The Culturable Mycobiota of Sediments and Associated Microplastics: From a Harbor to a Marine Protected Area, a Comparative Study

Fungi are an essential component of marine ecosystems, although little is known about their global distribution and underwater diversity, especially in sediments. Microplastics (MPs) are widespread contaminants worldwide and threaten the organisms present in the oceans. In this study, we investigated the fungal abundance and diversity in sediments, as well as the MPs, of three sites with different anthropogenic impacts in the Mediterranean Sea: the harbor of Livorno, the marine protected area "Secche della Meloria"; and an intermediate point, respectively. A total of 1526 isolates were cultured and identified using a polyphasic approach. For many of the fungal species this is the first record in a marine environment. A comparison with the mycobiota associated with the sediments and MPs underlined a "substrate specificity", highlighting the complexity of MP-associated fungal assemblages, potentially leading to altered microbial activities and hence changes in ecosystem functions. A further driving force that acts on the fungal communities associated with sediments and MPs is sampling sites with different anthropogenic impacts

Does salinity variation increase synergistic effects of triclosan and carbon nanotubes on Mytilus galloprovincialis? Responses on adult tissues and sperms

The use of carbon nanotubes (CNTs) is rapidly increasing and several scientific studies have addressed their toxicological properties. However, only a very small number of publications have deal with the interaction between CNTs and other molecules. Triclosan (TCS) is an antibacterial agent used in personal care and household products. Commonly detected in aquatic ecosystems, there is a strong evidence that aquatic biota is sensitive to this compound. Aside from emergent pollutants, aquatic organisms are continuously subjected to abiotic variations including salinities. Therefore, the main goal of the present study was to better understand how physio-chemical interactions of CNTs with TCS under different salinity levels (37, 28 and 19) affect the mussel species Mytilus galloprovincialis through the evaluation of biochemical alterations on gametes (sperms) and adult tissues, providing more ecologically relevant information on organisms' responses. The results showed toxicological effects in terms of sperm metabolic activity and intracellular reactive oxygen species production as well as cellular damage and alteration of metabolic capacity at the adult's stage when exposed to both contaminants acting alone and in combination, under tested salinities. Moreover, when the mussels were exposed to the combination of both contaminants, they showed major toxic impacts on both assessed biological levels (adult tissues and sperms) especially under control salinity. This suggests that toxicity upon mixture exposure compared to single-substance exposure may impair mussels' populations, affecting reproduction success and growth.publishe

Absence of Adiponutrin (PNPLA3) and Monoacylglycerol Lipase Synergistically Increases Weight Gain and Aggravates Steatohepatitis in Mice

Altered lipid metabolic pathways including hydrolysis of triglycerides are key players in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Whether adiponutrin (patatin-like phospholipase domain containing protein-3-PNPLA3) and monoacylglycerol lipase (MGL) synergistically contribute to disease progression remains unclear. We generated double knockout (DKO) mice lacking both Mgl and Pnpla3; DKO mice were compared to Mgl-/- after a challenge by high-fat diet (HFD) for 12 weeks to induce steatosis. Serum biochemistry, liver transaminases as well as histology were analyzed. Fatty acid (FA) profiling was assessed in liver and adipose tissue by gas chromatography. Markers of inflammation and lipid metabolism were analyzed. Bone marrow derived macrophages (BMDMs) were isolated and treated with oleic acid. Combined deficiency of Mgl and Pnpla3 resulted in weight gain on a chow diet; when challenged by HFD, DKO mice showed increased hepatic FA synthesis and diminished beta-oxidation compared to Mgl-/-.DKO mice exhibited more pronounced hepatic steatosis with inflammation and recruitment of immune cells to the liver associated with accumulation of saturated FAs. Primary BMDMs isolated from the DKO mice showed increased inflammatory activities, which could be reversed by oleic acid supplementation. Pnpla3 deficiency aggravates the effects of Mgl deletion on steatosis and inflammation in the liver under HFD challenge

