Enzyme Activity Research Articles

Dihydromyricetin Protects Against Hypoxia/Reoxygenation Injury in Cardiomyocytes by Activating miR-34a-Mediated Notch1 Pathway.

Dihydromyricetin (Dih), a naturally occurring flavonoid, has been identified to exert a protective effect against ischemia/reperfusion injury. However, the detailed mechanisms remain unclear. Here we investigated the biological role of Dih in preventing hypoxia/reoxygenation (H/R) injury in cardiomyocytes. The results showed that Dih protected cardiomyocytes against H/R-induced apoptosis, as proved by improved cell viability and decreased lactate dehydrogenase (LDH) release, cell apoptosis percentage, and caspase-3/7 activity. H/R-induced oxidative stress in cardiomyocytes was also prevented by Dih with increased activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), and decreased levels of malondialdehyde (MDA) and reactive oxygen species (ROS). Treatment with Dih prevented H/R-induced increase in the activities of myocardial enzymes aspartate aminotransferase (AST), creatine kinase-MB (CK-MB), and creatine kinase (CK). miR-34a expression was upregulated after H/R stimulation, which could be attenuated by Dih pretreatment. Besides, miR-34a overexpression attenuated the protective effects of Dih against H/R-caused increase in apoptosis, oxidative stress, and myocardial enzyme activities. Next, we demonstrated that Notch1 was a target molecule of miR-34a. Notch1 overexpression reversed the role of miR-34a in regulating the cardioprotective effect of Dih on H/R injury. These observations indicated that the cardioprotective effect of Dih against H/R injury was mediated by the miR-34a/Notch1 signaling. Dih may be a candidate agent for improving the clinical efficacy of cardiac ischemia/reperfusion injury treatment.

A flavin-containing monooxygenase from Schizosaccharomyces pombe: characterization and application in the synthesis of S-methyl-L-cysteine sulfoxide

S-methyl-L-cysteine sulfoxide (SMCO) is a non-protein sulfur-containing amino acid with a variety of functions. There are few reports on the enzymes catalyzing the biosynthesis of SMCO from S-methyl-L-cysteine (SMC). In this study, the flavin-containing monooxygenase gene derived from Schizosaccharomyces pombe (spfmo) was heterologously expressed in Escherichia coli BL21(DE3) and the enzymatic properties of the expressed protein were analyzed. The optimum catalytic conditions of the recombinant SpFMO were 30 ℃ and pH 8.0, under which the enzyme activity reached 72.77 U/g. An appropriate amount of Mg2+ improved the enzyme activity. The enzyme kinetic analysis showed that the Km and kcat/Km of SpFMO on the substrate SMC were 23.89 μmol/L and 61.71 L/(min·mmol), respectively. Under the optimal reaction conditions, the yield of SMCO synthesized from SMC catalyzed by SpFMO was 12.31% within 9 h. This study provides reference for the enzymatic synthesis of SMCO.

Versatility of Caenorhabditis elegans as a Model Organism for Evaluating Foodborne Neurotoxins and Food Bioactive Compounds in Nutritional Neuroscience.

Epidemiological evidence has shown that the regular ingestion of vegetables and fruits is associated with reduced risk of developing chronic diseases. The introduction of the 3Rs (replacement, reduction, and refinement) principle into animal experiments has led to the use of valid, cost-effective, and efficient alternative and complementary invertebrate animal models which are simpler and lower in the phylogenetic hierarchy. Caenorhabditis elegans (C. elegans), a nematode with a much simpler anatomy and physiology compared to mammals, share similarities with humans at the cellular and molecular levels, thus making it a valid model organism in neurotoxicology. This review explores the versatility of C. elegans in elucidating the neuroprotective mechanisms elicited by food bioactive compounds against neurotoxic effects of food- and environmental-related contaminants. Several signaling pathways linked to the molecular basis of neuroprotection exerted by bioactive compounds in chemically induced or transgenic C. elegans models of neurodegenerative diseases are also discussed. Specifically, the modulatory effects of bioactive compounds on the DAF-16/FoxO and SKN-1/Nrf2 signaling pathways, stress resistance- and autophagy-related genes, and antioxidant defense enzyme activities were highlighted. Altogether, C. elegans represent a valuable model in nutritional neuroscience for the identification of promising neuroprotective agents and neurotherapeutic targets which could help in overcoming the limitations of current therapeutic agents for neurotoxicity and neurodegenerative diseases.

