Responses of soil microbiome to copper-based materials (nano and bulk) for agricultural applications: An indoor-mesocosm experiment.

The foreseen increasing application of copper-based nanomaterials (Cu-NMs), replacing or complementing existing Cu-agrochemicals, may negatively impact the soil microbiome. Thus, we studied the effects on soil microbiome function and composition of nano copper oxide (nCuO) or copper hydroxide NMs in a commercial (Kocide®3000) or a lab-synthetized formulation (nCu(OH)2) or bulk copper hydroxide (Cu(OH)2-B), at the commonly recommended Cu dose of 50 mg(Cu)kg-1 soil. Microbial responses were studied over 28 days in a designed indoor mesocosm. On day-28, in comparison to non-treated soil (CT), all Cu-treatments led to a reduction in dehydrogenase (95% to 68%), arylsulfatase (41% to 27%), and urease (40% to 20%) activity. There was a 32% increase in the utilization of carbon substrates in the nCuO-treatment and an increased abundance of viable bacteria in the nCu(OH)2-treatment (75% of heterotrophic and 69% of P-solubilizing bacteria). The relative abundance of Acidobacteria [Kocide®3000, nCuO, and Cu(OH)2-B treatments] and Flavobacteriia [nCu(OH)2-treatment] was negatively affected by Cu exposure. The abundance of Cu-tolerant bacteria increased in soils treated with Kocide®3000 (Clostridia) and nCu(OH)2 (Gemmatimonadetes). All Cu-treated soils exhibited a reduced abundance of denitrification-related genes (0.05% of nosZ gene). The DTPA-extractable pool of ionic Cu(II) varied among treatments: Cu(OH)2-B > Kocide®3000 âˆ¼ nCuO>nCu(OH)2, which may explain changes on the soil microbiome composition, at the genera and OTU levels. Thus, our study revealed that Cu-materials (nano and bulk) influence the soil microbiome with implications on its ecological role. It highlights the importance of assessing the impact of Cu-materials under dynamic and complex exposure scenarios and emphasizes the need for specific regulatory frameworks for NMs.

Post-anthesis dry matter and nitrogen accumulation, partitioning, and translocation in maize under different nitrate-ammonium ratios in Northwestern China.

Introduction: An appropriate supply of ammonium (NH4+) in addition to nitrate (NO3-) can greatly improve plant growth and promote maize productivity. However, knowledge gaps exist regarding the mechanisms by which different nitrogen (N) fertilizer sources affect the enzymatic activity of nitrogen metabolism and non-structural carbohydrates during the post-anthesis period. Methods: A field experiment across 3-year was carried out to explore the effects of four nitrateammonium ratio (NO3-/NH4+ = 1:0 (N1), 1:1 (N2), 1:3 (N3), and 3:1 (N4)) on postanthesis dry matter (DM) and N accumulation, partitioning, transportation, and grain yield in maize. Results: NO3-/NH4+ ratio with 3:1 improved the enzymatic activity of N metabolism and non-structural carbohydrate accumulation, which strongly promoted the transfer of DM and N in vegetative organs to reproductive organs and improved the pre-anthesis DM and nitrogen translocation efficiency. The enzymatic activities of nitrate reductase, nitrite reductase, glutamine synthetase, glutamine oxoglutarate aminotransferase, and non-structural carbohydrate accumulation under N4 treatment were increased by 9.30%-32.82%, 13.19%-37.94%, 4.11%-16.00%, 11.19%-30.82%, and 14.89%-31.71% compared with the other treatments. Mixed NO3--N and NH4+-N increased the total DM accumulation at the anthesis and maturity stages, simultaneously decreasing the DM partitioning of stem, increasing total DM, DM translocation efficiency (DMtE), and contribution of pre-anthesis assimilates to the grain (CAPG) in 2015 and 2017, promoting the transfer of DM from stem to grain. Furthermore, the grain yield increased by 3.31%-9.94% (2015), 68.6%-26.30% (2016), and 8.292%-36.08% (2017) under the N4 treatment compared to the N1, N2, and N3 treatments. Conclusion: The study showed that a NO3-/NH4+ ratio of 3:1 is recommended for high-yield and sustainable maize management strategies in Northwestern China.

