Leucine Aminopeptidase Activity Research Articles

Biochar Influences Polyethylene Microplastic-Contaminated Soil Properties and Enzyme Activities

Microplastics (MPs) have emerged as a significant pollutant, threatening agricultural ecosystem sustainability and global food security. However, relatively few studies have investigated biochar remedial effects on plant growth and biochemical properties in soils contaminated with MPs. In polyethylene microplastic (PE-MP)-contaminated soil, we investigated corn stover biochar effects on soybean growth, soil nutrient content, enzyme activity, and microbial biomass and assessed its impact on soil microbial resource limitations. The addition of MPs inhibited soybean growth in various forms across four stages. Conversely, the addition of biochar to MPs improved soybean growth to some extent, where above-ground biomass increased by 5.82% after adding biochar to soils containing microplastics. In soil treated with MPs and biochar, nitrate nitrogen (N), available phosphorus (P), and available potassium (K) increased by 20.1, 27.4, and 57.2%, respectively, while available nitrogen significantly decreased to 128.3 mg kg−1 compared to the MP-only treatment. PE-MPs alone significantly reduced soil carbon (C), N, and P enzyme activities, as well as microbial biomass, with β-glucosidase, leucine aminopeptidase, and acid phosphatase activities decreasing by 29.9, 27.8, and 25.5%, respectively. Interestingly, biochar addition to MPs significantly alleviated these detrimental effects. Microbial biomass C, N, and P increased by up to 56.0, 22.5, and 96.6%, respectively, following biochar addition to soils containing MPs. Analysis of vector lengths, vector angles, and scatter plots indicated that the presence of MPs reduced soil N and P availability. Overall, while MPs inhibited soybean development, biochar addition alleviated this effect to some extent. Furthermore, partial least-squares path modeling revealed that MPs negatively affected soil chemical properties, microbial biomass, and enzyme activities, whereas biochar positively influenced soil enzyme activities.

Microplastic effects on soil nitrogen cycling enzymes: A global meta-analysis of environmental and edaphic factors

Microplastic accumulation in soil ecosystems poses significant environmental concerns, potentially impacting nitrogen cycling processes and ecosystem health. This meta-analysis of 147 studies (1138 data points) assessed the impact of microplastics (MPs) on soil nitrogen-acquisition enzymes. We found that MPs exposure significantly increased soil urease (UE) and leucine aminopeptidase activities by 7.6 % and 8.0 %, respectively, while N-acetyl-β-D-glucosaminidase activity was not significantly affected. Biodegradable MPs showed more pronounced effects compared to conventional MPs. Enzyme activities were influenced by MPs properties (e.g., polymer type, size, concentration), experimental conditions (e.g., field or laboratory setting, temperature, nitrogen fertilization), and soil properties (e.g., clay content, pH, organic carbon, total nitrogen). For instance, acidic soils enhanced UE activity, while neutral soils reduced it. These findings emphasize the complex interactions between MPs and soil ecosystems, highlighting the need for context-specific environmental management strategies and policy-making approaches to mitigate the impacts of MPs pollution on soil health.

Effects of dietary succinic acid supplementation on growth performance, digestive ability, intestinal development and immunity of large yellow croaker (Larimichthys crocea) larvae

The application of artificial micro-diet is an effective way to improve and standardize the quality of aquatic animal larvae. However, the widespread adoption of micro-diet faces a bottleneck due to the limited utilization capacity of the larvae. A 30-day feeding experiment was carried out to investigate the effect of dietary succinic acid (SA) on the growth performance, digestive ability, intestinal development, and immunity of large yellow croaker larvae (initial body weight 11.33 ± 0.57 mg). Four isonitrogenous and isolipidic diets were formulated, incorporating 0.00 %, 0.01 %, 0.02 % and 0.03 % SA separately. The results showed that a diet with 0.02 % SA significantly increased both the final body weight and the specific growth rate of the larvae. Regarding digestive ability, 0.01 % SA supplementation significantly enhanced trypsin activity in both intestinal and pancreatic segments. In addition, 0.01 % SA supplementation notably improved amylase activity in the intestinal segment, while diets with 0.01%–0.02 % SA significantly improved lipase activity in the pancreatic segment. In terms of intestinal development, 0.01 % SA supplementation remarkably boosted the activities of alkaline-phosphatase and leucine-aminopeptidase on brush border membrane in intestine. Furthermore, 0.03 % SA supplementation significantly increased the expression of occludin. In terms of immunity, larvae fed diets with 0.01%–0.02 % SA exhibited significantly higher lysozyme activity compared to the control group. Supplementation with 0.01 % SA also significantly increased both iNOS activity and NO content. In summary, the findings of this study suggested that supplementing 0.02 % SA can improve the growth performance of large yellow croaker larvae by improving digestive enzymes activities, promoting intestinal development, and enhancing nonspecific immunity.