Setting standards for brain collection procedures in metabolomic studies.

Current metabolomics technologies can measure hundreds of chemical entities in tissue extracts with good reliability. However, long-recognized requirements to halt enzyme activities during the initial moments of sample preparation are usually overlooked, allowing marked postmortem shifts in levels of labile metabolites representing diverse pathways. In brain many such changes occur in a matter of seconds. These comments overview the concern, contrast representative studies, and specify approaches to consider as standards in the field going forward. Comparison with established metabolite signatures of in vivo brain is an essential validation step when implementing any collection method.

Exposure to spaceflight enhances the virulence of Purpureocillium lilacinum against Tetranychus cinnabarinus: modulation of the host's enzyme activities and microbiome.

Multiple studies have been conducted to investigate the impact of space conditions on human, plant, and microbial life. This research investigated the virulence of spaceflight mutants of the entomopathogenic fungus Purpureocillium lilacinum (HP7, HP36, HP52) and its original strain (SP535) against Tetranychus cinnabarinus as well as examination of the T. cinnabarinus immune response, including alterations in enzyme profiles and microbiome composition post fungal application. Our observations revealed contrasting, time-specific differences in pathogenicity and tissue infection between the ground-based isolate and spaceflight mutant isolates. Analysis of detoxifying and antioxidant enzymes showed a significant reduction in enzyme activities T. cinnabarinus infected with the most virulent spaceflight mutants at 36h post-fungal infection, compared to ground-based isolates. Additionally, the microbiota was reduced due to a fungal infection, partly due to decreased antioxidant enzyme activities. Our findings indicate that changes in the microbiota of T. cinnabarinus following infection with P. lilacinum (both ground-based and spaceflight mutant isolates) resulted in variations in metabolism and genetic information-related KEGG pathways. This data can help identify potential changes in the host immune system that drive increased virulence after spaceflight mutation.

Cryo-EM structure and regulation of human NAD kinase.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is a crucial reducing cofactor for reductive biosynthesis and protection from oxidative stress. To fulfill their heightened anabolic and reductive power demands, cancer cells must boost their NADPH production. Progrowth and mitogenic protein kinases promote the activity of cytosolic NAD kinase (NADK), which produces NADP+, a limiting NADPH precursor. However, the molecular architecture and mechanistic regulation of human NADK remain undescribed. Here, we report the cryo-electron microscopy structure of human NADK, both in its apo-form and in complex with its substrate NAD+ (nicotinamide adenine dinucleotide), revealing a tetrameric organization with distinct structural features. We discover that the amino (N)- and carboxyl (C)-terminal tails of NADK have opposing effects on its enzymatic activity and cellular NADP(H) levels. Specifically, the C-terminal region is critical for NADK activity, whereas the N-terminal region exhibits an inhibitory role. This study highlights molecular insights into the regulation of a vital enzyme governing NADP(H) production.

Synergistic effects of allantoin and Achyranthes japonica-biochar profoundly alleviate lead toxicity during barley growth.

Lead (Pb), a toxic metal, causes severe health hazards to both humans and plants due to environmental pollution. Biochar addition has been efficiently utilized to enhance growth of plants as well as yield in the presence of Pb-induced stress. The present research introduces a novel use of biochar obtained from the weed Achyranthes japonica to enhance the growth of plants in Pb-contaminated soil. An experiment was performed with 7 treatments: Control, Pb2+ (10 mg kg-1) only, biochar (4 %) only, allantoin (4 g kg-1) only, biochar combined with Pb2+, allantoin combined with biochar, as well as a combination of allantoin and biochar with Pb2+. Lead toxicity alone markedly diminished plant growth metrics, including root and shoot length, biomass (wet and dry), chlorophyll concentration, and grain production. The application of biochar, allantoin, or their joint administration markedly enhanced the length of shoots (by 50.3 %, 29 %, and 70 %), length of roots (by 69 %, 50 %, and 69 %), and fresh biomass of shoots (by 5 %, 29 %, and 5 %), respectively. This enhancement is ascribed to improved soil characteristics and more efficient absorption of nutrients. The application of biochar, allantoin and their combination improved the tolerance against Pb2+ by increasing the total chlorophyll level by 12 %, 16 %, and 17 %, respectively, vs. the control. Likewise, these amendments significantly (p < 0.05) improved the activity of antioxidant enzymes, including SOD, POD, and CAT by 49 %, 29 %, and 49 %, respectively. The resistance towards Pb2+ was enhanced by biochar, allantoin, and their combined application, with lower Pb2+ concentrations in shoots (59.9 %, 40.1 %, and 49.8 %), roots (48.2 %, 24.1 %, and 58.3 %), and grains (60.2 %, 29.7 %, and 40.1 %) compared to solely Pb-stress, respectively. In summary, converting the weed Achyranthes japonica into biochar and integrating it with allantoin provides an eco-friendly approach to control its proliferation while efficiently alleviating Pb-induced toxicity in plants.

