Human Drug Pollution in the Aquatic System: The Biochemical Responses of Danio rerio Adults.

To date, drug pollution in aquatic systems is an urgent issue, and Danio rerio is a model organism to study the toxicological effects of environmental pollutants. The scientific literature has analyzed the effect of human drug pollution on the biochemical responses in the tissues of D. rerio adults. However, the information is still scarce and conflicting, making it difficult to understand its real impact. The scientific studies are not consistent with each other and, until now, no one has grouped their results to create a baseline of knowledge of the possible impacts. In this review, the analysis of literature data highlights that the effects of drugs on adult zebrafishes depend on various factors, such as the tissue analyzed, the drug concentration and the sex of the individuals. Furthermore, the most influenced biochemical responses concern enzymes (e.g., antioxidants and hydrolase enzymes) and total protein and hormonal levels. Pinpointing the situation to date would improve the understanding of the chronic effects of human drug pollution, helping both to reduce it in the aquatic systems and then to draw up regulations to control this type of pollution.

Comparative study of coelomocytes from Arbacia lixula and Lythechinus variegatus: Cell characterization and in vivo evidence of the physiological function of vibratile cells.

The knowledge on echinoderm coelomocytes has increased in recent years, but researchers still face a complex problem: how to obtain purified cells. Even flow cytometry being useful to address coelomocytes in suspension, the need for a method able to provide isolated cells is still noteworthy. Here, we use Imaging Flow Cytometry (IFC) to characterize the coelomocytes of two sea urchin species - Arbacia lixula and Lytechinus variegatus - and obtain gates to isolate cell populations. Then, we used these gates to study the physiological response of A. lixula coelomocytes during an induced immune challenge with Escherichia coli. An analysis of area and aspect ratio parameters of the flow cytometer allowed the identification of two main cell populations in the coelomic fluid: circular and elongated cells. A combination of this method with nucleus labeling using propidium iodide allowed the determination of gates containing isolated subpopulations of vibratile cells, red spherulocytes, and two phagocytes subpopulations in both species. We observed that during an induced bacterial immune challenge, A. lixula was able to modulate coelomocyte frequencies, increasing the phagocytes and decreasing red spherulocytes and vibratile cells. These results indicate that vibratile cells and red spherulocytes act by immobilizing and stoping bacterial growth, respectively, cooperating with phagocytes in the immune response. The use of IFC was fundamental not only to identify specific gates for the main coelomic subpopulations but also allowed the investigation on how echinoids modulate their physiological responses during immune challenges. Furthermore, we provide the first experimental evidence about the role of vibratile cells, corroborating its involvement with the immune system.

Identification of New Antimicrobial Peptides from Mediterranean Medical Plant Charybdis pancration (Steinh.) Speta.

The present work was designed to identify and characterize novel antimicrobial peptides (AMPs) from Charybdis pancration (Steinh.) Speta, previously named Urginea maritima, is a Mediterranean plant, well-known for its biological properties in traditional medicine. Polypeptide-enriched extracts from different parts of the plant (roots, leaves and bulb), never studied before, were tested against two relevant pathogens, Staphylococcus aureus and Pseudomonas aeruginosa. With the aim of identifying novel natural AMPs, peptide fraction displaying antimicrobial activity (the bulb) that showed minimum inhibitory concentration (MICs) equal to 30 µg/mL against the above mentioned strains, was analysed by high-resolution mass spectrometry and database search. Seventeen peptides, related to seven proteins present in the investigated database, were described. Furthermore, we focused on three peptides, which due to their net positive charge, have a better chance to be AMPs and they were investigated by molecular modelling approaches, in order to shed light on the solution properties of their equilibrium structures. Some of new detected peptides could represent a good platform for the development of new antimicrobials in the fight against antibiotic resistance phenomenon.

Allograft Inflammatory Factor AIF-1: early immune response in the Mediterranean sea urchin Paracentrotus lividus.

Echinoderms are a phylum of deuterostomic invertebrates that play a key role in maintaining the biodiversity of marine ecosystems. They represent a good study model for immunity because their coelomic fluid contains different types of cells involved in the inflammatory response: the coelomocytes. In the case of the sea urchin Paracentrotus lividus, the coelomocyte population is mainly represented by amoebocytes and uncoloured spherulocytes that implement a defence program through phagocytosis, encapsulation, cytotoxicity, and production of antimicrobial agents. The purpose of this study was to find evidence of a possible modulating effect of lipopolysaccharide LPS on the expression of the AIF-1 gene and therefore to determine whether or not there is a correlation between the P. lividus immune response and the expression of this gene when the homeostasis of the animals is disturbed by a bacterial infection which, in this case, was simulated with treatment with LPS. AIF-1 (Allograft Inflammatory Factor 1) is a 17 kDa calcium-binding protein that, in vertebrates, is involved in the activation of macrophages. Similarly, in Echinoids and in general in invertebrates, the expression of this gene increases considerably after a bacterial attack and this suggests that it plays a key role during the immune responses.