Biocompatible Flash Chemiluminescent Assay Enabled by Sterically Hindered Spiro-Strained-Oxetanyl-1,2-Dioxetane.

Chemiluminescence is the emission of light that occurs as a result of a chemical reaction. Depending on the rate of chemiexcitation, light emission can occur as a long-lasting, low-intensity, glow-type reaction or a rapid, highly intense flash-type reaction. Assays using a flash-type mode of action provide enhanced detection sensitivity compared to those using a glow-type mode. Recently, our group discovered that applying spiro-strain to 1,2-dioxetanes significantly increases their chemiexcitation rate, thereby transforming glow-type chemiluminescence into flash-type chemiluminescence. However, further examination of the structure-activity relationships revealed that the spiro-strain severely compromises the chemical stability of the 1,2-dioxetanes. We hypothesized that a combination of spiro-strain, steric hindrance, and an electron-withdrawing effect, will result in a chemically stable spiro-strained dioxetane with an accelerated chemiexcitation rate. Indeed, spiro-fused tetramethyl-oxetanyl exhibited a 128-fold faster chemiexcitation rate compared to adamantyl while maintaining similar chemical stability, with a half-life of over 400 hours in PBS 7.4 buffer at room temperature. Turn-on probes composed of tetramethyl-oxetanyl spiro-dioxetane exhibited significantly improved chemical stability in bacterial and mammalian cell media compared to previously developed dioxetane probes fused to a cyclobutyl unit. The superior chemical stability enables a tetramethyl-oxetanyl dioxetane probe to detect β-Galactosidase (β-gal) activity with enhanced sensitivity in Escherichia coli (E. coli) bacterial assays and leucine aminopeptidase activity in tumoral cell lines. Overall, the development of the tetramethyl-oxetanyl dioxetane luminophore enables us to enhance the detection sensitivity of chemiluminescent probes while maintaining high chemical stability. The results obtained in this study should assist in designing improved chemiluminescent probes and underscore the significance of strain-release techniques in enhancing the detection sensitivity of chemiluminescence assays.

Composted maize straw under fungi inoculation reduces soil N2O emissions and mitigates the microbial N limitation in a wheat upland

Enhancement of microbial assimilation of inorganic nitrogen (N) by straw addition is believed to be an effective pathway to improve farmland N cycling. However, the effectiveness of differently pretreated straws on soil N2O emissions and soil N-acquiring enzyme activities remains unclear. In this study, a pot experiment with four treatments (I, no addition, CK; II, respective addition of maize straw, S; III, composted maize straw under no fungi inoculation, SC; and IV, composted maize straw under fungi inoculation, SCPA) at the same quantity of carbon (C) input was conducted under the same amount of inorganic N fertilization. Results showed that the seasonal cumulative N2O emissions following the SCPA treatment were the lowest at 4.03 kg N ha−1, representing a significant reduction of 19 % compared with the CK treatment. The S and SC treatments had no significant effects on N2O emissions. The decrease of soil N2O emissions following the SCPA treatment was mainly attributed to the increase of microbial N assimilation and the increased abundance of functional genes related to N2O reductase. The SCPA treatment significantly decreased soil alkaline phosphatase (ALP) activity and increased leucine aminopeptidase (LAP) activity at the basal fertilization, while increased soil ALP and LAP activity, decreased soil N-Acetyl-β-D-Glucosidase (NAG) activity at harvest. Compared with the CK treatment, the S, SC, and SCPA treatment significantly increased soil β-glucosidase (β-GC) activity at harvest. The decrease in the (NAG+LAP)/ALP ratio following the SCPA treatment indicated that the composted maize straw under fungi inoculation alleviated microbial N limitation at harvest. Moreover, PICRUSt analysis also suggested that the SCPA treatment increased the abundance of bacterial genes associated with N assimilation and N2O reduction, whereas the S and SC treatment did not significantly affect the abundance of N2O reduction genes compared with the CK treatment. Our results suggest that the composted maize straw under fungi inoculation would reduce the risk of N2O emissions and effectively mitigate the microbial N limitation in dryland wheat system.