Regulatory and retrograde signaling networks in the chlorophyll biosynthetic pathway.

Plants, algae and photosynthetic bacteria convert light into chemical energy by means of photosynthesis, thus providing food and energy for most organisms on Earth. Photosynthetic pigments, including chlorophylls (Chls) and carotenoids, are essential components that absorb the light energy necessary to drive electron transport in photosynthesis. The biosynthesis of Chl shares several steps in common with the biosynthesis of other tetrapyrroles, including siroheme, heme and phycobilins. Given that many tetrapyrrole precursors possess photo-oxidative properties that are deleterious to macromolecules and can lead to cell death, tetrapyrrole biosynthesis (TBS) requires stringent regulation under various developmental and environmental conditions. Thanks to decades of research on model plants and algae, we now have a deeper understanding of the regulatory mechanisms that underlie Chl synthesis, including (i) the many factors that control the activity and stability of TBS enzymes, (ii) the transcriptional and post-translational regulation of the TBS pathway, and (iii) the complex roles of tetrapyrrole-mediated retrograde signaling from chloroplasts to the cytoplasm and the nucleus. Based on these new findings, Chls and their derivatives will find broad applications in synthetic biology and agriculture in the future.

Effects of Cu(Ⅱ) stress on embryonic development, oxidative stress response, cholinergic system and energy metabolism in the Sepiella maindroni.

In order to investigate the causes of population degradation and resource decline, this thesis investigated the ecotoxicological effects of heavy metal Cu(Ⅱ) on the embryonic development of Sepiella maindroni. Results indicate significant effects of Cu(Ⅱ) concentrations on the developmental toxicity, teratogenicity, and lethality of S. maindroni embryos. Different concentrations of Cu(Ⅱ) caused varying degrees of malformations in embryos, altered developmental rates, reduced hatchability and hatchling quality, and increased malformation and mortality of hatchlings. At the same time, Cu(Ⅱ) exposure led to an increase in the content of the lipid peroxidation product malondialdehyde (MDA) and a significant decrease in the activity of antioxidant enzymes (superoxide dismutase [SOD], catalase [CAT]), energy-metabolizing enzymes (adenylate kinase [AK]), and cholinergic-related enzymes (acetylcholinesterase [AChE], choline acetyltransferase [ChAT]). In conclusion, when the concentration of Cu(Ⅱ) in the environment is ≥ 0.01 mg/L, it causes significant lethality toxicity, developmental toxicity and teratogenicity in S. maindroni embryos. These effects are likely related to Cu(Ⅱ)-induced stress impacting the antioxidant capacity, energy metabolism, and cholinergic system. Ultimately, these toxic effects may lead to population degradation and resource decline in fishery organisms by affecting the early replenishment process of fisheries.

Laminated Layer Extract from Echinococcus Granulosus cyst Attenuates Ocular Damages and Inflammatory Responses in an Experimental Autoimmune Uveitis Model.

