Urease Assay Research Articles

Exploring fungal potential for microbial-induced calcite precipitation (MICP) in bio-cement production

IntroductionMicrobial-induced calcite precipitation (MICP) involves various microorganisms, such as bacteria, fungi, and algae. This study focuses on producing bio-cement using fungal species and selecting potential candidates isolated from alkaline soil of different regions of Punjab, namely, Majha, Malwa, and Doaba.MethodsThe selection of fungi isolates capable of bio-cement production involves several tests, including a urease assay and calcium precipitation. Isolates having high urease enzyme production and the ability to perform calcite precipitation are selected for instrumental analyses such as X-ray diffraction (XRD) and scanning electron microscopy (SEM). The isolates selected for further analysis are S1 (3) with 8.879 ± 2.94 µg/ml, S1 (18) with 8.421 ± 0.13 µg/ml, and S4 (1) with 10.057 ± 0.45 µg/ml urease activity and least free calcium ions that are 2.337 ± 0.5 µg/ml, 3.339 ± 0.5 µg/ml, and 4.074 ± 0.1 µg/ml respectively.Results and discussionCalcite precipitation is confirmed through XRD and field emission scanning electron microscopy (FESEM). XRD images showing calcite precipitation with sharp crystalline peaks for S1 (3), S1 (18), and S4 (1) are shown. The calcite precipitation is evident in the micrographs of FESEM. These combined results confirm the potential of urease-positive fungi to facilitate calcite production, which could lead to bio-cement development in future research.

Exploring Acyl Thiotriazinoindole Based Pharmacophores: Design, Synthesis, and SAR Studies with Molecular Docking and Biological Activity Profiling against Urease, α-amylase, α-glucosidase, Antimicrobial, and Antioxidant Targets.

A diminutive chemical library of acyl thiotriazinoindole (ATTI) based bioactive scaffolds was synthesized, instigated by taking the economical starting material Isatin, through a series of five steps. Isatin was first nitrated followed by the attachment of pentyl moiety via nucleophilic substitution reaction. The obtained compound was reacted with thiosemicarbazide to obtain thiosemicarbazone derivative, which was eventually cyclized using basic conditions in water as solvent. Finally, the reported series was obtained through reaction of nitrated thiotriazinoindole moiety with differently substituted phenacyl bromides. The synthesized compounds were characterized using NMR spectroscopy and elemental analysis. Finally, the synthesized motifs were scrutinized for their potential to impede urease, α-glucosidase, DPPH, and α-amylase. Compound 5h with para cyano group manifested the most pivotal biological activity among all, displaying IC50 values of 29.7 ± 0.8, 20.5 ± 0.5 and 36.8 ± 3.9µM against urease, α-glucosidase, and DPPH assay, respectively. Simultaneously, for α-amylase compound 5g possessing a p-CH3 at phenyl ring unfolded as most active, with calculated IC50 values 90.3 ± 1.1µM. The scaffolds were additionally gauged for their antifungal and antibacterial activity. Among the tested strains, 5d having bromo as substituent exhibited the most potent antibacterial activity, while it also demonstrated the highest potency against Aspergillus fumigatus. Other derivatives 5b, 5e, 5i, and 5j also exhibited dual inhibition against both antibacterial and antifungal strains. The interaction pattern of derivatives clearly displayed their SAR, and their docking scores were correlated with their IC50 values. In molecular docking studies, the importance of interactions like hydrogen bonding was further asserted. The electronic factors of various substituents engendered variety of interactions between the ligands and targets implying their importance in the structures of the synthesized heterocyclic scaffolds. To conclude, the synthesized compounds had satisfactory biological activity against various important targets. Further studies are therefore encouraged by attachment of different substitutions in the structure at various positions to enhance the activity of these compounds.

