Enzymatic Rate Research Articles

In Vitro Hepatic Metabolism Input Parameters Support Toxicokinetic Simulations for the Formation of Methoxy Propionic Acid From β-Isomer Propylene Glycol Methyl Ether.

Propylene glycol ethers (PGEs) are organic solvents commonly found as technical grade on the commercial market, as mixtures of secondary (α-isomer) and primary (β-isomer, generally < 5%) alcohols. After handling products containing PGEs, they readily enter the human body where they are metabolized. The minor β-isomer is oxidized by alcohol dehydrogenase (ADH) followed by aldehyde dehydrogenase (ALDH) to a potentially harmful metabolite. Although the enzymatic rate is needed to estimate both parent and metabolite internal exposures, kinetic data for many PGEs are still scarce. Therefore, we generated invitro hepatic intrinsic clearance data for propylene glycol methyl ether β-isomer (β-PGME) and its metabolite methoxy propionic acid (2-MPA) and integrated these data into an in silico toxicokinetic (TK) model. Hepatic clearance values for the model were generated using an established invitro 3D culture of the human HepaRG cell line and human S9 liver fraction. Our results showed the presence of ADH and ALDH and consequently, the formation of 2-MPA in the 3D HepaRG and S9 fraction, which was slow to medium. We integrated the hepatic clearance values into the TK model to predict urinary 2-MPA concentrations. The simulated urinary 2-MPA concentrations fitted well (within twofold error from observed experimental data) for both liver systems, showing that they were both able to reliably predict the hepatic clearance of β-PGME. Although S9 is suitable for short-term studies, 3D cell culture models maintain metabolic competence over days and weeks. This opens the opportunity for long-term metabolism studies applying the 3D HepaRG model alone or in multi-organ systems.

Antimalarial Effects of Nano Chloroquine Loaded Curcumin In vivo.

Malaria is still the deadliest parasitic disease caused by Plasmodium spp. Due to drug resistance and their unpleasant side effects, of conventional researchers are enormously seeking to achieve antimalarial drugs with more curative effective, less toxic and cost-affordable drugs using more advanced technology such as nanodrugs. The present study aimed to examine the antimalarial effects of a novel synthesized nonochloroquine-loaded curcumin relying on dendrimer G2 in susceptible mice. Antimalarial activity and toxicity of the nanocomposite were examined on BALB/C mice with microscopy, checking RBCs morphology and related enzymatic activity rate. The maximum inhibitory effect of the nanocomposite was seen at 10 mg/kg, killing 98% of P. berghei compared to sole chloroquine, whereas ED50 was reported at 5.5 mg/kg. The safety of the synthesized nanocomposite was confirmed with biochemical tests with no detrimental effects on mice. The sustainability and longevity of the nanodrug increased significantly with the NDC-CQ assay compared to the control groups. The study showed that nonochloroquine-loaded curcumin had a promising inhibitory effect on P. berghei growth in infected mice compared to standard drugs. However, further studies and clinical trials with large samples are recommended to study different aspects of using nanodrug.

Design, Synthesis, Physicochemical Properties, and Biological Activity of Thymidine Compounds Attached to 5,8-Quinolinedione Derivatives as Potent DT-Diaphorase Substrates.

After heart disease, cancer is the second-leading cause of death worldwide. The most effective method of cancer treatment is target therapy. One of the potential goals of therapy could be DT-diaphorase, which reduces quinone moiety to hydroquinone, and reactive oxygen species are create as a byproduct. The obtaining of hybrid compounds containing the quinone moiety and other bioactive compounds leads to new derivatives which can activate DT-diaphorase. The aim of this research was the synthesis and characterization of new hybrids of 5,8-quinolinedione with thymidine derivatives. The analysis of the physicochemical properties shows a strong relationship between the structure and properties of the tested compounds. The enzymatic assay shows that hybrids are good substrates of NQO1 protein. The analysis of the structure-activity relationship shows that the localization of nitrogen atoms influences the enzymatic conversion rate. The analysis was supplemented by a molecular docking study. Comparing the results of the enzymatic assay and the molecular docking presents a strong correlation between the enzymatic conversion rate and the scoring value.

