Leucine Aminopeptidase Research Articles

Effects of exogenous copper on microbial metabolic function and carbon use efficiency of Panax notoginseng planting soil.

Soil copper (Cu) pollution is a serious environmental risk in the Panax notoginseng planting area. However, the effect of Cu on soil microbial metabolism and nutrient cycling in this area remains unknown. Therefore, Biolog ECO-plate and enzyme stoichiometry methods were utilized in this study to investigate the impact of exogenous Cu (control: 0 mg·kg-1; Cu100: 100 mg·kg-1; Cu400: 400 mg·kg-1; and Cu600: 600 mg·kg-1) on the metabolic function of soil microbial and nutrient limitation in the P. notoginseng soil. The results indicated that Cu100 significantly increased soil organic carbon (SOC), total phosphorus (TP), soil C:N, microbial biomass carbon (MBC), and microbial biomass nitrogen (MBN) 9.89%, 15.65%, 17.91%, 61.87%, and 90.56% higher than the control, respectively. Moreover, the carbon source utilization ratio of carbohydrates, amino acids, and amphiphilic compounds of Cu100 also increased by 7.16%, 25.47%, and 84.68%, respectively, compared with the control. The activities of β-1,4-glucosidase, cellobiohyrolase, leucine amino peptidase, β-1,4-N-acetylglucosaminidase, and phosphatase significantly decreased with increasing Cu concentration. Soil enzyme stoichiometry showed that all treatments were limited by nitrogen (vector angle < 45°; 19.045-22.081). Cu600 led to the lowest carbon limitation (1.798) and highest carbon use efficiency (CUE:0.267). The PLS-SEM model also showed that MBC, MBN, MBP, and microbial diversity positively affected carbon and nitrogen limitation (0.654 and 0.424). Soil carbon, nitrogen, phosphorus, stoichiometric ratio, MBC, MBN, and MBP positively affected CUE (0.527 and 0.589). The microbial diversity index significantly negatively affected CUE (-1.490). Multiple linear stepwise regression analyses showed that CUE was mainly influenced by MBC, AP, C:P, and LAP. Thus, P. notoginseng soil can benefit soil microbial carbon and nitrogen limitations at low Cu concentrations. Clarifying the metabolic activity and nutritional status of microorganisms under Cu stress can provide some theoretical basis for realizing China's comprehensive and effective management and control policies for environmental risks from metals by 2035.

Dual Enzyme-Locked Activation Reporter for Accurate Liver Cancer Surveillance.

Activatable probes with a higher signal-to-background ratio and accuracy are essential for monitoring liver cancer as well as intraoperative fluorescence navigation. However, the presence of only one biomarker is usually not sufficient to meet the high requirement of a signal-to-background ratio in cancer surveillance, leading to the risk of misdiagnosis. In this work, a dual-locked activation response probe, Si-NTR-LAP, for nitroreductase and leucine aminopeptidase was reported. This dual-locked probe provides better tumor recognition and a higher signal-to-noise ratio than that of single-locked probes (Si-LAP and Si-NTR). In both the subcutaneous tumor model and the more complex orthotopic hepatocellular carcinoma model, the probe was able to identify tumor tissue with high specificity and accurately differentiate the boundaries between tumor tissue and normal tissue. Therefore, the dual-locked probe may provide a new and practical strategy for applying to real patient tumor tissue samples.

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.

The effects of postfire regeneration patterns on soil microbial metabolic limitation based on eco-enzyme stoichiometry in the boreal forest of China