Bioassay guided triterpene isolation and its biological evaluation using branches extract of a significant medicinal plant; Monotheca buxifolia

ObjectiveThe present study focuses on column chromatography of ethyl acetate fractions and re fractions of M.buxifolia branches for potential compound isolation alongside their biological and phytochemical evaluation and TLC analysis. MethodsThe obtained EtOAc fractions were subjected to phytochemical (TPC, TFC), antioxidant (TRP, TAC, DPPH, MC, ABTS), antibacterial and enzyme inhibition (α-amylase, lipase and urease) assays. Fractions indicating the presence of a compound were selected for running successive columns and their factions also analyzed employing the above mentioned bioassays. ResultsA collective none-to-low antibacterial activity was observed. MbBE11 presented the highest antidiabetic potential (79.92%±3.9) besides moderate phytochemical and antioxidant activity. Highest phenolic content (75.85±3.6 μgGAE/mg), flavonoid content (45.39±2μgQE/mg), antioxidant capacity (99.9±3.7 μgAAE/mg), reducing power (94.82±3.9 μgAAE/mg) and radical scavenging activity (DPPH; 86.28±3%, ABTS; 98.9±2.3%, MC; 59.01±2.7%) was reported by MbBE16 making it a potential source of antioxidant agents. Column chromatography of MbBE11 produced two refractions (MbBE21 and MbBE22) which reported moderate phytochemical and high antioxidant activity. High α-amylase and urease inhibition with low lipase inhibition was observed. MbBE21 depicted the largest ZOI against S.enterica (SE) (12±0.7 mm) and MbBE22 against Methicillin-resistant S.aureus (MRSA) (12±0.5 mm). Column chromatography of MbBE16 produced four refractions (MbBE31 – MbBE34) with moderate phytochemical and antioxidant activity without a single dominant fraction. Antibacterial activity remained low with the average ZOI sized at 7±0.5 mm. MbBE15 reported the highest anti-urease at 95.7%±4.7 and significant lipase inhibition at 85.5%±3. MbBE15, MbBE22 MbBE32 resulted in crystal formation later characterized as Lupeol with moderate phytochemical and antioxidant activity. ConclusionThe study concludes that M.buxifolia is a potential source of bioactive components and can be further explored through modern strategies.

Enrichment, characterization, and sand consolidation application of urease active calcite-producing bacteria.

Minerals such as calcium carbonate, which is prevalent in marble and limestone, are present naturally in rocks. Both physicochemical processes and microbial processes can result in the creation of calcium carbonate in nature, as is well documented. In this study, microbiologically induced calcite precipitation potential of three different Travertine-type water sources (Pamukkale Travertine Spring (PTS), Pamukkale Travertine Terraces (PTT), and Red Travertine of Karahayit (RTK)) using three different incubation media (NB, NB3, and ATCC1832) were investigated. After enrichment with ATCC1832 media, urease assays were positive for all of the microbial sources. The PTS and PTT were cultured with ATCC1832 medium for 48h, which showed the best results for urease activity and microbial growth among other samples. Metagenome analyses indicated that PTT enriched with ATCC1832 media contains > 99% Firmicutes, while PTS enriched with ATCC1832 contains > 99% Proteobacteria at the Phylum level. Results from SEM-EDX and XRD analysis revealed that calcite and/or vaterite were the minerals that emerged from the mineralization of the PTS and PTT during incubation. The type of calcium carbonate crystals tended to change from one form to another when the incubation period extends from 72 to 120h. Both the PTS and the PTT were able to precipitate calcite within the sand column. However, the bacteria from the PTT (26% CaCO3) outperformed those from the PTS (18% CaCO3) in terms of calcium carbonate deposition on the 21st day of incubation.

Virulence genotype and phenotype of two clinical isolates of Arcobacter butzleri obtained from patients with different pathologies

The surge in human arcobacteriosis has increased interest in determining the mechanisms involved in the pathogenesis of Arcobacter butzleri. Here, genomic analyses and in vitro Caco-2 infection, motility, urease and antimicrobial susceptibility testing (AST) assays were used to characterise the virulence and antimicrobial resistance (AMR) determinants of strains HC-1, isolated from a patient with travellers’ diarrhoea, and HC-2, isolated from another with pruritus. AMR determinants conferring resistance to tetracycline (tetO, present in both genomes) and to ampicillin and amoxicillin–clavulanic acid (bla3, present in HC-2) were identified. The same determinants associated with flagellum, chemotaxis, adhesion and invasion were detected in both, but HC-1 lacked eight flagellar genes. The urease cluster was only present in HC-1. Motility and urease tests confirmed the genetic differences between strains, but no genetic marker related to the inability of HC-2 to adhere and invade was identified. This inability could be conditioning the patient’s pathology.