Salmo salar fish waste oil: Fatty acids composition and antibacterial activity.

BACKGROUND AND AIMS: Fish by-products are generally used to produce fishmeal or fertilizers, with fish oil as a by-product. Despite their importance, fish wastes are still poorly explored and characterized and more studies are needed to reveal their potentiality. The goal of the present study was to qualitatively characterize and investigate the antimicrobial effects of the fish oil extracted from Salmo salar waste samples and to evaluate the potential use of these compounds for treating pathogen infections. METHODS: Salmo salar waste samples were divided in two groups: heads and soft tissues. Fatty acids composition, and in particular the content in saturated (SAFAs), mono-unsaturated (MUFAs) and Polyunsaturated (PUFAs) fatty acids, was characterized through GC/MS Thermo Focus GC-DSQ II equipped with a ZB-5 fused silica capillary tubes column. The antimicrobial activity of the salmon waste oils was evaluated through the Minimum Inhibitory Concentration assay and the antibiotics contamination was determined by Liquid Chromatography with tandem Mass Spectrometry (LC-MS/MS) analysis. All experiments were done at least in triplicate. RESULTS: GC/MS analysis has shown the specific fatty acid composition of the salmon waste oils and their enrichment in MUFAs and PUFAs, with special reference to omega-3, -6, -7, -9 fatty acids. Furthermore, our study has highlighted the antimicrobial activity of the fish waste oil samples against two Gram+ and Gram- bacterial strains. CONCLUSIONS: These data confirm that the fish waste is still quantitatively and qualitatively an important source of available biological properties that could be extracted and utilized representing an important strategy to counteract infective diseases in the context of the circular economy.

Changes in the proteome of sea urchin Paracentrotus lividus coelomocytes in response to LPS injection into the body cavity.

BACKGROUND: The immune system of echinoderm sea urchins is characterised by a high degree of complexity that is not completely understood. The Mediterranean sea urchin Paracentrotus lividus coelomocytes mediate immune responses through phagocytosis, encapsulation of non-self particles, and production of diffusible factors including antimicrobial molecules. Details of these processes, and molecular pathways driving these mechanisms, are still to be fully elucidated. PRINCIPAL FINDINGS: In the present study we treated the sea urchin P. lividus with the bacterial lipopolysaccharide (LPS) and collected coelomocytes at different time-points (1, 3, 6 and 24 hours). We have shown, using label-free quantitative mass spectrometry, how LPS is able to modulate the coelomocyte proteome and to effect cellular pathways, such as endocytosis and phagocytosis, as soon as the immunomodulating agent is injected. The present study has also shown that treatment can modulate various cellular processes such as cytoskeleton reorganisation, and stress and energetic homeostasis. CONCLUSIONS: Our data demonstrates, through mass spectrometry and the following functional annotation bioinformatics analysis, how the bacterial wall constituent is sufficient to set off an immune response inducing cytoskeleton reorganisation, the appearance of clusters of heat shock proteins (Hsp) and histone proteins and the activation of the endocytic and phagocytic pathways. Data are available via ProteomeXchange with identifier PXD008439.

Underwater high frequency noise: Biological responses in sea urchin Arbacia lixula (Linnaeus, 1758).

Marine life is extremely sensitive to the effects of environmental noise due to its reliance on underwater sounds for basic life functions, such as searching for food and mating. However, the effects on invertebrate species are not yet fully understood. The aim of this study was to determine the biochemical responses of Arbacia lixula exposed to high-frequency noise. Protein concentration, enzyme activity (esterase, phosphatase and peroxidase) and cytotoxicity in coelomic fluid were compared in individuals exposed for three hours to consecutive linear sweeps of 100 to 200 kHz lasting 1 s, and control specimens. Sound pressure levels ranged between 145 and 160 dB re 1µPa. Coelomic fluid was extracted and the gene and protein expression of HSP70 with RT-PCR was evaluated on coelomocytes. A significant change was found in enzyme activity and in the expression of the HSP70 gene and protein compared to the control. These results suggested that high-frequency stimuli elicit a noise-induced physiological stress response in A. lixula, confirming the vulnerability of this species to acoustic exposure. Furthermore, these findings provide the first evidence that cell-free coelomic fluid can be used as a signal to evaluate noise exposure in marine invertebrates.

