Genipin and glutaraldehyde based laccase two-layers immobilization with improved properties: New biocatalysts with high potential for enzymatic removal of trace organic contaminants.

This research proposes the preparation of a two-layer laccase biocatalyst using genipin or/and glutaraldehyde as cross-linking agents. The multilayer biocatalysts were prepared using different combinations of genipin and glutaraldehyde in the individual preparation of the first and second laccase layers. First, chitosan was treated with genipin or glutaraldehyde, followed by the immobilization of the first laccase layer to form a single-layer biocatalyst. Then, the immobilized laccases were coated once again with genipin or glutaraldehyde, and a new laccase layer was immobilized onto the system, resulting in the final two-layer biocatalyst. Compared to the single-layer biocatalysts, catalytic activity increased 1.7- and 3.4-fold when glutaraldehyde coating was used to prepare the second laccase layer. However, adding a second layer did not always produce more active biocatalysts, since the two-layer biocatalysts prepared with genipin (GenLacGenLac and GluLacGenLac) presented a decrease in activity of 65% and 28%, respectively. However, these two-layer biocatalysts prepared with genipin maintained 100% of their initial activity after 5 cycles of ABTS oxidation. Nevertheless, the two-layer, genipin-coated biocatalyst resulted in a higher removal of trace organic contaminants, since it removed 100% of mefenamic acid and 66% of acetaminophen, compared with the glutaraldehyde-coated biocatalyst, which removed 20% of mefenamic acid, and 18% of acetaminophen.

Pulcherriminic acid modulates iron availability and protects against oxidative stress during microbial interactions.

Siderophores are soluble or membrane-embedded molecules that bind the oxidized form of iron, Fe(III), and play roles in iron acquisition by microorganisms. Fe(III)-bound siderophores bind to specific receptors that allow microbes to acquire iron. However, certain soil microbes release a compound (pulcherriminic acid, PA) that, upon binding to Fe(III), forms a precipitate (pulcherrimin) that apparently functions by reducing iron availability rather than contributing to iron acquisition. Here, we use Bacillus subtilis (PA producer) and Pseudomonas protegens as a competition model to show that PA is involved in a peculiar iron-managing system. The presence of the competitor induces PA production, leading to precipitation of Fe(III) as pulcherrimin, which prevents oxidative stress in B. subtilis by restricting the Fenton reaction and deleterious ROS formation. In addition, B. subtilis uses its known siderophore bacillibactin to retrieve Fe(III) from pulcherrimin. Our findings indicate that PA plays multiple roles by modulating iron availability and conferring protection against oxidative stress during inter-species competition.

Size-Reduced Macrocyclic Analogues of [Pyr1]-apelin-13 Showing Negative Gα12 Bias Still Produce Prolonged Cardiac Effects.

We previously reported a series of macrocyclic analogues of [Pyr1]-apelin-13 (Ape13) with increased plasma stability and potent APJ agonist properties. Based on the most promising compound in this series, we synthesized and then evaluated novel macrocyclic compounds of Ape13 to identify agonists with specific pharmacological profiles. These efforts led to the development of analogues 39 and 40, which possess reduced molecular weight (MW 1020 Da vs Ape13, 1534 Da). Interestingly, compound 39 (Ki 0.6 nM), which does not activate the Gα12 signaling pathway while maintaining potency and efficacy similar to Ape13 to activate Gαi1 (EC50 0.8 nM) and ß-arrestin2 recruitment (EC50 31 nM), still exerts cardiac actions. In addition, analogue 40 (Ki 5.6 nM), exhibiting a favorable Gα12-biased signaling and an increased in vivo half-life (t1/2 3.7 h vs <1 min of Ape13), produces a sustained cardiac response up to 6 h after a single subcutaneous bolus injection.

Impact of Pharmaceutical Compounds in the Bioremediation of Municipal Biosolids by the White-Rot-Fungi Trametes hirsuta.

