ABSTRACT
Autonomous nanorobots represent an advanced tool for precision therapy to improve therapeutic efficacy. However, current nanorobotic designs primarily rely on inorganic materials with compromised biocompatibility and limited biological functions. Here, we introduce enzyme-powered bacterial outer membrane vesicle (OMV) nanorobots. The immobilized urease on the OMV membrane catalyzes the decomposition of bioavailable urea, generating effective propulsion for nanorobots. This OMV nanorobot preserves the unique features of OMVs, including intrinsic biocompatibility, immunogenicity, versatile surface bioengineering for desired biofunctionalities, capability of cargo loading and protection. We present OMV-based nanorobots designed for effective tumor therapy by leveraging the membrane properties of OMVs. These involve surface bioengineering of robotic body with cell-penetrating peptide for tumor targeting and penetration, which is further enhanced by active propulsion of nanorobots. Additionally, OMV nanorobots can effectively safeguard the loaded gene silencing tool, small interfering RNA (siRNA), from enzymatic degradation. Through systematic in vitro and in vivo studies using a rodent model, we demonstrate that these OMV nanorobots substantially enhanced siRNA delivery and immune stimulation, resulting in the utmost effectiveness in tumor suppression when juxtaposed with static groups, particularly evident in the orthotopic bladder tumor model. This OMV nanorobot opens an inspiring avenue to design advanced medical robots with expanded versatility and adaptability, broadening their operation scope in practical biomedical domains.
Subject(s)
Bacterial Outer Membrane , Animals , Humans , Bacterial Outer Membrane/metabolism , Mice , Robotics/methods , Urease/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolismABSTRACT
A hallmark of Proteus mirabilis infection of the urinary tract is the formation of stones. The ability to induce urinary stone formation requires urease, a nickel metalloenzyme that hydrolyzes urea. This reaction produces ammonia as a byproduct, which can serve as a nitrogen source and weak base that raises the local pH. The resulting alkalinity induces the precipitation of ions to form stones. Transcriptional regulator UreR activates expression of urease genes in a urea-dependent manner. Thus, urease genes are highly expressed in the urinary tract where urea is abundant. Production of mature urease also requires the import of nickel into the cytoplasm and its incorporation into the urease apoenzyme. Urease accessory proteins primarily acquire nickel from one of two nickel transporters and facilitate incorporation of nickel to form mature urease. In this study, we performed a comprehensive RNA-seq to define the P. mirabilis urea-induced transcriptome as well as the UreR regulon. We identified UreR as the first defined regulator of nickel transport in P. mirabilis. We also offer evidence for the direct regulation of the Ynt nickel transporter by UreR. Using bioinformatics, we identified UreR-regulated urease loci in 15 Morganellaceae family species across three genera. Additionally, we located two mobilized UreR-regulated urease loci that also encode the ynt transporter, implying that UreR regulation of nickel transport is a conserved regulatory relationship. Our study demonstrates that UreR specifically regulates genes required to produce mature urease, an essential virulence factor for P. mirabilis uropathogenesis. IMPORTANCE: Catheter-associated urinary tract infections (CAUTIs) account for over 40% of acute nosocomial infections in the USA and generate $340 million in healthcare costs annually. A major causative agent of CAUTIs is Proteus mirabilis, an understudied Gram-negative pathogen noted for its ability to form urinary stones via the activity of urease. Urease mutants cannot induce stones and are attenuated in a murine UTI model, indicating this enzyme is essential to P. mirabilis pathogenesis. Transcriptional regulation of urease genes by UreR is well established; here, we expand the UreR regulon to include regulation of nickel import, a function required to produce mature urease. Furthermore, we reflect on the role of urea catalysis in P. mirabilis metabolism and provide evidence for its importance.
Subject(s)
Proteus Infections , Urinary Tract Infections , Animals , Mice , Proteus mirabilis/genetics , Urease/metabolism , Nickel/metabolism , Bacterial Proteins/genetics , Escherichia coli/genetics , Urea/metabolismABSTRACT
Here, we report DNA-based synthetic nanostructures decorated with enzymes (hereafter referred to as DNA-enzyme swimmers) that self-propel by converting the enzymatic substrate to the product in solution. The DNA-enzyme swimmers are obtained from tubular DNA structures that self-assemble spontaneously by the hybridization of DNA tiles. We functionalize these DNA structures with two different enzymes, urease and catalase, and show that they exhibit concentration-dependent movement and enhanced diffusion upon addition of the enzymatic substrate (i.e., urea and H2O2). To demonstrate the programmability of such DNA-based swimmers, we also engineer DNA strands that displace the enzyme from the DNA scaffold, thus acting as molecular "brakes" on the DNA swimmers. These results serve as a first proof of principle for the development of synthetic DNA-based enzyme-powered swimmers that can self-propel in fluids.