Stress and immune response to bacterial LPS in the sea urchin Paracentrotus lividus (Lamarck, 1816).

The immune system of the sea urchin species Paracentrotus lividus is highly complex and, as yet, poorly understood. P. lividus coelomocytes mediate immune response through phagocytosis and encapsulation of non-self particles, in addition to the production of antimicrobial molecules. Despite this understanding, details of exactly how these processes occur and the mechanisms which drive them are still in need of clarification. In this study, we show how the bacterial lipopolysaccharides (LPS) is able to induce a stress response which increases the levels of the heat shock proteins HSP70 and HSP90 only a few hours after treatment. This study also shows that LPS treatment increases the expression of the ß-thymosin-derivated protein paracentrin, the precursor of antimicrobial peptides.

Antimicrobial and Antibiofilm Activity of a Recombinant Fragment of ß-Thymosin of Sea Urchin Paracentrotus lividus.

With the aim to obtain new antimicrobials against important pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa, we focused on antimicrobial peptides (AMPs) from Echinoderms. An example of such peptides is Paracentrin 1 (SP1), a chemically synthesised peptide fragment of a sea urchin thymosin. In the present paper, we report on the biological activity of a Paracentrin 1 derivative obtained by recombination. The recombinant paracentrin RP1, in comparison to the synthetic SP1, is 22 amino acids longer and it was considerably more active against the planktonic forms of S. aureus ATCC 25923 and P. aeruginosa ATCC 15442 at concentrations of 50 µg/mL. Moreover, it was able to inhibit biofilm formation of staphylococcal and P. aeruginosa strains at concentrations equal to 5.0 and 10.7 µg/mL, respectively. Molecular dynamics (MD) simulations allowed to rationalise the results of the experimental investigations, providing atomistic insights on the binding of RP1 toward models of mammalian and bacterial cell membranes. Overall, the results obtained point out that RP1 shows a remarkable preference for bacterial membranes, in excellent agreement with the antibacterial activity, highlighting the promising potential of using the tested peptide as a template for the development of novel antimicrobial agents.

Cytotoxic activity of Holothuria tubulosa (Echinodermata) coelomocytes.

The immune system of marine invertebrates, in particular that of holothurians, still requires further study. Our research showed that coelomocyte cells contained in the coelomic fluid of the sea cucumber, Holothuria tubulosa, are able to lyse, in vitro, red blood cells in rabbits and sheep. A plaque-forming assay showed spherule cells to be the effector cells, able to release cytotoxic molecules after xenogenic cell contact. The coelomocyte lysate supernatant, analysed by polyacrylamide gel electrophoresis overlay technique, using rabbit and sheep erythrocytes, showed two different haemolytic protein patterns: one calcium dependent and the other calcium independent. The fractions of each pattern were resolved on a polyacrylamide gel and calcium-dependent and independent coelomocyte lysate patterns were compared.

Anesthetic efficacy of ketamine-diazepam, ketamine-xylazine, and ketamine-acepromazine in Caspian Pond turtles (Mauremys caspica).

OBJECTIVES: The objective of this study was to assess the efficacy of different anesthetic drug combinations on the Caspian Pond turtles (Mauremys caspica). SUBJECTS AND METHODS: Three groups of the Caspian Pond turtles (n = 6) were anesthetized with three different drug combinations. Initially, a pilot study was conducted to determine the best drug doses for the anesthetization of the turtles, and according to these results, ketamine-diazepam (120 mg/kg ketamine hydrochloride [5%] and 2 mg/kg diazepam [5%]), ketamine-acepromazine (120 mg/kg ketamine hydrochloride [5%] and 1 mg/kg acepromazine [1%]), and ketamine-xylazine (120 mg/kg ketamine hydrochloride [5%] and 1 mg/kg xylazine [2%]) were injected intramuscularly. The onset times of anesthetization and the recovery time were measured. Statistical analysis of the data was performed using one-way analysis of variance followed by t-tests, and P < 0.05 was considered statistically significant. RESULTS: There were statistically significant differences in the mean of the onset times of anesthesia and recovery time among the three drug combinations depending on the treatment used. The onset of anesthesia of the animals treated with the ketamine-diazepam combination was 60% and 42% shorter, for male and female turtles, respectively, compared to that obtained with the ketamine-acepromazine combination and 64% (male turtles) and 50% (female turtles) shorter than that obtained with the ketamine-xylazine combination. Further, the recovery time, in male turtles, was 17% shorter in animals treated with the first drug combination than those treated with the ketamine-acepromazine combination and 37% shorter than those treated with the ketamine-xylazine combination. The recovery time, in female turtles, did not seem to be significantly different among treatments. CONCLUSIONS: The study showed that the ketamine-diazepam drug combination is the anesthetic combination with the fastest onset time and shortest recovery time.