Monoacylglycerol Lipase Inhibition Protects From Liver Injury in Mouse Models of Sclerosing Cholangitis

Background and Aims Monoacylglycerol lipase (MGL) is the last enzymatic step in triglyceride degradation, hydrolyzing monoglycerides into glycerol and fatty acids (FAs) and converting 2-arachidonoylglycerol into arachidonic acid, thus providing ligands for nuclear receptors as key regulators of hepatic bile acid (BA)/lipid metabolism and inflammation. We aimed to explore the role of MGL in the development of cholestatic liver and bile duct injury in mouse models of sclerosing cholangitis, a disease so far lacking effective pharmacological therapy. Approach and Results To this aim we analyzed the effects of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding to induce sclerosing cholangitis in wild-type (WT) and knockout (MGL(-/-)) mice and tested pharmacological inhibition with JZL184 in the multidrug resistance protein 2 knockout (Mdr2(-/-)) mouse model of sclerosing cholangitis. Cholestatic liver injury and fibrosis were assessed by serum biochemistry, liver histology, gene expression, and western blot characterization of BA and FA synthesis/transport. Moreover, intestinal FAs and fecal microbiome were analyzed. Transfection and silencing were performed in Caco2 cells. MGL(-/-) mice were protected from DDC-induced biliary fibrosis and inflammation with reduced serum liver enzymes and increased FA/BA metabolism and beta-oxidation. Notably, pharmacological (JZL184) inhibition of MGL ameliorated cholestatic injury in DDC-fed WT mice and protected Mdr2(-/-) mice from spontaneous liver injury, with improved liver enzymes, inflammation, and biliary fibrosis. In vitro experiments confirmed that silencing of MGL decreases prostaglandin E-2 accumulation in the intestine and up-regulates peroxisome proliferator-activated receptors alpha and gamma activity, thus reducing inflammation. Conclusions Collectively, our study unravels MGL as a metabolic target, demonstrating that MGL inhibition may be considered as potential therapy for sclerosing cholangitis

Hepatocyte-specific deletion of adipose triglyceride lipase (adipose triglyceride lipase/patatin-like phospholipase domain containing 2) ameliorates dietary induced steatohepatitis in mice

Background and Aims: Increased fatty acid (FA) flux from adipose tissue to the liver contributes to the development of NAFLD. Because free FAs are key lipotoxic triggers accelerating disease progression, inhibiting adipose triglyceride lipase (ATGL)/patatin-like phospholipase domain containing 2 (PNPLA2), the main enzyme driving lipolysis, may attenuate steatohepatitis. Approach and Results: Hepatocyte-specific ATGL knockout (ATGL LKO) mice were challenged with methionine-choline–deficient (MCD) or high-fat high-carbohydrate (HFHC) diet. Serum biochemistry, hepatic lipid content and liver histology were assessed. Mechanistically, hepatic gene and protein expression of lipid metabolism, inflammation, fibrosis, apoptosis, and endoplasmic reticulum (ER) stress markers were investigated. DNA binding activity for peroxisome proliferator-activated receptor (PPAR) α and PPARδ was measured. After short hairpin RNA–mediated ATGL knockdown, HepG2 cells were treated with lipopolysaccharide (LPS) or oleic acid:palmitic acid 2:1 (OP21) to explore the direct role of ATGL in inflammation in vitro. On MCD and HFHC challenge, ATGL LKO mice showed reduced PPARα and increased PPARδ DNA binding activity when compared with challenged wild-type (WT) mice. Despite histologically and biochemically pronounced hepatic steatosis, dietary-challenged ATGL LKO mice showed lower hepatic inflammation, reflected by the reduced number of Galectin3/MAC-2 and myeloperoxidase-positive cells and low mRNA expression levels of inflammatory markers (such as IL-1β and F4/80) when compared with WT mice. In line with this, protein levels of the ER stress markers protein kinase R–like endoplasmic reticulum kinase and inositol-requiring enzyme 1α were reduced in ATGL LKO mice fed with MCD diet. Accordingly, pretreatment of LPS-treated HepG2 cells with the PPARδ agonist GW0742 suppressed mRNA expression of inflammatory markers. Additionally, ATGL knockdown in HepG2 cells attenuated LPS/OP21-induced expression of proinflammatory cytokines and chemokines such as chemokine (C-X-C motif) ligand 5, chemokine (C-C motif) ligand (Ccl) 2, and Ccl5. Conclusions: Low hepatic lipolysis and increased PPARδ activity in ATGL/PNPLA2 deficiency may counteract hepatic inflammation and ER stress despite increased steatosis. Therefore, lowering hepatocyte lipolysis through ATGL inhibition represents a promising therapeutic strategy for the treatment of steatohepatitis