Subject(s)
Catalase , DNA , Urease , DNA/chemistry , DNA/metabolism , Urease/chemistry , Urease/metabolism , Catalase/chemistry , Catalase/metabolism , Nanostructures/chemistry , Biocatalysis , Hydrogen Peroxide/chemistry , Hydrogen Peroxide/metabolismABSTRACT
BACKGROUND: Rhododendron delavayi is a natural shrub that is distributed at different elevations in the karst region of Bijie, China, and that has an important role in preventing land degradation in this region. In this study, we determined the soil mineral element contents and soil enzyme activities. The composition of the soil bacterial community of R. delavayi at three elevations (1448 m, 1643 m, and 1821 m) was analyzed by high-throughput sequencing, and the interrelationships among the soil bacterial communities, mineral elements, and enzyme activities were determined. RESULTS: The Shannon index of the soil bacterial community increased and then decreased with increasing elevation and was highest at 1643 m. Elevations increased the number of total nodes and edges of the soil bacterial community network, and more positive correlations at 1821 m suggested stronger intraspecific cooperation. Acidobacteria, Actinobacteria and Proteobacteria were the dominant phyla at all three elevations. The Mantel test and correlation analysis showed that Fe and soil urease significantly affected bacterial communities at 1448 m; interestingly, Chloroflexi was positively related to soil urease at 1448 m, and Actinobacteria was positively correlated with Ni and Zn at 1821 m. Fe and soil urease significantly influenced the bacterial communities at lower elevations, and high elevation (1821 m) enhanced the positive interactions of the soil bacteria, which might be a strategy for R. delavayi to adapt to high elevation environments. CONCLUSION: Elevation significantly influenced the composition of soil bacterial communities by affecting the content of soil mineral elements and soil enzyme activity.
Subject(s)
Bacteria , Forests , Rhododendron , Soil Microbiology , Soil , Soil/chemistry , Rhododendron/microbiology , China , Bacteria/classification , Bacteria/genetics , Bacteria/enzymology , Bacteria/isolation & purification , Metals/analysis , Actinobacteria/genetics , Actinobacteria/enzymology , Actinobacteria/isolation & purification , Actinobacteria/classification , Microbiota , Urease/metabolism , Acidobacteria/genetics , Acidobacteria/isolation & purification , Acidobacteria/enzymology , Acidobacteria/classification , RNA, Ribosomal, 16S/genetics , Phylogeny , High-Throughput Nucleotide SequencingABSTRACT
Emerging evidence suggests differential antagonism of lactic acid-producing bacteria (LAB) to Helicobacter pylori, posing challenges to human health and food safety due to unclear mechanisms. This study assessed 21 LAB strains from various sources on H. pylori growth, urease activity, and coaggregation. Composite scoring revealed that Latilactobacillus sakei LZ217, derived from fresh milk, demonstrates strong inhibitory effects on both H. pylori growth and urease activity. L. sakei LZ217 significantly reduced H. pylori adherence of gastric cells in vitro, with inhibition ratios of 47.62%. Furthermore, in vivo results showed that L. sakei LZ217 alleviated H. pylori-induced gastric mucosa damage and inflammation in mice. Metabolomic exploration revealed metabolic perturbations in H. pylori induced by L. sakei LZ217, including reduced amino acid levels (e.g., isoleucine, leucine, glutamate, aspartate, and phenylalanine) and impaired carbohydrate and nucleotide synthesis, contributing to the suppression of ureA (28.30%), ureE (84.88%), and ureF (59.59%) expressions in H. pylori. This study underscores the efficacy of LAB against H. pylori and highlights metabolic pathways as promising targets for future interventions against H. pylori growth and colonization.
Subject(s)
Gastric Mucosa , Helicobacter Infections , Helicobacter pylori , Urease , Urease/metabolism , Animals , Helicobacter Infections/microbiology , Gastric Mucosa/microbiology , Gastric Mucosa/metabolism , Mice , Humans , Bacterial Adhesion , Female , Probiotics , MaleABSTRACT
BACKGROUND: Theoretically, a rapid urease test (RUT) using a swab of the gastric wall (Swab-RUT) for Helicobacter pylori (H. pylori) is safe. However, the validity and utility of Swab-RUT remain unclear. Therefore, we assessed the validity and utility of Swab-RUT compared to RUT using mucosal forceps of the gastric wall (Forceps-RUT) and 13C-urea breath test (UBT). METHODS: This study was a multicenter prospective observational study. When the examinees were suspected of H. pylori infection during esophagogastroduodenoscopy, we performed Swab-RUT and Forceps-RUT continuously. When the examinees were not suspected of H. pylori infection, we performed Swab-RUT alone. We validated the status of H. pylori infection using UBT. RESULTS: Ninety-four examinees were enrolled from four institutions between May 2016 and December 2020 (median age [range], 56.5 [26-88] years). In this study, the sensitivity, specificity, and accuracy of Swab-RUT to UBT were 0.933 (95% confidence interval: 0.779-0.992), 0.922 (0.827-0.974), and 0.926 (0.853-0.970), respectively. The Kappa coefficient of Swab-RUT to UBT was 0.833, and that of Swab-RUT to forceps-RUT was 0.936. No complications were observed in this study. CONCLUSIONS: Swab-RUT is a valid examination for the status of H. pylori infection compared to the conventional Forceps-RUT.