The potential of microorganisms for the treatment of municipal biosolids is continuously growing. The present studies evaluated the potency of Trametes hirsuta for the reduction in biosolid mass, production of extracellular enzymes, and removal of pharmaceutical compounds (PhACs) in biosolid slurry in the presence and absence of spiked PhACs [5 non-steroidal anti-inflammatories (NSAIs) and 2 psychoactive compounds (PACs)]. Toxicity after 35 days of fungal treatment was also assessed. Results showed that the growth of T. hirsuta is limited above 25% and wholly inhibited above 50% of biosolids in the slurry. At 12% of biosolid concentration, biosolid mass was reduced by 90%, NSAIs were entirely removed, but PACs' removal was only ~20%. Increasing biosolid content to 25% did not markedly affect biosolid reduction but significantly enhanced the removal of PACs (>50%). Results also showed that both PhACs and biosolids induced the production of oxidative enzymes. In 12% biosolids in the slurry, the oxidative potential measured by the ABTS assay (OABTS) reached 5,000 mM of OABTS in the presence of PhACs, and 2,500 mM of OABTS without PhACs, as compared to 1,200 mM of OABTS in control culture. Finally, we report that white rot fungi (WRF) treatment significantly decreased the toxicity of the biosolids.

Pharmacodynamic and pharmacokinetic profiles of a neurotensin receptor type 2 (NTS2) analgesic macrocyclic analog.

The current opioid crisis highlights the urgent need to develop safe and effective pain medications. Thus, neurotensin (NT) compounds represent a promising approach, as the antinociceptive effects of NT are mediated by activation of the two G protein-coupled receptor subtypes (i.e., NTS1 and NTS2) and produce potent opioid-independent analgesia. Here, we describe the synthesis and pharmacodynamic and pharmacokinetic properties of the first constrained NTS2 macrocyclic NT(8-13) analog. The Tyr11 residue of NT(8-13) was replaced with a Trp residue to achieve NTS2 selectivity, and a rationally designed side-chain to side-chain macrocyclization reaction was applied between Lys8 and Trp11 to constrain the peptide in an active binding conformation and limit its recognition by proteolytic enzymes. The resulting macrocyclic peptide, CR-01-64, exhibited high-affinity for NTS2 (Ki 7.0 nM), with a more than 125-fold selectivity over NTS1, as well as an improved plasma stability profile (t1/2 > 24 h) compared with NT (t1/2 ~ 2 min). Following intrathecal administration, CR-01-64 exerted dose-dependent and long-lasting analgesic effects in acute (ED50 = 4.6 µg/kg) and tonic (ED50 = 7.1 µg/kg) pain models as well as strong mechanical anti-allodynic effects in the CFA-induced chronic inflammatory pain model. Of particular importance, this constrained NTS2 analog exerted potent nonopioid antinociceptive effects and potentiated opioid-induced analgesia when combined with morphine. At high doses, CR-01-64 did not cause hypothermia or ileum relaxation, although it did induce mild and short-term hypotension, all of which are physiological effects associated with NTS1 activation. Overall, these results demonstrate the strong therapeutic potential of NTS2-selective analogs for the management of pain.

Assessing pesticides exposure effects on the reproductive performance of a declining aerial insectivore.

In the context of increasing global environmental changes, it has become progressively important to understand the effects of human activity on wildlife populations. Declines in several avian populations have been observed since the 1970s, especially with respect to many farmland and grassland birds, which also include some aerial insectivores. Changes in farming practices referred to as agricultural intensification coincide with these major avian declines. Among those practices, increased pesticide use is hypothesized to be a key driver of avian population declines as it can lead to both toxicological and trophic effects. While numerous laboratory studies report that birds experience acute and chronic effects upon consuming pesticide treated food, little is known about the effects of the exposure to multiple pesticides on wildlife in natural settings. We monitored the breeding activities of Tree Swallows (Tachycineta bicolor) on 40 farms distributed over a gradient of agricultural intensification in southern Québec, Canada, to evaluate the presence of pesticides in their diet and quantify the exposure effects of those compounds on their reproductive performance between 2013 and 2018. We first assessed the presence of 54 active agents (or derivatives) found in pesticides in 2,081 food boluses (insects) delivered to nestlings by parents and documented their spatial distribution within our study area. Second, we assessed the effect of pesticide exposure through food (number of active agents detected and number of contaminated boluses on a given farm for a given year, while controlling for sampling effort) on clutch size as well as hatching and fledging successes and nestling's mass upon fledging. Pesticides were ubiquitous in our study system and nearly half (46%) of food boluses were contaminated by at least one active agent. Yet we found no relationship between our proxies of food contamination by pesticides and Tree Swallow reproductive performance. More studies are needed to better understand the putative role of pesticides in the decline of farmland birds and aerial insectivores as potential sublethal effects of pesticides can carry over to later life stages and impact fitness.

