Mini-Collagens in Hydra Nematocytes

We have isolated and characterized four collagen-related c-DNA clones (N-COL 1, N-COL 2, N-COL 3, N-COL 4) that are highly expressed in developing nematocytes in hydra. All four c-DNAs as well as their corresponding transcripts are small in size (600-1,000 bp). The deduced amino acid sequences show that they contain a central region consisting of 14 to 16 Gly-X-Y triplets. This region is flanked amino-terminal by a stretch of 14-23 proline residues and carboxy-terminal by a stretch of 6-9 prolines. At the NH2- and COOH-termini are repeated patterns of cysteine residues that are highly conserved between the molecules. A model is proposed which consists of a central stable collagen triple helix of 12-14 nm length from which three 9-22 nm long polyproline II type helices emerge at both ends. Disulfide linkage between cysteine- rich segments in these helices could lead to the formation of oligomeric network structures. Electrophoretic characterization of nematocyst extracts allows resolution of small proline-rich polypeptides that correspond in size to the cloned sequences

Taylor dispersion of nanoparticles

The ability to detect and accurately characterize particles is required by many fields of nanotechnology, including materials science, nanotoxicology, and nanomedicine. Among the most relevant physicochemical properties of nanoparticles, size and the related surface-to-volume ratio are fundamental ones. Taylor dispersion combines three independent phenomena to determine particle size: optical extinction, translational diffusion, and sheer-enhanced dispersion of nanoparticles subjected to a steady laminar flow. The interplay of these defines the apparent size. Considering that particles in fact are never truly uniform nor monodisperse, we rigorously address particle polydispersity and calculate the apparent particle size measured by Taylor dispersion analysis. We conducted case studies addressing aqueous suspensions of model particles and large-scale-produced “industrial” particles of both academic and commercial interest of various core materials and sizes, ranging from 15 to 100 nm. A comparison with particle sizes determined by transmission electron microscopy confirms that our approach is model-independent, non-parametric, and of general validity that provides an accurate account of size polydispersity—independently on the shape of the size distribution and without any assumption required a priori

Dynamic and biocompatible thermo-responsive magnetic hydrogels that respond to an alternating magnetic field

Magnetic thermo-responsive hydrogels are a new class of materials that have recently attracted interest in biomedicine due to their ability to change phase upon magnetic stimulation. They have been used for drug release, magnetic hyperthermia treatment, and can potentially be engineered as stimuli-responsive substrates for cell mechanobiology. In this regard, we propose a series of magnetic thermo-responsive nanocomposite substrates that undergo cyclical swelling and de-swelling phases when actuated by an alternating magnetic field in aqueous environment. The synthetized substrates are obtained with a facile and reproducible method from poly-N- isopropylacrylamide and superparamagnetic iron oxide nanoparticles. Their conformation and the temperature-related, magnetic, and biological behaviors were characterized via scanning electron microscopy, swelling ratio analysis, vibrating sample magnetometry, alternating magnetic field stimulation and indirect viability assays. The nanocomposites showed no cytotoxicity with fibroblast cells, and exhibited swelling/de-swelling behavior near physiological temperatures (around 34 °C). Therefore these magnetic thermo-responsive hydrogels are promising materials as stimuli-responsive substrates allowing the study of cell-behavior by changing the hydrogel properties in situ

Integrating silver compounds and nanoparticles into ceria nanocontainers for antimicrobial applications