Postfire regeneration is essential for maintaining the stability and functionality of forest ecosystems following wildfires. However, it is unclear whether regeneration patterns affect carbon (C), nitrogen (N), or phosphorus (P) limitations on microbial metabolism in the soil. In this study, we investigated the soil extracellular enzyme activities (EEAs) in different forest types regenerated by five patterns (Dahurian larch monocultures, Dahurian larch and birch mixed plantations, Mongolian pine monocultures, Mongolian pine and birch mixed plantations, and naturally regenerated forests). Based on the principles of ecoenzymatic stoichiometry, we elucidated the spatiotemporal variation in energy (C) and nutrient (N or P) limitations on soil microbial metabolism by collecting a total of 135 composite soil samples across three seasons in the five regeneration patterns. The regeneration pattern had a significant effect on the activities of C-, N- and P-acquiring soil extracellular enzymes (P<0.05). The P-acquiring enzyme activity was 2.71 times greater than the C-acquiring enzyme activity and 3.44 times greater than the N-acquiring enzyme activity across all forest types and seasons. The activities of microbial metabolic C-, N-, and P-acquiring enzymes showed similar seasonal dynamic patterns, with the exception of L-leucine aminopeptidase. In all forest types, the activities of enzymes responsible for acquiring C, N, and P were linearly correlated with one another according to the findings from standard major axis regression analysis (P<0.001). The values of the enzymatic vectors were greater than 45°, indicating that P rather than N was the dominant constraint on soil microbial metabolism. Forests with mixed regeneration patterns had greater P limitations than did the corresponding monoculture forests. Soil temperature was the common soil variable affecting the activities of EEAs across seasons, while the contents of soil organic carbon, dissolved carbon, ammonium nitrogen, and nitrate nitrogen were the significant driving factors of EEAs in a single season. However, forest productivity, including tree density, stand basal area, and forest stand volume had a predominant influence on microbial metabolic P limitation. This study suggests a pronounced role of P in soil microbial metabolism within boreal forests regenerated after wildfires.

Priming and balance of soil organic carbon differ with additive C:N ratios and long-term green manuring

The input of organic C strongly alters the magnitude and direction of the microbial mineralization of soil organic matter (SOM), a phenomenon known as the “priming effect” (PE). The C:N ratios of additives are expected to affect the intensity of PE as it balances the C and N requirements of microbial growth. Growing of green manure (GM) crops in agricultural system strongly regulates PE intensity as it enhances the level and stability of SOM. However, the driving factors of additive C:N ratios and long-term GM crops on PE remain unclear. We addressed this knowledge gap by performing a 92-day incubation of 10-year summer fallow-wheat and GM-wheat soils by adding mixtures of 13C-labelled glucose and ammonium sulfate differing in the C:N ratio (15 vs. 50). The PE was increased by 148–288 % due to the high C:N ratio of the added mixtures, indicating that the quality of input exerted effects on the PE across the two soils. The PE of green manured (GMd) soil was increased by 23 % (p < 0.05) compared with that of summer fallow soil, as microorganisms produce extracellular enzymes such as β-glucosidase and leucine aminopeptidase to co-metabolize SOM. Nevertheless, compared with the summer fallow soil, 26 % more glucose-C was sequestered in GMd soil to compensate for C loss. We propose a conceptual model of “N mining” and “co-metabolism” to explain the effect of additive C:N ratio on PE in the soils under different GM practices. The “N mining” is the main cause of PE when the additive C:N ratio is high, and the “co-metabolism” becomes the dominant factor in long-term GMd soil with high SOM content and stability. Our findings demonstrate the importance of long-term incorporation of GM-driven changes in organic C inputs and SOM content and stability in regulating PE and soil C dynamics. Understanding the C dynamics under long-term GM practices contributes to formulate optimized agricultural strategies for promoting C sequestration and accurately predict soil C dynamics in the future.

Enzyme stoichiometry reveals microbial nitrogen limitation in stony soils

Resource limitation for soil microorganisms is the crucial factor in nutrient cycling and vegetation development, which are especially important in arid climate. Given that rock fragments strongly impact hydrologic and geochemical processes in arid areas, we hypothesized that microbial resource (C and N) limitation will increase along the rock fragment content (RFC) gradient. We conducted a field experiment in Minjiang river arid valleys with four RFC content (0 %, 25 %, 50 %, and 75 %, V V−1) and four vegetation types (Artemisia vestita, Bauhinia brachycarpa, Sophora davidii, and the soil without plants). Activities of C (β-1,4-glucosidase, BG), N (β-1,4-N-acetyl-glucosaminidase, NAG; L-leucine aminopeptidase, LAP), and P (acid phosphatase, ACP) acquiring enzymes were investigated to assess the limitations by C, N or P. In unplanted soil, the C acquiring enzyme activity decreased by 43 %, but N acquiring enzyme activity increased by 72 % in 75 % RFC than those in rock-free soils (0 % RFC). Increasing RFC reduced C:N and C:P enzymatic ratios, as well as vector length and vector angle (< 45°). Plants increased the activities of C and N acquiring enzymes in soils, as well as C:P and N:P enzyme activities, as well as vector length (by 5.6 %–25 %), but decreased vector angle (by 13 %–21 %). Enzyme stoichiometry was dependent on biotic and abiotic factors, such as soil water content, soil C:N, and total content of phospholipid fatty acids, reflecting microbial biomass content. Increased RFC shifted enzymatic stoichiometry toward lower C but stronger N limitation for microorganisms. Vegetation increased microbial C and N limitation, and impacted the enzymatic activities and stoichiometry depending on shrub functional groups. Consequently, the direct effects of vegetation, nutrient availability and microbial biomass content, as well as indirect effects of soil properties collectively increased microbial resource limitations along the RFC gradient.