Characterization of unique EDTA-insensitive methylthioalkylmalate synthase from Eutrema japonicum and its potential application in synthetic biology.

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC), a derivative of glucosinolate with a six-carbon chain, is a compound found in wasabi and has diverse health-promoting properties. The biosynthesis of glucosinolates from methionine depends on a crucial step catalyzed methylthioalkylmalate synthases (MAMs), which are responsible for the generation of glucosinolates with varying chain lengths. In this study, our primary focus was the characterization of two methylthioalkyl malate synthases, MAM1-1 and MAM1-2, derived from Eutrema japonicum, commonly referred to as Japanese wasabi. Eutremajaponicum MAMs (EjMAMs) were expressed in an Escherichiacoli expression system, subsequently purified, and in vitro enzymatic activity was assayed. We explored the kinetic properties, optimal pH conditions, and cofactor preferences of EjMAMs and compared them with those of previously documented MAMs. Surprisingly, EjMAM1-2, categorized as a metallolyase family enzyme, displayed 20% of its maximum activity even in the absence of divalent metal cofactors or under high concentrations of EDTA. Additionally, we utilized AlphaFold2 to generate structural homology models of EjMAMs, and used in silico analysis and mutagenesis studies to investigate the key residues participating in catalytic activity. Moreover, we examined in vivo biosynthesis in E. coli containing Arabidopsis thaliana branched-chain amino acid transferase 3 (AtBCAT3) along with AtMAMs or EjMAMs and demonstrated that EjMAM1-2 exhibited the highest conversion rate among those MAMs, converting l-methionine to 2-(2-methylthio) ethyl malate (2-(2-MT)EM). EjMAM1-2 shows a unique property in vitro and highest activity on converting l-methionine to 2-(2-MT)EM in vivo which displays high potential for isothiocyanate biosynthesis in E. coli platform.

Standard Radio-Iodine Labeling Protocols Impaired the Functional Integrity of Mesenchymal Stem/Stromal Cell Exosomes.

Mesenchymal stem/stromal cells (MSCs) are an extensively studied cell type in clinical trials due to their easy availability, substantial ex vivo proliferative capacity, and therapeutic efficacy in numerous pre-clinical animal models of disease. The prevailing understanding suggests that their therapeutic impact is mediated by the secretion of exosomes. Notably, MSC exosomes present several advantages over MSCs as therapeutic agents, due to their non-living nature and smaller size. However, despite their promising therapeutic potential, the clinical translation of MSC exosomes is hindered by an incomplete understanding of their biodistribution after administration. A primary obstacle to this lies in the lack of robust labels that are highly sensitive, capable of directly and easily tagging exosomes with minimal non-specific labeling artifacts, and sensitive traceability with minimal background noise. One potential candidate to address this issue is radioactive iodine. Protocols for iodinating exosomes and tracking radioactive iodine in live imaging are well-established, and their application in determining the biodistribution of exosomes has been reported. Nevertheless, the effects of iodination on the structural or functional activities of exosomes have never been thoroughly examined. In this study, we investigate these effects and report that these iodination methods abrogate CD73 enzymatic activity on MSC exosomes. Consequently, the biodistribution of iodinated exosomes may reflect the biodistribution of denatured exosomes rather than functionally intact ones.

Functional prediction of the potential NGLY1 mutations associated with rare disease CDG.

