Microbial degradation of pyrene in holm oak (Quercus ilex) phyllosphere: Role of particulate matter in regulating bioaccessibility.

In this study we first measured the mineralization of pyrene on leaves of urban holm oak (Quercus ilex) by autochthonous microorganisms and an inoculated PAH degrading bacterium (i.e., Mycobacterium gilvum), selected as a model phyllosphere species, as well as the leaf-water (KLW) and leaf-air (KLA) partition coefficients for this chemical. Mineralization was investigated in two different experimental systems in terms of leaf and microorganism environment. Additionally, the influence on pyrene partitioning and mineralization when particulate matter (PM) was present on the leaf surface or removed was studied. Mineralization of 14C-labeled pyrene by autochthonous microorganisms was lower than 1% after approximately two weeks, while M. gilvum mineralized 5% to 17% of pyrene. These extents corresponded to mineralization half-lives that ranged between ~30 to ~200 days. We proposed that PM present at the leaf surface reduced the accumulation of pyrene by inner compartments (cuticle) distantly located from microbial cells and enhanced the bioaccessibility of pyrene, speeding up microbial activity and therefore mineralization. These results highlight that plant-phyllosphere microorganism interaction is more complex than currently established and deserves additional studies to further comprehend the air purification ecosystem service of phyllosphere microorganisms.

Role of photo- and biodegradation of two PAHs on leaves: Modelling the impact on air quality ecosystem services provided by urban trees.

Urban trees provide important ecosystem services, including air quality improvement. Polycyclic aromatic hydrocarbons (PAHs) are among the most important pollutants in air, due to their elevated concentrations and toxicity. Plants can act as filters of PAHs and as "chemical reactors" for pollutant removal, therefore reducing air concentrations. Here, the first assessment of photo- vs. biodegradation of PAHs on leaves of urban trees is presented. A dynamic air-vegetation-soil model (SoilPlusVeg) was improved to simulate the fate of two representative PAHs with contrasting physico-chemical properties (phenanthrene and benzo[a]pyrene). Simulations were performed for two different environmental scenarios from Italy (Como and Naples), selected for their dissimilar meteorological parameters, plant species and emission levels. The effect of photo- and biodegradation on leaf concentrations and fluxes towards air and soil was investigated comparing deciduous (maple, cornel and hazelnut) and evergreen (holm oak) broadleaf woods. The results showed that biodegradation in the phyllosphere could not be neglected when evaluating the ecosystem services provided by urban trees, as this process contributed significantly to the reductions (up to 25% on average) in PAH leaf concentrations and fluxes to air and soil; however, the reductions revealed ample variations with time (up to more than two orders of magnitude) showing the dependence on meteorological parameters, air compartment structure, as well as type of woods. These findings permitted to improve the ecological realism of the simulations and obtain more accurate results when predicting organic contaminant uptake and release by plant leaves, including potential for food chain transfer and long-range transport.

Data for Tandem Mass Tag (TMT) proteomic analysis of the pancreas during the early phase of experimental pancreatitis.

The quantitative proteomics data reported here pertain to the research article entitled "A Tandem Mass Tag (TMT) proteomic analysis during the early phase of experimental pancreatitis reveals new insights in the disease pathogenesis" (García-Hernández et al., 2018) [1]. The development of acute pancreatitis (AP, an important pathological inflammatory state of the exocrine pancreas) would be based on early changes in protein expression and signaling pathways whose unmasking would be crucial for deciphering AP at the molecular level. We reported here a Tandem Mass Tag (TMT)-based proteomics analysis of rat subcellular fractions of the pancreas during the early phase of experimental AP, using a sixplex isobaric chemical labeling technique. We identified 997 unique proteins, of which 353 were significantly different (22, 276 or 55 in both, the soluble or the membrane fractions, respectively). Accordingly, using TMT proteomics and bioinformatic tools, in García-Hernández et al., 2018- [1] we were able to detect significant changes in protein expression related to many pathobiological pathways of AP as from the early phase of the disease, including some changes never described before in this disease. Proteomics data are publicly available in ProteomeXchange via PRIDE through the identifier PXD007096.

A tandem mass tag (TMT) proteomic analysis during the early phase of experimental pancreatitis reveals new insights in the disease pathogenesis.