Dimethylaminododecyl Methacrylate-Incorporated Dental Materials Could Be the First Line of Defense against Helicobacter pylori.

Oral cavity is an essential reservoir for H. pylori. We aimed to investigate the antibacterial effects of dimethylaminododecyl methacrylate (DMADDM) against H. pylori. Modified giomers were prepared by introducing 0%, 1.25% and 2.5% DMADDM monomers. Broth microdilution assay, spot assay, Alamer Blue assay, PMA-qPCR, crystal violet staining, scanning electron microscopy observation and live/dead bacterial staining were performed to evaluate the antibacterial and antibiofilm effects of DMADDM and modified giomers in vitro. Urease assay, qPCR, hematoxylin-eosin staining and ELISA were performed to evaluate the inflammation levels and colonization of H. pylori in vivo. In vitro experiments indicated that the minimum inhibitory concentration and minimum bactericidal concentration of DMADDM were 6.25 μg/mL and 25 μg/mL, respectively. It inhibited H. pylori in a dose- and time-dependent manner, and significantly reduced the expression of cagA, vacA, flaA and ureB. DMADDM-modified giomers inhibited the formation of H. pylori biofilm and reduced live cells within it. In vivo experiments confirmed that the pretreatment with DMADDM-modified dental resin effectively reduced the gastric colonization of oral-derived H. pylori, suppressed systemic and local gastric inflammation. DMADDM monomers and DMADDM-modified giomers possessed excellent antibacterial and antibiofilm effects on H. pylori. Pretreatment with DMADDM-modified giomers significantly inhibited the gastric infection by H. pylori.

Efficacy of Qingre Huashi decoction on infection of : inhibiting adhesion, antioxidant, and anti-inflammation.

To investigate the phytochemical profile of Qingre Huashi decoction (, QHD) and evaluate the mechanisms rationalizing the use of QHD against ()-associated gastritis. QHD is composed of 11 herbs, which was prepared by a fixed Pharmacy and concentrated into clear paste. High-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (HPLC-QTOF/MS) was used to detect the phytochemical profile of QHD. The toxicity of QHD against and human gastric epithelial cells was evaluated by the toxicology test and cell counting kit-8 assay. The adhesion model was constructed by incubating and gastric mucosal epithelial cells for 2 h. The urease assay was used to examine the anti-adhesion effects of QHD, and gene expression of adhesins was evaluated by quantitative polymerase chain reaction. The antioxidant activity was assessed by DCFH-DA labeling. To evaluate the anti-inflammatory effect, the levels of pro-inflammatory cytokines in the culture supernatant were detected by enzyme-linked immunosorbent assay. HPLC-QTOF/MS profiling indicated the presence of primary compounds 1-20 in QHD. Drug concentration was determined as 1, 2, and 5 mg/mL by the toxic concentration of QHD against and human gastric epithelial cells. QHD prevented adhesion to the human gastric epithelial cells and reduced levels of reactive oxygen species. QHD also reduced the level of interleukin-8 and other pro-inflammatory cytokines that were upregulated by infection. QHD could inhibit adhesion, and exert antioxidant and anti-inflammatory effects .

Establishment of Noninvasive Methods for the Detection of Helicobacter pylori in Mongolian Gerbils and Application of Main Laboratory Gerbil Populations in China.