Since extract of the laminated layer (LL) from E. granulosus showed immuno-modulatory effects in vitro and in vivo, we sought to determine its effect on the onset, development, and evolution of experimental auto-immune uveitis (EAU). The latter is a model of some human diseases with ocular inflammation that can cause blindness. E. granulosus LL extract was either injected before EAU induction for the pretreated group or later for treated group. Ocular exploration was made by retinal histological and immunohistological (CD86, CD4, CD8) analysis. Myeloperoxidase (MPO), Superoxide dismutase (SOD), Catalase enzymatic activities (CAT), and Malondialdehyde (MDA), Nitric oxide (NO), Urea, and TNF-α levels were measured in plasma. LL injection attenuated retinal histological damage and reduced cells infiltration. Also, LL decreased systemic inflammatory and oxidative markers as well as TNF-α production and increased antioxidant parameters. Interestingly, we observed a protective effect of E. granulosus LL extract during EAU. LL appears to ameliorate retinal damage by down-regulating inflammatory responses. Our results support LL immunomodulatory effects during autoimmune diseases and offer a promising prospect for helminthic therapy during autoimmune uveitis.

Chemopreventive role of β-caryophyllene in DMBA-induced skin cancer: Modulation of apoptotic pathways and PI3K/Akt signaling in Swiss albino mice.

The skin, with its robust structural integrity and advanced immune defense system, serves as a critical protective barrier against environmental toxins and carcinogenic compounds. Despite this, it remains vulnerable to the harmful effects of certain hazardous agents. This study aimed to investigate the chemopreventive potential of β-caryophyllene (BCP) in mitigating 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin carcinogenesis, focusing on the modulation of apoptosis and PI3K/AKT signaling pathways. Swiss albino mice were utilized to assess the preventive effects of BCP in DMBA-induced skin cancer. Skin carcinogenesis was initiated by topical DMBA application, followed by promotion using croton oil. To evaluate the chemopreventive efficacy of BCP, a 50 mg/kg oral dose was administered 3 times a week for 16 weeks. The BCP treatment in DMBA-induced skin cancer mice significantly reduced tumor incidence, tumor burden and the total number of papillomas compared to untreated DMBA-exposed mice. Notably, BCP administration (p < 0.05) resulted in a marked increase in body weight and improvement in antioxidant enzyme activity. Additionally, BCP treatment led to significant reductions in lipid peroxidation and enhanced detoxification enzyme function. Histological examination of DMBA-induced skin tissues revealed the presence of keratin pearls, well-differentiated tumor cells and neutrophil infiltration. In contrast, BCP-treated mice showed only mild hyperplasia, dysplasia and moderate keratosis, suggesting a lower degree of tissue damage. Furthermore, BCP demonstrated a protective effect on liver histology, counteracting the toxic effects of DMBA exposure. Gene expression analysis revealed that BCP treatment significantly (p < 0.05) upregulated the pro-apoptotic genes Bax, p53, caspase-3 and caspase-9, while downregulating the anti-apoptotic Bcl-2 expression. Additionally, BCP treatment led to a marked reduction in the expression of proliferating cell nuclear antigen (PCNA), cyclin D1 and PI3K/Akt signaling pathways, which are key regulators of cell proliferation and survival. This study provides compelling evidence that the antioxidant and pro-apoptotic effects of β-caryophyllene contribute to its chemopreventive properties in DMBA-induced skin carcinogenesis in mice. The modulation of key apoptotic signaling pathways and the suppression of the PI3K/Akt pathway by BCP underscores its potential as a therapeutic agent for preventing skin cancer. These findings pave the way for further exploration of BCP as a promising candidate for skin cancer prevention and therapy.

Glycoside-specific metabolomics reveals the novel mechanism of glycinebetaine-induced cold tolerance by regulating apigenin glycosylation in tea plants.

Glycosylation is a key modification that affects secondary metabolites under stress and is influenced by glycinebetaine (GB) to regulate plant stress tolerance. However, the complexity and detection challenges of glycosides hinder our understanding of the regulatory mechanisms of their metabolic interaction with GB during stress. A glycoside-specific metabolomic approach utilizing cone voltage-induced in-source dissociation was developed, achieving precise and high-throughput detection of glycosides in tea plants by narrowing the target ion range by 94.3%. Combined with enzyme activity assays, exogenous spraying, and gene silencing, this approach helps investigate the role of GB-glycosides cascade effect in enhancing cold tolerance of tea plants. Our method demonstrated that silencing betaine aldehyde dehydrogenase (CsBADH1) in tea plants altered 60 glycoside ions while reducing GB content and cold tolerance, indicating that glycosylation affects GB-mediated cold tolerance. By combining glycoside-specific with conventional metabolomics, isorhoifolin, a GB-regulated cold response metabolite was discovered, and its precursor apigenin was found to be a new cold tolerance metabolite that enhanced cold tolerance by scavenging reactive oxygen species. This study reveals a new mechanism by which GB mediated cold tolerance in tea plants through regulating apigenin glycosylation, broadening our understanding of the role of glycosylation in plant cold tolerance.