Changes in the protein expression occurring within the initiation phase of acute pancreatitis (AP) might be vital in the development of this complex disease. However, the exact mechanisms involved in the onset of AP remains elusive and most of our knowledge about the pathobiology of AP comes from animal models. We performed in a rat pancreatitic model a high-throughput shotgun proteomic profiling of the soluble and whole membrane fractions from the pancreas during the early phase of cerulein (Cer)-induced AP. We identified 997 proteins, of which 353 were significantly different (22, 276 or 55 in both, the soluble or the membrane fractions, respectively). Gene Ontology and KEGG PATHWAY analyses revealed that these proteins were implicated in molecular mechanisms relevant to AP pathogenesis, including vesicle-mediated and protein transport, lysosomal and mitochondrial impairment or proteolysis. Numerous metabolic processes were downregulated apparently to reduce energy consumption, and a remarkable increase in inflammatory and stress responses was also highlighted. The proteomic data were verified by immunoblotting of 11 and 7 different soluble or membrane-associated proteins, either novel (VPS29 and MCTS1) or known factors in AP. Also, our first observation of the imbalance of some COP proteins during AP early phase deserves further characterization. BIOLOGICAL SIGNIFICANCE: AP is one of the most important pathological inflammatory states of the exocrine pancreas but its pathophysiology remains incompletely understood, especially the early acinar events. Proteomic analysis of pancreatic subcellular fractions simplifies protein maps and helps in the identification of new protein alterations and biomarkers characterizing pancreatic tissue damage. Our shotgun approach has not been previously used to profile the early proteomic alterations of the disease, which are considered crucial for its development and for the founding of clinical procedures. Furthermore, our subcellular fractionation protocol allowed us to detect changes in membrane proteins so far overlooked in the proteomic study of AP. Accordingly, using TMT proteomics and bioinformatic tools, we were able to detect significant changes in protein expression related to many pathobiological pathways of acute pancreatitis as from the early phase of the disease. To our knowledge, some of these changes, such as the imbalance of some COP proteins, have never been described in this disease.

Changes in the expression of LIMP-2 during cerulein-induced pancreatitis in rats: Effect of inhibition of leukocyte infiltration, cAMP and MAPKs early on in its development.

Lysosomal integral membrane protein-2 (LIMP-2) is an important protein in lysosomal biogenesis and function and also plays a role in the tissue inflammatory response. It is known that lysosomes play a central role in acute pancreatitis, with inflammatory cell infiltration triggering the disease early on. In this study we report increases in pancreatic LIMP-2 protein and mRNA levels as early events that occur during the development of cerulein (Cer)-induced acute pancreatitis (AP) in rats. GdCl3, a macrophage inhibitor, but not FK506, a T lymphocyte inhibitor, was able to reverse the increase in LIMP-2 expression after Cer treatment, although such reversion was abolished if the animals were depleted of neutrophils due to a vinblastine sulfate pre-treatment. Immunostaining revealed that the cellular source of LIMP-2 was mainly acinar cells. Additionally, pre-treatments with the MAPKs inhibitors SP600125 and PD98059, inhibitors of JNK and ERK½ activation, respectively, but not of rolipram, a type IV phosphodiesterase inhibitor, suppressed the increase in the expression of LIMP-2 after Cer administration. Together, these results indicate that neutrophils are able to drive a macrophage activation that would regulate the increase in LIMP-2 expression during the early phase of Cer-induced AP, with the stress kinases JNK and ERK½ also playing a coordinated role in the increase of LIMP-2 expression due to Cer.

From Bioavailability Science to Regulation of Organic Chemicals.

The bioavailability of organic chemicals in soil and sediment is an important area of scientific investigation for environmental scientists, although this area of study remains only partially recognized by regulators and industries working in the environmental sector. Regulators have recently started to consider bioavailability within retrospective risk assessment frameworks for organic chemicals; by doing so, realistic decision-making with regard to polluted environments can be achieved, rather than relying on the traditional approach of using total-extractable concentrations. However, implementation remains difficult because scientific developments on bioavailability are not always translated into ready-to-use approaches for regulators. Similarly, bioavailability remains largely unexplored within prospective regulatory frameworks that address the approval and regulation of organic chemicals. This article discusses bioavailability concepts and methods, as well as possible pathways for the implementation of bioavailability into risk assessment and regulation; in addition, this article offers a simple, pragmatic and justifiable approach for use within retrospective and prospective risk assessment.

Modulation in the expression of SHP-1, SHP-2 and PTP1B due to the inhibition of MAPKs, cAMP and neutrophils early on in the development of cerulein-induced acute pancreatitis in rats.

The protein tyrosine phosphatases (PTPs) SHP-1, SHP-2 and PTP1B are overexpressed early on during the development of cerulein -induced acute pancreatitis (AP) in rats, and their levels can be modulated by some species of mitogen-activated protein kinases (MAPKs), the intracellular levels of cAMP and by general leukocyte infiltration, the latter at least for SHP-2 and PTP1B. In this study we show that cerulein treatment activates extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) but not p38 MAPK during the early phase of cerulein-induced AP (2h after the first injection of cerulein). Therefore, by using the MAPK inhibitors SP600125 (a specific JNK inhibitor) and PD98059 (a specific ERK inhibitor), we have unmasked the particular MAPK that underlies the modulation of the expression levels of these PTPs. JNK would act by preventing SHP-1 protein expression from increasing beyond a certain level. ERK 1/2 was the main MAPK involved in the increase in SHP-2 protein expression due to cerulein. JNK negatively modulated the SH2-domain containing PTPs. Both MAPKs played a role in the increase in PTP1B protein expression due to cerulein. Finally, by using the white blood cell inhibitors vinblastine sulfate, gadolinium chloride and FK506 (tacrolimus), we show that the macrophage activity or T-lymphocytes does not modulate the expression of any of the PTPs, although neutrophil infiltration was found to be a regulator of SHP-2 and PTP1B protein expression due to cerulein.