Identifying Helicobacter pylori (H. pylori, Hp) infection in animals before and after artificial infection influences the subsequent experiment. We established effective and noninvasive detection methods, including the gastric fluid nested polymerase chain reaction (PCR) method and the 13C-urea breath test, which can detect Hp before modeling Hp infection in Mongolian gerbils. We designed a gas collection equipment for gerbils. Hp nested PCR was also performed on gastric fluid, gastric mucosa, duodenal contents, and faeces of gerbils challenged with Hp. Conventional Hp detection methods, including rapid urease assay and immunohistochemistry, were compared. Moreover, we assessed the natural infection of Hp in 135 gerbils that had never been exposed to Hp artificially from the major laboratory gerbil groups in China. In 10 Hp infected gerbils, the positive detection results were 100%, 100%, 90%, and 10% in gastric fluid, gastric mucosa, duodenal contents, and faeces with nested PCR, respectively. A rapid urease test performed on gastric mucosa showed that all animals were infected with Hp. Immunohistochemical detection and bacteria culture of gastric mucosa samples that were positive by the nested PCR method also confirmed the presence of Hp. 9% (3/35) and 6% (2/31) natural infection rates were found in conventional gerbil groups from the Capital Medical University and Zhejiang Laboratory Animal Center. In conclusion, we established two noninvasive Hp detection methods that can be performed before modelingHp infection, including the gastric fluid nested PCR method and the 13C-urea breath test.

Anti-Helicobacter pylori potential, antioxidant capacity, and anti-inflammatory activity of Xylopia sericea A. St.-Hil. (Annonaceae) leaves

Background: Xylopia sericea A. St.-Hil. (Annonaceae), a Brazilian pepper, popularly known as “pindaíba,” has ethnopharmacological relevance as a useful remedy for gastric disorders; however, few reports are available in the literature to explain its therapeutic effects. Purpose: This study aimed to evaluate the in vitro effects of X. sericea leaf ethanol extract (EE) on Helicobacter pylori (HP) growth, cytokine production, antioxidant capacity, and cytotoxicity in human gastric adenocarcinoma cell line (AGS). Study design: Using a cell model system (murine macrophage LPS-stimulated) and in vitro assays, EE bioactivity, with regard to antioxidant capacity, inhibition of cytokine production, and urease inhibition, was assessed. The EE chemical profile was obtained using chromatography and biomarker quantification. Methods: Antioxidant and urease assays were performed using colorimetric methods, H. pylori growth was evaluated using the minimal inhibitory concentration assay, cytokine production was measured using enzyme-linked immunosorbent assay, cytotoxicity was determined using the MTT method, EE chromatographic profile was obtained using HPLC-DAD-UV analysis, and biomarker quantification was obtained using calibration curves. Results: Our findings demonstrated that EE has an antioxidant capacity similar to that of gallic acid in the hypochlorous acid inhibition assay (EC50 = 61.09 µg/ml) and higher activity (p <0.05) in the superoxide radical assay (EC50 = 25.56 µg/ml) compared to the same antioxidant standard. In RAW 264.7 LPS stimulated cells, EE (100 µg/ml) inhibited the production of NO (76%) and IL-6 (85%) but did not reduce TNF-α and superoxide levels at all tested concentrations (6.25 to 100 µg/ml). EE demonstrated urease enzyme inhibition similar to boric acid, the standard inhibitor, and inhibited H. pylori non-resistant strain growth at 512 µg/mL. EE did not reduce the AGS cell viability at the tested concentrations (6.25 to 100 µg/ml). The EE flavonoid content was 198.50 µg of rutin equivalents in each mg of extract, and the HPLC analyses showed four majoritarian components as quercetin glycosides. Conclusions: This study partially explains the role of X. sericea in defense mechanisms associated with gastric diseases, and provides support for further research on the potential of X. sericea in gastric disease treatments.

Incubation Effect of Selected Organic Mulches on Various Biochemical Soil Health Indicators of Two Taro Soils Under Semi-Controlled Environment

Organic matter plays an important role in the nutrient recycling that helps improve the soil physical, chemical, and biological properties of the soil. This study looks at the characteristics of the two taro soils as affected by the application of four organic amendments used as treatments (Biochar, Mucuna, Erythrina, and grass). The effects of nutrients released from these four treatments were analyzed in the soil laboratory using various soil tests such as Labile Carbon, Fluorescein diacetate (FDA) hydrolysis activity, Potentially mineralizable nitrogen (PMN), Mineral nitrogen, Urease assay, and Phosphatase assay. The results showed that Erythrina and Mucuna are the two cover crops that significantly responded to the two soils. In terms of carbon mineralization, Mucuna and Erythrina fallow mulches had higher means compared to grass and biochar. The difference in the carbon mineralization could be attributed to the characteristics of Mucuna and Erythrina are both legume mulches which have lower C: N ratio. Therefore, they have faster rates of mineralization compared to grass and biochar fallows. However, at the highest rate of application (45 tons/ha), Mucuna was found to have faster rate of mineralization compared to Erythrina. From the other tests that were being carried out, Grass and Erythrina cover crops showed the highest levels of mean biological activity at their highest rate of application (45 tons/ha) on both soils. Mucuna and biochar covers, on the other hand, showed highest levels of mean biological activity at the lowest rate of application (15 tons/ha).

