Action of Varespladib (LY-315920), a Phospholipase A2 Inhibitor, on the Enzymatic, Coagulant and Haemorrhagic Activities of Lachesis muta rhombeata (South-American Bushmaster) Venom

Varespladib (VPL) was primarily developed to treat inflammatory disturbances associated with high levels of serum phospholipase A2 (PLA2). VPL has also demonstrated to be a potential antivenom support agent to prevent PLA2-dependent effects produced by snake venoms. In this study, we examined the action of VPL on the coagulant, haemorrhagic and enzymatic activities of Lachesis muta rhombeata (South-American bushmaster) venom. Conventional colorimetric enzymatic assays were performed for PLA2, caseinolytic and esterasic activities; in vitro coagulant activities for prothrombin time (PT) and activated partial thromboplastin time (aPTT) were performed in rat citrated plasma through a quick timer coagulometer, whereas the dimensions of haemorrhagic haloes obtained after i.d. injections of venom in Wistar rats were determined using ImageJ software. Venom (1 mg/ml) exhibited accentuated enzymatic activities for proteases and PLA2in vitro, with VPL abolishing the PLA2 activity from 0.01 mM; VPL did not affect caseinolytic and esterasic activities at any tested concentrations (0.001–1 mM). In rat citrated plasma in vitro, VPL (1 mM) alone efficiently prevented the venom (1 mg/ml)-induced procoagulant disorder associated to extrinsic (PT) pathway, whereas its association with a commercial antivenom successfully prevented changes in both intrinsic (aPTT) and extrinsic (PT) pathways; commercial antivenom by itself failed to avoid the procoagulant disorders by this venom. Venom (0.5 mg/kg)-induced hemorrhagic activity was slightly reduced by VPL (1 mM) alone or combined with antivenom (antivenom:venom ratio 1:3 ‘v/w’) in rats, with antivenom alone producing no protective action on this parameter. In conclusion, VPL does not inhibit other major enzymatic groups of L. m. rhombeata venom, with its high PLA2 antagonize activity efficaciously preventing the venom-induced coagulation disturbances.

Proteogenic Dipeptides Are Characterized by Diel Fluctuations and Target of Rapamycin Complex-Signaling Dependency in the Model Plant Arabidopsis thaliana

As autotrophic organisms, plants capture light energy to convert carbon dioxide into ATP, nicotinamide adenine dinucleotide phosphate (NADPH), and sugars, which are essential for the biosynthesis of building blocks, storage, and growth. At night, metabolism and growth can be sustained by mobilizing carbon (C) reserves. In response to changing environmental conditions, such as light-dark cycles, the small-molecule regulation of enzymatic activities is critical for reprogramming cellular metabolism. We have recently demonstrated that proteogenic dipeptides, protein degradation products, act as metabolic switches at the interface of proteostasis and central metabolism in both plants and yeast. Dipeptides accumulate in response to the environmental changes and act via direct binding and regulation of critical enzymatic activities, enabling C flux distribution. Here, we provide evidence pointing to the involvement of dipeptides in the metabolic rewiring characteristics for the day-night cycle in plants. Specifically, we measured the abundance of 13 amino acids and 179 dipeptides over short- (SD) and long-day (LD) diel cycles, each with different light intensities. Of the measured dipeptides, 38 and eight were characterized by day-night oscillation in SD and LD, respectively, reaching maximum accumulation at the end of the day and then gradually falling in the night. Not only the number of dipeptides, but also the amplitude of the oscillation was higher in SD compared with LD conditions. Notably, rhythmic dipeptides were enriched in the glucogenic amino acids that can be converted into glucose. Considering the known role of Target of Rapamycin (TOR) signaling in regulating both autophagy and metabolism, we subsequently investigated whether diurnal fluctuations of dipeptides levels are dependent on the TOR Complex (TORC). The Raptor1b mutant (raptor1b), known for the substantial reduction of TOR kinase activity, was characterized by the augmented accumulation of dipeptides, which is especially pronounced under LD conditions. We were particularly intrigued by the group of 16 dipeptides, which, based on their oscillation under SD conditions and accumulation in raptor1b, can be associated with limited C availability or photoperiod. By mining existing protein-metabolite interaction data, we delineated putative protein interactors for a representative dipeptide Pro-Gln. The obtained list included enzymes of C and amino acid metabolism, which are also linked to the TORC-mediated metabolic network. Based on the obtained results, we speculate that the diurnal accumulation of dipeptides contributes to its metabolic adaptation in response to changes in C availability. We hypothesize that dipeptides would act as alternative respiratory substrates and by directly modulating the activity of the focal enzymes.