Subject(s)
Breath Tests , Helicobacter Infections , Helicobacter pylori , Sensitivity and Specificity , Urease , Humans , Breath Tests/methods , Breath Tests/instrumentation , Helicobacter Infections/diagnosis , Helicobacter Infections/microbiology , Middle Aged , Prospective Studies , Urease/analysis , Urease/metabolism , Male , Female , Aged , Helicobacter pylori/isolation & purification , Helicobacter pylori/enzymology , Adult , Aged, 80 and over , Gastric Mucosa/microbiology , Endoscopy, Digestive System , Reproducibility of Results , Carbon Isotopes , Surgical Instruments/microbiologyABSTRACT
OBJECTIVES: Dandelion has been shown to exert anti-inflammatory and anti-bacterial effects. Our study aimed to identify the effect and mechanism of dandelion flower extracts on H.â pylori-induced gastritis and screen for novel antimicrobial substances. METHODS: Anti-H.â pylori activities of water extracts(WEDF) and ethanol extracts (EEDF) of dandelion flowers were performed with disk diffusion method assay, MIC, and MBC. The H.â pylori-induced model was constructed to examine the gastroprotective of EEDF using RUT, pathological analysis, and ELISA. RESULTS: EEDF exhibited better anti- H.â pylori and urease inhibition activities than WEDF. In vivo studies, EEDF can reduce the adhesion of H.â pylori to the gastric mucosa, alleviate gastric damage, and concurrently reduce the levels of TNF-α and IL-6 in gastric tissues. The six phenolic compounds showed urease inhibition effect (IC50: 2.99±0.15 to 66.08±6.46â mmol/mL). Among them, chlorogenic acid, caffeic acid, and luteolin also had anti-H.â pylori activity (MIC: 64-256â µg/mL). CONCLUSION: EEDF exhibited anti-H.â pylori, gastroprotective and anti-inflammatory effects. Chicoric acid and luteolin may be the main active compounds of dandelion flowers to exert anti-H.â pylori, and worthy of further investigation.
Subject(s)
Anti-Bacterial Agents , Flowers , Helicobacter pylori , Microbial Sensitivity Tests , Plant Extracts , Taraxacum , Urease , Taraxacum/chemistry , Helicobacter pylori/drug effects , Flowers/chemistry , Plant Extracts/pharmacology , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/isolation & purification , Urease/antagonists & inhibitors , Urease/metabolism , Animals , Gastric Mucosa/drug effects , Gastric Mucosa/pathology , Gastric Mucosa/metabolism , Male , Helicobacter Infections/drug therapy , Dose-Response Relationship, Drug , MiceABSTRACT
This study was carried out to examine the interaction of enzyme activities, microbial biomass carbon, and CO2 respiration with heavy metals under different land uses in terms of quality and sustainability of the soil. There is a statistically significant positive correlation between dehydrogenase enzyme activity and Mn, Pb, Cd, and Co, while it was negative between Cr. There was a positive correlation between catalase enzyme activity and Mn and Pb and between urease and Co. The higher interaction of dehydrogenase activity with heavy metals, which is included in the endo enzyme group, has been explained as a much stronger effect of heavy metals on living microorganisms and endoenzymes than extracellular enzymes stabilized on clay minerals and organic matter. The high clay content of the soil is thought to reduce some of the negative effects of heavy metals on enzymes. The results of this study may be good indicators of enzyme activities, especially dehydrogenase, catalase, and urease, for soil health and quality, chemical degradation and restoration processes, and ecosystem functioning in soils contaminated or to be contaminated with heavy metals. It shows that the activities of these enzymes are very sensitive and can decrease rapidly in case of high concentrations of heavy metals.
Soil health and quality, chemical degradation and restoration processes, and soils contaminated with heavy metals or potentially polluted can be good indicators of ecosystem functioning. This study was carried out with the belief that the interaction of enzymes with heavy metals in this type of soil will be revealed in detail and will shed light on such studies to be done in the future.
