Recreating human skin in vitro: should the microbiota be taken into account?

Skin plays crucial roles in the human body: besides protecting the organism from external threats, it acts as a thermal regulator, is responsible for the sense of touch, hosts microbial communities (the skin microbiota) involved in preventing the invasion of foreign pathogens, contains immunocompetent cells that maintain a healthy immunogenic/tolerogenic balance, and is a suitable route for drug administration. In the skin, four defense levels can be identified: besides the physical, chemical, and immune barriers that are inherent to the tissue, the skin microbiota (i.e., the numerous microorganisms living on the skin surface) provides an additional barrier. Studying the skin barrier function or the effects of drugs or cosmetic agents on human skin is a difficult task since snapshot evidence can only be obtained using bioptic samples where dynamic processes cannot properly be followed. To overcome these limitations, many different in vitro models of human skin have been developed that are characterized by diverse levels of complexity in terms of chemical, structural, and cellular composition. The aim of this review is to summarize and discuss the advantages and disadvantages of the different human skin models so far available and to underline how the insertion of a proper microbiota would positively impact an in vitro human skin model in an attempt to better mimic conditions in vivo

Best Procedures for Leaf and Stem Water Potential Measurements in Grapevine: Cultivar and Water Status Matter

: The pressure chamber is the most used tool for plant water status monitoring. However, species/cultivar and seasonal effects on protocols for reliable water potential determination have not been properly tested. In four grapevine cultivars and two times of the season (early season, Es; late season, Ls, under moderate drought), we assessed the maximum sample storage time before leaf water potential (Ψleaf) measurements and the minimum equilibration time for stem water potential (Ψstem) determination, taking 24 h leaf cover as control. In 'Pinot gris', Ψleaf already decreased after 1 h leaf storage in both campaigns, dropping by 0.4/0.5 MPa after 3 h, while in 'Refosk', it decreased by 0.1 MPa after 1 and 2 h in Es and Ls, respectively. In 'Merlot' and 'Merlot Kanthus', even 3 h storage did not affect Ψleaf. In Es, the minimum Ψstem equilibration was 1 h for 'Refošk' and 10 min for 'Pinot gris' and 'Merlot'. In Ls, 'Merlot Kanthus' required more than 2 h equilibration, while 1 h to 10 min was sufficient for the other cultivars. The observed cultivar and seasonal differences indicate that the proposed tests should be routinely performed prior to experiments to define ad hoc procedures for water status determination

Early Antiangiogenic Activity of SU11248 Evaluated <i>In vivo</i> by Dynamic Contrast-Enhanced Magnetic Resonance Imaging in an Experimental Model of Colon Carcinoma

Abstract Purpose: To compare two dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) techniques in terms of their ability in assessing the early antiangiogenic effect of SU11248, a novel selective multitargeted tyrosine kinase inhibitor, that exhibits direct antitumor and antiangiogenic activity via inhibition of the receptor tyrosine kinases platelet-derived growth factor receptor, vascular endothelial growth factor receptor, KIT, and FLT3. Experimental Design: A s.c. tumor model of HT29 human colon carcinoma in athymic mice was used. Two DCE-MRI techniques were used based, respectively, on macromolecular [Gd-diethylenetriaminepentaacetic acid (DTPA)-albumin] and low molecular weight (Gd-DTPA) contrast agents. The first technique provided a quantitative measurement of transendothelial permeability and fractional plasma volume, accepted surrogate markers of tumor angiogenesis. With the second technique, we quantified the initial area under the concentration-time curve, which gives information related to tumor perfusion and vascular permeability. Experiments were done before and 24 hours after a single dose administration of SU11248. Results: The early antiangiogenic effect of SU11248 was detected by DCE-MRI with macromolecular contrast agent as a 42% decrease in vascular permeability measured in the tumor rim. The effect was also detected by DCE-MRI done with Gd-DTPA as a 31% decrease in the initial area under the concentration-time curve. Histologic slices showed a statistically significant difference in mean vessel density between the treated and control groups. Conclusions: The early antiangiogenic activity of SU11248 was detected in vivo by DCE-MRI techniques using either macromolecular or low molecular weight contrast agents. Because DCE-MRI techniques with low molecular weight contrast agents can be used in clinical studies, these results could be relevant for the design of clinical trials based on new paradigms