Serum Leucine Aminopeptidase Activity Patterns Across Various Disease States: Potential Implications for Bleeding and Thrombosis Risk.

Disruptions in the pathways for activating and deactivating proteases in the bloodstream can lead to thrombosis and bleeding issues. Leucine aminopeptidases (LAPs), which are exopeptidases essential for regulating protein and peptide activities, are recognized as clinical biomarkers for liver diseases. However, the relationship between serum LAP activity and the risks of bleeding or thrombosis, as well as the identification of the specific tissues or organs that control LAP levels, is not well understood. We performed a retrospective study to evaluate serum LAP activities in 149,360 patients with 47 different diseases and 9,449 healthy individuals. The analysis was conducted using SPSS V2.6, RStudio V.1.3.1073, and libraries in Python 3.8. Our research revealed that 21 of the 47 diseases studied showed increased median serum LAP activities, while 26 diseases were associated with significantly lower activities, especially those related to thrombosis. Furthermore, most diseases were found to have an increased risk of bleeding and thrombosis, indicated by higher Q25 and lower Q75 LAP activities compared to the control group. Receiver operating characteristic curve analysis confirmed the effectiveness of LAP activities as biomarkers for specific conditions like hepatic encephalopathy, liver cancer, pancreatitis, and pancreatic cancer. Diseases were categorized into clusters with similar bleeding or thrombotic tendencies through principal component analysis. This study highlighted regulatory influence of the liver and pancreas on LAP levels. The established link between serum LAP concentrations and the risk of bleeding or thrombosis paved the way for the development of diagnostic and preventative approaches for various medical conditions.

Phosphorus addition stimulates overall carbon acquisition enzymes but suppresses overall phosphorus acquisition enzymes: A global meta-analysis

Of the total land worldwide, 43 % is phosphorus (P)-constrained, which is significantly greater than the proportion of nitrogen-constrained land (18 %). However, there are some uncertainties regarding the responses of enzyme activities to P addition on a global scale. We collected data from 99 publications and performed a meta-analysis to assess the responses of 11 extracellular enzymes, including carbon, nitrogen, and phosphorus acquisition enzymes, and oxidases. P addition significantly increased the activities of β-1, 4-glucosidase, sucrase, cellobiohydrolase, and alkaline phosphatase, while lowering the activities of acid phosphatase and phosphodiesterase. The responses of enzyme activities to P addition differed considerably across the three ecosystems (forest, grassland, and cropland). P addition significantly decreased the activities of acid phosphatase, phosphodiesterase, and P-acquisition enzymes in forests, but increased the activities of leucine aminopeptidase, carbon acquisition enzymes, and nitrogen acquisition enzymes in grasslands. Sucrase, β-1,4-glucosidase, N-acetyl-β-glucosaminidase, alkaline phosphatase, and carbon acquisition enzymes were significantly stimulated in croplands. In addition, all P addition rates significantly increased the activity of alkaline phosphatase, a P-acquisition enzyme, in croplands. Ca(H2PO4)2 had a greater effect on enzyme activity than other fertilizer types, possibly because Ca2+ can improve the soil structure and enhance microbial metabolic activities. Furthermore, structural equation models revealed that mean annual precipitation and ecosystem type had significant impacts on the response of P-acquisition enzymes, but no factor could directly influence the response of carbon-acquisition enzymes. Our investigation of enzyme activities in response to P addition complements the biogeochemical model and helps forecast soil nutrient dynamics. We propose that rather than focusing on a single factor, P fertilizer addition programs should take into account the effects of mean annual precipitation, ecosystem type, and nutrient interactions, which could also reduce P pollution.