Silver compounds and nanoparticles (NPs) are gaining increasing interest in medical applications, specifically in the treatment and prevention of biomaterial-related infections. However, the silver release from these materials, resulting in a limited antimicrobial activity, is often difficult to control. In this paper, ceria nanocontainers were synthesized by a template-assisted method and were then used to encapsulate silver nitrate (AgNO₃/CeO₂ nanocontainers). Over the first 30 days, a significant level of silver was released, as determined using inductively coupled plasma optical emission spectroscopy (ICP-OES). A novel type of ceria container containing silver NPs (AgNP/CeO₂ containers) was also developed using two different template removal methods. The presence of AgNPs was confirmed both on the surface and in the interior of the ceria containers by X-ray diffraction (XRD), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Upon removal of the template by calcination, the silver was released over a period exceeding three months (>90 days). However, when the template was removed by dissolution, the silver release was shortened to ≤14 days. The antimicrobial activity of the silver-containing CeO₂ containers was observed and the minimum bactericidal concentration (MBC) was determined using the broth dilution method. Investigation on human cells, using a model epithelial barrier cell type (A549 cells), highlighted that all three samples induced a heightened cytotoxicity leading to cell death when exposed to all containers in their raw form. This was attributed to the surface roughness of the CeO₂ nanocontainers and the kinetics of the silver release from the AgNO₃/CeO₂ and AgNP/CeO₂ nanocontainers. In conclusion, despite the need for further emphasis on their biocompatibility, the concept of the AgNP/CeO₂ nanocontainers offers a potentially alternative long-term antibactericidal strategy for implant materials

Size-dependent accumulation of particles in lysosomes modulates dendritic cell function through impaired antigen degradation

Introduction: Nanosized particles may enable therapeutic modulation of immune responses by targeting dendritic cell (DC) networks in accessible organs such as the lung. To date, however, the effects of nanoparticles on DC function and downstream immune responses remain poorly understood. Methods: Bone marrow–derived DCs (BMDCs) were exposed in vitro to 20 or 1,000 nm polystyrene (PS) particles. Particle uptake kinetics, cell surface marker expression, soluble protein antigen uptake and degradation, as well as in vitro CD4⁺ T-cell proliferation and cytokine production were analyzed by flow cytometry. In addition, co-localization of particles within the lysosomal compartment, lysosomal permeability, and endoplasmic reticulum stress were analyzed. Results: The frequency of PS particle–positive CD11c⁺/CD11b⁺ BMDCs reached an early plateau after 20 minutes and was significantly higher for 20 nm than for 1,000 nm PS particles at all time-points analyzed. PS particles did not alter cell viability or modify expression of the surface markers CD11b, CD11c, MHC class II, CD40, and CD86. Although particle exposure did not modulate antigen uptake, 20 nm PS particles decreased the capacity of BMDCs to degrade soluble antigen, without affecting their ability to induce antigen-specific CD4⁺ T-cell proliferation. Co-localization studies between PS particles and lysosomes using laser scanning confocal microscopy detected a significantly higher frequency of co-localized 20 nm particles as compared with their 1,000 nm counterparts. Neither size of PS particle caused lysosomal leakage, expression of endoplasmic reticulum stress gene markers, or changes in cytokines profiles. Conclusion: These data indicate that although supposedly inert PS nanoparticles did not induce DC activation or alteration in CD4⁺ T-cell stimulating capacity, 20 nm (but not 1,000 nm) PS particles may reduce antigen degradation through interference in the lysosomal compartment. These findings emphasize the importance of performing in-depth analysis of DC function when developing novel approaches for immune modulation with nanoparticles.

Visual Cycle: Dependence of Retinol Production and Removal on Photoproduct Decay and Cell Morphology

The visual cycle is a chain of biochemical reactions that regenerate visual pigment following exposure to light. Initial steps, the liberation of all-trans retinal and its reduction to all-trans retinol by retinol dehydrogenase (RDH), take place in photoreceptors. We performed comparative microspectrophotometric and microfluorometric measurements on a variety of rod and cone photoreceptors isolated from salamander retinae to correlate the rates of photoproduct decay and retinol production. Metapigment decay rate was spatially uniform within outer segments and 50–70 times faster in the cells that contained cone-type pigment (SWS2 and M/LWS) compared to cells with rod-type pigment (RH1). Retinol production rate was strongly position dependent, fastest at the base of outer segments. Retinol production rate was 10–40 times faster in cones with cone pigments (SWS2 and M/LWS) than in the basal OS of rods containing rod pigment (RH1). Production rate was approximately five times faster in rods containing cone pigment (SWS2) than the rate in basal OS of rods containing the rod pigment (RH1). We show that retinol production is defined either by metapigment decay rate or RDH reaction rate, depending on cell type or outer segment region, whereas retinol removal is defined by the surface-to-volume ratio of the outer segment and the availability of retinoid binding protein (IRBP). The more rapid rates of retinol production in cones compared to rods are consistent with the more rapid operation of the visual cycle in these cells