Enzyme polymer engineered structure strategy to enhance cross-linked enzyme aggregate stability: a step forward in laccase exploitation for cannabidiol removal from wastewater.

Despite all its advantages and potential, cross-linking enzyme aggregate (CLEA) technology is still not applied at an industrial scale for enzyme insolubilization for bioremediation purposes. In this study, the enzyme polymer engineered structure (EPES) method was used to enhance CLEA stability and reuse. A crude laccase from Trametes hirsuta was successfully insolubilized to form EPES-CLEAs. The polymeric network provided excellent stability (> 90%) to CLEAs after a 24-h incubation in a non-buffered municipal wastewater effluent (WW), and the biocatalysts were recycled using a centrifugation process. While CLEAs activity dropped to 17%, EPES-CLEAs showed a laccase activity retention of 67% after five cycles of 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) oxidation. After 8 h of treatment in WW, the EPES-CLEAs were equally as effective in removing cannabidiol (CBD) as the free-LAC (~ 37%). This research demonstrates that the EPES method is a promising alternative for CLEA stabilization and reuse in environmental conditions.

Constraining the Side Chain of C-Terminal Amino Acids in Apelin-13 Greatly Increases Affinity, Modulates Signaling, and Improves the Pharmacokinetic Profile.

Side-chain-constrained amino acids are useful tools to modulate the biological properties of peptides. In this study, we applied side-chain constraints to apelin-13 (Ape13) by substituting the Pro12 and Phe13 positions, affecting the binding affinity and signaling profile on the apelin receptor (APJ). The residues 1Nal, Trp, and Aia were found to be beneficial substitutions for Pro12, and the resulting analogues displayed high affinity for APJ (Ki 0.08-0.18 nM vs Ape13 Ki 0.7 nM). Besides, constrained (d-Tic) or α,α-disubstituted residues (Dbzg; d-α-Me-Tyr(OBn)) were favorable for the Phe13 position. Compounds 47 (Pro12-Phe13 replaced by Aia-Phe, Ki 0.08 nM) and 53 (Pro12-Phe13 replaced by 1Nal-Dbzg, Ki 0.08 nM) are the most potent Ape13 analogues activating the Gα12 pathways (53, EC50 Gα12 2.8 nM vs Ape13, EC50 43 nM) known to date, displaying high affinity, resistance to ACE2 cleavage as well as improved pharmacokinetics in vitro (t1/2 5.8-7.3 h in rat plasma) and in vivo.

Monitoring of prenatal exposure to organic and inorganic contaminants using meconium from an Eastern Canada cohort.

Evaluating in utero exposure to inorganic and multiclass organic contaminants is critical to better evaluate potential harmful effects on prenatal and postnatal development. The analysis of meconium, the first bowel discharge of the newborn, has been proposed as a non-invasive way to assess cumulative prenatal exposure. The aim of this study was to implement an analytical method for quantifying 72 targeted organic compounds, including pesticides, pharmaceutical compounds and daily life xenobiotics, in meconium in addition to selected elements (17 elements). We report initial monitoring results based on the analysis of 396 meconium samples from an Eastern Canada cohort (Quebec, Canada). Element contents in meconium were analysed by mass spectrometry after digestion in nitric acid and peroxide. Targeted organic compounds were extracted and purified from meconium samples by a solid-liquid extraction followed by a dispersive-SPE purification before tandem mass spectrometry analysis. Concentrations of targeted elements were within the range of concentration reported in European and US studies but were lower than concentrations found in a developing country cohort (i.e., Pb, Cd). Out of the 72 targeted organic compounds, 31 were detected at least once and 30 were quantified. Compounds with the highest frequency of detection were caffeine, detected in all samples (from 2.80 to 6186 ng g-1), followed by acetaminophen detected in 53% of the samples (up to ~402 µg g-1) and methyl paraben detected in 20% of the samples (up to ~10 µg g-1). Pesticides were detected in low frequencies (< 2%) and low concentration (< 35 ng g-1). Results show that meconium can be used to monitor prenatal exposure of foetus to a wide array of inorganic and organic contaminants.