A transformable and self-oxygenated smart probe for enhanced tumor sonodynamic therapy

Sonodynamic therapy (SDT) is emerging as a promising modality for cancer treatment. However, improving the tumor bioavailability and anti-hypoxia capability of sonosensitizers faces a big challenge. In this work, we present a tumor microenvironment (TME)-mediated nanomorphology transformation and oxygen (O2) self-production strategy to enhance the sonodynamic therapeutic efficacy of tumors. A smart probe Ce6-Leu@Mn2+ that consists of a glutathione (GSH) and leucine amino peptidase (LAP) dual-responsive unit, a 2-cyanobenzothiazole (CBT) group, and a Mn2+-chelated Ce6 as sonosensitizer for tumor SDT was synthesized, and its SDT potential for liver tumor HepG2 in living mice was systematically studied. It was found that the probes could self-assemble into large nanoparticles in physiological condition and spontaneously transformed into small particles under the dual stimulation of GSH and LAP in TME resulting in enhanced tumor accumulation and deep penetration. More notably, Ce6-Leu@Mn2+ could convert endogenous hydrogen peroxide to O2, thereby alleviating the hypoxia and achieving effective SDT against hypoxic tumors under the excitation of ultrasound. We thus believe this smart TME-responsive probe may provide a noninvasive and efficient means for malignant tumor treatment. Statement of significanceSonodynamic therapy (SDT) is emerging as a promising therapeutic modality for cancer treatment. However, how to improve the tumor bioavailability and anti-hypoxia capability of sonosensitizers remains a huge challenge. Herein, we rationally developed a theranostic probe Ce6-Leu@Mn2+ that can transform into small-size nanoparticles from initial large particles under the dual stimulation of LAP and GSH in tumor microenvironment (TME) resulting in enhanced tumor accumulation, deep tissue penetration as well as remarkable O2 self-production for enhanced sonodynamic therapy of human liver HepG2 tumor in living mice. This smart TME-responsive probe may provide a noninvasive and efficient means for hypoxic tumor treatment.

Soil nitrogen and phosphorus regulate decomposition of organic nitrogen compounds in the rothamsted experiment

The effects of long-term N and P fertilisation on soil organic nitrogen (SON) turnover are unclear. We sampled soils fertilised by N and/or P since 1843 at Rothamsted Research to investigate the effects of long-term N and P fertilisation on high- or low-molecular-weight SON decomposition and gross N mineralisation. Short-term assays with added 14C-labelled proteins, peptides, and amino acids and gross organic N mineralisation were measured, and the microbial communities and functions were also assessed in parallel. Long-term N fertilisation increased the contents of soil extractable organic N and peptides but decreased organic N-hydrolysing enzyme activities (N-acetyl-β-D-glucosaminidase and leucine aminopeptidase). Fertilisation with P and N accelerated and reduced the decomposition of 14C-labelled organic N compounds, respectively. The decomposition of peptides and amino acids, as labile SON components, was mainly regulated by soil P content because microbial biomass and activity were more sensitive to P fertilisation than to N fertilisation. Gross NH4+/NO3− production and consumption were accelerated by 41%–60% under N fertilisation but remained unchanged under P fertilisation compared to the unfertilised treatment. Metagenomic sequencing showed that N fertilisation increased microbial diversity and enriched functional genes associated with organic N decomposition, compared to the unfertilised treatment. P fertilisation had no effect on the abundance of these functional genes. In agricultural practices, it is essential to comprehensively consider the interaction between N and P fertilisation to optimise the cycling and utilisation of SON.

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.

