Mixotrophic flagellate ingestion boosts microplastic accumulation in ascidians.

Microplastics are contaminants of global environmental concern. They can be ingested by a variety of organisms when they enter the food web. Several studies have reported trophic transfer of microplastics from low trophic levels to higher ones. Bioaccumulation has been suggested to occur but few studies have demonstrated it for marine environments. In this article, in controlled laboratory conditions, we exposed filter-feeder ascidian juveniles to microplastics in the presence or in absence of mixotrophic cryptomonad flagellates. Cryptomonads can efficiently ingest microbeads, and their presence significantly increased the concentration of microplastics in the digestive tract of the ascidians. Our results demonstrate the occurrence of microplastic bioaccumulation in the lower levels of the marine trophic chain and suggest that unicellular organisms can be key actors in microplastic trophic transfer at the microscale level.

A New Disease Caused by an Unidentified Etiological Agent Affects European Salamanders.

New pathologies are causing dramatic declines and extinctions of multiple amphibian species. In 2013, in one fire salamander population of Northern Italy, we found individuals with undescribed cysts at the throat level, a malady whose existence has not previously been reported in amphibians. With the aim of describing this novel disease, we performed repeated field surveys to assess the frequency of affected salamanders from 2014 to 2020, and integrated morphological, histological, and molecular analyses to identify the pathogen. The novel disease affected up to 22% of salamanders of the study population and started spreading to nearby populations. Cysts are formed by mucus surrounding protist-like cells about 30 µm long, characterized by numerous cilia/undulipodia. Morphological and genetic analyses did not yield a clear match with described organisms. The existence of this pathogen calls for the implementation of biosecurity protocols and more studies on the dynamics of transmission and the impact on wild populations.

Fluorescence Properties of a Novel Isoquinoline Derivative Tested in an Invertebrate Chordate, Ciona intestinalis.

3-Hydroxyisoquinolines (ISOs) and their tautomeric isoquinolin-3-ones are heterocycles with attractive biological properties. Here we reported the revisited synthesis of a highly functionalized ISO that showed blue fluorescence and the characterization of its biological properties in an invertebrate animal model, the ascidian Ciona intestinalis. Larvae exposed to ISO at concentrations higher than 1 µM showed an intense fluorescence localized in the cell nuclei of all tissues. Moreover, exposure to ISO interfered with larval ability to swim; this neuromuscular effect was reversible. Overall, these results suggested that ISOs can have promising applications as novel fluorescent dyes of the cell nuclei.

Exploring miR-9 Involvement in Ciona intestinalis Neural Development Using Peptide Nucleic Acids.

The microRNAs are small RNAs that regulate gene expression at the post-transcriptional level and can be involved in the onset of neurodegenerative diseases and cancer. They are emerging as possible targets for antisense-based therapy, even though the in vivo stability of miRNA analogues is still questioned. We tested the ability of peptide nucleic acids, a novel class of nucleic acid mimics, to downregulate miR-9 in vivo in an invertebrate model organism, the ascidian Ciona intestinalis, by microinjection of antisense molecules in the eggs. It is known that miR-9 is a well-conserved microRNA in bilaterians and we found that it is expressed in epidermal sensory neurons of the tail in the larva of C. intestinalis. Larvae developed from injected eggs showed a reduced differentiation of tail neurons, confirming the possibility to use peptide nucleic acid PNA to downregulate miRNA in a whole organism. By identifying putative targets of miR-9, we discuss the role of this miRNA in the development of the peripheral nervous system of ascidians.

miR-7 Knockdown by Peptide Nucleic Acids in the Ascidian Ciona intestinalis.

Peptide Nucleic Acids (PNAs) are synthetic mimics of natural oligonucleotides, which bind complementary DNA/RNA strands with high sequence specificity. They display numerous advantages, but in vivo applications are still rare. One of the main drawbacks of PNAs application is the poor cellular uptake that could be overcome by using experimental models, in which microinjection techniques allow direct delivery of molecules into eggs. Thus, in this communication, we investigated PNAs efficiency in miR-7 downregulation and compared its effects with those obtained with the commercially available antisense molecule, Antagomir (Dharmacon) in the ascidian Ciona intestinalis. Ascidians are marine invertebrates closely related to vertebrates, in which PNA techniques have not been applied yet. Our results suggested that anti-miR-7 PNAs were able to reach their specific targets in the developing ascidian embryos with high efficiency, as the same effects were obtained with both PNA and Antagomir. To the best of our knowledge, this is the first evidence that unmodified PNAs can be applied in in vivo knockdown strategies when directly injected into eggs.

N-mixture models reliably estimate the abundance of small vertebrates.