Human health risk assessment by exposure to contaminants from an urban reservoir: a pilot study in the Madin Dam (México).

Water contamination greatly impacts human health. The Metropolitan Area of the Valley of Mexico (MAVM) is one of the most densely inhabited and polluted places globally, with a significant problem being the rising water demand. The research aims to assess the impact of metals such as iron, aluminum, lead, cadmium, and total chromium, among others, in the water of the Madin Dam, a key reservoir in the area's water supply. The assessment concentrated on individuals who were in good health and had been exposed to these pollutants. It analyzed factors such as levels of oxidized proteins; changes in the function of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase); and their genetic expression, the occurrence of micronuclei, and the amount of DNA methylation. The findings were linked to the metals present in the MAVM drinking water, and the risk was evaluated. The research included four groups: two associated with the Madin Dam, consisting of persons living nearby or using water from the reservoir, and two acting as a control. The study highlighted a significant link between long-term exposure to pollutants in drinking water and elevated levels of oxidized proteins, increased micronuclei frequency, changes in antioxidant enzyme activity and gene expression, and a higher percentage of 5-methylcytosine. The risk assessment showed that people who use drinking water and/or consume fish from Madin Dam have a potential higher risk associated with metal contamination.

Effects of Low-Severity Fire on the Composition and Stability of Soil Organic Carbon in Permafrost Peatlands (Northeast China).

Climate change and human activity are increasing the frequency of wildfires in peatlands and threatening permafrost peatland carbon pools. In Northeast China, low-severity prescribed fires are conducted annually on permafrost peatlands to reduce the risk of wildfires. These fires typically do not burn surface peat but lead to the loss of surface vegetation and introduction of pyrogenic carbon. However, the long-term effects of repeated low-severity fires on soil carbon stability in these ecosystems remain unclear. Thus, we conducted low-severity prescribed fire experiments over 3 years in the permafrost peatlands of the Great Khingan Mountains. Our findings showed a gradual decline in the total carbon content, primarily due to the reduction in free particulate organic matter (fPOM). Initially, fPOM was higher in the burned sites but decreased with repeated burning. Chemical analyses revealed a 32% increase in the aromaticity of the fPOM at the burned sites, which diminished the thermal stability of the soil. Furthermore, both prescribed fires and the addition of pyrogenic carbon reduced biological stability while increasing enzyme activity and CO2 production, which was attributed to the introduction of post-fire pyrogenic carbon. These results suggest that low-severity fires compromise the stability of permafrost peatlands, particularly because the pyrogenic carbon input alters the chemical composition of the soil carbon fraction.

Combined Effects of Arsenic and Bisphenol-A on Locomotor Activity and Oxidative Stress Mediated Neurotoxicity in Mice.

Bisphenol A (BPA) is a monomer of plastic that can leach into water from scratched containers when used for an extended period. Arsenic (As) is an environmental toxicant, and people are exposed to both arsenic and BPA through drinking water and through scratched plastic containers used in contaminated areas. However, the combined effects of As and BPA on locomotor performance and neurobehavioral changes are yet to be investigated. Thus, this study was designed to assess the combined effect of As and BPA on locomotor activity and neurotoxicity through a mouse model. The neurobehavioral changes in experimental mice were evaluated using the different maze tests. Mice exposed to As or BPA exhibited higher anxiety-like behavior, decreased locomotor activity, and impaired learning and memory including social interaction compared with control mice. However, As + BPA-exposed mice showed a significantly reduced anxiety-like behavior, improved learning and memory including locomotor activity, and social interaction compared to individual As-exposed mice. Furthermore, mice exposed to As or BPA showed lower levels of antioxidant and cholinesterase enzymes activity, nuclear factor erythroid-2-related factor-2 (Nrf2), heme-oxygenase-1 (HO-1), and interleukin-10 (IL-10) in the brain and higher levels of interleukin-6 (IL-6) in the brain and lactate dehydrogenase (LDH) in the serum compared to control mice. However, combined exposure augmented antioxidant and cholinesterase enzymes activity, Nrf2, HO-1, IL-10 levels in the brain and reduced serum LDH activity and IL-6 in the brain compared to As exposure. Therefore, this study suggests that As and BPA may have antagonistic effects, and BPA could attenuate the As-induced neurobehavioral and biochemical changes in co-exposed mice.