A Correlative Imaging Study of in vivo and ex vivo Biodistribution of Solid Lipid Nanoparticles

Purpose: Solid lipid nanoparticles are largely used in biomedical research and are characterized by high stability and biocompatibility and are also able to improve the stability of various loaded molecules. In vitro studies demonstrated that these nanoparticles are low cytotoxic, while in vivo studies proved their efficiency as nanocarriers for molecules characterized by a low bioavailability. However, to our knowledge, no data on the systemic biodistribution and organ accumulation of solid lipid nanoparticles in itself are presently available. Methods: In this view, we investigated the solid lipid nanoparticles biodistribution by a multimodal imaging approach correlating in vivo and ex vivo analyses. We loaded solid lipid nanoparticles with two different fluorophores (cardiogreen and rhodamine) to observe them with an optical imager in the whole organism and in the excised organs, and with fluorescence microscopy in tissue sections. Light and transmission electron microscopy analyses were also performed to evaluate possible structural modification or damage due to nanoparticle administration. Results: Solid lipid nanoparticles loaded with the two fluorochromes showed good optic characteristics and stable polydispersity. After in vivo administration, they were clearly detectable in the organism. Four hours after the injection, the fluorescent signal occurred in anatomical districts corresponding to the liver and this was confirmed by the ex vivo acquisitions of excised organs. Brightfield, fluorescence and transmission electron microscopy confirmed solid lipid nanoparticles accumulation in hepatocytes without structural damage. Conclusion: Our results support the systemic biocompatibility of solid lipid nanoparticles and demonstrate their detailed biodistribution from the whole organism to organs until the cells

An in vivo study of quantum dots tissue accumulation

Nanotechnology represents a new frontier for the science progress and there are great expectations in relation to diagnostic and therapeutic envelopes [1]. Living organisms are built of cells that are typically 10 \u3bcm across. However, the cell parts are much smaller and are in the sub-micron size domain, for example a red blood cell is approximately 7,000 nm wide. Even smaller are the proteins with a typical size of just 5 nm, which is comparable with the dimensions of smallest manmade nanoparticles. This simple size comparison gives an idea of using nanoparticles as very small probes that would allow us to spy at the cellular machinery without introducing too much interference. Understanding of biological processes on the nanoscale level is a strong driving force behind development of nanotechnologyOur current knowledge of the toxicology of nanoparticles in vivo is poor [2] but suggests that nanoparticles may able to have adverse effects at their portal of entry , for example, the lungs, but that some nanoparticles may also escape the normal defences and translocate from their portal of entry to have diverse effects in other target organs [3].There is no cut-off below witch particles suddenly become harmful, in the lung at least. This is because harmful particles have their effects as a consequence of two factors that act together to determine their potential to cause harm: their large surface area, and the reactivity or intrinsic toxicity of the surface. It is self evident that the smaller particles are, the more surface area they have per unit mass; therefore any intrinsic toxicity of the particles surface will be emphasised. Some of the most complex nanoparticles are likely to be produced for therapeutic purposes, furthermore nanoparticles binding to protein may result in a series of consequences not expected to occur when proteins bind to large particles. Very small particles may be not detected by the normal phagocytic defences, allowing them to gain access to the blood or nervous system [4]. Very small particles are smaller than some molecules and could act like haptens to modify protein structures, either altering their function or rendering them antigenic, raising the potential for autoimmune effects.Tracers that we have used are nanoparticles with optical properties, fluorescent semiconductors, that absorb photons of light and re-emit photons at a different wavelength They are known as quantum dots (QDs), nanocrystals that are nanometres-scale (10-20nm, roughly protein-sized) atom clusters, containing from a few hundred to a few thousand atoms of a semiconductor material (cadmium mixed with selenium), which has been coated with an additional semiconductor shell (zinc sulfide) to improve the optical properties of the material. These nanoparticles fluoresce in a different way than do traditional fluorophores, they exhibit some important differences as compared to organic fluorescent dyes and naturally fluorescent proteins: they have an extinction coefficient 10-50 times bigger than them. These nanoparticles projected around their optical properties: stable , bright and photo-stable fluorescence, observed and measured for hours, and that persists also into isolated tissues. Nanoparticles like QDs, could be targeted and not targeted and provided several unique features and capabilities[5, 6]: the size-effect does the QDs cancer biomarkers and there is the possibility to functionalize their surface area with a several numbers of functional groups that can be linked with multiple diagnostic (e.g. radio-isotopic or magnetic) and therapeutic agents. The aim of the study is to monitor nanoparticles behaviour into blood system: kinetic, T1/2, bio distribution, and tissues accumulation. We would extrapolate from optics parameters physiological ones, in specific districts so as liver and lungs that are the most probably targets of toxicity