Fire-deposited charcoal enhances soil microbial biomass in a recently harvested subtropical plantation forest

Charcoal, a byproduct of biomass burning, is widely and heterogeneously distributed in fire-affected ecosystems. However, few field studies have been conducted to evaluate the effects of fire-deposited charcoal on the post-fire soil quality. In this study, we aimed to investigate whether charcoal generated during post timber harvest broadcast burning influenced the recovery of individual soil properties and overall soil quality in subtropical forest plantations. Broadcast burning was conducted on experimental Pinus massoniana plantation timber harvest sites in southern China. Surface soils (0–10 cm) were collected in plots established on the burnt sites five years after the disturbance event. Plots were established immediately after burning with three different levels of charcoal input (C0, removal of all visible charcoal; C1, charcoal retained in-situ; C2, charcoal removed from C0 added to C2) and an unburnt control (UB). Thirty-two soil indicators representing soil physical, chemical, and biological properties were determined. The results showed that 11 indicators exhibited significant differences among the treatments, with significantly lower SOM, microbial biomass carbon (MBC), peroxidase (POD) and leucine aminopeptidase (LAP) activities for the C0 and C1 plots than the UB plots (P < 0.05), while no significant difference was observed between the UB and C2 plot (P > 0.05). The soil available N, P and exchangeable nutrient contents were not significantly different among the treatments (P > 0.05). Our results indicate that SOM and microbial attributes did not recover five years after the disturbance event and that charcoal appeared to play a positive role in the post-fire soil restoration. Whether the accelerated recovery of soil restoration induced by charcoal might contribute to higher forest productivity needs to be testified in combination with plant growth and performance analysis in the future.

Enzyme Activity Stoichiometry Suggests That Fertilization, Especially Nitrogen Fertilization, Alleviates Nutrient Limitation of Soil Microorganisms in Moso Bamboo Forests

Rational application of N fertilizer is essential for maintaining the long-term productivity of Moso bamboo forests. Microbial activity is a crucial indicator of soil quality. Changes in soil nutrient resources due to N addition can lead to microbial nutrient limitations, thereby impeding the maintenance of soil quality. Currently, there is limited research on the effects of N application on microbial nutrient limitations in Moso bamboo forest soils. To examine the changes in extracellular enzyme activity and microbial nutrient limitations in Moso bamboo forest soils following N application, we conducted an N application experiment in northern Guizhou. The findings revealed that the N3 treatment (726 kg·N·hm−2·yr−1) significantly reduced β-glucosidase (BG) activity by 27.61% compared to the control group (no fertilization). The N1 (242 kg·N·hm−2·yr−1), N2 (484 kg·N·hm−2·yr−1), and N3 treatments notably increased the activities of leucine aminopeptidase (LAP) and N-acetyl-β-D-glucosidase (NAG) by 11.45% to 15.79%. Acid phosphatase (ACP) activity remained unaffected by fertilization. N application treatments significantly decreased the C:Ne and C:Pe ratios, while the N:Pe ratio was less influenced by N fertilizer application. Scatter plots and vector characteristics of enzyme activity stoichiometry suggested that microorganisms in the study area were limited by C and N, and N fertilizer application reduced the vector length and increased the vector angle, indicating that N application alleviated the C and N limitation of microorganisms in Moso bamboo forests. Redundancy Analysis (RDA) demonstrated that microbial biomass phosphorus (MBP) was the most critical factor affecting extracellular enzyme activity and stoichiometry. Furthermore, Random Forest Regression analysis identified MBP and the N:Pm ratio as the most significant factors influencing microbial C and N limitation, respectively. The study demonstrated that N application modulates the microbial nutrient acquisition strategy by altering soil nutrient resources in Moso bamboo forests. Formulating fertilizer application strategies based on microbial nutrient requirements is more beneficial for maintaining soil quality and sustainably managing Moso bamboo forests. Additionally, our study offers a theoretical reference for understanding carbon cycling in bamboo forest ecosystems in the context of substantial N inputs.

Persulfidome of Sweet Pepper Fruits during Ripening: The Case Study of Leucine Aminopeptidase That Is Positively Modulated by H2S.

Protein persulfidation is a thiol-based oxidative posttranslational modification (oxiPTM) that involves the modification of susceptible cysteine thiol groups present in peptides and proteins through hydrogen sulfide (H2S), thus affecting their function. Using sweet pepper (Capsicum annuum L.) fruits as a model material at different stages of ripening (immature green and ripe red), endogenous persulfidated proteins (persulfidome) were labeled using the dimedone switch method and identified using liquid chromatography and mass spectrometry analysis (LC-MS/MS). A total of 891 persulfidated proteins were found in pepper fruits, either immature green or ripe red. Among these, 370 proteins were exclusively present in green pepper, 237 proteins were exclusively present in red pepper, and 284 proteins were shared between both stages of ripening. A comparative analysis of the pepper persulfidome with that described in Arabidopsis leaves allowed the identification of 25% of common proteins. Among these proteins, glutathione reductase (GR) and leucine aminopeptidase (LAP) were selected to evaluate the effect of persulfidation using an in vitro approach. GR activity was unaffected, whereas LAP activity increased by 3-fold after persulfidation. Furthermore, this effect was reverted through treatment with dithiothreitol (DTT). To our knowledge, this is the first persulfidome described in fruits, which opens new avenues to study H2S metabolism. Additionally, the results obtained lead us to hypothesize that LAP could be involved in glutathione (GSH) recycling in pepper fruits.