Synthesis of K+ channel radioligand [18F]5-methyl-3-fluoro-4-aminopyridine and PET imaging in mice

[18F]3-fluoro-4-aminopyridine ([18F]3F4AP) is the first positron emission tomography (PET) radioligand that targets voltage-gated potassium (K+) channels in the brain for imaging demyelination. [18F]3F4AP exhibits high brain penetration, favorable kinetics for PET imaging, and high sensitivity to demyelinating lesions. However, recent studies in awake human subjects indicate lower metabolic stability than in anesthetized animals, resulting in reduced brain uptake. Therefore, there is a need for novel radioligands for K+ channels with suitable pharmacological properties and enhanced metabolic stability. Recent in vitro studies demonstrate that 5-methyl-3-fluoro-4-aminopyridine (5Me3F4AP) exhibits comparable binding affinity to K+ channels, pKa, logD, and membrane permeability as 3F4AP, and a slower enzymatic metabolic rate, suggesting its potential as a K+ channel PET tracer. In this study, we describe the radiochemical synthesis of [18F]5Me3F4AP using an isotope exchange method from the corresponding 3-fluoro-5-methyl-4-nitropyridine N-oxide, followed by a palladium on carbon mediated hydrogenation of the nitro and N-oxide groups. This method yielded [18F]5Me3F4AP with high purity and acceptable molar activity. PET/CT studies using naïve mice demonstrate that [18F]5Me3F4AP effectively crosses the blood–brain barrier and has comparable kinetics to [18F]3F4AP. These findings strongly suggest that [18F]5Me3F4AP is a promising candidate for neuroimaging applications and warrant further studies to investigate its sensitivity to lesions and in vivo metabolic stability.

Biomimetic bilayer scaffold from Bombyx mori silk materials for small diameter vascular applications in tissue engineering.

Enhancing the biocompatibility and mechanical stability of small diameter vascular scaffolds remain significant challenges. To address this challenge, small-diameter tubular structures were electrospun from silk fibroin (SF) from silk textile industry discarded materials to generate bilayer scaffolds that mimic native blood vessels, but derived from a sustainable natural material resource. The inner layer was obtained by directly dissolving SF in formic acid, while the middle layer (SF-M) was achieved through aqueous concentration of the protein. Structural and biological properties of each layer as well as the bilayer were evaluated. The inner layer exhibited nano-scale fiber diameters and 57.9% crystallinity, and degradation rates comparable with the SF-M layer. The middle layer displayed micrometer-scale fibers diameters with an ultimate extension of about 274%. Both layers presented contact angles suitable for cell growth and cytocompatibility, while the bilayer material displayed an intermediate mechanical response and a reduced enzymatic degradation rate when compared to each individual layer. The bilayer material emulates many of the characteristics of native small-diameter vessels, thereby suggesting further studies towards in vivo opportunities.

Preparation and recovery performance of modified cellulase with pH-sensitive separation properties

The efficient utilization of biomass resources holds significant importance for sustainable development in the energy, materials, and chemical industries. Therefore, it has garnered widespread attention. In this study, we have successfully achieved pH-sensitive separation and recycling of cellulase in an aqueous system. Firstly, the cellulase was modified by introducing a polyethylene glycol modifier with an aromatic aldehyde group. This modification altered the conformation of the cellulase and created a homogeneous-like system due to the hydrophilicity of the ethoxyl group in the modifier. As a result, the accessibility of the enzyme to the substrate was improved, which led to an increase in the enzyme activity of 116.12 %. The aromatic aldehyde group in the modifier and the primary amino group in the polyethyleneimine (PEI) formed a dynamic Schiff base covalent bond, which had pH-sensitive properties to ensure the completion of the recycling of the enzyme in the aqueous system. It was found that the molecular weight of PEI affected the recovery properties of the enzyme. Under the condition that the enzymatic activity was not damaged, the recovery rate of enzymatic activity increased as the molecular weight of the PEI increased. Particularly, when the molecular weight of PEI was 1800, the modified cellulase still retained an enzymatic activity recovery rate of 80 % after 5 cycles. This research provides a novel solution to improve the efficient hydrolysis of cellulose and the recovery of cellulase.