Genetic diseases are currently diagnosed by functional mutations. However, only some mutations are associated with disease. It is necessary to establish a quick prediction model for clinical screening. Pathogenic mutations in NGLY1 cause a rare autosomal recessive disease known as congenital disorder of deglycosylation (NGLY1-CDDG). Although NGLY1-CDDG can be diagnosed through gene sequencing, clinical relevance of a detected mutation in NGLY1 needs to be further confirmed. In this study, taken NGLY1-CDDG as an example, a comprehensive and practical predictive model for pathogenic mutations on NGLY1 through an NGLY1/Glycopeptide complex model was constructed, the binding sites of NGLY1 and glycopeptides were simulated, and an in vitro enzymatic assay system was established to facilitate quick clinical decisions for NGLY1-CDDG patients. The docking model covers 42 % of reported NGLY1-CDDG missense mutations (5/12). All reported mutations were subjected to in vitro enzymatic assay in which 18 mutations were dysfunctional (18/30). In addition, a full spectrum of functional R328 mutations was assayed and 11 mutations were dysfunctional (11/19). In this study, a model of NGLY1 and glycopeptides was built for potential functional mutations in NGLY1. In addition, the effect of potential regulatory compounds, including N-acetyl-l-cysteine and dithiothreitol, on NGLY1 was examined. The established in vitro assay may serve as a standard protocol to facilitate rapid diagnosis of all mutations in NGLY1-CDDG. This method could also be applied as a comprehensive and practical predictive model for the other rare genetic diseases.

Extraction of Soluble Proteins from Leaves.

Protein biochemistry can provide valuable answers to better understand plant performance and responses to the surrounding environment. In this chapter, we describe the process of extracting proteins from plant leaf samples. We highlight the key aspects to take into consideration to preserve protein integrity, from sample collection to extraction and preparation or storage for subsequent analysis of protein abundance and/or enzymatic activities.

Investigating the synergistic effects of biochar, trans-zeatin riboside, and Azospirillum brasilense on soil improvement and enzymatic activity in water-stressed wheat.

BACKGROUND: Water stress is a major danger to crop yield, hence new approaches to strengthen plant resilience must be developed. To lessen the negative effects of water stress on wheat plants, present study was arranged to investigate the role of synergistic effects of biochar, trans-zeatin riboside (t-ZR), and Azospirillum brasilense on soil improvement and enzymatic activity in water-stressed wheat. RESULTS: In a three-replication experiment comprising of four treatments (T0: Control, T1: Drought stress (DS), T2: DS + t-ZR with biochar, T3: DS + A. brasilense with biochar), we observed notable improvements in soil quality and enzymatic activities in water-stressed wheat plants with the application of t-ZR and A. brasilense with biochar. In drought stress, Treatment having the application of A. brasilense with biochar performs best as compared to the other and significant increased the enzymatic activities such as peroxidase (7.36%), catalase (8.53%), superoxide dismutase (6.01%), polyphenol oxidase (14.14%), and amylase (16.36%) in wheat plants. Different enzymatic activities showed different trends of results. Soil organic C, dissolved organic C, dissolved organic N also enhanced 29.46%, 8.59%, 22.70% respectively with the application of A. brasilense with biochar under drought stress condition. CONCLUSIONS: The synergistic action of A. brasilense and biochar creates an effective microbiological environment that supports essential plant physiological processes during drought stress. This enhancement is attributed to improved soil fertility and increased organic matter content, highlighting the potential of these novel strategies in mitigating water stress effects and enhancing crop resilience.

Recent advances in design and application of synthetic membraneless organelles.

Membraneless organelles (MLOs) formed by liquid-liquid phase separation (LLPS) have been extensively studied due to their spatiotemporal control of biochemical and cellular processes in living cells. These findings have provided valuable insights into the physicochemical principles underlying the formation and functionalization of biomolecular condensates, which paves the way for the development of versatile phase-separating systems capable of addressing a variety of application scenarios. Here, we highlight the potential of constructing synthetic MLOs with programmable and functional properties. Notably, we organize how these synthetic membraneless compartments have been capitalized to manipulate enzymatic activities and metabolic reactions. The aim of this review is to inspire readerships to deeply comprehend the widespread roles of synthetic MLOs in the regulation enzymatic reactions and control of metabolic processes, and to encourage the rational design of controllable and functional membraneless compartments for a broad range of bioengineering applications.