Accurate measures of species abundance are essential to identify conservation strategies. N-mixture models are increasingly used to estimate abundance on the basis of species counts. In this study we tested whether abundance estimates obtained using N-mixture models provide consistent results with more traditional approaches requiring capture (capture-mark recapture and removal sampling). We focused on endemic, threatened species of amphibians and reptiles in Italy, for which accurate abundance data are needed for conservation assessments: the Lanza's Alpine salamander Salamandra lanzai, the Ambrosi's cave salamander Hydromantes ambrosii and the Aeolian wall lizard Podarcis raffonei. In visual counts, detection probability was variable among species, ranging between 0.14 (Alpine salamanders) and 0.60 (cave salamanders). For all the species, abundance estimates obtained using N-mixture models showed limited differences with the ones obtained through capture-mark-recapture or removal sampling. The match was particularly accurate for cave salamanders in sites with limited abundance and for lizards, nevertheless non-incorporating heterogeneity of detection probability increased bias. N-mixture models provide reliable abundance estimates that are comparable with the ones of more traditional approaches, and offer additional advantages such as a smaller sampling effort and no need of manipulating individuals, which in turn reduces the risk of harming animals and spreading diseases.

Teratogenic potential of nanoencapsulated vitamin A evaluated on an alternative model organism, the tunicate Ciona intestinalis.

Nano-encapsulation is a technology used to pack substances in order to enhance their stability and bioavailability, but this packing may interact with living systems, causing unexpected toxicity. Vitamin A (vit A) is a substance that has received attention, because in developed countries, the increasing availability of supplements is leading to its excessive intake. This study aims to compare teratogenic effects caused by exposure to the traditional formulation of vit A versus nano-encapsulated vit A. We used ascidian embryos as an alternative model. Ascidians are marine organisms closely related to vertebrates that share with them a body plan and developmental programme, including the morphogenetic role of retinoic acid (RA). Our data showed that the adverse effects of exposure to the same concentration of the two formulations were different, suggesting that the nano-encapsulation increased the bioavailability of the molecule, which could be better absorbed and metabolised to RA, the effective teratogenic substance.

Thermal equilibrium and temperature differences among body regions in European plethodontid salamanders.

Information on species thermal physiology is extremely important to understand species responses to environmental heterogeneity and changes. Thermography is an emerging technology that allows high resolution and accurate measurement of body temperature, but until now it has not been used to study thermal physiology of amphibians in the wild. Hydromantes terrestrial salamanders are strongly depending on ambient temperature for their activity and gas exchanges, but information on their body temperature is extremely limited. In this study we tested if Hydromantes salamanders are thermoconform, we assessed whether there are temperature differences among body regions, and evaluated the time required to reach the thermal equilibrium. During summers of 2014 and 2015 we analysed 56 salamanders (Hydromantes ambrosii and Hydromantes italicus) using infrared thermocamera. We photographed salamanders at the moment in which we found them and 1, 2, 3, 4, 5 and 15min after having kept them in the hands. Body temperature was equal to air temperature; salamanders attained the equilibrium with air temperature in about 8min, the time required to reach equilibrium was longer in individuals with large body size. We detected small temperature differences between body parts, the head being slightly warmer than the body and the tail (mean difference: 0.05°C). These salamanders quickly reach the equilibrium with the environment, thus microhabitat measurement allows obtaining accurate information on their tolerance limits.

Screening of the chemical composition and bioactivity of Waldheimia glabra (Decne.) Regel essential oil.

BACKGROUND: This research aimed at improving knowledge as to the chemical composition and the antibacterial and anti-cancer activities of the essential oil of Waldheimia glabra, a wild plant from the Himalayan Mountains. RESULTS: The results obtained by GC-MS showed that spathulenol, 9-tetradecenol, thujopsene, α-thujone, santolina alcohol and terpinen-4-ol were the main constituents of Waldheimia glabra essential oil. These results were confirmed by HS-SPME GC-MS analysis that also reported high amounts of artemisia alcohol and camphor. Disc diffusion assay suggested a mild antibacterial activity against both Escherichia coli and Staphylococcus aureus. Finally, a dose-response correlation was observed between Waldhemia glabra essential oil concentration and viability of human breast adenocarcinoma cells MDA-MB-231 and MCF-7. CONCLUSIONS: Together with the GC-MS method, HS-SPME GC-MS proved to be a reliable technique to characterise the chemical composition of essential oil obtained from aromatic plants. Further studies will focus on W. glabra phytochemicals and their biological activity, in order to support traditional uses of the plant. © 2015 Society of Chemical Industry.

Inducible factors with antimicrobial activity after immune challenge in the haemolymph of Red Palm Weevil (Insecta).