Response of a simulated aquatic fungal community to nanoplastics exposure and functional consequence on leaf decomposition

Nanoplastics pose a potential threat to a wide variety of aquatic organisms. Despite the awareness of this existing hazard, the impact of nanoplastics on natural fungal communities remains a research gap. In this study, five dominant fungi species, isolated from a stream ecosystem, were used to explore the effects of different nano-polystyrene (nano-PS) particles concentrations on a simulated fungal community. Specifically, the evaluation was conducted regarding the fungal growth, reproductivity, structural composition, and ecological function in leaf litter decomposition. A 15-day exposure experiment showed that 100 μg/L nano-PS significantly reduced the microcosm pH. The extracellular enzyme activities of β-glucosidase, leucine-aminopeptidase, and peroxidase were significantly promoted by nano-PS exposure for 5 days or 15 days. Total sporulation rate significantly decreased after the 15-day exposure to 1 and 100 μg/L nano-PS and significantly increased under 10 μg/L nano-PS. In contrast, nano-PS concentrations had no effects on fungal biomass. In addition, the reduced relative abundance of Geotrichum candidum lowered its contribution to leaf decomposition, resulting in a decreased litter decomposition rate of a 24.5–27.9 % after exposure. This suggests that 1–100 μg/L nano-PS inhibited leaf decomposition by inhibiting fungal reproduction and reducing the contribution of specific fungal species. In addition, the findings highlight the importance of exploring the potential mechanisms of the interaction between nanoplastics and fungal species.

Long-term soil warming decreases soil microbial necromass carbon by adversely affecting its production and decomposition.

Microbial necromass carbon (MNC) accounts for a large fraction of soil organic carbon (SOC) in terrestrial ecosystems. Yet our understanding of the fate of this large carbon pool under long-term warming is uncertain. Here, we show that 14 years of soil warming (+4°C) in a temperate forest resulted in a reduction in MNC by 11% (0-10 cm) and 33% (10-20 cm). Warming caused a decrease in the content of MNC due to a decline in microbial biomass carbon and reduced microbial carbon use efficiency. This reduction was primarily caused by warming-induced limitations in available soil phosphorus, which, in turn, constrained the production of microbial biomass. Conversely, warming increased the activity of soil extracellular enzymes, specifically N-acetylglucosaminidase and leucine aminopeptidase, which accelerated the decomposition of MNC. These findings collectively demonstrate that decoupling of MNC formation and decomposition underlie the observed MNC loss under climate warming, which could affect SOC content in temperate forest ecosystems more widespread.

A novel near-infrared fluorescent probe for real-time monitoring of leucine aminopeptidase activity and metastatic tumor progression

This article discusses the importance of early tumor detection, particularly in liver cancer, and the role of leucine aminopeptidase (LAP) as a potential marker for liver cancer diagnosis and prognosis assessment. The article highlights the limitations of current tumor markers and the need for new markers and multi-marker approaches to improve accuracy. The authors introduce a novel near-infrared fluorescent probe, NTAP, designed for LAP detection. They describe the synthesis of the probe and evaluate its spectral properties, including the LOD was 0.0038 U/mL, and QY was 0.32 %. The kinetic properties of NTAP, such as the relationship between LAP concentration (0–0.08 U/mL), reaction time (3 min), and fluorescence excitation spectra (475 nm) and emission spectra (715 nm) are investigated. The article also discusses the stability and selectivity of the probe and its ability to detect LAP in complex samples. Cellular imaging experiments demonstrate the NATP specificity and selectivity in detecting LAP activity and its inhibition. Animal models of liver and lung metastasis are used to evaluate the probe's imaging capabilities, showing its ability to accurately locate and detect metastatic lesions. The article concludes by emphasizing the potential applications of the NTAP probe in early tumor diagnosis, treatment monitoring, and the study of tumor metastasis mechanisms.