Exogenous dopamine application ameliorates chilling injury and preserves quality of kiwifruit during cold storage

This study investigated the mechanisms employed by exogenous dopamine application in alleviating chilling injury in kiwifruits during storage at 1 °C for 120 days. Our results indicated that dopamine treatment at 150 µM alleviated chilling injury in kiwifruits during storage at 1 °C for 120 days. By 150 µM dopamine application, higher SUMO E3 ligase (SIZ1) and target of rapamycin (TOR) genes expression accompanied by lower poly(ADP-Ribose) polymerase 1 (PARP1) and sucrose non-fermenting 1-related kinase 1 (SnRK1) genes expression was associated with higher salicylic acid, ATP, NADPH and proline accumulation in kiwifruits during storage at 1 °C for 120 days. In addition, higher 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging capacity arising from higher phenols and flavonoids accumulation in kiwifruits treated with 150 µM dopamine could be ascribed to higher phenylalanine ammonia-lyase (PAL) enzyme activity. Additionally, lower endogenous hydrogen peroxide (H2O2) accumulation along with higher ascorbic acid accumulation in kiwifruits treated with 150 µM dopamine could be attributed to lower superoxide dismutase (SOD) along with higher catalase (CAT) enzymes activity. Moreover, lower phospholipase D (PLD) and lipoxygenase (LOX) genes expression in kiwifruits treated with 150 µM dopamine was accompanied with membrane integrity preservation as evidenced by lower electrolyte leakage and malondialdehyde (MDA) accumulation. Therefore, exogenous dopamine could be employed as a potential technique for alleviating chilling injury in kiwifruits during cold storage.

Intrinsic fluorescence hydrogels for ON/OFF screening of antidiabetic drugs: assessing α-glucosidase inhibition by acarbose.

Diabetes remains one of the most prevalent chronic diseases globally, significantly impacting mortality ratetables. The development of effective treatments for controlling glucose level in blood is critical to improve the quality of life of patients with diabetes. In this sense, smart optical sensors using hydrogels, responsive to external stimuli, have emerged as a revolutionary approach to diabetes care. In this study, changes in the optical properties of a hydrogel are employed for monitoring α-glucosidase activity, a critical enzyme involved in diabetes mellitus type II due to its role in breaking terminal α-glycosidic bonds, releasing α-glucose. The enzyme is encapsulated within a triazine-based hydrogel that exhibits intrinsic blue fluorescence. Upon hydrolysis of the substrate p-nitrophenyl-α-D-glucopyranoside (p-NPG) by α-glucosidase, the fluorescence is quenched due to the release of p-nitrophenol (PNP). However, when exposed to potential antidiabetic drugs, the enzyme's activity is inhibited, and the hydrogel's fluorescence remains intact. This ON/OFF fluorescence-based assay enables rapid screening of drug candidates by evaluating their ability to inhibit α-glucosidase enzymatic activity. Sensor optimization involves conducting swelling studies, fluorescent assays, reusability tests and a trial with a real antidiabetic drug. This innovative approach holds potential for enhancing antidiabetic drug screening and management, offering a more accessible and efficient solution compared to traditional biosensors.

Nanomachine Networks: Functional All-Enzyme Hydrogels from Photochemical Cross-Linking of Glucose Oxidase.