Subject(s)
Metals, Heavy , Soil Pollutants , Soil/chemistry , Catalase , Ecosystem , Clay , Urease/metabolism , Lead , Soil Pollutants/metabolism , Biodegradation, Environmental , Metals, Heavy/analysisABSTRACT
Helicobacter pylori (Hp) infections pose a global health challenge demanding innovative therapeutic strategies by which to eradicate them. Urease, a key Hp virulence factor hydrolyzes urea, facilitating bacterial survival in the acidic gastric environment. In this study, a multi-methodological approach combining pharmacophore- and structure-based virtual screening, molecular dynamics simulations, and MM-GBSA calculations was employed to identify novel inhibitors for Hp urease (HpU). A refined dataset of 8,271,505 small molecules from the ZINC15 database underwent pharmacokinetic and physicochemical filtering, resulting in 16% of compounds for pharmacophore-based virtual screening. Molecular docking simulations were performed in successive stages, utilizing HTVS, SP, and XP algorithms. Subsequent energetic re-scoring with MM-GBSA identified promising candidates interacting with distinct urease variants. Lys219, a residue critical for urea catalysis at the urease binding site, can manifest in two forms, neutral (LYN) or carbamylated (KCX). Notably, the evaluated molecules demonstrated different interaction and energetic patterns in both protein variants. Further evaluation through ADMET predictions highlighted compounds with favorable pharmacological profiles, leading to the identification of 15 candidates. Molecular dynamics simulations revealed comparable structural stability to the control DJM, with candidates 5, 8 and 12 (CA5, CA8, and CA12, respectively) exhibiting the lowest binding free energies. These inhibitors suggest a chelating capacity that is crucial for urease inhibition. The analysis underscores the potential of CA5, CA8, and CA12 as novel HpU inhibitors. Finally, we compare our candidates with the chemical space of urease inhibitors finding physicochemical similarities with potent agents such as thiourea.
Subject(s)
Helicobacter pylori , Helicobacter pylori/metabolism , Urease/metabolism , Molecular Dynamics Simulation , Molecular Docking Simulation , Urea/pharmacologyABSTRACT
Urease, a pivotal enzyme in nitrogen metabolism, plays a crucial role in various microorganisms, including the pathogenic Helicobacter pylori. Inhibiting urease activity offers a promising approach to combating infections and associated ailments, such as chronic kidney diseases and gastric cancer. However, identifying potent urease inhibitors remains challenging due to resistance issues that hinder traditional approaches. Recently, machine learning (ML)-based models have demonstrated the ability to predict the bioactivity of molecules rapidly and effectively. In this study, we present ML models designed to predict urease inhibitors by leveraging essential physicochemical properties. The methodological approach involved constructing a dataset of urease inhibitors through an extensive literature search. Subsequently, these inhibitors were characterized based on physicochemical properties calculations. An exploratory data analysis was then conducted to identify and analyze critical features. Ultimately, 252 classification models were trained, utilizing a combination of seven ML algorithms, three attribute selection methods, and six different strategies for categorizing inhibitory activity. The investigation unveiled discernible trends distinguishing urease inhibitors from non-inhibitors. This differentiation enabled the identification of essential features that are crucial for precise classification. Through a comprehensive comparison of ML algorithms, tree-based methods like random forest, decision tree, and XGBoost exhibited superior performance. Additionally, incorporating the "chemical family type" attribute significantly enhanced model accuracy. Strategies involving a gray-zone categorization demonstrated marked improvements in predictive precision. This research underscores the transformative potential of ML in predicting urease inhibitors. The meticulous methodology outlined herein offers actionable insights for developing robust predictive models within biochemical systems.
Subject(s)
Enzyme Inhibitors , Machine Learning , Urease , Urease/antagonists & inhibitors , Urease/chemistry , Urease/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Helicobacter pylori/enzymology , Helicobacter pylori/drug effects , Algorithms , HumansABSTRACT
An overexploitation of earth resources results in acid deposition in soil, which adversely impacts soil ecosystems and biodiversity and affects conventional heavy metal remediation using immobilization. A series of column experiments was conducted in this study to compare the cadmium (Cd) retention stability through biotic and abiotic carbonate precipitation impacted by simulated acid rain (SAR), to build a comprehensive understanding of cadmium speciation and distribution along soil depth and to elucidate the biogeochemical bacteria-soil-heavy metal interfaces. The strain of Sporosarcina pasteurii DSM 33 was used to trigger the biotic carbonate precipitation and cultivated throughout the 60-day column incubation. Results of soil pH, electrical conductivity (EC), and quantitative CdCO3/CaCO3 analysis concluded that the combination of biotic and abiotic soil treatment could reinforce soil buffering capacity as a strong defense mechanism against acid rain disturbance. Up to 1.8 ± 0.04 U/mg urease enzyme activity was observed in combination soil from day 10, confirming the sustained effect of urease-mediated microbial carbonate precipitation. Cadmium speciation and distribution analyses provided new insights into the dual stimulation of carbonate-bound and Fe/Mn-bound phases of cadmium immobilization under microbially induced carbonate precipitation (MICP). As confirmed by the microbial community analysis, outsourcing urea triggered diverse microbial metabolic responses, notably carbonate precipitation and dissimilatory iron metabolism, in both oxygen-rich topsoil and oxygen-depleted subsurface layers. The overall investigation suggests the feasibility of applying MICP for soil Cd remediation under harsh environments and stratagem by selecting microbial functionality to overcome environmental challenges.