Organic contamination and multi-biomarker assessment in watersheds of the southern Brazil: an integrated approach using fish from the Astyanax genus.

We aimed to examine the responses of pollution biomarkers in feral fish from Astyanax genus collected at three hydrographic regions in southern Brazil and the capacity of these tools to differentiate between various levels of contamination. To achieve this, levels of organochlorine pesticides (liver), as well as the biomarkers AChE (muscle and brain), TBARS (liver), and EROD (liver) were assessed. Collections were conducted in four municipalities (Alegrete, Caraá, Lavras, and Santa Vitória) during 1 year, encompassing winter and summer. Fish from Alegrete were the most contaminated overall, but animals sampled in Caraá, and Lavras also displayed elevated levels of current-use pesticides. Elevated levels of endosulfans, DDTs, HCHs, and current-use pesticides were accompanied by elevated levels of TBARS in the liver. Conversely, fish from Santa Vitória exhibited the highest levels of PAHs, accompanied by elevated levels of EROD in the liver and reduced levels of AChE in muscle and brain. TBARS proved to be a reliable biomarker for assessing impacts arising from pesticide accumulation, while EROD and AChE served as valuable indicators of impacts resulting from PAHs accumulation. Ultimately, the results obtained in this study demonstrate the reliable use of the proposed biomarkers for tracking biological impacts stemming from aquatic pollution using feral Astyanax as biomonitoring species.

Human fecal alpha-glucosidase activity and its relationship with gut microbiota profiles and early stages of intestinal mucosa damage.

OBJECTIVES: We investigated potential relationships among initial lesions of the intestinal mucosa, fecal enzymatic activities and microbiota profiles. METHODS: Fecal samples from 54 volunteers were collected after recruitment among individuals participating in a colorectal cancer (CRC) screening program in our region (Northern Spain) or attending for consultation due to clinical symptoms; intestinal mucosa samples were resected during colonoscopy. Enzymatic activities were determined in fecal supernatants by a semi-quantitative method. The fecal microbiota composition was determined by 16S rRNA gene-based sequencing. The results were compared between samples from clinical diagnosis groups (controls and polyps), according with the type of polyp (hyperplastic polyps or conventional adenomas) and considering the grade of dysplasia for conventional adenomas (low and high grade dysplasia). RESULTS: High levels of α-glucosidase activity were more frequent among samples from individuals diagnosed with intestinal polyps, reaching statistical significance for conventional adenomas and for low grade dysplasia adenomas when compared to controls. Regarding the microbiota profiles, higher abundance of Christensenellaceae_R-7 group and Oscillospiraceae_UCG-002 were found in fecal samples displaying low α-glucosidase activity as compared with those with higher activity as well as in controls with respect to conventional adenomas. A relationship was evidenced among intestinal mucosal lesions, gut glucosidase activities and intestinal microbiota profiles. CONCLUSIONS: Our findings suggest a relationship among altered fecal α-glucosidase levels, the presence of intestinal mucosal lesions, which can be precursors of CRC, and shifts in defined microbial groups of the fecal microbiota.

Galactokinase-like protein from Leishmania donovani: Biochemical and structural characterization of a recombinant protein.