Decoupling the shape parameter to assess gold nanorod uptake by mammalian cells

The impact of nanoparticles (NPs) upon biological systems can be fundamentally associated with their physicochemical parameters. A further often-stated tenet is the importance of NP shape on rates of endocytosis. However, given the convoluted parameters concerning the NP–cell interaction, it is experimentally challenging to attribute any findings to shape alone. Herein we demonstrate that shape, below a certain limit, which is specific to nanomedicine, is not important for the endocytosis of spherocylinders by either epithelial or macrophage cells in vitro. Through a systematic approach, we reshaped a single batch of gold nanorods into different aspect ratios resulting in near-spheres and studied their cytotoxicity, (pro-)inflammatory status, and endocytosis/exocytosis. It was found that on a length scale of ∼10–90 nm and at aspect ratios less than 5, NP shape has little impact upon their entry into either macrophages or epithelial cells. Conversely, nanorods with an aspect ratio above 5 were preferentially endocytosed by epithelial cells, whereas there was a lack of shape dependent uptake following exposure to macrophages in vitro. These findings have implications both in the understanding of nanoparticle reshaping mechanisms, as well as in the future rational design of nanomaterials for biomedical applications

Feed Restriction Modulates the Fecal Microbiota Composition, Nutrient Retention, and Feed Efficiency in Chickens Divergent in Residual Feed Intake

Publication histroy: Accepted - 23 October 2018; Published - 19 November 2019.There is a great interest to understand the impact of the gut microbiota on host’s nutrient use and FE in chicken production. Both chicken’s feed intake and gut bacterial microbiota differ between high and low-feed efficient chickens. To evaluate the impact of the feed intake level on the feed efficiency (FE)-associated variation in the chicken intestinal microbiota, differently feed efficient chickens need to eat the same amount of feed, which can be achieved by feeding chickens restrictively. Therefore, we investigated the effect of restrictive vs. ad libitum feeding on the fecal microbiome at 16 and 29 days posthatch (dph), FE and nutrient retention in chickens of low and high residual feed intake (RFI; metric for FE). Restrictively fed chickens were provided the same amount of feed which corresponded to 85% of the ad libitum fed group from 9 dph. FE was determined for the period between 9 and 30 dph and feces for nutrient retention were collected on 31 to 32 dph. From the 112 chickens (n = 56 fed ad libitum, and n = 56 fed restrictively), 14 low RFI and 15 high RFI ad libitum fed chickens, and 14 low RFI (n = 7 per sex) and 14 high RFI restrictively fed chickens were selected as the extremes in RFI and were retrospectively chosen for data analysis. Bray-Curtis dissimilarity matrices showed significant separation between time points, and feeding level groups at 29 dph for the fecal bacterial communities. Relevance networking indicated positive associations between Acinetobacter and feed intake at 16 dph, whereas at 29 dph Escherichia/Shigella and Turicibacter positively and Lactobacillus negatively correlated to chicken’s feed intake. Enterobacteriaceae was indicative for low RFI at 16 dph, whereas Acinetobacter was linked to high RFI across time points. However, restrictive feeding-associated changes in the fecal microbiota were not similar in low and high RFI chickens, whichmay have been related to the higher nutrient retention and thus lower fecal nutrient availability in restrictively fed high RFI chickens. Thismay also explain the decreased RFI value in restrictively fed high RFI chickens indicating improved FE, with a stronger effect in females.This project (ECO-FCE) has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 311794