Labile carbon content and nutrient availability determines microbial composition in topsoil and subsoil following land‐use change in subtropical China

AbstractEvaluating the impact of land‐use intensification on soil microbial communities is essential for recognizing the implications on microbiome stability and ecosystem function. The microbial biomass and enzyme activity in the topsoil have been found to decrease as a consequence of natural forest conversion; however, the impact of land‐use conversion on the microbes in subsoil remains largely unclear. Here, we examined the effect of primary forest conversion to plantations and cultivated lands on microbial communities at three sites with similar soil, climate, and landform. The forest conversion was set as the experimental treatment, and the primary forest as the control. A linear mixed‐effect model was applied to investigate the role of environmental parameters in shaping the soil microbial biomass and community determined by the phospholipid fatty acids analysis in both the topsoil (0–20 cm) and subsoil (20–40 cm). Compared to the primary forest, the total microbial biomass, β‐1,4‐N‐acetylglucosaminidase, leucine aminopeptidase, acid phosphatase activities, and labile organic C fraction contents in both topsoil and subsoil were reduced in cultivated lands. The ratios of gram‐positive bacteria to gram‐negative bacteria and arbuscular mycorrhizal fungi to saprotrophic fungi in subsoil decreased by 45%–53% and increased by 29%–151%, respectively, following the primary forest conversion to cultivated lands. The response ratio (the percentage of microbial and enzyme indicator response to the primary forest conversion) ranged from −80% to 140% depending on the soil depth, specific microbial group, and converted land‐use type. Microbial biomass and enzyme activity are primarily controlled by the labile organic C content and nutrients availability in both topsoil and subsoil. This study suggests that the primary forest conversion exerts an adverse effect on the microbial biomass, enzyme activity, and substrate availability in both topsoil and subsoil, highlighting the degradation of subsoil health in subtropical China.

Substrate specificity of exopeptidases in small intestinal mucosa determines the structure of food-derived collagen peptides in rat lumen and blood

Gly-Pro-Hyp-Gly is the most common motif in collagen. Ingestion of collagen hydrolysate (CH) has been reported to significantly increase Pro-Hyp and Hyp-Gly in human peripheral blood. However, the elevation of Gly-Pro content remains negligible. This study seeks to elucidate the underlying reasons behind the bioavailability of CH-derived peptides using in vivo and in vitro digestion. After oral administration of CH (800 mg/kg body weight) to rats, Pro-Hyp significantly increased in the lumen, small intestinal tissue, and blood from the abdominal vein, while only negligible amounts of Gly-Pro were detected. In vitro digestion of CH by digestive exopeptidases, including aminopeptidase N, leucine aminopeptidase, and carboxypeptidase A predominantly generated Gly-Pro. Interestingly, the digestion of CH with crude extract of small intestinal mucosa predominantly generated Pro-Hyp and negligible amounts of Gly-Pro. Furthermore, synthetic Gly-Pro was rapidly degraded by crude extract of the intestinal mucosa, while Pro-Hyp showed higher resistance. These facts indicate prolidase activity in the mucosa plays a crucial role in determining collagen-derived peptides in the body.

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.

Identification and Validation of Compounds Targeting Leishmania major Leucyl-Aminopeptidase M17.

Leishmaniasis is a neglected tropical disease; there is currently no vaccine and treatment is reliant upon a handful of drugs suffering from multiple issues including toxicity and resistance. There is a critical need for development of new fit-for-purpose therapeutics, with reduced toxicity and targeting new mechanisms to overcome resistance. One enzyme meriting investigation as a potential drug target in Leishmania is M17 leucyl-aminopeptidase (LAP). Here, we aimed to chemically validate LAP as a drug target in L.major through identification of potent and selective inhibitors. Using RapidFire mass spectrometry, the compounds DDD00057570 and DDD00097924 were identified as selective inhibitors of recombinant Leishmania major LAP activity. Both compounds inhibited in vitro growth of L.major and L.donovani intracellular amastigotes, and overexpression of LmLAP in L.major led to reduced susceptibility to DDD00057570 and DDD00097924, suggesting that these compounds specifically target LmLAP. Thermal proteome profiling revealed that these inhibitors thermally stabilized two M17 LAPs, indicating that these compounds selectively bind to enzymes of this class. Additionally, the selectivity of the inhibitors to act on LmLAP and not against the human ortholog was demonstrated, despite the high sequence similarities LAPs of this family share. Collectively, these data confirm LmLAP as a promising therapeutic target for Leishmania spp. that can be selectively inhibited by drug-like small molecules.