Leishmaniasis is a spectrum of conditions caused by infection with the protozoan Leishmania spp. parasites. Leishmaniasis is endemic in 98 countries around the world, and resistance to current anti-leishmanial drugs is rising. Our work has identified and characterised a previously unstudied galactokinase-like protein (GalK) in Leishmania donovani, which catalyses the MgATP-dependent phosphorylation of the C-1 hydroxyl group of d-galactose to galactose-1-phosphate. Here, we report the production of the catalytically active recombinant protein in E. coli, determination of its substrate specificity and kinetic constants, as well as analysis of its molecular envelope using in solution X-ray scattering. Our results reveal kinetic parameters in range with other galactokinases with an average apparent Km value of 76 µM for galactose, Vmax and apparent Kcat values with 4.46376 × 10-9 M/s and 0.021 s-1, respectively. Substantial substrate promiscuity was observed, with galactose being the preferred substrate, followed by mannose, fructose and GalNAc. LdGalK has a highly flexible protein structure suggestive of multiple conformational states in solution, which may be the key to its substrate promiscuity. Our data presents novel insights into the galactose salvaging pathway in Leishmania and positions this protein as a potential target for the development of pharmaceuticals seeking to interfere with parasite substrate metabolism.

Plant-soil interactions alter nitrogen and phosphorus dynamics in an advancing subarctic treeline.

Treelines advance due to climate warming. The impacts of this vegetation shift on plant-soil nutrient cycling are still uncertain, yet highly relevant as nutrient availability stimulates tree growth. Here, we investigated nitrogen (N) and phosphorus (P) in plant and soil pools along two tundra-forest transects on Kola Peninsula, Russia, with a documented elevation shift of birch-dominated treeline by 70 m during the last 50 years. Results show that although total N and P stocks in the soil-plant system did not change with elevation, their distribution was significantly altered. With the transition from high-elevation tundra to low-elevation forest, P stocks in stones decreased, possibly reflecting enhanced weathering. In contrast, N and P stocks in plant biomass approximately tripled and available P and N in the soil increased fivefold toward the forest. This was paralleled by decreasing carbon (C)-to-nutrient ratios in foliage and litter, smaller C:N:P ratios in microbial biomass, and lower enzymatic activities related to N and P acquisition in forest soils. An incubation experiment further demonstrated manifold higher N and P net mineralization rates in litter and soil in forest compared to tundra, likely due to smaller C:N:P ratios in decomposing organic matter. Overall, our results show that forest expansion increases the mobilization of available nutrients through enhanced weathering and positive plant-soil feedback, with nutrient-rich forest litter releasing greater amounts of N and P upon decomposition. While the low N and P availability in tundra may retard treeline advances, its improvement toward the forest likely promotes tree growth and forest development.

Salt stress influences the proliferation of Fusarium solani and enhances the severity of wilt disease in potato.

Soil salinity has emerged as a critical abiotic stress in potato production, whereas wilt disease, caused by Fusarium solani, is the significant biotic stress. An experiment was performed to decipher the occurrence of wilt incidence by F. solani FJ1 under the influence of salinity in both in vitroand pot culture conditions. High salt concentration negatively influenced root and shoot development in the variety "Kufri Jyoti" but positively affected the mycelial growth and sporulation behaviours of F. solani FJ1. There was abundant whitish mycelial growth with enhanced biomass and high sporulation (microconidia production) in F. solani FJ1 cultured on salt-supplemented media. Moreover, under high salinity conditions (EC 2-8 dS m-1), severe wilting and rotting of vascular bundles were observed in plants artificially inoculated with F. solani FJ1. The mortality rate of potato plants was significantly higher under individual and combined stresses as compared to control. The wilt index of individual and combined stressed plants was also substantially higher compared to the control. Additionally, compared to the control, there was a significant decrease in total chlorophyll content and membrane stability index of the leaves under combined stress. However, the total phenols were increased under stress conditions. The total sugar content of potato plants decreased in infected plants, but increased when exposed to salt stress or a combination of salt stress and pathogen infection. F. solani infection also increased the activity of peroxidase (POX) and decreased the activity of phenylalanine ammonia-lyase (PAL) and catalase (CAT). These results suggest that Fusarium wilt and dry rot will be a more severe disease for potato cultivation in saline soils.