Correlation of Enzymatic Depolymerization Rates with the Structure of Polyethylene-Like Long-Chain Aliphatic Polyesters.

Long-chain aliphatic polyesters are emerging sustainable materials that exhibit polyethylene-like properties while being amenable to chemical recycling and biodegradation. However, varying polyester chemical structures results in markedly different degradation rates, which cannot be predicted from commonly correlated bulk polyester properties, such as polymer melting temperature. To elucidate these structure-degradability relationships, long-chain polyesters varying in their monomer composition and crystallinity were subjected to enzymatic hydrolysis, the rates of which were quantified via detection of formed monomers. Copolymers with poorly water-soluble, long-chain diol monomers (e.g., 1,18-octadecanediol) demonstrated strongly reduced depolymerization rates compared to copolymers with shorter chain length diol monomers. This was illustrated by, e.g., the 20× faster hydrolysis of PE-4,18, consisting of 1,4-butanediol and 1,18-octadecanedicarboxylic acid monomers, relative to PE-18,4. The insoluble long-chain diol monomer released upon hydrolysis was proposed to remain attached to the bulk polymer surface, decreasing the accessibility of the remaining ester bonds to enzymes for further hydrolysis. Tuning of polyester crystallinity via the introduction of branched monomers led to variable hydrolysis rates, which increased by an order of magnitude when crystallinity decreased from 72% to 45%. The results reported enables the informed design of polyester structures with balanced material properties and amenability to depolymerization.

Effect of the crystal orientation on the enzymatic degradation rate of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) fibers

In this study, the relationship between the crystal orientation and enzymatic degradation rate of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) was investigated. The enzymatic degradation rates of melt-spun PHBH fibers were quantified via PHB depolymerase from Ralstonia pickettii T1, and the degradation rate decreased with increasing drawing ratio. The solid structure was investigated via differential scanning calorimetry and small-angle X-ray scattering, and kinetic analysis was performed, indicating that the degradation rate was affected not only by crystallinity and lamellar thickness but also by highly ordered structure. The highly ordered structure was examined via wide-angle X-ray diffraction. The enzymatic degradation rate from the fiber cross-sections was then quantified for the first time, and it increased with drawing ratio and was larger than that from the fiber sides. This result showed that the degradation rate varied depending on the crystal orientation. Furthermore, an enzymatic degradation test of PHBH ring-banded spherulites was conducted, and it was found that the degradation rate of flat-on lamellar crystals was greater than that of edge-on lamellar crystals. This is the first study to quantitatively evaluate the relationship between the crystal orientation and the enzymatic degradation rate.

The role of β‐glucanase in modifying dough characteristics, texture and in vitro starch digestibility of highland barley‐enriched bread

SummaryThis study aims to evaluate how β‐glucanase impacts dough characteristics, and the in vitro starch digestibility and quality of breads from mixed wheat and highland barley flour at different wheat/highland barley flour weight ratios (70/30 and 50/50). The results showed that β‐glucanase reduced the dough's viscoelasticity and enhanced its extensibility, softness, α‐helix (2.52%–16.12%), β‐sheet (11.10%–31.66%) and free sulfhydryl content (22.70%–34.49%). This helped to mitigate the negative effects of a high β‐glucan content on the wheat/highland barley dough (P &lt; 0.05). Moreover, β‐glucanase improved the specific volume (44.67%–56.56%) and springiness (5.56%–40.41%) as well as reduced the hardness of wheat/highland barley bread (7.50%–71.32%). Furthermore, as the higher content of slowly digestible starch (20.81%–30.84%) reduced the enzymatic rate (k value), β‐glucanase‐treated wheat/highland barley bread exhibited lower starch digestibility (66.17%–71.16%) than white bread (75.37%). Therefore, this study indicated that β‐glucanase played an offsetting role on highland barley bread while maintaining relatively low starch digestibility.