Changes in Digestive Enzyme Activities during Larval Development of Spotted Seatrout (Cynoscion nebulosus)

The spotted seatrout (Cynoscion nebulosus)—an important commercial species—has a high potential for aquaculture in the Gulf of Mexico. To optimize its feeding during larval rearing, this study aims to evaluate the primary gastric (pepsin), intestinal (leucine aminopeptidase and alkaline phosphatase), and pancreatic (alkaline protease, trypsin, chymotrypsin, amylase, and lipase) enzyme activities from hatching to day 30. A multivariate analysis identified three digestive enzyme development stages during the spotted seatrout larval transformation. The first stage occurred between 1 (mean ± standard error (SE) = 1.73 ± 0.14 millimeter (mm) standard length (SL)) and 3 (2.14 ± 0.07 mm SL) days after hatching (DAH); a period of digestive stability showed the highest activity in amylase and bile salt-dependent lipase. The second stage (from 4 (2.53 ± 0.09 mm SL) to 20 (10.92 ± 0.51 mm SL) DAH) was a period of digestive transition, during which leucine aminopeptidase, chymotrypsin, and alkaline proteases were identified as the predominant enzymes from 4 to 5 DAH. In the third stage—a period of digestive stability—pepsin was the major enzyme that occurred between 25 (16.51 ± 0.81 mm SL) and 30 (25.91 ± 0.82 mm SL) DAH. These results indicate that the spotted seatrout larvae have a digestive system adapted to lipids and carbohydrates at the onset of feeding, with an immediate transition to protein digestion when exogenous feeding begins. Additionally, the digestive system of the spotted seatrout may be considered mature at 25 DAH. Further research is needed to elucidate the mechanisms of digestive tract development in the spotted seatrout larvae.

Proteolytic reaction-based versatile electrochemical biosensor chip for simple and quantitative point-of-care testing

Electrochemical protease biosensors are well-suited for simple and quantitative point-of-care testing. However, there has been limited development of versatile chip formats similar to lateral-flow and glucose test strips for electrochemical proteases biosensors. This paper presents a versatile electrochemical biosensor chip for the wash-free and sensitive detection of proteases. The chip comprises (i) a reaction well, (ii) micropatterned electrodes on a glass substrate, and (iii) target protease-dependent filter paper. All (bio)chemicals required for the proteolytic reaction and signal amplification were predried on filter paper, which was then placed inside the reaction well. Upon injecting the sample solution into the reaction well, most of the dried (bio)chemicals dissolved rapidly. Wash-free and sensitive protease detection was achieved using electrochemical-enzymatic (EN) redox cycling of a redox-active species (4-amino-1-naphthol, AN) liberated from a peptide substrate through a rapid proteolytic reaction. The EN redox cycling involves a working electrode, AN, glucose dehydrogenase, and glucose. To enhance dissolvability and stability, (bio)chemicals were dried using a surfactant and stabilizer and stored at 4 °C with vacuum sealing. To minimize the effect of background variation among the different samples, a high concentration of glucose (200 mM) was used, and the difference between the two charge values obtained before and after the incubation period was measured. The biosensor chips successfully detected three proteases: thrombin, leucine aminopeptidase (LAP), and 3CL protease from coronavirus SARS-CoV-2 (Mpro). The detection limits of thrombin in artificial serum, Mpro in artificial saliva, and LAP in human serum were approximately 100, 100 pg/mL, and 8 U/L, respectively.

Summer cover crop and temporary legume-cereal intercrop effects on soil microbial indicators, soil water and cash crop yields in a semi-arid environment

ContextFew studies have investigated summer cover crops as a replacement for bare fallow in semi-arid temperate regions, and summer cover crop impacts on soil biological function, water balances and subsequent winter crop yields in these regions remain largely unknown. ObjectiveThe 4-year field study aimed to determine the consequences of replacing a summer fallow with a summer growing cover crop on soil function, water balances and subsequent winter cash crop yields in a semi-arid region (486 mm mean annual rainfall) of the Australian southern cropping zone. MethodsA canola (Brassica napus)-wheat (Triticum aestivum L.) rotation with summer fallow was compared to the same rotation with either i) a single species (buckwheat, Fagopyrum esculentum L.) summer cover crop; ii) a four-species mixed brassica + cereal summer cover crop or iii) a cropping sequence with a temporary legume intercrop in the wheat phase of the rotation. Cover crop biomass production, soil water to a depth of 90 cm at cash crop sowing, crop yields and soil functional indices were quantified. ResultsCover crops produced 500–700 kg biomass ha−1 in the 2020 and 2021 seasons and 1000–2400 kg biomass ha−1 in 2022. The four-species mixed cover crop significantly lowered soil water (around 30 mm; P < 0.05) at the time of sowing the cash crop compared to the control and resulted in 15% reduction (P = 0.12) in canola seed yield in 2020, but no significant effect of cover crops of soil water and mineral N at sowing, or cash crop yields, were observed in subsequent seasons. In 2021 the temporary vetch intercrop produced > 1000 kg ha−1 biomass over 9 weeks, and led to a (non-significant) 10% wheat yield reduction. There was no significant effect of cover crop or temporary intercrop treatments on soil functions in the 2020 or 2021 seasons, but activities of several soil enzymes (leucine aminopeptidase, arylsulfatase and B-glucosidase; P < 0.05) and water-soluble carbon (P = 0.059) were significantly higher in the summer cover crop plots compared to control and intercrop plots in March 2022 following termination of the cover crops. These changes coincided with a significant increase in saturated hydraulic conductivity in the four-species cover crop plots in March 2022 compared to control plots. ImplicationsThe study did not provide any compelling evidence for benefits of summer cover crops over fallow in the short term (3–4 years). The relatively low cover crop biomass production over the 4-year study suggests summer cover crops are unlikely to substantially improve soil carbon levels in temperate, semi-arid cropping systems.