A hybrid bioreactor based on insolubilized tyrosinase and laccase catalysis and microfiltration membrane remove pharmaceuticals from wastewater.

The increasing presence of pharmaceutical products (PPs) and other organic contaminants of emerging concern (CECs) in aquatic systems has become one of the major global environmental contamination concerns. Sewage treatment plants (STPs) are one of the major sources of PPs discharge into natural waters due to the deficiencies of conventional treatment processes to deal with these micropollutants. Numerous new treatment processes and technologies have been investigated for the removals of CECs in wastewaters with more or less success. In the present study, we investigated the efficiency of a hybrid bioreactor (HBR) of a combined crosslinked tyrosinase and laccase aggregates and hollow fiber microfiltration (MF) membrane to remove a mixture of 14 PPs from municipal wastewater at environmentally relevant concentration of 10 µg/L. After a 5-day continuous operation, the HBR achieved complete removal of all tested PPs. Results also highlight that these high performances result from a synergistic action of the MF membrane and the insoluble enzymes. The biocatalyst retained nearly 70% of its initial enzymatic activity over the treatment period. The removal of PPs is unlikely to result from their sole sorption on the membrane. Overall, the results suggest that the HBR is well suited to the biocatalysts (i.e. insolubilized tyrosinase and laccase). The results invite to further investigate how the HBR can be tailored with various types of enzymes and membranes for either specific or non-specific target substrates and to further explore the applicability of this technology for the continuous treatment of wastewater at environmentally relevant concentration of PPs.

A Systematic Exploration of Macrocyclization in Apelin-13: Impact on Binding, Signaling, Stability, and Cardiovascular Effects.

The apelin receptor generates increasing interest as a potential target across several cardiovascular indications. However, the short half-life of its cognate ligands, the apelin peptides, is a limiting factor for pharmacological use. In this study, we systematically explored each position of apelin-13 to find the best position to cyclize the peptide, with the goal to improve its stability while optimizing its binding affinity and signaling profile. Macrocyclic analogues showed a remarkably higher stability in rat plasma (half-life >3 h versus 24 min for Pyr-apelin-13), accompanied by improved affinity (analogue 15, Ki 0.15 nM and t1/2 6.8 h). Several compounds displayed higher inotropic effects ex vivo in the Langendorff isolated heart model in rats (analogues 13 and 15, maximum response at 0.003 nM versus 0.03 nM of apelin-13). In conclusion, this study provides stable and active compounds to better characterize the pharmacology of the apelinergic system.

Intracellular Enzymes Contribution to the Biocatalytic Removal of Pharmaceuticals by Trametes hirsuta.

The use of white rot fungi (WRF) for bioremediation of recalcitrant trace organic contaminants (TrOCs) is becoming greatly popular. Biosorption and lignin modifying enzymes (LMEs) are the most often reported mechanisms of action. Intracellular enzymes, such as cytochrome P450 (CYP450), have also been suggested to contribute. However, direct evidence of TrOCs uptake and intracellular transformation is lacking. The aim of this study was to evaluate the relative contribution of biosorption, extracellular LMEs activity, TrOCs uptake, and intracellular CYP450 on the removal of six nonsteroidal anti-inflammatories (NSAIs) by Trametes hirsuta. Results show that for most tested NSAIs, LMEs activity and biosorption failed to explain the observed removal. Most tested TrOCs are quickly taken up and intracellularly transformed. Fine characterization of intracellular transformation using ketoprofen showed that CYP450 is not the sole intracellular enzyme responsible for intracellular transformation. The contribution of CYP450 in further transformation of ketoprofen byproducts is also reported. These results illustrate that TrOCs transformation by WRF is a more complex process than previously reported. Rapid uptake of TrOCs and intracellular transformation through diverse enzymatic systems appears to be important components of WRF efficiency toward TrOCs.