Excellent performance of Ag6Si2O7 oxidase mimic endowed by self-released ions for rapid colorimetric determination with reduced operational interference

Increasing attention has been directed towards oxidase mimics due to their catalytic ability toward enzyme substrates in the absence of H2O2, offering great colorimetric sensing potential in assays and diagnostics. However, slow electron-transfer and unstable absorbance over time often lead to unsatisfactory enzymatic reaction rates and unreliable readouts, significantly reducing detection efficiency and accuracy. Herein, we report on an Ag6Si2O7 nanozyme utilizing 3,3′,5,5′-tetramethylbenzidine (TMB) as a chromogenic substrate, demonstrating excellent oxidase-like activity and long-term stable colorimetric signals. Experimental results reveal that the excellent catalytic kinetics of Ag6Si2O7 (Km = 0.197 mM, Vmax = 5.1 × 10−7 M/s) are attributed to ultrafast electron transfer between Ag+ and TMB, mediated by Ag2O intermediates formed from self-released alkaline ions. Additionally, self-released H2SiO42− improves the stability of the oxidized substrate, resulting in a long-term stable colorimetric signal (∼20 min), which exhibits reliable accuracy regardless of the time point at which analyte is added within 20 min, thus eliminating the interference of individual operation time errors. Ascorbic acid (AA) is selected as the target molecule to assess the performance of the Ag6Si2O7-TMB colorimetric sensing system. This approach demonstrates a wide detection range from 5 to 300 μM and a detection limit of 0.452 μM. The developed colorimetric sensing strategy exhibits good stability, specificity, rapidity, and potential application in real sample detection.

Rapid determination of the antimicrobial properties of surfaces using an enzymatic activity surrogate

AbstractTypical approaches for assessing the antimicrobial activity of metals‐based surfaces involve the contact of a bacterial culture with the surface for a period of time, followed by culturing on agar plates to assess the decrease in microbial viability versus controls. This is a time‐consuming methodology requiring at least 24 h to produce a set of results, which can be a bottleneck for productivity in novel materials development. An enzyme‐based method was shown to be a satisfactory and much more rapid surrogate test for this application. A β‐galactosidase solution was applied to copper, silver, and zinc‐based antimicrobial surfaces for up to 1 h, and then the rate of colour development at 578 nm was monitored for a few minutes after addition of the chromogenic enzyme substrate chlorophenol red‐β‐d‐galactopyranoside (CPRG). Highly active antimicrobial surfaces were detected by a lack of colour development, due to enzyme inhibition by the metals. The enzymatic reaction rates were quantified and compared, demonstrating that the copper sample showed the greatest inhibition effect followed by the silver and zinc samples. The antimicrobial activity was quantified using bacteria and the plate count method, and the results correlated well with this enzyme assay, demonstrating that the metals‐based antimicrobial activities of both hard and soft (textile) surfaces could be quickly assessed with this enzyme‐based methodology.

Using ion exchange chromatography for the purification of glyoxylate reductase from corn leaves and studying its characteristics

The purpose of this study was to obtain a purified preparation of the studied enzyme from corn leaves and to study its characteristics. In this work, kinetic and regulatory parameters of glyoxylate reductase were calculated for leaves of 14-day-old corn (Zea mays) seedlings grown hydroponically at 25°C. The following methods were used during the study: sample homogenisation, four-step purification including ammonium sulphate for desalting, gel filtration on G-25 columns, and ion exchange chromatography using DEAE-sephacel, as well as electrophoresis on polyacrylamide gels and quantitative analysis of protein. To study the properties of the enzyme, we used electrophoretically homogenous preparations. The influence of pH, substrate concentration, and cofactor on the rate of the enzymatic reaction was determined by a series of measurements with different values of the enzymatic reaction rate. As a result of four-stage purification, we obtained a homogeneous preparation with a specific activity of167 E/mg of protein. Ion exchange chromatography was important for purification; we obtained 1 peak of enzyme activity upon desorption in 104 mM sodium chloride. Studying the properties of glyoxylate reductase showed a significant dependence of activity on pH. The optimal pH value was 6.5 units.