Assessing the Fungal Simultaneous Removal Efficiency of Carbamazepine, Diclofenac and Ibuprofen in Aquatic Environment

Unused pharmaceutical compounds (PhCs) discharged into the aquatic environment have been regarded as emerging pollutants due to potential harmful effects on humans and the environment. Microbial bioremediation is considered as a viable option for their removal from wastewater. The aim of this study was to assess the simultaneous removal of carbamazepine (CBZ), diclofenac (DCF) and ibuprofen (IBP) by previously isolated fungi (Aspergillus niger, Mucor circinelloides, Trichoderma longibrachiatum, Trametes polyzona, and Rhizopus microsporus). The tolerance to PhCs was conducted by tracking the fungal mycelium mat diameters in solid media and its dry biomass in liquid media, at the drug concentration range of 0.1 to 15 mg/L. The fungal enzymatic activities were determined for lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase (Lac), respectively. The PhC removal efficiency of the fungi was assessed in aerated batch flasks and the drug concentrations and intermediate compounds formation were determined by using SPE-UPLC/MS. A tolerance over 70% was recorded for all the fungi at drug concentration of 0.1 mg/L. Manganese peroxidase was produced by all the fungi with very low amount of LiP, while all the enzymes were produced by T. polyzona. The pH of 4.3, temperature 37 ± 1.5°C and incubation time of 6 days were the optimum parameters for the fungal enzymatic activities. The best removal of CBZ (87%) was achieved by R. microsporus after 10 days. Between 78 and 100% removal of DCF was observed by all the fungi after 24 h, while 98% of IBP was removed after 2 days by M. circinelloides. Only a few intermediate compounds were identified after 3 days and disappeared after 10 days of incubation. This study demonstrated that apart from the basidiomycetes, the ascomycetes and zygomycetes are also producers of ligninolytic enzymes and have the ability to biodegrade emerging pollutants such as PhCs.

Quorum Sensing Regulates the Hydrolytic Enzyme Production and Community Composition of Heterotrophic Bacteria in Coastal Waters

Heterotrophic microbial communities play a central role in biogeochemical cycles in the ocean by degrading organic matter through the synthesis of extracellular hydrolytic enzymes. Their hydrolysis rates result from the community’s genomic potential and the differential expression of this genomic potential. Cell-cell communication pathways such as quorum sensing (QS) could impact both aspects and, consequently, structure marine ecosystem functioning. However, the role of QS communications in complex natural assemblages remains largely unknown. In this study, we investigated whether N-acylhomoserine lactones (AHLs), a type of QS signal, could regulate both hydrolytic activities and the bacterial community composition (BCC) of marine planktonic assemblages. To this extent, we carried out two microcosm experiments, adding five different AHLs to bacterial communities sampled in coastal waters (during early and peak bloom) and monitoring their impact on enzymatic activities and diversity over 48 h. Several specific enzymatic activities were impacted during both experiments, as early as 6 h after the AHL amendments. The BCC was also significantly impacted by the treatments after 48 h, and correlated with the expression of the hydrolytic activities, suggesting that changes in hydrolytic intensities may drive changes in BCC. Overall, our results suggest that QS communication could participate in structuring both the function and diversity of marine bacterial communities.

Pharmacological Effects of Marine-Derived Enterococcus faecium EA9 against Acute Lung Injury and Inflammation in Cecal Ligated and Punctured Septic Rats

Microorganisms obtained from the marine environment may represent a potential therapeutic value for multiple diseases. This study explored the possible protective role of marine-derived potential probiotic Enterococcus faecium EA9 (E. faecium) against pulmonary inflammation and oxidative stress using the cecal ligation and puncture (CLP) model of sepsis in Wistar rats. Animals were pretreated with E. faecium for 10 days before either sham or CLP surgeries. Animals were sacrificed 72 hours following the surgical intervention. The histological architecture of lung tissues was evaluated as indicated by the lung injury score. In addition, the extend of pulmonary edema was determined as wet/dry weight ratio. The inflammatory cytokines were estimated in lung tissues, including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β) using the enzyme-linked-immunosorbent-assay (ELISA) technique. Moreover, markers for lipid peroxidation such as thiobarbituric acid reaction substances (TBARs), and endogenous antioxidants, including reduced glutathione (GSH) were determined in lung tissues. Finally, the enzymatic activities of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) were assayed in the lungs. Pretreatment with E. faecium markedly attenuated CLP-induced lung injury and pulmonary edema. Markers for inflammation, including TNF-α, IL-6, and IL-1β were augmented in the lung tissues of CLP animals, while E. faecium ameliorated their augmented levels. E. faecium pretreatment also restored the elevated TBARS levels and the prohibited CAT, SOD, and GPx enzymatic activities in CLP animals. GSH levels were corrected by E. faecium in CLP animals. The inflammatory and lipid peroxidation mediators were positively correlated, while antioxidant enzymatic activities were negatively correlated with CLP-induced lung injury and pulmonary edema. Collectively, marine-derived Enterococcus faecium EA9 might be considered as a prospective therapeutic tool for the management of pulmonary dysfunction associated with sepsis.