Volatile and phenolic profiling of a traditional medicinal plant, Hypericum empetrifolium with in vitro biological activities

Ethnopharmacological relevanceHypericum empetrifolium Willd is a member of the Hypericaceae family, mainly known in southern Greece, and western Turkey. Being a centuries-old medicinal plant, the aerial parts of the plant have been used for the treatment of herpes, kidney stones, gastric ulcer, and also for their anti-helminthic, and diuretic purposes traditionally. Aim of the studyThe current study aimed to investigate the phytochemical profiles of the essential oil, and two ethanol extracts prepared from the aerial parts (H. empetrifolium aerial parts extract – HEA), and roots of the plant (H. empetrifolium roots extract – HER), and to provide data on antioxidant, anticholinesterase, antityrosinase, antiurease, cytotoxic, and antimicrobial activities of the extracts. Materials and methodsIn this study, volatile and phenolic compounds of the HEA and HER were analyzed by GC-MS and LC-MS/MS, respectively. Antioxidant potential of the extracts was clarified by using DPPH radical scavenging assay, ABTS cation radical assay, and the CUPRAC assay. Acetylcholinesterase, butyrylcholinesterase, urease, and tyrosinase inhibitory activity assays were used to determine enzyme inhibition capacity of the extracts. Cytotoxic activity of the extracts was established by using XTT assay. The antimicrobial activity of the extracts was determined by the microbroth dilution technique. ResultsThe major compounds of the essential oil were revealed as alloaromodendrene (24.7%), α-pinene (14.7%), β-pinene (10.7%), and α-terpineol (7.7%) by the GC-MS analysis. According to the LC-MS/MS analysis results, quinic acid was the most abundant constituent in both extracts with 20612.42 ± 169.02 μg/g extract in HEA extract, and with 2254.34 ± 18.49 μg/g extract in HER extract, respectively. The HEA extract was also found to be rich in terms of chlorogenic acid (5583.14 ± 38.52 μg/g extract), isoquercitrin (3076.77 ± 40.92 μg/g extract), and malic acid (2822.90 ± 31.90 μg/g extract). HEA extract exhibited a significant antioxidant effect with an IC50 value of 11.98 ± 0.22 μg/mL according to the DPPH radical scavenging assay. Similar results were obtained with the ABTS cation radical assay, and the CUPRAC assay. HER extract showed a strong butyrylcholinesterase inhibitory effect with 88.69 ± 0.62% at 200 μg/mL concentration. Both extracts were considered to have moderate anti-tyrosinase activity compared to the standard at 200 μg/mL. The best antimicrobial activity was obtained for HEA against C. parapsilosis with an MIC value of 4.88 μg/mL. HEA also exhibited antifungal activity against C. tropicalis with 19.53 μg/mL. Only HER exhibited low cytotoxicity on A498 renal cell lines with 60.6% cell viability. ConclusionUnquestionably, H. empetrifolium Willd has ethnopharmacological importance according to these results, and further investigations are required to evaluate other therapeutic properties of the plant.

Chilean Rhubarb, Gunnera tinctoria (Molina) Mirb. (Gunneraceae): UHPLC-ESI-Orbitrap-MS Profiling of Aqueous Extract and its Anti-Helicobacter pylori Activity.