Enzymes are attractive as catalysts due to their specificity and biocompatibility; however, their use in industrial and biomedical applications is limited by stability. Here, we present a facile approach for enzyme immobilization within "all-enzyme" hydrogels by forming photochemical covalent cross-links between the enzyme glucose oxidase. We demonstrate that the mechanical properties of the enzyme hydrogel can be tuned with enzyme concentration and the data suggests that the dimeric nature of glucose oxidase results in unusual gel formation behavior which suggests a degree of forced induced dimer dissociation and unfolding. We confirm and quantify the enzyme activity of the hydrogel using the Trinder assay and a 1D modeling approach and show that 50% enzymatic activity is retained upon hydrogel formation. These observed effects may be due to the forces experienced by the individual nanoscale enzymes during mesoscale network formation. We have therefore demonstrated that photochemical cross-linking can be readily employed to produce functional all-enzyme glucose oxidase hydrogels with easily tunable mechanical properties and specific catalytic activity. This approach provides enormous potential for producing biocatalytic materials with tunable mechanical properties, responsive biological functionality and high volumetric productivity which may inform the future design of biomedical devices with enhanced sensitivity and activity.

Toll/interleukin-1 receptor-only genes contribute to immune responses in maize.

Proteins with Toll/interleukin-1 receptor (TIR) domains are widely distributed in both prokaryotes and eukaryotes, serving as essential components of immune signaling. Although monocots lack the major TIR-nucleotide-binding (NB)-leucine-rich repeat (LRR)-type (TNL) immune receptors, they possess a small number of TIR-only proteins, the function of which remains largely unknown. In the monocot maize (Zea mays), there are three conserved TIR-only genes in the reference genome, namely ZmTIR1 to ZmTIR3. A genome-wide scan for TIR genes and comparative analysis revealed that these genes exhibit low sequence diversity and do not show copy number variation among 26 diverse inbred lines. ZmTIR1 and ZmTIR3, but not ZmTIR2, specifically trigger cell death and defense gene expression when overexpressed in Nicotiana benthamiana leaves. These responses depend on the critical glutamic acid and cysteine residues predicted to be essential for TIR-mediated NADase and 2',3'-cAMP/cGMP synthetase activity, respectively, as well as the key TIR downstream regulator Enhanced Disease Susceptibility 1 (EDS1). Overexpression of ZmTIR3 in N. benthamiana produces signaling molecules, including 2'cADPR, 2',3'-cAMP and 2',3'-cGMP, a process that requires the enzymatic glutamic acid and cysteine residues of ZmTIR3. ZmTIR expression in maize is barely detectable under normal conditions, but is substantially induced by different pathogens. Importantly, the maize Zmtir3 knockout mutant exhibits enhanced susceptibility to the fungal pathogen Cochliobolus heterostrophus, highlighting the role of ZmTIR3 in maize immunity. Overall, our results unveil the function of the maize ZmTIRs. We propose that the pathogen-inducible ZmTIRs play an important role in maize immunity, likely through their enzymatic activity and via EDS1-mediated signaling.

Valorization of Cocoa and Peach-Palm Wastes for the Production of Amylases by Pleurotus pulmonarius CCB19 and Its Application as an Additive in Commercial Detergents.

In the context of agribusiness, the agricultural and livestock sectors generate a considerable quantity of waste on a daily basis. Solid-state fermentation (SSF) represents a potential alternative for mitigating the adverse effects of residue accumulation and for producing high-value products such as enzymes. Pleurotus pulmonarius is capable of producing a number of commercial enzymes, including amylases. Accordingly, the present study sought to produce, characterize, and apply amylases obtained from solid-state fermentation of cocoa and peach-palm waste by the fungus Pleurotus pulmonarius CCB19. The highest amylase production by P. pulmonarius was observed after 3days of solid-state fermentation of the cocoa shells, with an activity of 83.90 U/gds. The physicochemical characterization of the crude amylase using the artificial neural network (ANN) revealed that the highest activity was observed at pH 9 and a temperature of 20°C (120.7 U/gds). Furthermore, the amylase demonstrated stability in the majority of the tested conditions, maintaining up to 80% of its residual activity for up to 120min of incubation. With regard to the impact of ions and reagents on enzymatic activity, a positive effect was observed in the presence of Co+ ions at concentrations of 1 and 5mM, whereas Cu+ ions at 5mM demonstrated an inhibitory effect. The addition of SDS and EDTA reagents did not affect the observed activity. Furthermore, the extract was tested in commercial detergent formulations and demonstrated enhanced compatibility (110%) and efficacy (270% with boiled detergent) in removing starch stains from fabrics with Ariel liquid detergent. In conclusion, amylase derived from the fungus Pleurotus pulmonarius CCB19 exhibited favorable properties that make it a suitable candidate for use as an additive in laundry detergent formulations.