biorecognition, appropriate targeting vectors directed against selected tumour biomarkers are a key prerequisite. The diversity of such vector molecules ranges from proteins, including antibodies and fragments thereof, through aptamers and glycans to short peptides and small molecules. Here, we analyse the specific nanoparticle targeting capabilities of two previously suggested peptides (D4 and GE11) and a small camelid single-domain antibody (sdAb), representing potential recognition agents for the epidermal growth factor receptor (EGFR). We investigate specificity by way of receptor RNA silencing techniques and look at increasing complexity in vitro by introducing increasing concentrations of human or bovine serum. Peptides D4 and GE11 proved problematic to employ and conjugation resulted in non-receptor specific uptake into cells. Our results show that sdAb-functionalized particles can effectively target the EGFR, even in more complex bovine and human serum conditions where targeting specificity is largely conserved for increasing serum concentration. In human serum however, an inhibition of overall nanoparticle uptake is observed with increasing protein concentration. For highly affine targeting ligands such as sdAbs, targeting a receptor such as EGFR with low serum competitor abundance, receptor recognition function can still be partially realised in complex conditions. Here, we stress the value of evaluating the targeting efficiency of nanoparticle constructs in realistic biological milieu, prior to more extensive in vivo studies.

The effect of selenium supplementation on vaccination response and immune function in adult horses.

Selenium status has been reported to affect immune function across many different species. Yet few studies have focused on the effect of Se status on the equine immune system. This study examined the effect of Se supplementation on vaccination response and immune function in mature horses. Twenty-eight horses were blocked by age and sex and were randomly allocated to 1 of 4 dietary treatment groups: low Se (LS), adequate Se (AS), Se-yeast (SP), and sodium selenite (SS). For 35 wk, horses allocated to LS, SP, and SS received a low-Se diet (0.06 mg/kg DM) with the intention to lower Se stores, whereas AS received an adequate Se diet (0.12 mg/kg DM). A 29-wk repletion phase was as follows: LS and AS were kept on the diets fed during the depletion period, whereas SP and SS received the depletion diet plus their respective Se supplements to achieve a dietary Se concentration of 0.3 mg/kg DM. The Se status of the horses was monitored using whole blood Se and glutathione peroxidase (GSH-Px) activity as indicators. At wk 22 and 25 of the repletion phase, horses were vaccinated intramuscularly with 10 mg ovalbumin (OVA). Horses were also vaccinated against equine influenza at wk 25. Blood samples were collected for 7 wk after initial vaccination for serum separation and at 0, 3, and 5 wk postvaccination for peripheral blood mononuclear cell (PBMC) isolation and whole blood cytokine mRNA evaluation. At wk 22 of the repletion phase, both Se and GSH-Px were greater for SP and SS compared with AS and LS (P < 0.001). Serum vitamin E was similar between treatments. Response to OVA vaccination, evaluated as OVA-specific IgG production, cytokine mRNA expression of PBMC stimulated with OVA in vitro, and lymphocyte proliferation, was unaffected by Se status. Similarly, memory response to the influenza vaccine was not affected by Se status. However, decreased mRNA expression of selected cytokines was observed in PBMC stimulated with phorbol 12-myristate 13-acetate for LS compared with AS, SP, and SS (P < 0.05). Whole blood mRNA expression of IL-10 was greater for SS compared with LS, AS, and SP (P = 0.043). Although the OVA and influenza vaccination responses were unaffected by Se status, other measures of immune function did indicate that low Se status could adversely affect cell-mediated immunity.

Measures of antioxidant status of the horse in response to selenium depletion and repletion.

Selenium plays a role in the antioxidant mechanism via the selenoenzyme glutathione peroxidase (GSH-Px). Change in Se status because of Se depletion or supplementation is associated with a change in GSH-Px activity and could potentially affect antioxidant status. This study evaluated the impact of change in Se status on measures of antioxidant status and oxidative stress in adult horses. Twenty-eight horses were blocked by age and gender and were randomly allocated to 1 of 4 dietary treatment groups: low Se (LS), adequate Se (AS), high organic Se (SP), and high inorganic Se (SS). For 196 d, LS, SP, and SS received a low-Se diet (0.06 mg Se/kg DM) to allow for depletion of Se stores, whereas AS received an adequate Se diet (0.12 mg Se/kg DM). Then, for the next 189 d, LS and AS were maintained on the same diets, whereas SP was supplemented with Se-yeast and SS with sodium selenite to allow for a total dietary Se intake of 0.3 mg Se/kg DM. Blood samples were collected throughout the study. Variables of interest included whole blood Se and GSH-Px activity, serum vitamin E concentration, total antioxidant capacity (TAC), serum malondialdehyde (MDA), and triiodothyronine and thyroxine concentrations. Data were analyzed using ANOVA with repeated measures. Whole blood Se and GSH-Px activity decreased in LS, SP, and SS during the depletion phase and increased in SP and SS with supplementation (treatment × time, P < 0.001). At the conclusion of the supplementation period, GSH-Px activity was greater in SP and SS compared with AS and LS (P < 0.05). Vitamin E status remained adequate throughout the study, and no differences existed between treatments. Serum TAC did not change in response to Se depletion or repletion. Serum MDA was greater for AS than LS during depletion (P < 0.05) but similar across treatments after supplementation. Overall, change in Se status did not have a large impact on TAC or MDA, possibly because the horses maintained an adequate vitamin E status. However, Se supplementation at 0.3 mg/kg DM increased GSH-Px activity above that of the horses fed an adequate diet based on the 2007 NRC recommendations, indicating a potential benefit to feeding greater Se diets to horses kept in low-Se areas.