Subject(s)
Acid Rain , Metals, Heavy , Cadmium/chemistry , Soil/chemistry , Calcium Carbonate/chemistry , Ecosystem , Urease/metabolism , Carbonates/chemistry , Bacteria/metabolism , Oxygen/metabolismABSTRACT
As part of the multifaceted strategies developed to shape the common environmental policy, considerable attention is now being paid to assessing the degree of environmental degradation in soil under xenobiotic pressure. Bisphenol A (BPA) has only been marginally investigated in this ecosystem context. Therefore, research was carried out to determine the biochemical properties of soils contaminated with BPA at two levels of contamination: 500 mg and 1000 mg BPA kg-1 d.m. of soil. Reliable biochemical indicators of soil changes, whose activity was determined in the pot experiment conducted, were used: dehydrogenases, catalase, urease, acid phosphatase, alkaline phosphatase, arylsulfatase, and ß-glucosidase. Using the definition of soil health as the ability to promote plant growth, the influence of BPA on the growth and development of Zea mays, a plant used for energy production, was also tested. As well as the biomass of aerial parts and roots, the leaf greenness index (SPAD) of Zea mays was also assessed. A key aspect of the research was to identify those of the six remediating substances-molecular sieve, zeolite, sepiolite, starch, grass compost, and fermented bark-whose use could become common practice in both environmental protection and agriculture. Exposure to BPA revealed the highest sensitivity of dehydrogenases, urease, and acid phosphatase and the lowest sensitivity of alkaline phosphatase and catalase to this phenolic compound. The enzyme response generated a reduction in the biochemical fertility index (BA21) of 64% (500 mg BPA) and 70% (1000 mg BPA kg-1 d.m. of soil). The toxicity of BPA led to a drastic reduction in root biomass and consequently in the aerial parts of Zea mays. Compost and molecular sieve proved to be the most effective in mitigating the negative effect of the xenobiotic on the parameters discussed. The results obtained are the first research step in the search for further substances with bioremediation potential against both soil and plants under BPA pressure.
Subject(s)
Acid Phosphatase , Benzhydryl Compounds , Phenols , Soil Pollutants , Soil , Zea mays , Phenols/chemistry , Benzhydryl Compounds/chemistry , Soil Pollutants/chemistry , Zea mays/chemistry , Soil/chemistry , Acid Phosphatase/metabolism , Arylsulfatases/metabolism , Alkaline Phosphatase/metabolism , Zeolites/chemistry , Oxidoreductases/metabolism , Urease/metabolism , Catalase/metabolism , Biodegradation, Environmental , Magnesium Silicates/chemistry , Starch/chemistry , beta-Glucosidase/metabolism , Composting/methodsABSTRACT
Introducing and establishing new food requires a detailed evaluation of its safety, nutritional value and functionality, thus the control and probiotic-rich adzuki and mung bean sprouts were studied in an in vivo rats model. However, the total feed intake did not differ significantly between the groups, the highest body weight gain and body weight change were recorded in the control AIN diet. At the same time, the addition of legume sprouts caused a reduction of these parameters (up to 25% in the variant with probiotic-rich adzuki bean sprouts). There was no significant effect on serum morphology, except white blood cells (ca. 20% reduction in the control sprout-supplemented diets). Serum and liver antiradical properties were significantly elevated by consuming mung bean sprouts (no effect of the probiotics). The faecal lactic acid bacteria were already increased by the control sprouts (a 2.8- and 2.1-fold increase for adzuki and mung bean sprouts, respectively). The probiotic-rich sprouts further improved this parameter. The diets enriched with mung bean sprouts significantly decreased the urease (by ca. 65%) and ß-glucuronidase activities (by ca. 30%). All the tested diets caused also a significant reduction of faecal tryptophanase activity (the effect was intensified by Lactiplantibacillus plantarum 299v). The functional components did not affect negatively the nutritional parameters and blood morphological characteristics. They improved also the antioxidant potential and significantly decreased the activities of colon cancer-related enzymes (urease and tryptophanase). The results confirmed that these new probiotic carriers may be a valuable, safe and functional element of a healthy diet.