The full UHPLC-MS metabolome fingerprinting and anti-Helicobacter pylori effect of Gunnera tinctoria (Molina) Mirb. (Nalca) total extract (GTE) and fractions prepared from its edible fresh petioles were evaluated. The activity of G. tinctoria against H. pylori strains ATCC 45504 and J99 was assessed in vitro by means of agar diffusion assay, Minimum Inhibition Concentration (MIC), and Minimum Bactericidal Concentration (MBC), while killing curve and transmission electronic microscopy (TEM) were conducted in order to determine the effect of the plant extract on bacterial growth and ultrastructure. Additionally, the inhibitory effect upon urease was evaluated using both the Jack Bean and H. pylori enzymes. To determine which molecules could be responsible for the antibacterial effects, tentative identification was done by ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-Q-Orbitrap®-HR-MS). Furthermore, the total G. tinctoria extract was fractionated using centrifugal partition chromatography (CPC), giving four active fractions (1–4). It was determined that the crude extract and centrifugal partition chromatography fractions of G. tinctoria have a bactericidal effect being the lowest MIC and MBC = 32 μg/ml. In the killing curves, fraction one acts faster than control amoxicillin. In the urease assay, F3 exhibited the lowest IC50 value of 13.5 μg/ml. Transmission electronic microscopy showed that crude G. tinctoria extract promotes disruption and separation of the cellular wall and outer membrane detachment on H. pylori causing bacterial cell death.

Field detection of urease and carbonic anhydrase activity using rapid and economical tests to assess microbially induced carbonate precipitation.

Microbial precipitation of calcium carbonate is a widespread environmental phenomenon that has diverse engineering applications, from building and soil restoration to carbon sequestration. Urease-mediated ureolysis and CO2 (de)hydration by carbonic anhydrase (CA) are known for their potential to precipitate carbonate minerals, yet many environmental microbial community studies rely on marker gene or metagenomic approaches that are unable to determine in situ activity. Here, we developed fast and cost-effective tests for the field detection of urease and CA activity using pH-sensitive strips inside microcentrifuge tubes that change colour in response to the reaction products of urease (NH3 ) and CA (CO2 ). The urease assay proved sensitive and useful in the field to detect in situ activity in biofilms from a saline lake, a series of calcareous fens, and ferrous springs, finding relatively high urease activity in lake samples. Incubations of lake microbes with urea resulted in significantly higher CaCO3 precipitation compared to incubations with a urease inhibitor, showing that the rapid assay indicated an on-site active metabolism potentially mediating carbonate precipitation. The CA assay, however, showed less sensitivity compared to the urease test. While its sensitivity limits its utility, the assay may still be useful as a preliminary indicator given the paucity of other means for detecting CA activity in the field. Field urease, and potentially CA, activity assays complement molecular approaches and facilitate the search for carbonate-precipitating microbes and their in situ activity, which could be applied toward agriculture, engineering and carbon sequestration technologies.

The Effects of Sulglycotide on the Adhesion and the Inflammation of Helicobacter Pylori.

Helicobacter pylori (H. pylori) is a primary etiologic factor in gastric diseases. Sulglycotide is a glycopeptide derived from pig duodenal mucin. Esterification of its carbohydrate chains with sulfate groups creates a potent gastroprotective agent used to treat various gastric diseases. We investigated the inhibitory effects of sulglycotide on adhesion and inflammation after H. pylori infection in human gastric adenocarcinoma cells (AGS cells). H. pylori reference strain 60190 (ATCC 49503) was cultured on Brucella agar supplemented with 10% bovine serum. Sulgylcotide-mediated growth inhibition of H. pylori was evaluated using the broth dilution method. Inhibition of H. pylori adhesion to AGS cells by sulglycotide was assessed using a urease assay. Effects of sulglycotide on the translocation of virulence factors was measured using western blot to detect cytotoxin-associated protein A (CagA) and vacuolating cytotoxin A (VacA) proteins. Inhibition of IL-8 secretion was measured using enzyme-linked immunosorbent assay (ELISA) to determine the effects of sulglycotide on inflammation. Sulglycotide did not inhibit the growth of H. pylori, however, after six and 12 hours of infection on AGS cells, H. pylori adhesion was significantly inhibited by approximately 60% by various concentrations of sulglycotide. Sulglycotide decreased H. pylori virulence factor (CagA and VacA) translocation to AGS cells and inhibited IL-8 secretion. Sulglycotide inhibited H. pylori adhesion and inflammation after infection of AGS cells in vitro. These results support the use of sulglycotide to treat H. pylori infections.