Unraveling iron oxides as abiotic catalysts of organic phosphorus recycling in soil and sediment matrices

In biogeochemical phosphorus cycling, iron oxide minerals are acknowledged as strong adsorbents of inorganic and organic phosphorus. Dephosphorylation of organic phosphorus is attributed only to biological processes, but iron oxides could also catalyze this reaction. Evidence of this abiotic catalysis has relied on monitoring products in solution, thereby ignoring iron oxides as both catalysts and adsorbents. Here we apply high-resolution mass spectrometry and X-ray absorption spectroscopy to characterize dissolved and particulate phosphorus species, respectively. In soil and sediment samples reacted with ribonucleotides, we uncover the abiotic production of particulate inorganic phosphate associated specifically with iron oxides. Reactions of various organic phosphorus compounds with the different minerals identified in the environmental samples reveal up to twenty-fold greater catalytic reactivities with iron oxides than with silicate and aluminosilicate minerals. Importantly, accounting for inorganic phosphate both in solution and mineral-bound, the dephosphorylation rates of iron oxides were within reported enzymatic rates in soils. Our findings thus imply a missing abiotic axiom for organic phosphorus mineralization in phosphorus cycling.

Development of Albendazole Nanodispersion for the Effective Treatment of Helminthiasis

Albendazole is a drug used to treat parasitic worm infections. ALB results in the tegument and intestinal cells of the worm undergoing degenerative changes, which ultimately lead the parasite to become immobile and die. The purpose of the study was to improve the solubility and effectiveness of an ALB-loaded solid nano dispersion for the effective treatment of helminthiasis.ALBFs were generated by a solvent evaporation procedure involving PVPK30 and carbopol, which may improve the stability of controlled drug release rates. The newly found ALBFs are more soluble than pure albendazole and have a better bioavailability. ALBFs allow for the optimization of therapeutic efficacy while lowering potential side effects and modulating enzymatic resistance, degradation, and drug release rate. Thus, the created ALBFs demonstrated successful helminthiasis treatment with lower dose frequency.

Mushroom Tyrosinase: Six Isoenzymes Catalyzing Distinct Reactions.

"Mushroom tyrosinase" from the common button mushroom is the most frequently used source of tyrosinase activity, both for basic and applied research. Here, the complete tyrosinase family from Agaricus bisporus var. bisporus (abPPO1-6) was cloned from mRNA and expressed heterologously using a single protocol. All six isoenzymes accept a wide range of phenolic and catecholic substrates, but display pronounced differences in their specificity and enzymatic reaction rate. AbPPO3 ignores γ-l-glutaminyl-4-hydroxybenzene (GHB), a natural phenol present in mM concentrations in A. bisporus, while AbPPO4 processes 100 μM GHB at 4-times the rate of the catechol l-DOPA. All six AbPPOs are biochemically distinct enzymes fit for different roles in the fungal life cycle, which challenges the traditional concept of isoenzymes as catalyzing the same physiological reaction and varying only in secondary properties. Transferring this approach to other enzymes and organisms will greatly stimulate both the study of the in vivo function(s) of enzymes and the application of these highly efficient catalysts.

Pretreatment of multiple lignocellulosic batches with recoverable and fermentable deep eutectic solvent (FDES) for co-production of sugar, lignin and lipid