Liquid chromatography-tandem mass spectrometry determination for multiclass pesticides from insect samples by microwave-assisted solvent extraction followed by a salt-out effect and micro-dispersion purification.

The effects of phyto-pharmaceutic compounds (PPCs), such as neonicotinoids, on wildlife reproduction and survival are a rising concern. Yet, understanding the biological consequences of PPC use is particularly complex given the large diversity of PPCs and their derivatives to which wildlife can be exposed. Here, we present a simple and sensitive method for the simultaneous detection and quantification of multiclass PPCs (54 molecules) in single insect boluses (<0.05 g dry mass) by ultra-high pressure liquid chromatography coupled to a tandem mass spectrometer (LC-MS/MS). A key part of this new method is the use of a two-step extraction method combining (i) the high efficiency of a microwave-assisted solvent extraction (MAE) for extracting analytes that might be tightly bound to environmental matrices and (ii) the versatility of a salt-out effect adapted from the QuEChERS methodology allowing the extraction and purification of a wide array of analytes. This microwave-assisted salt-out extraction (MASOE) approach was compared to classical extraction methods including matrix solid phase dispersion (MSPD), microwave-assisted extraction (MAE), and the QuEChERS method. Average recoveries for 54 analytes ranged from 49% to 106%, (relative standard deviations <22%). The limits of detection (LODs) and quantification (LOQs) were in the ranges of 0.10-3.00 ng g(-1) and 0.40-7.00 ng g(-1), respectively. We applied this method to analyse 881 insect boluses collected from Tree Swallow (Tachycineta bicolour) nestlings along an agricultural intensification gradient in southern Québec (Canada). We detected 25 PPCs out of the 54 considered. We detected at least one PPC in 30% of samples and were able to quantify at least one of them in 17% of samples. Our study shows that the MASOE method should prove to be a powerful tool for studying the fate and impacts of PPCs on wildlife.

Evaluation of the efficiency of Trametes hirsuta for the removal of multiple pharmaceutical compounds under low concentrations relevant to the environment.

An evaluation of the efficiency of the White-rot fungi (WRF) Trametes hirsuta to remove multi-classes pharmaceutical active compounds (17 PhACs) at low and environmentally realistic concentrations (20-500 ng L(-1)) was performed. The importance of biosorption over enzymatic activity on PhACs removal was also evaluated. Results highlight the importance to consider environmentally relevant PhACs concentrations while evaluating the removal capacities of WRF in wastewaters treatment processes, as PhACs concentration strongly influence both the enzymatic activity profile and the removal efficiency. Results also show that under tested experimental conditions, laccase was the only active extracellular lignin modifying enzyme and that biosorption and possibly intracellular enzymes also contribute to the removal of some PhACs.

Synthesis and characterization of combined cross-linked laccase and tyrosinase aggregates transforming acetaminophen as a model phenolic compound in wastewaters.

Laccase (EC 1.10.3.2) and tyrosinases (EC 1.14.18.1) are ubiquitous enzymes present in nature as they are known to originate from bacteria, fungi, plants, etc. Both laccase and tyrosinase are copper-containing phenoloxidases requiring readily available O2 without auxiliary cofactor for their catalytic transformation of numerous phenolic substrates. In the present study, laccase and tyrosinase have been insolubilized as combined crosslinked enzyme aggregates (combi-CLEA) using chitosan, a renewable and biodegradable polymer, as crosslinker. The combi-CLEA, with specific activity of 12.3 U/g for laccase and 167.4 U/g for tyrosinase, exhibited high enzymatic activity at pH5-8 and temperature at 5-30°C, significant resistance to denaturation and no diffusional restriction to its active site based upon the Michaelis-Menten kinetic parameters. Subsequently, the combi-CLEA was applied to the transformation of acetaminophen as a model phenolic compound in samples of real wastewaters in order to evaluate the potential efficiency of the biocatalyst. In batch mode the combi-CLEA transformed more than 80% to nearly 100% of acetaminophen from the municipal wastewater and more than 90% from the hospital wastewater. UPLC-MS analysis of acetaminophen metabolites showed the formation of its oligomers as dimers, trimers and tetramers due to the laccase and 3-hydroxyacetaminophen due to the tyrosinase.