Proteomic analysis of the soluble and the lysosomal+mitochondrial fractions from rat pancreas: Implications for cerulein-induced acute pancreatitis.

Alterations in protein expression within the initiation phase of acute pancreatitis (AP) might play an important role in the development of this disease, lysosomes being involved in its pathophysiology. The use of pancreatic subcellular fractions in proteomic analysis, simplifies protein maps and helps in the identification of new protein changes and biomarkers characterizing tissue damage. The present study aims to determine the differentially expressed acidic proteins in the pancreatic soluble and lysosomal+mitochondrial (L+M) fractions from rats during the early phase of the experimental model of cerulein (Cer)-induced AP. Subcellular pancreatic extracts from diseased and control rats were analyzed by 2-DE (3-5.6 pH range) and MALDI-TOF/TOF MS. Comparative analysis afforded the conclusive identification of 13 (soluble fraction) and 7 (L+M fraction) proteins or protein fragments occuring in different amounts between diseased and control pancreas, some of them being newly described in AP. In the soluble fraction, we detected changes related to inflammation and apoptosis (α1-inhibitor-3, α-1 antitrypsin, α-1 macroglobulin, haptoglobin, STRAP), oxidative stress and stress response (peroxiredoxin-2, thioredoxin-like 1, GRP94/TRA1, heat shock cognate 71kDa protein), digestive proteases (elastase 3B), serine protease inhibition (serpins B6 and A3L) and translation processes (EF 1-δ). In the L+M fraction, we detected changes mainly related to energy generation or cellular metabolism (ATP synthase ß subunit, chymotrypsinogen B, triacylglycerol lipase), cell redox homeostasis (iodothyronine 5´monodeiodinase) and digestive proteases (carboxypeptidase B1). The data should provide valuable information for unraveling the early pathophysiologic mechanisms of Cer-induced AP.

Bacterial chemotaxis towards aromatic hydrocarbons in Pseudomonas.

Bacterial chemotaxis is an adaptive behaviour, which requires sophisticated information-processing capabilities that cause motile bacteria to either move towards or flee from chemicals. Pseudomonas putida DOT-T1E exhibits the capability to move towards different aromatic hydrocarbons present at a wide range of concentrations. The chemotactic response is mediated by the McpT chemoreceptor encoded by the pGRT1 megaplasmid. Two alleles of mcpT are borne on this plasmid and inactivation of either one led to loss of this chemotactic phenotype. Cloning of mcpT into a plasmid complemented not only the mcpT mutants but also its transfer to other Pseudomonas conferred chemotactic response to high concentrations of toluene and other chemicals. Therefore, the phenomenon of chemotaxis towards toxic compounds at high concentrations is gene-dose dependent. In vitro experiments show that McpT is methylated by CheR and McpT net methylation was diminished in the presence of hydrocarbons, what influences chemotactic movement towards these chemicals.

Changes in the morphology and lability of lysosomal subpopulations in caerulein-induced acute pancreatitis.

BACKGROUND AND AIMS: Lysosomes play an important role in acute pancreatitis (AP). Here we developed a method for the isolation of lysosome subpopulations from rat pancreas and assessed the stability of lysosomal membranes. METHODS: AP was induced by four subcutaneous injections of 20 µg caerulein/kg body weight at hourly intervals. The animals were killed 9h after the first injection. Marker enzymes [N-acetyl-ß-D-glucosaminidase (NAG), cathepsin B and succinate dehydrogenase (SDH)] were assayed in subcellular fractions from control pancreas and in pancreatitis. Lysosomal subpopulations were separated by Percoll density gradient centrifugation and observed by electron microscopy. NAG molecular forms were determined by DEAE-cellulose chromatography. RESULTS: AP was associated with: (i) increases in the specific activity of lysosomal enzymes in the soluble fraction, (ii) changes in the size and alterations in the morphology of the organelles from the lysosomal subpopulations, (iii) the appearance of large vacuoles in the primary and secondary lysosome subpopulations, (iv) the increase in the amount of the NAG form associated with the pancreatic lysosomal membrane as well as its release towards the soluble fraction. CONCLUSIONS: Lysosome subpopulations are separated by a combination of differential and Percoll density gradient centrifugations. Primary lysosome membrane stability decreases in AP.