Subject(s)
Antioxidants , Lactobacillus plantarum , Probiotics , Vigna , Weight Gain , Animals , Probiotics/pharmacology , Vigna/chemistry , Antioxidants/metabolism , Male , Adansonia/chemistry , Rats , Rats, Wistar , Urease/metabolism , Glucuronidase/metabolism , Feces/microbiology , Feces/chemistry , Diet , Gastrointestinal Microbiome/drug effects , Nutritive Value , LiverABSTRACT
BACKGROUND: Tillage measures have been effectively adopted for mitigating waterlogging damage in field crops, yet little is known about the role of tillage measures in crop responses to waterlogging. A field experiment was performed to investigate the effect of conventional planting (CK), small ridge planting (SR), big ridge planting (BR) and film side planting (FS) on soil available nutrients and enzymatic activity, chlorophyll contents, leaf nutrients, soluble protein, soluble sugar, nitrate reductase, antioxidant enzyme activity, lipid peroxidation, agronomic traits and yield of rapeseed under waterlogging stress conditions. RESULTS: Tillage measures remarkably improved rapeseed growth and yield parameters under waterlogging stress conditions. Under waterlogging conditions, rapeseed yield was significantly increased by 33.09 and 22.70% in the SR and BR groups, respectively, compared with CK. Correlation analysis showed that NO3--N, NH4+-N, and urease in soils and malonaldehyde (MDA), superoxide dismutase (SOD), and nitrate reductase in roots were the key factors affecting rapeseed yield. The SR and BR groups had significantly increased NO3--N by 180.30 and 139.77%, NH4+-N by 115.78 and 66.59%, urease by 41.27 and 26.45%, SOD by 6.64 and 4.66%, nitrate reductase by 71.67 and 26.67%, and significantly decreased MDA content by 14.81 and 13.35% under waterlogging stress, respectively, compared with CK. In addition, chlorophyll and N content in leaves, soluble sugar and POD in roots, and most agronomic traits were also significantly enhanced in response to SR and BR under waterlogging conditions. CONCLUSION: Overall, SR and BR mitigated the waterlogging damage in rapeseed mainly by reducing the loss of soil available nitrogen, decreasing the MDA content in roots, and promoting urease in soils and SOD and nitrate reductase in roots. Finally, thorough assessment of rapeseed parameters indicated that SR treatment was most effective followed by BR treatment, to alleviate the adverse effects of waterlogging stress.
Subject(s)
Brassica napus , Brassica rapa , Brassica napus/metabolism , Urease/metabolism , Soil , Brassica rapa/metabolism , Superoxide Dismutase/metabolism , Chlorophyll/metabolism , Antioxidants/metabolism , Nitrate Reductase/metabolism , Sugars/metabolismABSTRACT
BACKGROUND: Helicobacter pylori (Hp) infects the stomach of 50% of the world's population. Importantly, chronic infection by this bacterium correlates with the appearance of several extra-gastric pathologies, including neurodegenerative diseases. In such conditions, brain astrocytes become reactive and neurotoxic. However, it is still unclear whether this highly prevalent bacterium or the nanosized outer membrane vesicles (OMVs) they produce, can reach the brain, thus affecting neurons/astrocytes. Here, we evaluated the effects of Hp OMVs on astrocytes and neurons in vivo and in vitro. METHODS: Purified OMVs were characterized by mass spectrometry (MS/MS). Labeled OMVs were administered orally or injected into the mouse tail vein to study OMV-brain distribution. By immunofluorescence of tissue samples, we evaluated: GFAP (astrocytes), ßIII tubulin (neurons), and urease (OMVs). The in vitro effect of OMVs in astrocytes was assessed by monitoring NF-κB activation, expression of reactivity markers, cytokines in astrocyte-conditioned medium (ACM), and neuronal cell viability. RESULTS: Urease and GroEL were prominent proteins in OMVs. Urease (OMVs) was present in the mouse brain and its detection coincided with astrocyte reactivity and neuronal damage. In vitro, OMVs induced astrocyte reactivity by increasing the intermediate filament proteins GFAP and vimentin, the plasma membrane αVß3 integrin, and the hemichannel connexin 43. OMVs also produced neurotoxic factors and promoted the release of IFNγ in a manner dependent on the activation of the transcription factor NF-κB. Surface antigens on reactive astrocytes, as well as secreted factors in response to OMVs, were shown to inhibit neurite outgrowth and damage neurons. CONCLUSIONS: OMVs administered orally or injected into the mouse bloodstream reach the brain, altering astrocyte function and promoting neuronal damage in vivo. The effects of OMVs on astrocytes were confirmed in vitro and shown to be NF-κB-dependent. These findings suggest that Hp could trigger systemic effects by releasing nanosized vesicles that cross epithelial barriers and access the CNS, thus altering brain cells.