Digestive proteases of Morelet’s crocodile (Crocodylus moreletii) in three life stages

Introduction: Morelet’s crocodile (Crocodylus moreletii) is a species distributed in the Mexican southeast and threatened due to multiple pressures. Objective: To characterize the digestive proteases in the acid phase (stomach) and alkaline phase (intestine) of three life stages of C. moreletii in captivity (hatchling, juvenile, and adult). Methods: Total alkaline and acid protease activities were quantified using casein and haemoglobin as substrates. Trypsin, chymotrypsin, leucine aminopeptidase, and elastase activities were quantified using synthetic substrates. Protease profiles were analysed by SDS-PAGE and Native-PAGE. Results: The specific activity of acid and alkaline proteases showed differences between the three stages, finding the highest activity in the juveniles. Trypsin, chymotrypsin, leucine aminopeptidase, and elastase activities were higher in hatchlings. There were differences in optimum pH and temperature of acid and alkaline proteases, trypsin, and leucine aminopeptidase between the three stages, demonstrating the diversification of the enzymes according to different stages, as well as the presence of specific isoforms in each stage of C. moreletii. The acid phase zymogram showed four bands with pepsin-like acid activity in the hatchling and juvenile crocodile, while in the adult only two of the four bands were detected. The alkaline zymogram showed that the hatchling had the highest number of activity bands compared to the other stages, corresponding to the high specific activity reported in the alkaline phase. Conclusions: Digestive proteases of Morelet’s crocodile differ in their biochemical characteristics and the number of proteases between hatchling, juvenile, and adult. This could help in the future design of balanced diets as well to the sustainable management and production of this species.

A novel magnetic graphene-loaded biochar gel for the remediation of arsenic- and antimony-contaminated mining soil

Metalloid co-contamination such as arsenic (As) and antimony (Sb) in soils has posed a significant threat to ecological balance and human well-being. In this study, a novel magnetic graphene-loaded biochar gel (FeBG) was developed, and its remediation potential for the reclamation of AsSb spoiled soil was assessed through a six-month soil incubation experiment. Results showed that the incorporation of iron substances and graphene imparted FeBG with enhanced surface characteristics, such as the formation of a new FeO bond and an enlarged surface area compared to the pristine biochar (BC) (80.5 m2 g−1 vs 57.4 m2 g−1). Application of FeBG significantly decreased Na2HPO4-extractable concentration of As in soils by 9.9 %, whilst BC addition had a non-significant influence on As availability, compared to the control. Additionally, both BC (8.2 %) and FeBG (16.4 %) treatments decreased the Na2HPO4-extractable concentration of Sb in soils. The enhanced immobilization efficiency of FeBG for As/Sb could be attributed to FeBG-induced electrostatic attraction, complexation (Fe–O(H)–As/Sb), and π-π electron donor-acceptor coordination mechanisms. Additionally, the FeBG application boosted the activities of sucrase (9.6 %) and leucine aminopeptidase (7.7 %), compared to the control. PLS-PM analysis revealed a significant negative impact of soil physicochemical properties on the availability of As (β = −0.611, P < 0.01) and Sb (β = −0.848, P < 0.001) in soils, in which Sb availability subsequently led to a suppression in soil enzyme activities (β = −0.514, P < 0.01). Overall, the novel FeBG could be a potential amendment for the simultaneous stabilization of As/Sb and the improvement of soil quality in contaminated soils.

Restoring Subtropical Forests: Alleviating P Limitation and Introducing C Limitation Using Evergreen Broad-Leaved Tree Species