Insects are capable of innate immune responses elicited after microbial infection. In this process, the receptor-mediated recognition of foreign bodies and the subsequent activation of immunocompetent cells lead to the synthesis ex novo of a peptide pool with antimicrobial activity. We investigated the inducible immune response of a coleopteran, Rhynchophorus ferrugineus, challenged with both Gram-negative and Gram-positive bacteria. After immunization, we evaluated the presence of antimicrobial peptides using either biochemical analyses or microbiological techniques. The antimicrobial properties of the newly synthesized protein pool, detectable in haemolymph fractions of low molecular mass, showed strong antibacterial activity against various bacterial strains (Escherichia coli, Pseudomonas sp. OX1, Bacillus subtilis and Micrococcus luteus). In addition to the preliminary study of the mechanism of action of the pool of antimicrobial peptides, we also investigated its effects on bacterial cell walls by means of fluorescence microscopy and scanning electron microscopy. The data suggest that the main effects seem to be directed at destabilizing and damaging the bacterial wall. This study provides data that help us to understand some aspects of the inducible innate immunity in a system model that lacks anticipatory responses. However, the weevil has finely tuned its defensive strategies to counteract effectively microbial infection.

Gold nanoparticles capped by a GC-containing peptide functionalized with an RGD motif for integrin targeting.

Gold nanoparticles were obtained by reduction of a tetrachloroaurate aqueous solution in the presence of a RGD-(GC)(2) peptide as stabilizer. As comparison, the behavior of the (GC)(2) peptide has been studied. The (GC)(2) and RGD-(GC)(2) peptides were prepared ad hoc by Fmoc synthesis. The colloidal systems have been characterized by UV-visible, TGA, ATR-FTIR, mono and bidimensional NMR techniques, confocal and transmission (TEM) microscopy, ζ-potential, and light scattering measurements. The efficient cellular uptake of Au-RGD-(GC)(2) and Au-(GC)(2) stabilized gold nanoparticles into U87 cells (human glioblastoma cells) were investigated by confocal microscopy and compared with the behavior of (GC)(2) capped gold nanoparticles. A quantitative determination of the nanoparticles taken up has been carried out by measuring the pixel brightness of the images, a measure that highlighted the importance of the RGD termination of the peptide. Insight in the cellular uptake mechanism was investigated by TEM microscopy. Various important evidences indicated the selective uptake of RGD-(GC)(2) gold nanoparticles into the nucleus.

Growth factors, CD34 positive cells, and fibrin network analysis in concentrated growth factors fraction.

An interesting clinical option for optimizing healing tissue is the use of platelet concentrate. Platelets contain high quantities of growth factors, among these TGF-ß1 and VEGF, which are known to be implicated in tissue regeneration. CGF is produced by processing blood samples with a special centrifuge device; three layers are formed: top acellular plasma (PPP), middle CGF and bottom red blood cells (RBC) layers. Given that to date there are no data concerning the biological characteristic of CGF, the aim of this study was to evaluate the presence of TGF-ß1 and VEGF in CGF and also in PPP and RBC layers. In addition, since circulating stem cells are recruited from blood to injured tissue for healing we also evaluated the presence of CD34 positive cells. Our data show the presence of TGF-ß1 and VEGF in CGF and RBC layers. In addition, we show CD34 positive cells in CGF.

Phagocytosis of biocompatible gold nanoparticles.

We report the evidence for the cellular uptake of gold nanoparticles via the phagocytosis mechanism in murine macrophage cells strongly supported by TEM and optical microscopy. Nanoparticles were prepared using several biocompatible molecules of choice (5-aminovaleric acid, l-DOPA, melatonin, and serotonin hydrochloride) as stabilizers for gold colloids. Their surface chemistry was fully characterized by UV-vis, ATR-FTIR, (1)H NMR, and HR-MAS (1)H NMR spectroscopies, and size distribution was determined by CPS disc centrifuge and TEM. Differences in coatings were evaluated against cellular uptake, and a preferential movement of macrophages toward 5-aminovaleric acid-modified gold nanoparticles was shown, leading to the fast accumulation of nanoparticles in the cytosol.

Gold nanoparticles prepared using cape aloe active components.

A novel use of two components of Cape aloe, aloin A and aloesin, acting as stabilizers in the preparation of gold and silver nanoparticles, is reported. Stable water-soluble particles of different size and shape are prepared by varying the reaction conditions, temperature, reaction time, and reducing agents. Characterization of the obtained particles is performed using UV-visible, attenuated total reflection Fourier transform infrared (ATR-FTIR), and 1H NMR spectroscopies and transmission electron microscopy (TEM). The efficient cellular uptake of 50 nm sized aloin A and aloesin stabilized gold particles into macrophages and HeLa cells was investigated, proposing these particles as nanovehicles.

Gold-ligand interaction studies of water-soluble aminoalcohol capped gold nanoparticles by NMR.

Novel reproducible preparations of gold nanoparticles capped by aminoalcohols have been set up by reduction of HAuCl4 with NaBH4 in aqueous solution. The characterization of Au@aminoalcohol nanoparticles performed by 1H NMR, ATR-FTIR, UV-vis spectroscopies, and TEM microscopy highlighted the binding site and the nature of the Au-NH bond.