Subject(s)
Helicobacter pylori , Mice , Animals , Helicobacter pylori/metabolism , Astrocytes , Urease/metabolism , Urease/pharmacology , NF-kappa B/metabolism , Complement Factor B/metabolism , Complement Factor B/pharmacology , Disease Models, Animal , Tandem Mass Spectrometry , NeuronsABSTRACT
In this work, a series of chalcones (1a-d, 2a-d, 3a-d, 4a-d, and 5a-d) were designed and synthesized by Claisen-Schmidt condensation. Also, their chemical structures were elucidated using UV-Vis, FT IR, 1 H NMR, 13 C NMR, MS spectral data, and elemental analyses. Subsequently, the anticholinesterase, tyrosinase, urease inhibitory activities and antioxidant activities of all chalcones were evaluated. The inhibitory potential of all chalcones in terms of IC50 value was observed to range from 7.18 ± 0.43 to 29.62 ± 0.30 µM against BChE by comparing with Galantamine (IC50 46.06 ± 0.10 µM) as a reference drug. Also, compounds 2c, 3c, 4c, 4b, and 4d exhibited high anticholinesterase activity against both AChE and BChE enzymes. The tyrosinase inhibitory activity results revealed that three compounds (IC50 1.75 ± 0.83 µM for 2b, IC50 2.24 ± 0.11 µM for 3b, and IC50 1.90 ± 0.64 µM for 4b) displayed good inhibitory activity against tyrosinase compared with kojic acid (IC50 0.64 ± 0.12 µM). In addition, other different three chalcones (IC50 22.34 ± 0.25 µM for 2c, IC50 20.98 ± 0.08 µM for 3c, and IC50 18.26 ± 0.13 µM for 4c) showed excellent inhibitory activity against the urease by comparing with thiourea (IC50 23.08 ± 0.19 µM). Compounds 3c and 4c showed the best potency in all antioxidant activity tests. In light of these findings, the structure-activity relationship for compounds was also described. Furthermore, molecular modeling studies, including molecular docking, absorption, distribution, metabolism, excretion, and toxicity (ADMET), and pharmacophore analyses of compounds, gave important information about the interactions and drug-likeness properties. As a result, all chalcones exhibited suitable ADMET findings, predicting good oral bioavailability.
Subject(s)
Chalcones , Cholinesterase Inhibitors , Molecular Docking Simulation , Cholinesterase Inhibitors/chemistry , Cholinesterase Inhibitors/pharmacology , Chalcones/chemistry , Monophenol Monooxygenase/metabolism , Urease/metabolism , Structure-Activity Relationship , Antioxidants/chemistry , Molecular StructureABSTRACT
Cellular metal homeostasis is a critical process for all organisms, requiring tight regulation. In the major pathogen Helicobacter pylori, the acquisition of nickel is an essential virulence determinant as this metal is a cofactor for the acid-resistance enzyme, urease. Nickel uptake relies on the NixA permease and the NiuBDE ABC transporter. Till now, bacterial metal transporters were reported to be controlled at their transcriptional level. Here we uncovered post-translational regulation of the essential Niu transporter in H. pylori. Indeed, we demonstrate that SlyD, a protein combining peptidyl-prolyl isomerase (PPIase), chaperone, and metal-binding properties, is required for the activity of the Niu transporter. Using two-hybrid assays, we found that SlyD directly interacts with the NiuD permease subunit and identified a motif critical for this contact. Mutants of the different SlyD functional domains were constructed and used to perform in vitro PPIase activity assays and four different in vivo tests measuring nickel intracellular accumulation or transport in H. pylori. In vitro, SlyD PPIase activity is down-regulated by nickel, independently of its C-terminal region reported to bind metals. In vivo, a role of SlyD PPIase function was only revealed upon exposure to high nickel concentrations. Most importantly, the IF chaperone domain of SlyD was shown to be mandatory for Niu activation under all in vivo conditions. These data suggest that SlyD is required for the active functional conformation of the Niu permease and regulates its activity through a novel mechanism implying direct protein interaction, thereby acting as a gatekeeper of nickel uptake. Finally, in agreement with a central role of SlyD, this protein is essential for the colonization of the mouse model by H. pylori.