Cellular target effects of adipokines in obesity: action of osteopontin and adiponectin on monocytes/macrophages and hepatic stellate cells, respectively

Adipositas geht mit einer chronischen, niedrig-gradigen Entzündung einher, die durch die Infiltration von aktivierten Makrophagen ins Fettgewebe charakterisiert ist. Diese vertreten die wichtigsten Produzenten von beteiligten Zytokinen bei der Entstehung und Aufrechterhaltung der Insulinresistenz und damit des Typ-2- Diabetes. Wohingegen einzelne Adipokine wie Adiponektin, produziert durch das weiße Fettgewebe, die systemische Insulin-Resistenz verringern. Das Zytokin Osteopontin (OPN) wird bei einer vorliegenden Entzündung exprimiert und ist beispielsweise im Fettgewebe bei Adipositas stark hochreguliert, im Gegensatz zu Adiponektin, welches bei Adipositas runterreguliert ist. In vorranggegangen Studien zeigte sich bereits die kritische Rolle der lokalen Makrophagenproliferation bei Fettgewebsentzündung und auch unsere Daten liefern einen Anhaltspunkt, dass bei Adipositas die Lebensfähigkeit von Makrophagen verstärkt und die Apoptose vermindert wird. Entzündetes Fettgewebe spielt eine zusätzliche Rolle bei Lebererkrankungen, die mit Fettakkumulation und Fibrose in Verbindung stehen. Diese Prozesse umfassen die Glycerolaufnahme in die Leber und Kanalproteine wie Aquaporine (AQPs), welche die Diffusion von Glycerol durch die Zellmembran begünstigen. Daher habe ich die Hypothese aufgestellt, dass OPN zur lokalen Makrophagen-Proliferation beitragen könnte. Darüber hinaus könnten Adipokine die Funktion von AQPs und die Aktivierung von hepatischen Sternzellen beeinflussen. Das Ziel meiner Arbeit war die Untersuchung der Effekte von Adipositas-assoziierten Veränderungen der Zytokinproduktion durch die Messung von OPN Effekten auf die Monozyten- und Makrophagenproliferation und deren Relevanz in Bezug auf die Fettgewebsentzündung. Das zweite Ziel war die Erforschung von hepatischer AQP Expression und die Verbindung mit der Adiponektinproduktion bei Adipositas. Ich konnte zeigen, dass humane Monozyten verstärkt überleben, wenn sie mit OPN behandelt werden und gleichzeitig die Apoptose vermindert wird. Zusätzlich bedingte OPN eine Erhöhung der Proliferationsraten von humanen und murinen Monozyten/Makrophagen. Weiters korrelierten die hepatischen AQP Genexpressionen mit dem Adiponektin im Serum. Zellversuche zeigten, dass eine Adiponektin-induzierte Hochregulierung von AQP3 in hepatischen Sternzellen (HSC) zu einer Erhöhung der Fettspeicherung und der Glycerolaufnahme führt. Diese Ergebnisse zeigen, dass OPN die Proliferation und die Überlebensfähigkeit von Makrophagen und Monozyten vorantreibt und dass Adiponektin einen Effekt auf die AQP Genexpression in hepatischen Sternzellen hat. Zusammengefasst könnten