Analysis of potential volatile organic compounds in Sitophilus zeamais (Coleoptera: Curculionidae)-infested wheat based on lipid oxidation-a reference to early and rapid detection method.

The maize weevil, Sitophilus zeamais Motschulsky, is a hidden pest that presents serious risk to grain quality during postharvest storage. Lipid-derived volatile detection is considered a key reference for early prediction of S. zeamais infestation. However, the exact compositions of fatty acids and volatile organic compounds (VOCs) in S. zeamais-infested wheat are yet to be determined. In this study, we aimed to explore the effect of S. zeamais infestation on lipid metabolism in wheat infested with S. zeamais eggs (4 days), larvae (20 days), pupae (35 days), and adults (45 days). Compared to those in the control group, the activities of lipid oxidation enzymes, such as lipase, lipoxygenase, and alcohol dehydrogenase, increased by 82.73%, 105.12%, and 487.86%, respectively, during the storage period of 1 life cycle of S. zeamais. Additionally, the fatty acid composition of S. zeamais-infested wheat was significantly altered (palmitic acid [1.10-fold], oleic acid [1.07-fold], and linoleic acid [0.95-fold]). Furthermore, 91 VOCs were identified in all wheat samples; then, multivariate statistical analyses categorized these samples into 4 groups: uninfested, longer storage, lightly infested, and heavily infested. Moreover, 31, 26, and 45 potential VOCs were identified to distinguish uninfested wheat from those in the other 3 groups. These results demonstrated that S. zeamais infestation induces an elevation in lipid-related enzymatic activities, which potentially leads to a decrease in lipid content alongside the production of specific VOCs (undecan-4-olide, heptaldehyde, and 2-nonenal). These findings provide novel insights for rapidly identifying grains infested by hidden pests and effectively managing these pests during grain storage.

The Impact of Rootstock on "Big Top" Nectarine Postharvest Concerning Chilling Injury, Biochemical and Molecular Parameters.

Peaches and nectarines have a short shelf life even when harvested at appropriate physiological maturity. Market life is increased by storage at low temperatures. However, chilling injury symptoms can appear, causing physiological disorders and limiting shipping potential. The rootstock effect on the post-harvest quality has hardly been explored. Thus, the principal aim of this work was to study the influence of seven different Prunus rootstocks on the "Big Top" nectarine cv, considering harvest and post-harvest quality parameters and their correlation with chilling injury disorders. Basic fruit quality traits, individual sugars and organic acids analyzed by HPLC and other biochemical compounds such as relative antioxidant capacity, total phenolics content, flavonoids, anthocyanins, vitamin C and related enzyme activities (PAL, POD, PPO) were considered. In addition, correlations with possible candidate genes for chilling injury (CI) tolerance were searched by qPCR. Although a low susceptibility to CI symptoms has been found in "Big Top", rootstocks "PADAC 9902-01", "PADAC 99-05" and "ReplantPAC" exhibited lower CI symptoms. A statistically significant influence of the evaluated rootstocks was found concerning the parameters of this study. Phenols and anthocyanins seem to be important parameters to be considered in the prevention of chilling injury disorders. Moreover, PAL1, PPO4, PG2 and LDOX genes relative expressions were positively associated with chilling injury susceptibility. This study opens new perspectives for understanding peach fruit adaptation and response to cold storage temperatures during the post-harvest period.

Performance evaluation and metagenomic analysis of sequencing batch reactor under transient 2,4,6-trichlorophenol shock.