Subject(s)
Bacterial Proteins/metabolism , Helicobacter Infections/metabolism , Helicobacter pylori/metabolism , Metallochaperones/metabolism , Nickel/metabolism , Peptidylprolyl Isomerase/metabolism , Animals , Helicobacter Infections/microbiology , Mice , Urease/metabolismABSTRACT
BACKGROUND: Two important virulence factors, urease and cagA, play an important role in Helicobacter pylori (H. pylori) gastric cancer. Aim of this study was to investigate the expression level and function of ureB and cagA using small interfering RNAs (siRNA). METHODS: SS1 strain of H. pylori was considered as host for natural transformation. siRNA designed for ureB and cagA genes were inserted in pGPU6/GFP/Neo siRNA plasmid vector to evaluate using phenotypic and genotypic approaches. Then, qPCR was performed for determining inhibition rate of ureB and cagA gene expression. RESULTS: The expression levels of siRNA-ureB and siRNA-cagA in the recombinant strain SS1 were reduced by about 5000 and 1000 fold, respectively, compared to the native H. pylori strain SS1. Also, preliminary evaluation of siRNA-ureB in vitro showed inhibition of urea enzyme activity. These data suggest that siRNA may be a powerful new tool for gene silencing in vitro, and for the development of RNAi-based anti-H. pylori therapies. CONCLUSION: Our results show that targeting ureB and cagA genes with siRNA seems to be a new strategy to inhibit urease enzyme activity, reduce inflammation and colonization rate.
Subject(s)
Helicobacter Infections , Helicobacter pylori , Humans , Helicobacter pylori/genetics , Helicobacter pylori/metabolism , Urease/genetics , Urease/metabolism , RNA, Small Interfering/genetics , Bacterial Proteins/genetics , Antigens, Bacterial/geneticsABSTRACT
CRISPR/Cas enables targeted genome editing in many different plant and algal species including the model diatom Thalassiosira pseudonana. However, efficient gene targeting by homologous recombination (HR) to date is only reported for photosynthetic organisms in their haploid life-cycle phase. Here, a CRISPR/Cas construct, assembled using Golden Gate cloning, enabled highly efficient HR in a diploid photosynthetic organism. Homologous recombination was induced in T. pseudonana using sequence-specific CRISPR/Cas, paired with a dsDNA donor matrix, generating substitution of the silacidin, nitrate reductase and urease genes by a resistance cassette (FCP:NAT). Up to c. 85% of NAT-resistant T. pseudonana colonies screened positive for HR by nested PCR. Precise integration of FCP:NAT at each locus was confirmed using an inverse PCR approach. The knockout of the nitrate reductase and urease genes impacted growth on nitrate and urea, respectively, while the knockout of the silacidin gene in T. pseudonana caused a significant increase in cell size, confirming the role of this gene for cell-size regulation in centric diatoms. Highly efficient gene targeting by HR makes T. pseudonana as genetically tractable as Nannochloropsis and Physcomitrella, hence rapidly advancing functional diatom biology, bionanotechnology and biotechnological applications targeted on harnessing the metabolic potential of diatoms.
Subject(s)
Diatoms , Diatoms/genetics , Diatoms/metabolism , CRISPR-Cas Systems/genetics , Urease/genetics , Urease/metabolism , Gene Editing , Homologous RecombinationABSTRACT
Uropathogens have adaptation strategies to survive in the host urinary tract by efficiently utilizing and tolerating the urinary metabolites. Many uropathogens harbour the enzyme urease for the breakdown of urea and the enzymatic breakdown of urea increases the pH and facilitate the struvite crystallization. In this study, the differential urease activity of uropathogenic Escherichia coli and Pseudomonas aeruginosa strains was investigated under different nutritional conditions. The experiments included measurement of growth, pH, urease activity, NH4-N generation and urease gene (ureC) expression among the bacterial strains under different conditions. Further, the implications of urea breakdown on the struvite crystallization in vitro and biofilm formation were also assessed. The study included urease positive isolates and for comparison urease negative isolates were included. Compared to the urease negative strains the urease positive strains formed higher biofilms and motility. The urease positive P. aeruginosa showed significantly higher (p < 0.01) pH and urease activity (A557-A630) compared to E. coli under experimental conditions. Further, supplementation of glucose to the growth media significantly increased the urease activity in P. aeruginosa and in contrast, it was significantly lower in E. coli. The expression profile of urease gene (ureC) was significantly higher (p < 0.001) in P. aeruginosa compared to E. coli and was consistent with the biochemical results of the urease activity under the nutritional conditions. The differential urease activity under two nutritional conditions influenced the biogenic struvite crystallization. It correlated with the urease activity showing higher crystallization rate in P. aeruginosa compared to E. coli. The results highlight the differential urease activity in two common uropathogens under different nutritional conditions that may have significant role on the regulation of virulence, pathogenicity and in the kidney stone disease.