diese Adipositas-gesteuerten Mechanismen einerseits die Fettgewebsentzündung durch OPN und auf der anderen Seite die Leberfibrose, die zu einer Veränderung der HCSs mit Hilfe von Adiponektin, beeinflussen.Obesity is pathology associated with chronic low-grade inflammation, which is characterized by infiltration of adipose tissue with activated macrophages. Resident macrophages are able to produce cytokines which are key molecules in the onset of insulin resistance and, consequently, type 2 diabetes. Whereas, adipokine such as adiponectin produced by the white adipose tissue, reduce systemic insulin resistance. Osteopontin (OPN) is a cytokine expressed in the site of inflammation known to be strongly upregulated in obese adipose tissue, in contrast to adiponectin, which is well known to be downregulated by obesity. Recent works point towards a critical role of local macrophage proliferation in adipose tissue inflammation and my own data suggest that in obesity viability of macrophages is enhanced and apoptosis diminished. Inflamed adipose tissue is also key player in liver pathologies connected with fat accumulation and fibrosis. These processes involve glycerol uptake in liver with channel proteins such as aquaporins (AQPs) facilitating the transport of the latter through the cell membrane. Therefore, I hypothesised that OPN may contribute to local macrophage proliferation. Moreover, adipokines might influence AQPs function and hepatic stellate cells activation. In this thesis I aimed at investigating effects of obesity-induced alterations of cytokine production by determining OPN effects on monocyte and macrophage proliferation and their relevance in the context of adipose tissue inflammation. As a second approach, liver AQPs expression and function was studied in relation to the obesity-induced decrease of adiponectin production. Survival of human monocytes was enhanced by OPN, while apoptosis was diminished. Proliferation rates in human and mice monocytes/macrophage increased by OPN in parallel. In liver, serum adiponectin correlated with AQPs gene expression in human. Moreover, AQP3 expression was strongly induced by adiponectin, increasing lipid content and glycerol uptake in hepatic stellate cells (HSC). In conclusion, OPN increases macrophage abundance in obese adipose tissue also by inhibiting apoptosis and enhancing their local proliferation. Second, adiponectin may counteract the progress of fatty liver disease to fibrosis by leading HSC into a quiescent functional state. Hence in this thesis, I could demonstrate novel effects of important adipokines with potential impact on the prognosis of obese patients.submitted by Matteo Tardelli, MScZusammenfassung in deutscher SpracheMedizinische Universität Wien, Dissertation, 2016OeBB(VLID)192232