The transient chlorophenol shock under some emergency conditions might directly affect the pollutant removal of bioreactor. Therefore, the recovery of bioreactor performance after transient chlorophenol shock is a noteworthy issue. In the present research, the performance, antioxidant response, microbial succession and functional genes of sequencing batch reactor (SBR) were evaluated under transient 2,4,6-trichlorophenol (2,4,6-TCP) shock. The chemical oxygen demand (COD) and ammonia nitrogen (NH4+-N) removal efficiencies decreased sharply in the first 4 days after 60 mg/L 2,4,6-TCP shock for 24 h and gradually recovered to normal in the subsequent 8 days. The nitrogen removal rates and their corresponding enzymatic activities rapidly decreased after transient 2,4,6-TCP shock and then gradually increased to normal. The increase of antioxidant enzymatic activity, Cu-Zn SOD genes and Fe-Mn SOD genes contributed to the recovery of SBR performance. The abundance of genes encoding ammonia monooxygenase and hydroxylamine dehydrogenase decreased after transient 2,4,6-TCP shock, including amoA, amoC and nxrA. Thauera, Dechloromonas and Candidatus_Competibacter played key roles in the restorative process, which provided stable abundances of narG, norB , norC and nosZ. The results will deeply understand into the effect of transient 2,4,6-TCP shock on bioreactor performance and provide theoretical basis to build promising recoveries strategy of bioreactor performance.

Enzymatic activity of cGAS in the presence of three types of DNAs: limited cGAS stimulation by single-stranded HIV-1 SL2 DNA.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is activated by binding to DNA. Activated cGAS produces 2'3'-cGAMP, which subsequently binds to the adaptor protein STING (stimulator of interferon genes). This interaction triggers the cGAS/STING signaling pathway, leading to the production of type I interferons. Three types of DNA, namely double-stranded DNA longer than 40 base pairs, a 70-nucleotide single-stranded HIV-1 DNA known as SL2, and Y-form DNA with unpaired guanosine trimers (G3 Y-form DNA), induce interferon production by activating cGAS/STING signaling. However, the extent of cGAS activation by each specific DNA type remains unclear. The comparison of cGAS stimulation by various DNAs is crucial for understanding the mechanisms underlying cGAS-mediated type I interferon production in the innate immune response. Here, we revealed that cGAS produces 2'3'-cGAMP at a significantly lower rate in the presence of single-stranded SL2 DNA than in the presence of double-stranded DNA or G3 Y-form DNA. Furthermore, the guanine-to-cytosine mutations and the deletion of unpaired guanosine trimers significantly reduced the 2'3'-cGAMP production rate and the binding of cGAS to Y-form DNA. These studies will provide new insights into the cGAS-mediated DNA-sensing in immune response.

A GMR enzymatic assay for quantifying nuclease and peptidase activity.

Hydrolytic enzymes play crucial roles in cellular processes, and dysregulation of their activities is implicated in various physiological and pathological conditions. These enzymes cleave substrates such as peptide bonds, phosphodiester bonds, glycosidic bonds, and other esters. Detecting aberrant hydrolase activity is vital for understanding disease mechanisms and developing targeted therapeutic interventions. This study introduces a novel approach to measuring hydrolase activity using giant magnetoresistive (GMR) spin valve sensors. These sensors change resistance in response to magnetic fields, and here, they are functionalized with specific substrates for hydrolases conjugated to magnetic nanoparticles (MNPs). When a hydrolase cleaves its substrate, the tethered magnetic nanoparticle detaches, causing a measurable shift in the sensor's resistance. This design translates hydrolase activity into a real-time, activity-dependent signal. The assay is simple, rapid, and requires no washing steps, making it ideal for point-of-care settings. Unlike fluorescent methods, it avoids issues like autofluorescence and photobleaching, broadening its applicability to diverse biofluids. Furthermore, the sensor array contains 80 individually addressable sensors, allowing for the simultaneous measurement of multiple hydrolases in a single reaction. The versatility of this method is demonstrated with substrates for nucleases, Bcu I and DNase I, and the peptidase, human neutrophil elastase. To demonstrate a clinical application, we show that neutrophil elastase in sputum from cystic fibrosis patients hydrolyze the peptide-GMR substrate, and the cleavage rate strongly correlates with a traditional fluorogenic substrate. This innovative assay addresses challenges associated with traditional enzyme measurement techniques, providing a promising tool for real-time quantification of hydrolase activities in diverse biological contexts.