Characterization of combined cross-linked enzyme aggregates from laccase, versatile peroxidase and glucose oxidase, and their utilization for the elimination of pharmaceuticals.

In order to transform a wide range of pharmaceutically active compounds (PhACs), the three oxidative enzymes laccase (Lac) from Trametes versicolor, versatile peroxidase (VP) from Bjerkandera adusta and glucose oxidase (GOD) from Aspergillus niger were concomitantly cross-linked after aggregation, thus, making a combined cross-linked enzyme aggregate (combi-CLEA) that was versatile and involved in an enzymatic cascade reaction. From the initial enzymes about 30% of initial laccase activity was recovered along with 40% for each of VP and GOD. The combi-CLEA showed good results in conditions close to those of real wastewater (neutral pH and medium temperature) as well as a good ability to resist to denaturing conditions such as high temperature (60°C) and low pH (3). Batch experiments were realized to test the free enzyme's ability to degrade, a PhACs cocktail, mainly in a synthetic wastewater containing acetaminophen, naproxen, mefenamic acid, indometacin, diclofenac, ketoprofen, caffeine, diazepam, ciprofloxacin, trimethoprim, fenofibrate and bezafibrate, carbamazepine and its by-product 10-11 epoxy-carbamazepine. High removal was achieved (more than 80%) for the five first compounds. Then, the elimination ability of the combi-CLEA with or without hydrogen peroxide, glucose or manganese sulfate was determined. Globally, our results demonstrated that VP has a wider removal spectrum than Lac. These removal features are enhanced under more specific conditions, whereas the combi-CLEA combined advantages of both VP and laccase. Finally, the elimination of PhACs in a municipal wastewater treatment plant effluent using the combi-CLEA was marginally investigated. Concentrations of most of the selected PhACs were below the limit of quantification (lower than 20 ng/L) except for acetaminophen. Its combi-CLEA-mediated removal reached up to 25%.

Photocatalytic degradation of carbamazepine and three derivatives using TiO2 and ZnO: effect of pH, ionic strength, and natural organic matter.

Removal of pharmaceuticals (PhCs) by photocatalysis is a promising avenue in water treatment. The efficiency of these treatments on PhC derivatives compared to their parent molecules remains poorly documented. The present study investigates the efficiency of photodegradation catalyzed by TiO2 and ZnO nanoparticles on the removal of carbamazepine (CBZ) and three of its derivatives; carbamazepine epoxide (CBZ-E), acridine (AI), and acridone (AO). The effects of environmental parameters such as pH, ionic strength, and natural organic matter content on photodegradation efficiency (transformation after 6h and kinetics) were tested. We report that the efficiency of the catalysts (TiO2 and ZnO) can be very different when comparing CBZ and its derivatives (CBZ-E, AI and AO). TiO2 was more efficient than ZnO at degrading CBZ and CBZ-E. For AI and AO, no significant differences were observed between the two catalysts. We also report that environmental parameters have contrasting effects on the efficiency of the photodegradation of CBZ compared to its derivatives. Changing pH and organic matter content had the most contrasted effects; the photodegradation of CBZ and CBZ-E was significantly affected by pH (especially in presence of TiO2 NPs) and by the presence of natural organic matter. In contrast, the photodegradation of AI and AO was not affected by pH and organic matter. Only the photodegradation of CBZ was clearly affected by IS and solely at very high IS (1M). Overall, our results highlight that TiO2 and ZnO catalysts present contrasted efficiency on the removal of CBZ when compared to its derivatives (CBZ-E, AI and AO). Our results also show that the effect of environmental parameters on the efficiency of the photodegradation of CBZ derivatives cannot be predicted based on the behavior of the parent molecule (CBZ).