Hepatic Medicine: Evidence and Research / PNPLA3 expression and its impact on the liver: current perspectives

A single-nucleotide polymorphism occurring in the sequence of the human patatin-like phospholipase domain-containing 3 gene (PNPLA3), known as I148M variant, is one of the best characterized and deeply investigated variants in several clinical scenarios, because of its tight correlation with increased risk for developing hepatic steatosis and more aggressive part of the disease spectrum, such as nonalcoholic steatohepatitis, advanced fibrosis and cirrhosis. Further, the I148M variant is positively associated with alcoholic liver diseases, chronic hepatitis Crelated cirrhosis and hepatocellular carcinoma. The native gene encodes for a protein that has not yet a fully defined role in liver lipid metabolism and, according to recent observations, seems to be divergently regulated among distinct liver cells type, such as hepatic stellate cells. Therefore, the aim of this review is to collect the latest data regarding PNPLA3 expression in human liver and to analyze the impact of its genetic variant in human hepatic pathologies. Moreover, a description of the current biochemical and metabolic data pertaining to PNPLA3 function in both animal models and in vitro studies is summarized to allow a better understanding of the relevant pathophysiological role of this enzyme in the progression of hepatic diseases.(VLID)488575

Nuclear Receptor Regulation of Aquaglyceroporins in Metabolic Organs

Nuclear receptors, such as the farnesoid X receptor (FXR) and the peroxisome proliferator-activated receptors gamma and alpha (PPAR-, -), are major metabolic regulators in adipose tissue and the liver, where they govern lipid, glucose, and bile acid homeostasis, as well as inflammatory cascades. Glycerol and free fatty acids are the end products of lipid droplet catabolism driven by PPARs. Aquaporins (AQPs), a family of 13 small transmembrane proteins, facilitate the shuttling of water, urea, and/or glycerol. The peculiar role of AQPs in glycerol transport makes them pivotal targets in lipid metabolism, especially considering their tissue-specific regulation by the nuclear receptors PPAR and PPAR. Here, we review the role of nuclear receptors in the regulation of glycerol shuttling in liver and adipose tissue through the function and expression of AQPs.(VLID)471770

The serpulid Ficopomatus enigmaticus (Fauvel, 1923) as candidate organisms for ecotoxicological assays in brackish and marine waters

Ficopomatus enigmaticus is an ubiquitous fouling reef-forming species, easy to sample and recognize, diecious with gamete spawning along different seasons in different salinity conditions. Due to its characteristics it could become a good candidate for the monitoring of both marine and brackish waters. The suitability of F. enigmaticus as a promising model organism in ecotoxicological bioassays was evaluated by a sperm toxicity and a larval development assay. The fertilization rate in different salinity conditions (range 5-35‰) was first assessed in order to detect the salinity threshold within which profitably perform the assays. Afterward copper (Cu2+), cadmium (Cd2+), sodium dodecyl sulfate (SDS) and 4-n-nonylphenol (NP) were used as reference toxicants in exposure experiments with spermatozoids (sperm toxicity assay) and zygotes (larval development assay). A dose-response effect was obtained for all tested toxicants along all salinity conditions except for 5‰ salinity condition where a too low (<30%) fertilization rate was observed. NP showed the highest degree of toxicity both in sperm toxicity and larval development assay. In some cases the results, expressed as EC50 values at 35‰ salinity condition, were similar to those observed in the literature for marine organisms such as the sea urchin (Paracentrotus lividus) and the marine serpulid Hydroides elegans, while the exposure of F. enigmaticus spermatozoids' to Cd2+ and NP resulted in toxicity effects several orders of magnitude higher than observed in P. lividus. Spermatozoids resulted to be slightly more sensitive then zygotes to all different toxicants

Soluble esterases as biomarkers of neurotoxic compounds in the widespread serpulid Ficopomatus enigmaticus (Fauvel, 1923)

The characterization of soluble cholinesterases (ChEs) together with carboxylesterases (CEs) in Ficopomatus enigmaticus as suitable biomarkers of neurotoxicity was the main aim of this study. ChEs of F. enigmaticus were characterized considering enzymatic activity, substrate affinity (acetyl-, butyryl-, propionylthiocholine), kinetic parameters (Km and Vmax) and in vitro response to model inhibitors (eserine hemisulfate, iso-OMPA, BW284C51), and carbamates (carbofuran, methomyl, aldicarb, and carbaryl). CEs were characterized based on enzymatic activity, kinetic parameters and in vitro response to carbamates (carbofuran, methomyl, aldicarb, and carbaryl). Results showed that cholinesterases from F. enigmaticus showed a substrate preference for acetylthiocholine followed by propionylthiocholine; butyrylthioline was not hydrolyzed differently from other Annelida species. CE activity was in the same range of cholinesterase activity with acetylthiocholine as substrate; the enzyme activity showed high affinity for the substrate p-nytrophenyl butyrate. Carbamates inhibited ChE activity with propionylthiocholine as substrate to a higher extent than with acetylthiocoline. Also CE activity was inhibited by all tested carbamates except carbaryl. In vitro data highlighted the presence of active forms of ChEs and CEs in F. enigmaticus that could potentially be inhibited by pesticides at environmentally relevant concentration.publishe