Antibacterial Activity of Desert Truffles from Saudi Arabia Against Staphylococcus aureus and Pseudomonas aeruginosa.

Medicinal mushrooms represent an unlimited source of polysaccharides with nutritional, antitumoral, antibacterial, and immune-stimulating properties. Traditional studies of epigeous higher Basidiomycetes have recently been joined by studies of hypogeous fungi and, in particular, of so-called desert truffles. With the aim to obtain novel agents against bacteria of clinical importance, we focused on the edible desert truffle mushrooms Tirmania pinoyi, Terfezia claveryi, and Picoa juniperi as sources of new antimicrobial agents. In particular, we investigated the in vitro antibacterial activity of acid-soluble protein extracts (aqueous extracts) of these 3 species against the Gram-positive human pathogenic reference strain Staphylococcus aureus ATCC 29213 and the Gram-negative strain Pseudomonas aeruginosa ATCC 15442. The acid-soluble protein extracts of T. pinoyi and T. claveryi showed minimum inhibitory concentrations of 50 µg/mL against tested pathogens. We believe that such preliminary results are promising to obtain a valuable antibiotic alternative to fight antibiotic-resistant pathogens.

A Shotgun Proteomics Approach Reveals a New Toxic Role for Alzheimer's Disease Aß Peptide: Spliceosome Impairment.

Proteomic changes have been described in many neurodegenerative diseases, including Alzheimer's disease (AD). However, the early events in the onset of the pathology are yet to be fully elucidated. A cell model system in which LAN5 neuroblastoma cells were incubated for a short time with a recombinant form of Aß42 was utilized. Proteins extracted from these cells were subjected to shotgun proteomics analysis by LTQ-Orbitrap-MS followed by label-free quantitation. By bioinformatics tools we found that the most significant of those found to be up-regulated were related to cytoskeletal dynamics (Rho related) and membrane-related processes. The most significant of the down-regulated proteins were hnRNP-related. In particular, hnRNPs involved in ribosomal biogenesis and in splicing were down-regulated. The latter of these processes stood out as it was highlighted ubiquitously and with the highest significance in the results of every analysis. Furthermore, our findings revealed down-regulation at every stage of the splicing process through down-regulation of every subunit of the spliceosome. Dysregulation of the spliceosome was also confirmed using a Western blot. In conclusion, these data suggest dysregulation of the proteins and processes identified as early events in pathogenesis of AD following Aß accumulation.

Curcumin as a possible lead compound against hormone-independent, multidrug-resistant breast cancer.

We examine the possible evidence that the phytochemical curcumin may overcome resistance to hormonal and cytotoxic agents in breast cancer. We present our observations on MCF-7R, a multidrug-resistant (MDR) variant of the MCF-7 breast cancer cell line. In contrast to MCF-7, MCF-7R lacks aromatase and estrogen receptor alpha (ERalpha) and overexpresses the multidrug transporter ABCB1 and the products of different genes implicated in cell proliferation and survival, like c-IAP-1, NAIP, survivin, and COX-2. Nevertheless, in cytotoxicity and cell death induction assays, we found that the antitumor activity of curcumin is substantial both in MCF-7 and in MCF-7R. We elaborated the diketone system of curcumin into different analogues; the benzyloxime and the isoxazole and pyrazole heterocycles showed remarkable increases in the antitumor potency both in the parental and in the MDR MCF-7 cells. Furthermore, curcumin or, more potently, the isoxazole analogue, produced early reductions in the amounts of relevant gene transcripts that were diverse (i.e., they were relative to Bcl-2 and Bcl-X(L) in MCF-7 and the inhibitory of apoptosis proteins and COX-2 in MCF-7R) in the two cell lines. Thus, the two compounds exhibited the remarkable property of being able to modify their molecular activities according to the distinct characteristics of the parental and MDR cells. We discuss also how curcumin may (1) exert antitumor effects in breast cancer through ER-dependent and ER-independent mechanisms; and (2) act as a drug transporter-mediated MDR reversal agent. Overall, the structure of curcumin may represent the basis for the development of new, effective anticancer agents in hormone-independent MDR breast cancer.