Investigation of non-structural carbohydrates and xylem anatomy in petiole of grapevine varieties during water limitation and after re-irrigation

Water shortage (WS) during growing of Vitis vinifera L. can limit shoot growth and affect yield and fruit quality, as well as allocation of carbon reserves into perennial organs for the upcoming years. Varietal anatomical differences, such as specific mean xylem vessel diameter in petiole, are expected to influence water transport in canes facing water limitation. Several authors have also evidenced that non-structural carbohydrates (NSC) of adjacent living parenchyma are involved in the repair mechanism of embolized vessels. In this work, we evaluated NSC level and xylem anatomy in petiole of Cabernet Sauvignon and Syrah varieties, subjected to WS and subsequent water refilling in the summer of 2017. The anatomical analysis highlighted that Syrah had high frequency of classes of large vessels, and that the xylem differentiation of vascular bundles was also affected by WS. Moreover, petiole NSC content was significantly influenced by WS and recovery, supporting the hypothesis that starch mobilization was associated to an elevated concentration in soluble NSC. This effect was determinant for Cabernet Sauvignon, whose stress response seemed to be based mainly on NSC metabolism. Finally, Syrah, differently to Cabernet Sauvignon, sustained the WS-induced increase in soluble NSC of petiole also 18 h after re-watering

Biodistribution of Near-Infrared Fluorescent Nanoparticles:an in vivo study

Introduction: Research in new fluorescent nanoparticles is today an important field for preclinical studies in the optical imaging technique area. Great expectation concerns new fluorescent markers engeneered for particular applications (conjugated with antibody or pharmaceutical compounds) or alone to study nano-compound intrinsic behaviour in living organisms. In particular, nanosized fluorescent particles (silica nanopaticles1 and quantum dots2-3) are promising tools for the development of luminescent probes and markers provided by great brightness and high photostability respect to traditional organic fluorophores. Here we present an in vivo study of biodistribution in a small laboratory animal model of silica-core / PEG-shell fluorescent nanoparticles (20-30nm) doped with a CY7 NIR emitting dye ((2-((E)-2-((E)-2-chloro-3-((Z)-2-(3-ethyl-1,1-dimethyl-1H-benzo[e]indol-2(3H)-ylidene) ethylidene)cyclohex-1-enyl)vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indolium iodide). Silica particles, due to the recognized low toxicity of their chemical composition, could be interesting for future clinical applications. Methods: Silica fluorescent nanoparticles biodistribution was studied. We have observed with Optical Imager the biodistribution kinetics and tissue accumulation during three hours immediately after fluorescent tracer administration, in gas anaesthetized mice. Optical images were acquired with IVIS\uae 200 (Xenogen Corporation, Alameda USA). Data were extracted using Living Image 2.6 software. Silica nanoparticles, with an emission peak around 810 nm, were excited with ICG exc. filter (710-760 nm) and the fluorescent emission acquired with ICG ems. filter (810-875 nm). Results: Biodistribution kinetics and accumulation of the silica nanoparticles was studied in all anatomical districts4 for three hours after injection using the fluorescent signal escaping from the animal surface and acquired in the optical images. The fluorescent emission was measured on anatomical Region of Interest (ROIs) traced on the optical images corresponding to the plane projection of the organs. and directly on the surgically extracted organs. Actually we are analysing section from explanted organs with the aim of histologically localizing the exact accumulation sites and to detect the (nanoparticles) fluorescent signal. Conclusions: Fluorescent silica-core / PEG-shell nanoparticles showed a very good fluorescent efficiency comparable with commercial semiconductors nanocrystals (quantum dots, QDs) actually used in preclinical research. They can be successfully used for in vivo applications allowing to follow the biodistribution for hours in a small animal model. The very low intrinsic toxicity of the chemical composition encourages the employ of such fluorescent markers for many in vivo applications in preclinical research and to investigate the possibility to engineering them with biomelcules for targeting bio-analytical applications