Artemisinin and its derivatives prevent Helicobacter pylori-induced gastric carcinogenesis via inhibition of NF-κB signaling

BackgroundGastric cancer has a high morbidity and is a leading cause of cancer-related mortality worldwide. Helicobacter pylori (H. pylori) infection is commonly found in the early stage of gastric cancer pathogenesis, which induces chronic gastritis. Artemisinin (ART) and its derivatives (ARTS, artesunate and DHA, dihydroartemisinin), a new class of potent antimalarials, have been reported to exert both preventive and anti-gastric cancer effects. However, the underlying mechanisms of the chemopreventive effects of ART and its derivatives in H. pylori infection induced-gastric cancer are not fully elucidated. PurposeWe investigated the effects of H. pylori infection in gastric cancer; and the preventive mechanisms of ART, ARTS and DHA. MethodsThe H. pylori growth was determined by the broth macro-dilution method, and its adhesion to gastric cancer cells was evaluated by using the urease assay. The protein and mRNA levels, reactive oxygen species (ROS) production, as well as the production of inflammatory cytokines were evaluated by Western blot, real-time PCR, flow cytometry and ELISA, respectively. Moreover, an in vivo MNU (N-methyl-N-nitroso-urea) and H. pylori-induced gastric adenocarcinoma mouse model was established for the investigation of the cancer preventive effects of ART and its derivaties, and the underlying mechanisms of action. ResultsART, DHA and ARTS inhibited the growth of H. pylori and gastric cancer cells,suppressed H. pylori adhesion to the gastric cancer cells, and reduced the H. pylori-enhanced ROS production. Moreover, ART, DHA and ARTS significantly reduced tumor incidence, number of tumor nodules and tumor size in the mouse model. Among these three compounds, DHA exerted the most potent chemopreventive effect. Mechanistic studies showed that ART and its derivatives potently inhibited the NF-κB activation. ConclusionART, DHA and ARTS have potent preventive effects in H. pylori-induced gastric carcinogenesis. These effects are, at least in part, attributed to the inhibition of NF-κB signaling pathway. Our findings provide a molecular justification of using ART and its derivatives for the prevention and treatment of gastric cancer.

Stabilizing effects on a Cd polluted coastal wetland soil using calcium polysulphide

In this study, different dosages of calcium polysulphide (CaSx) were used as an amendment to investigate effects on the immobilizing of Cd in a wetland soil by pot experiment. In addition to chemical analysis (pH and bioavailable Cd concentration), changes in soil enzyme activities, microbial carbon utilization capacity, metabolic and community diversity were examined to assess dynamic impacts on soil environmental quality and toxicity of Cd resulting from ameliorant dosing. Soil pH increased immediately upon CaSx amendment compared to the unamended control (CK), and then declined slowly to a level lower than CK. Diethylenetriamine pentaacetic acid (DTPA) extractable Cd concentration was determined to characterize the bioavailability of Cd in the soil. The CaSx dose-dependent effect observed that with increasing CaSx dosage, the immobilizing efficiency decreased. Soil urease and catalase activity assays and Biolog EcoPlate assay indicated that early stage addition of CaSx significantly inhibited soil microbial activities. However, mid and late stage time periods showed the inhibition effects were alleviated, and the microbial activities could be recovered in 1% and 2% CaSx treatments. Moreover, with increasing incubation time, microbial community diversity and richness were significantly recovered in 1% and 2% CaSx treatments compared to the CK. No considerable changes were observed in the 5% CaSx treatment. Conclusively, the 1% CaSx amendment was an efficient and safe dosage for the stabilization of Cd contaminated wetland soil. This study contributes to the development of in situ remediation ameliorants and technologies for heavy metal polluted wetland soils.

RovM and CsrA Negatively Regulate Urease Expression in Yersinia pseudotuberculosis.