Fecal Microbiota Transplant from Highly Feed-Efficient Donors Shows Little Effect on Age-Related Changes in Feed-Efficiency-Associated Fecal Microbiota from Chickens

peer-reviewedChickens with good or poor feed efficiency (FE) have been shown to differ in their intestinal microbiota composition. This study investigated differences in the fecal bacterial community of highly and poorly feed-efficient chickens at 16 and 29 days posthatch (dph) and evaluated whether a fecal microbiota transplant (FMT) from feed-efficient donors early in life can affect the fecal microbiota in chickens at 16 and 29 dph and chicken FE and nutrient retention at 4 weeks of age. A total of 110 chickens were inoculated with a FMT or a control transplant (CT) on dph 1, 6, and 9 and ranked according to residual feed intake (RFI; the metric for FE) on 30 dph. Fifty-six chickens across both inoculation groups were selected as the extremes in RFI (29 low, 27 high). RFI-related fecal bacterial profiles were discernible at 16 and 29 dph. In particular, Lactobacillus salivarius, Lactobacillus crispatus, and Anaerobacterium operational taxonomic units were associated with low RFI (good FE). Multiple administrations of the FMT only slightly changed the fecal bacterial composition, which was supported by weighted UniFrac analysis, showing similar bacterial communities in the feces of both inoculation groups at 16 and 29 dph. Moreover, the FMT did not change the RFI and nutrient retention of highly and poorly feedefficient recipients, whereas it tended to increase feed intake and body weight gain in female chickens. This finding suggests that host- and environment-related factors may more strongly affect chicken fecal microbiota and FE than the FMT.European Union Seventh Framework Programm

Feed Restriction Modifies Intestinal Microbiota-Host Mucosal Networking in Chickens Divergent in Residual Feed Intake

Publication history: Accepted online - 8 January 2019; Published online - 29 January 2019.Differences in chickens’ feed intake may be the underlying factor influencing feed-efficiency (FE)-associated variation in intestinal microbiota and physiology. In chickens eating the same amount of feed, quantitative feed restriction may create similar intestinal conditions and help clarify this cause-and-effect relationship. This study investigated the effect of ad libitum versus restrictive feeding (85% of ad libitum) on ileal and cecal microbiota, concentrations of short-chain fatty acids, visceral organ size, intestinal morphology, permeability, and expression of genes related to nutrient uptake, barrier function, and innate immune response in broiler chickens with divergent residual feed intake (RFI; metric for FE). On day 30 posthatch, 28 low-RFI (good FE) and 29 high-RFI (poor FE) chickens across both feedinglevel groups (n 112) were selected. Supervised multigroup data integration and relevance network analyses showed that especially Lactobacillus (negative) in ileal digesta, Turicibacter (positive) in cecal digesta, and Enterobacteriaceae (positive) in both intestinal segments depended on chicken’s feed intake, whereas the level of Anaerotruncus in cecal digesta was most discriminative for high RFI. Moreover, shallower crypts and fewer goblet cells in ceca indicated host-related energy-saving mechanisms with low RFI, whereas greater tissue resistance suggested a stronger jejunal barrier function in low-RFI chickens. Values corresponding to feed intake level RFI interactions indicated larger pancreas and lower levels of ileal and cecal short-chain fatty acids in restrictively fed high-RFI chickens than in the other 3 groups, suggesting host physiological adaptations to support greater energy and nutrient needs of high-RFI chickens compensating for the restricted feeding. IMPORTANCE The impact of the FE-associated differences in feed intake on intestinal bacterial and host physiological parameters has so far not been clarified. Understanding the underlying principles is essential for the development of cost-effective strategies to improve FE in chicken production. Under conditions of quantitative feed restriction, low- and high-RFI chickens ate the same amount of feed. Therefore, this research helps in distinguishing intestinal bacterial taxa and functions that were highly reliant on feed intake from those that were associated with physiological adaptations to RFI-associated differences in host nutritional needs and intestinal nutrient availability. This work provides a background for further research to assess manipulation of the intestinal microbiota, host physiology, and FE in chickens by dietary intervention.This project (ECO-FCE) has received funding from the European Union’s Seventh Framework Program for research, technological development, and demonstration under grant agreement 311794