Addressing the high cost of comprehensive utilization of lignocellulose, an efficient and green lignocellulosic biorefinery process was developed in this study, which can achieve the co-production of lignin, sugar and lipid. A series of FDES were prepared using glycerin, choline chloride (ChCl), and H2SO4 for the pretreatment of lignocellulose and ultimately for fermentation. After optimization, the hemicellulose removal reaches 98 %, and enzymatic hydrolysis rate of pretreated solids exceeds 90 %. The FDES was then recovered and reused for pretreatment, in total five batches of lignocellulose were pretreated, only a slight decrease in lignin and hemicellulose removal rate occurred, no significant decrease in lignin yield, and a decrease in solid enzyme hydrolysis from 83.26 % to 78.25 %. Finally, FDES after using five batches was used as a substrate for Rhodotorula glutinis with biomass up to 40.25 g/L and lipid concentration up to 8.33 g/L. In addition, the cellulose was probed with high solid-loading enzymatic hydrolysis, obtaining high concentration of glucose (137.4 g/L) for ethanol fermentation. In conclusion, this study developed a biorefinery technology for the efficient utilization of lignocellulosic fractions and pretreatment solvents, which reduces the cost of DES pretreatments of lignocellulose, and achieves the cogeneration of lignin, sugar and lipids.

Investigating the Inhibitory Factors of Sucrose Hydrolysis in Sugar Beet Molasses with Yeast and Invertase

Sugar beet molasses is a low-value byproduct from the sugar industry. It contains significant amounts of sucrose (approx. 50% (w/w)), which can be used for many different applications, for example, as feedstock for the production of fuel (as ethanol) and biobased chemicals such as 5-hydoxymethyl furfural (HMF). To produce platform chemicals, sucrose is hydrolyzed into its monomeric C6 sugars: glucose and fructose. When comparing the hydrolysis rates of molasses with a pure sucrose solution, the specific reaction rate is much slower (Qp/x,60min = 93 and 70 gprod L−1 h−1 gcell−1 for pure sucrose and crude molasses, respectively) at the same sucrose concentration (300 g/L) and process conditions. To clarify why molasses inhibits the enzymatic hydrolysis rate, the influence of its viscosity and inorganic and organic composition was investigated. Also, the effects of molasses and treated molasses on pure enzymes, invertase (from Saccharomyces cerevisiae, 0.05 mg/mL), compared with hydrolysis using whole cells of Baker’s yeast (3 mg/mL), were tested. The results indicate an inhibitory effect of potassium (Qp/x,60min = 76 gprod L−1 h−1 gcell−1), generally at high salt concentrations (Qp/x,60min = 67 gprod L−1 h−1 gcell−1), which could be correlated to the solution’s high salt concentrations and possibly the synergistic effects of different ions when applying concentrations that were four times that in the molasses. Also, the viscosity and sucrose purity seem to have an effect, where pure sucrose solutions and thick juice from the sugar mill yielded higher hydrolysis rates (Qp/x,60min = 97 gprod L−1 h−1 gcell−1) than molasses-type solutions with a higher viscosity (Qp/x,60min = 70–74 gprod L−1 h−1 gcell−1). Attempting to further understand the effects of different components on the invertase activity, an in silico investigation was performed, indicating that high salt concentrations affected the binding of sucrose to the active site of the enzyme, which can result in a lower reaction rate. This knowledge is important for future scale-up of the hydrolysis process, since reduced hydrolysis rates require larger volumes to provide a certain productivity, requiring larger process equipment and thereby higher investment costs.

Higher efficiency of vanadate iron in heterogeneous Fenton-like systems to pretreat sugarcane bagasse and its enzymatic saccharification.

Pretreatment is crucial for effective enzymatic saccharification of lignocellulose such as sugarcane bagasse (SCB). In the present study, SCB was pretreated with five kinds of heterogeneous Fenton-like systems (HFSs), respectively, in which α-FeOOH, α-Fe2O3, Fe3O4, and FeS2 worked as four traditional heterogeneous Fenton-like catalysts (HFCs), while FeVO4 worked as a novel HFC. The enzymatic reducing sugar conversion rate was then compared among SCB after different heterogeneous Fenton-like pretreatments (HFPs), and the optimal HFS and pretreatment conditions were determined. The mechanism underlying the difference in saccharification efficiency was elucidated by analyzing the composition and morphology of SCB. Moreover, the ion dissolution characteristics, variation of pH and Eh values, H2O2 and hydroxyl radical (·OH) concentration of FeVO4 and α-Fe2O3 HFSs were compared. The results revealed that the sugar conversion rate of SCB pretreated with FeVO4 HFS reached up to 58.25%, which was obviously higher than that under other HFPs. In addition, the surface morphology and composition of the pretreated SCB with FeVO4 HFS were more conducive to enzymatic saccharification. Compared with α-Fe2O3, FeVO4 could utilize H2O2 more efficiently, since the dissolved Fe3+ and V5+ can both react with H2O2 to produce more ·OH, resulting in a higher hemicellulose and lignin removal rate and a higher enzymatic sugar conversion rate. It can be concluded that FeVO4 HFP is a promising approach for lignocellulose pretreatment.