Determining which species to utilize for the artificial restoration of subtropical secondary forests has become a focal point in forestry and ecology. To compare the effects of the subtropical secondary forest artificial restoration model on soil microbial nutrient acquisition and limitation, we examined secondary forests (CKs), evergreen coniferous forests (GCPs), evergreen coniferous mixed broad-leaved forests (GCBMs), evergreen mixed broad-leaved forests (GBMs), and natural deciduous broad-leaved mixed forests (DBMs) as research subjects. Among them, GCPs, GCBMs, and GBMs were dominated by the species of the early, middle, and climax stages of subtropical forest succession, respectively. The activities and stoichiometry of β-1,4-glucosidase (BG), β-1,4-N-acetylglucosaminidase (NAG), leucine aminopeptidase (LAP), and acid phosphatase (ACP) in the topsoil were analyzed. The results showed that the forest type significantly affects the activities of BG and LAP rather than NAG or ACP. The BG activity in DBMs was the lowest, while the LAP activity in CKs was significantly higher than that in plantations. Furthermore, the nutrient limitation of microbes was quantitatively analyzed by using the vector analysis of enzyme stoichiometry. The soil microbes in the study area were co-limited by C and P, and the nutrient limitation was in the order of C &gt; P &gt; N. Among the forests, the enzyme stoichiometric ratios in GCPs and DBMs were closest to 1:1:1. From CKs to GBMs, the microbial C limitation was increased, while the P limitation was decreased. The C limitation in DBMs was slightly lower than that in CKs. Overall, the P limitation in evergreen plantations was less than that in CKs. The soil bulk density, C/P, and N/P significantly influenced enzyme activities and stoichiometry. These results suggest that the artificial restoration of subtropical forests using evergreen species alleviated P limitation, while using deciduous broad-leaved species offered potential for alleviating microbial C limitation. Compared with evergreen broad-leaved species, employing pioneer and mid-successional or deciduous broad-leaved species can better achieve balanced microbial nutrient requirements.

Characterization of leucine aminopeptidase (LAP) activity in sweet pepper fruits during ripening and its inhibition by nitration and reducing events

Key messagePepper fruits contain two leucine aminopeptidase (LAP) genes which are differentially modulated during ripening and by nitric oxide. The LAP activity increases during ripening but is negatively modulated by nitration.Leucine aminopeptidase (LAP) is an essential metalloenzyme that cleaves N-terminal leucine residues from proteins but also metabolizes dipeptides and tripeptides. LAPs play a fundamental role in cell protein turnover and participate in physiological processes such as defense mechanisms against biotic and abiotic stresses, but little is known about their involvement in fruit physiology. This study aims to identify and characterize genes encoding LAP and evaluate their role during the ripening of pepper (Capsicum annuum L.) fruits and under a nitric oxide (NO)-enriched environment. Using a data-mining approach of the pepper plant genome and fruit transcriptome (RNA-seq), two LAP genes, designated CaLAP1 and CaLAP2, were identified. The time course expression analysis of these genes during different fruit ripening stages showed that whereas CaLAP1 decreased, CaLAP2 was upregulated. However, under an exogenous NO treatment of fruits, both genes were downregulated. On the contrary, it was shown that during fruit ripening LAP activity increased by 81%. An in vitro assay of the LAP activity in the presence of different modulating compounds including peroxynitrite (ONOO−), NO donors (S-nitrosoglutathione and nitrosocyteine), reducing agents such as reduced glutathione (GSH), l-cysteine (l-Cys), and cyanide triggered a differential response. Thus, peroxynitrite and reducing compounds provoked around 50% inhibition of the LAP activity in green immature fruits, whereas cyanide upregulated it 1.5 folds. To our knowledge, this is the first characterization of LAP in pepper fruits as well as of its regulation by diverse modulating compounds. Based on the capacity of LAP to metabolize dipeptides and tripeptides, it could be hypothesized that the LAP might be involved in the GSH recycling during the ripening process.

Microbial nutrient limitation along a 2-million-year dune chronosequence

Long-term ecosystem development is characterized by a switch from nitrogen (N) to phosphorus (P) limitation of plant communities as soils age, which leads to changes in plant biomass, diversity, and foliar nutrient concentrations. Similar effects occur belowground, although the extent to which nutrient availability is the primary driver of long-term changes in soil microbial communities remains uncertain. To investigate this, we assessed proxies for soil microbial nutrient limitation along the Jurien Bay dune chronosequence in Western Australia, where a long-term decline in P availability is reflected in strong variation in plant and microbial community composition over 2 million years of soil development. We quantified carbon (C), N, and P in the soil microbial biomass, assayed soil enzymes involved in the acquisition of those nutrients from soil organic matter, and used microbial stoichiometric ratios and vector analysis of enzyme activities to assess whether long-term changes in soil nutrient availability are reflected in nutrient demand by soil microbes. Concentrations of microbial nutrients peaked in Holocene soils (1–6.5 ky), although differences in microbial P were not significant. There were no significant differences in microbial nutrient ratios (C:N, C:P, and N:P) along the chronosequence. Acid phosphatase activity increased continually throughout the chronosequence, while the activities of leucine aminopeptidase and β-glucosidase were greatest in Holocene soils and declined with increasing soil age. Corresponding vector lengths indicated greater investment in C-degrading enzymes in Holocene soils (≤6.5 ky), while vector angles indicated greater investment in the acquisition of P compared with N in Pleistocene soils (≥120 ky). Overall, these findings show that changes in nutrient availability during long-term pedogenesis at Jurien Bay are reflected in microbial investment in nutrient acquisition, but that the soil microbial biomass exhibits a remarkable degree of homeostasis despite major shifts in nutrient availability and community composition over 2 million years of ecosystem development.