Hyperthermic superparamagnetic nanoparticles modulate adipocyte metabolism

Adipocytes are the principal cellular component in adipose tissue and their excessive hyperplasia or hypertrophy is actively involved in regulating physiologic and pathologic processes such as inflammation, cardiovascular disease, obesity and tumour. The main depot of energy in adipocytes is represented by lipid droplets, intracellular organelles that play fundamental roles in regulation of metabolic processes. An accumulation of such droplets could be a potential biomarker of disease caused by metabolic dysregulation. Recent studies have demonstrated that heat shock is associated with alteration in energy metabolism: the aim of this study is to modulate the energy metabolism of the adipocytes via controlled administration of thermal energy to reduce the number of lipid droplets. We have investigated the effect of controlled heating of adipocytes using an alternating magnetic field (AMF) on samples loaded with superparamagnetic nanoparticles (MNP) as heating agent

Genetic diversity and connectivity of southern right whales (Eubalaena australis) found in the Brazil and Chile-Peru wintering grounds and the South Georgia (Islas Georgias del Sur) feeding ground

As species recover from exploitation, continued assessments of connectivity and population structure are warranted to provide information for conservation and management. This is particularly true in species with high dispersal capacity, such as migratory whales, where patterns of connectivity could change rapidly. Here we build on a previous long-term, large-scale collaboration on southern right whales (Eubalaena australis) to combine new (nnew) and published (npub) mitochondrial (mtDNA) and microsatellite genetic data from all major wintering grounds and, uniquely, the South Georgia (Islas Georgias del Sur: SG) feeding grounds. Specifically, we include data from Argentina (npub mtDNA/microsatellite=208/46), Brazil (nnew mtDNA/microsatellite=50/50), South Africa (nnew mtDNA/microsatellite=66/77, npub mtDNA/microsatellite=350/47), Chile-Peru (nnew mtDNA/microsatellite=1/1), the Indo-Pacific (npub mtDNA/microsatellite=769/126), and SG (npub mtDNA/microsatellite=8/0, nnew mtDNA/microsatellite=3/11) to investigate the position of previously unstudied habitats in the migratory network: Brazil, SG and Chile-Peru. These new genetic data show connectivity between Brazil and Argentina, exemplified by weak genetic differentiation and the movement of one genetically identified individual between the South American grounds. The single sample from Chile-Peru had a mtDNA haplotype previously only observed in the Indo-Pacific and had a nuclear genotype that appeared admixed between the Indo-Pacific and South Atlantic, based on genetic clustering and assignment algorithms. The SG samples were clearly South Atlantic, and were more similar to the South American than the South African wintering grounds. This study highlights how international collaborations are critical to provide context for emerging or recovering regions, like the SG feeding ground, as well as those that remain critically endangered, such as Chile-Peru

Antamanide, a Derivative of Amanita phalloides, Is a Novel Inhibitor of the Mitochondrial Permeability Transition Pore

Antamanide is a cyclic decapeptide derived from the fungus Amanita phalloides. Here we show that antamanide inhibits the mitochondrial permeability transition pore, a central effector of cell death induction, by targeting the pore regulator cyclophilin D. Indeed, (i) permeability transition pore inhibition by antamanide is not additive with the cyclophilin D-binding drug cyclosporin A, (ii) the inhibitory action of antamanide on the pore requires phosphate, as previously shown for cyclosporin A; (iii) antamanide is ineffective in mitochondria or cells derived from cyclophilin D null animals, and (iv) abolishes CyP-D peptidyl-prolyl cis-trans isomerase activity. Permeability transition pore inhibition by antamanide needs two critical residues in the peptide ring, Phe6 and Phe9, and is additive with ubiquinone 0, which acts on the pore in a cyclophilin D-independent fashion. Antamanide also abrogates mitochondrial depolarization and the ensuing cell death caused by two well-characterized pore inducers, clotrimazole and a hexokinase II N-terminal peptide. Our findings have implications for the comprehension of cyclophilin D activity on the permeability transition pore and for the development of novel pore-targeting drugs exploitable as cell death inhibitors