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.

Substitution of Inorganic Fertilizer with Organic Fertilizer Influences Soil Carbon and Nitrogen Content and Enzyme Activity under Rubber Plantation

Conventional fertilization practices can lead to many ecological problems, such as nutrient imbalance, soil acidity, and reduced soil fertility, in natural rubber plantations. To address these challenges, a field investigation was strategically carried out to substitute inorganic fertilizer with organic fertilizer, consisting of six treatments: no fertilization (CK), inorganic fertilizer (NPK), 25% replacement of inorganic through organic (25% manure (M)), 50% replacement of inorganic through organic (50% manure (M)), 75% replacement of inorganic through organic (75% manure (M)), and 100% organic fertilizer (100% manure). The soil physicochemical properties (soil organic carbon (SOC), total nitrogen (TN), mineral nitrogen (N), ammonium nitrogen (NH4+-N), and nitrate nitrogen (NO3−-N)), C:N, pH, and the carbon- and nitrogen-converting enzymes β-1,4-glucosidase (BG), N-acetylglucosaminidase (NAG) and L-leucine aminopeptidase (LAP) were all determined. The partial substitution of inorganic fertilizer with organic fertilizer (i.e., 75% M at surface soil layer) showed higher SOC (14.52 g·kg−1), TN (1.06 g·kg−1), N (20.07 mg·kg−1), C:N (14.63), NH4+-N (10.63 mg·kg−1), and NO3−-N (11.06 mg·kg−1) than NPK and CK. This increase in physicochemical properties after partial replacement of inorganic with organic fertilizer resulted from higher carbon and nitrogen enzyme activities (BG (143.17·nmol·g−1·h−1), NAG (153.96 nmol·g−1·h−1), and LAP (153.48 nmol·g−1·h−1)) compared to NPK and CK. Further, the Pearson correlation and redundancy analysis (RDA) analyses confirmed a significant positive correlation between SOC, N, and soil enzymes. This study presents a new strategy for assessing the impact of partially replacing inorganic fertilizer with organic fertilizer in rubber plantations in tropical regions, mainly by modifying the soil nutrient composition.

Cloning and catalytic profile of Hyalomma dromedarii leucine aminopeptidase

Ticks have harmful impacts on both human and animal health and cause considerable economic losses. Leucine aminopeptidase enzymes (LAP) play important roles during tick infestation to liberate vital amino acids necessary for growth. The aim of the current study is to identify, express and characterize the LAP from the hard tick Hyalomma dromedarii and elucidate its biochemical characteristics. We cloned an open reading frame of 1560 bp encoding a protein of 519 amino acids. The LAP full-length was expressed in Escherichia coli BL21 (DE3) and purified. The recombinant enzyme (H.d rLAP- 6×His) had a predicted molecular mass of approximately 55 kDa. Purification and the enzymatic characteristics of H.d rLAP- 6×His were studied. The purified enzyme showed maximum activity at 37 °C and pH 8.0–8.5 using Leu-p-nitroanilide as a substrate. The activity of H.d rLAP- 6×His was sensitive to β-mercaptoethanol, dl-dithiothreitol, 1,10- phenanthroline, bestatin HCl, and EDTA and completely abolished by 0.05 % SDS. In parallel, the enzymatic activity was enhanced by Ni2+, Mn2+ and Mg2+, partially inhibited by Na+, Cu2+, Ca2+ and completely inhibited by Zn2+.