Rolipram and SP600125 suppress the early increase in PTP1B expression during cerulein-induced pancreatitis in rats.

OBJECTIVES: To analyze the expression modulation of pancreatic protein tyrosine phosphatase (PTP)1B during the development of cerulein (Cer)-induced acute pancreatitis (AP) and the effect of inhibition of type 4 phosphodiesterase and c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 on its expression levels. METHODS: Acute pancreatitis was induced in rats by subcutaneous injections of 20 microg Cer per kilogram body weight at hourly intervals, and the animals were killed at 2, 4, or 9 hours after the first injection. Neutropenia was induced with vinblastine sulfate. Phosphodiesterase and the mitogen-activated protein kinases were inhibited with rolipram and SP600125, respectively, before the induction of AP. RESULTS: Protein tyrosine phosphatase 1B increases its expression at the levels of both protein and messenger RNA during the early phase of Cer-induced AP. The increase in protein expression persisted along the development of the disease, and neutrophil infiltration seemed to play a central role. Rolipram and SP600125 pretreatments mostly suppressed the increase in the expression of PTP1B during the early phase of AP. CONCLUSIONS: Cerulein-induced AP is associated with an increase in the expression of PTP1B in its early phase. An increase in cyclic adenosine monophosphate levels in inflammatory cells and the inhibition of c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 are able to suppress the increase in PTP1B protein level.

Ducts isolated from the pancreas of CFTR-null mice secrete fluid.

The pancreatic pathology in cystic fibrosis (CF) is normally attributed to the failure of ductal fluid secretion resulting from the lack of functional CF transmembrane conductance regulator (CFTR). However, murine models of CF show little or no pancreatic pathology. To resolve this dichotomy we analysed the transport mechanisms involved in fluid and electrolyte secretion by pancreatic ducts isolated from CFTR-null mice. Experiments were performed on cultured interlobular duct segments isolated from the pancreas of the Cftr(tm1Cam) strain of CFTR-null mouse. Fluid secretion to the closed luminal space was measured by video microscopy. The secretory response of ducts isolated from CF mice to cAMP-elevating agonists forskolin and secretin was significantly reduced compared with wild type but not abolished. The Cl(-)- and HCO(3) (-) -dependent components of the ductal secretion were affected equally by the absence of CFTR. The secretory response to carbachol stimulation was unaltered in CF ducts. Loading the ductal cells with the Ca2+ chelator BAPTA completely abolished carbachol-evoked secretion, but did not affect forskolin-evoked secretion in CF or wild-type ducts. We conclude that pancreatic duct cells from CF mice can secrete a significant amount of water and electrolytes by a cAMP-stimulated mechanism that is independent of CFTR and cannot be ascribed to the activation of calcium-activated chloride channels.

Changes in the expression and dynamics of SHP-1 and SHP-2 during cerulein-induced acute pancreatitis in rats.

Protein tyrosine phosphatases (PTPs) are important regulators of cell functions but data on different PTP expression and dynamics in acute pancreatitis (AP) are very scarce. Additionally, both c-Jun N-terminal kinases (JNK) and extracellular signal-regulated kinases (ERK1/2), together with intracellular cAMP levels in inflammatory cells, play an essential role in AP. In this study we have detected an increase in PTP SHP-1 and SHP-2 in the pancreas at the level of both protein and mRNA as an early event during the development of Cerulein (Cer)-induced AP in rats. Nevertheless, while SHP-2 protein returned to baseline levels in the intermediate or later phases of AP, SHP-1 protein expression remained increased throughout the development of the disease. The increase in SHP-2 protein expression was associated with changes in its subcellular distribution, with higher percentages located in the fractions enriched in lysosomes+mitochondria or microsomes. Furthermore, while the increase in SHP-2 protein was also observed in sodium-taurocholate duct infusion or bile-pancreatic duct obstruction AP, that of SHP-1 was specific to the Cer-induced model. Neutrophil infiltration did not affect the increase in SHP-1 protein, but favoured the return of SHP-2 protein to control levels, as indicated when rats were rendered neutropenic by the administration of vinblastine sulfate. Inhibition of JNK and ERK1/2 with SP600125 pre-treatment further increased the expression of both SHP-1 and SHP-2 proteins in the early phase of Cer-induced AP, while the inhibition of type IV phosphodiesterase with rolipram only suppressed the increase in SHP-2 protein expression during the same phase. Our results show that AP is associated with increases in the expression of SHP-1 and SHP-2 and changes in the dynamics of SHP-2 subcellular distribution in the early phase of Cer-induced AP. Finally, both JNK and ERK1/2 and intracellular cAMP levels are able to modulate the expression of these PTPs.