Urease acts as an important acid resistance system and virulence factor that is widespread among microorganisms. RovM is a global regulator that regulates a series of genes and pathways including acid survival systems in the enteric bacterium Yersinia pseudotuberculosis (Yptb). However, whether RovM regulates the urease activity in Yptb was still unknown. In this study, by using qualitative and quantitative urease assays, we show that the urease expression responds to nutrient conditions and the RovM protein represses urease expression by binding to its promoter. A previously reported positive regulator OmpR activates urease activity but RovM plays a dominant role in different nutrient conditions. In addition, carbon storage regulator system A (CsrA), the upstream regulator of RovM, dramatically down-regulates urease activity possibly by its binding to the Shine-Dalgarno (SD) sequence of the mRNA encoding the urease. In conclusion, this study demonstrates that urease activity is strictly controlled by nutrient conditions and is down-regulated by the CsrA-RovM pathway.

Urea transporters and sweat response to uremia.

In humans, urea is excreted in sweat, largely through the eccrine sweat gland. The urea concentration in human sweat is elevated when compared to blood urea nitrogen. The sweat urea nitrogen (UN) of patients with end‐stage kidney disease (ESRD) is increased when compared with healthy humans. The ability to produce sweat is maintained in the overwhelming majority of ESRD patients. A comprehensive literature review found no reports of sweat UN neither in healthy rodents nor in rodent models of chronic kidney disease (CKD). Therefore, this study measured sweat UN concentrations in healthy and uremic rats. Uninephrectomy followed by renal artery ligation was used to remove 5/6 of renal function. Rats were then fed a high‐protein diet to induce uremia. Pilocarpine was used to induce sweating. Sweat droplets were collected under oil. Sweat UN was measured with a urease assay. Serum UN was measured using a fluorescent ortho‐pthalaldehyde reaction. Immunohistochemistry (IHC) was accomplished with a horseradish peroxidase and diaminobenzidine technique. Sweat UN in uremic rats was elevated greater than two times compared to healthy pair‐fed controls (220 ± 17 and 91 ± 15 mmol/L, respectively). Post hoc analysis showed a significant difference between male and female uremic sweat UN (279 ± 38 and 177 ± 11 mmol/L, respectively.) IHC shows, for the first time, the presence of the urea transporters UT‐B and UT‐A2 in both healthy and uremic rat cutaneous structures. Future studies will use this model to elucidate how rat sweat UN and other solute excretion is altered by commonly prescribed diuretics.

Urease immobilized polymer hydrogel: Long-term stability and enhancement of enzymatic activity

A method has been developed in which an enzyme namely urease was immobilized inside hydrogel matrix to study the stability and enzymatic activity in room temperature (∼27–30°C). This urease coupled hydrogel (UCG) was obtained by amine-acid coupling reaction and this procedure is such that it ensured the wider opening of mobile flap of enzyme active site. A systematic comparison of urea–urease assay and the detailed kinetic data clearly revealed that the urease shows activity for more than a month when stored at ∼27–30°C in case of UCG whereas it becomes inactive in case of free urease (enzyme in buffer solution). The aqueous microenvironment inside the hydrogel, unusual morphological features and thermal behaviour were believed to be the reasons for unexpected behaviour. UCG displayed enzyme activity at basic pH and up to 60°C. UCG showed significant enhancement in activity against thermal degradation compared to free urease. In summary, this method is a suitable process to stabilize the biomacromolecules in standard room temperature for many practical uses.

Isolation and identification of Pseudomonas azotoformans for induced calcite precipitation.

Biomineralization is a process by which living organisms produce minerals. The extracellular production of these biominerals by microbes has potential for various bioengineering applications. For example, crack remediation and improvement of durability of concrete is an important goal for engineers and biomineral-producing microbes could be a useful tool in achieving this goal. Here we report the isolation, biochemical characterization and molecular identification of Pseudomonas azotoformans, a microbe that produces calcite and which potentially be used to repair cracks in concrete structures. Initially, 38 bacterial isolates were isolated from soil and cements. As a first test, the isolates were screened using a urease assay followed by biochemical tests for the rate of urea hydrolysis, calcite production and the insolubility of calcite. Molecular amplification and sequencing of a 16S rRNA fragment of selected isolates permitted us to identify P. azotoformans as a good candidate for preparation of biotechnological concrete. This species was isolated from soil and the results show that among the tested isolates it had the highest rate of urea hydrolysis, produced the highest amount of calcite, which, furthermore was the most adhesive and insoluble. This species is thus of interest as an agent with the potential ability to repair cracks in concrete.