Genomic characterization and expression profiling of the lytic polysaccharide monooxygenases AA9 family in thermophilic fungi Thermothelomyces fergusii in response to carbon source media.

Enhancing enzyme activity and stability in biomass degradation can improve substrate saccharification and, increases biorefinery efficiency. For the first time, we identified 20 lytic polysaccharide monooxygenases (LPMOs) AA9 genes in the genome of Thermothelomyces fergusii. Our results showed that TfAA9 was categorized into LPMOs1, LPMOs2, and LPMOs3 subgroups based on protein diversity. Protein- 3D structure analysis showed strong interactions between Myceliophthora thermophila AA9 proteins and 17 TfAA9 proteins. Gene ontology analysis indicated a high enrichment of cellulase activity in TfAA9 genes. KEGG pathways analysis revealed the role of TfAA9 proteins in the endohydrolysis of 1,4-beta-D-glucosidic linkages in cellulose. Numerous TfAA9s gene transcripts were up-regulated on avicel, cellobiose, and glucose, with a higher proportion on avicel. Protein concentration, endoglucanase, and cellulase activity were also boosted on avicel. However, limited fungal biomass was observed on avicel, despite the abundance of AA9 LPMOs in the T. fergusii genome. These findings expand our understanding of fungal AA9 genes and their role in lignocellulolytic degradation. The disparity between biomass and enzymatic activity suggests screening TfAA9 genes for highly active enzymes and redundant genes via heterologous expression. In short, functional characterization of these genes could contribute to improving the saccharification process of industrial raw materials.

Urbanization-induced soil organic carbon loss and microbial-enzymatic drivers: insights from aggregate size classes in Nanchang city, China.

Soil microorganisms and enzymes play crucial roles in soil organic carbon (SOC) sequestration by promoting soil aggregate formation and stability and by participating in SOC cycling and accumulation. However, the effects by which soil microorganisms and enzymes act as mediators driving dynamic changes in SOC during rapid urbanization remain unclear. Therefore, this study selected the built-up area of Nanchang City, China (505 km2), as the study area. Sampling surveys were conducted using 184 sample plots stratified based on the proportion of impermeable surface area to distinguish different urbanization levels. The driving factors of dynamic changes in SOC of different aggregates during the process of urbanization were analyzed using the soil microbial community and enzyme activities. The results demonstrated that with an increase in urbanization intensity, both SOC content and stock exhibited a significant decline (p < 0.05). The highest SOC stock and contribution rate were observed in the 0.25-1 mm aggregates, and they were significantly influenced by urbanization (p < 0.05). In addition, the biomass of gram-positive bacteria (G+) and actinomycetota, and the activities of N-acetylglucosaminidase and acid phosphatase (AP) were significantly higher in low-urbanization areas than in high-urbanization areas (p < 0.05). SOC of each aggregate was positively correlated with fungi, arbuscular mycorrhizal fungi, G+, gram-negative bacteria, actinomycetota, protozoa, ß-1,4-glucosidase, N-acetylglucosaminidase, AP, urease, and catalase. Compared to soil enzymes, soil microorganisms exhibited a greater role in SOC sequestration (22.7%). Additionally, a structural equation model indicated that urbanization can directly or indirectly lead to a decrease in SOC of aggregates by altering soil physicochemical properties and affecting microbial and enzyme dynamics. However, the larger vegetation characteristics index mitigate the negative impacts of urbanization on SOC. Overall, urbanization had a negative impact on soil carbon storage. In the future, it is important to consider strategies that focus on improving soil nutrients, maintaining soil structure, protecting existing urban trees, and enhancing plant diversity during the urbanization process. These measures can help increase soil microbial biomass and enzyme activity, thereby improving soil and aggregate-related SOC content. The study could contribute to enhancing carbon sequestration in urban greenspaces.