Accumulation of polyethylene microplastics in river biofilms and effect on the uptake, biotransformation and toxicity of the antimicrobial triclosan

The interaction of multiple stressors in freshwater ecosystems may lead to adverse effects on aquatic communities and their ecological functions. Microplastics (MPs) are a class of contaminants of emerging concern that can exert both direct and indirect ecotoxicological effects. A growing number of studies have investigated MPs-attached microbial communities, but the interaction between MPs and substrate-associated biofilm (i.e., on natural river substrates, such as stones and sediments) remains poorly studied. In this work, the combined effects of polyethylene MPs (PE-MPs) with a particle size of 10–45 μm (2 mg/L) and the antimicrobial triclosan (TCS) (20 μg/L) were investigated on river biofilms through a short-term exposure experiment (72 h). To the best of authors’ knowledge, this is the first time that the combined effects of MPs and chemical contaminants in substrate-associated river biofilms were assessed. Different response parameters were evaluated, including (i) exposure assessment and ii) contaminants effects at different levels: bacterial community composition, antibiotic resistance, extracellular polymeric substances (EPS), photosynthetic efficiency (Yeff), and leucine aminopeptidase activity (LAPA). Triclosan was accumulated in river biofilms (1189–1513 ng/g dw) alongside its biotransformation product methyl-triclosan (20−29 ng/g dw). Also, PE-MPs were detected on biofilms (168−292 MP/cm2), but they had no significant influence on the bioaccumulation and biotransformation of TCS. A moderate shift in bacterial community composition was driven by TCS, regardless of PE-MPs co-exposure (e.g., increased relative abundance of Sphingomonadaceae family). Additionally, Yeff and EPS content were significantly disrupted in TCS-exposed biofilms. Therefore, the most remarkable effects on river biofilms were related to the antimicrobial TCS, whereas single PE-MPs exposure did not alter any of the evaluated parameters. These results demonstrate that biofilms might act as environmental sink of MPs. Although no interaction between PE-MPs and TCS was observed, the possible indirect impact of other MPs-adsorbed contaminants on biofilms should be further assessed.

Methionine sulfoxide reductase 2 regulates Cvt autophagic pathway by altering the stability of Atg19 and Ape1 in Saccharomyces cerevisiae

The reversible oxidation of methionine plays a crucial role in redox regulation of proteins. Methionine oxidation in proteins causes major structural modifications that can destabilize and abrogate their function. The highly conserved methionine sulfoxide reductases protect proteins from oxidative damage by reducing their oxidized methionines, thus restoring their stability and function. Deletion or mutation in conserved methionine sulfoxide reductases leads to aging and several human neurological disorders and also reduces yeast growth on nonfermentable carbon sources. Despite their importance in human health, limited information about their physiological substrates in humans and yeast is available. For the first time, we show that Mxr2 interacts invivo with two core proteins of the cytoplasm to vacuole targeting (Cvt) autophagy pathway, Atg19, and Ape1 in Saccharomyces cerevisiae. Deletion of MXR2 induces instability and early turnover of immature Ape1 and Atg19 proteins and reduces the leucine aminopeptidase activity of Ape1 without affecting the maturation process of Ape1. Additonally, Mxr2 interacts with the immature Ape1, dependent on Met17 present within the propeptide of Ape1 as a single substitution mutation of Met17 to Leu abolishes this interaction. Importantly, Ape1 M17L mutant protein resists oxidative stress-induced degradation in WT and mxr2Δ cells. By identifying Atg19 and Ape1 as cytosolic substrates of Mxr2, our study maps the hitherto unexplored connection between Mxr2 and the Cvt autophagy pathway and sheds light on Mxr2-dependent oxidative regulation of the Cvt pathway.