The transcription factor WRKY22 modulates ethylene biosynthesis and root development through transactivating the transcription of ACS5 and ACO5 in Arabidopsis.

The WRKY transcription factor (TF) genes form a large family in higher plants, with 72 members in Arabidopsis (Arabidopsis thaliana). The gaseous phytohormone ethylene (ET) regulates multiple physiological processes in plants. It is known that 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACSs, EC 4.4.1.14) limit the enzymatic reaction rate of ethylene synthesis. However, whether WRKY TFs regulate the expression of ACSs and/or ACC oxidases (ACOs, EC 1.14.17.4) remains largely elusive. Here, we demonstrated that Arabidopsis WRKY22 positively regulated the expression of a few ACS and ACO genes, thus promoting ethylene production. Inducible overexpression of WRKY22 caused shorter hypocotyls without ACC treatment. A qRT-PCR screening demonstrated that overexpression of WRKY22 activates the expression of several ACS and ACO genes. The promoter regions of ACS5, ACS11, and ACO5 were also activated by WRKY22, which was revealed by a dual luciferase reporter assay. A follow-up chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) and electrophoretic mobility shift assay (EMSA) showed that the promoter regions of ACS5 and ACO5 could be bound by WRKY22 directly. Moreover, wrky22 mutants had longer primary roots and more lateral roots than wild type, while WRKY22-overexpressing lines showed the opposite phenotype. In conclusion, this study revealed that WRKY22 acts as a novel TF activating, at least, the expression of ACS5 and ACO5 to increase ethylene synthesis and modulate root development.

First report on pollutant accumulation and associated microbial communities in the freshwater sponge Spongilla lacustris (Linnaeus, 1759) from the sub-Arctic Pasvik River (Norway).

This explorative study was aimed at first characterizing the sponge Spongilla lacustris (Linnaeus, 1759) from the sub-Arctic Pasvik River (Northern Fennoscandia), in terms of associated microbial communities and pollutant accumulation. Persistent organic pollutants were determined in sponge mesohyl tissues, along with the estimation of the microbial enzymatic activity rates, prokaryotic abundance and morphometric traits, and the analysis of the taxonomic bacterial diversity by next-generation sequencing techniques. The main bacterial groups associated with S. lacustris were Alphaproteobacteria and Gammaproteobacteria, followed by Chloroflexi and Acidobacteria. The structure of the S. lacustris-associated bacterial communities was in sharp contrast to those of the bacterioplankton, being statistically close to those found in sediments. Dieldrin was measured at higher concentrations in the sponge tissues (3.1 ± 0.4ng/g) compared to sediment of the same site (0.04 ± 0.03 ng/g). Some taxonomic groups were possibly related to the occurrence of certain contaminants, as was the case of Patescibacteria and dieldrin. Obtained results substantially contribute to the still scarce knowledge of bacterial community diversity, activities, and ecology in freshwater sponges. PRACTITIONER POINTS: Microbial community associated with Spongilla lacustris is probably shaped by the occurrence of certain contaminants, mainly dieldrin and heavy metals. A higher accumulation of dieldrin in the sponge mesohyl tissues than in sediment was determined. S. lacustris is suggested as sponge species to be used as a sentinel of pesticide pollution in the Pasvik River. S. lacustris, living in tight contact with soft substrates, harbored communities more similar to sediment than water communities.