Action Of Urease Research Articles

A hydrogel sensor based on cellulose nanofiber/polyvinyl alcohol with colorimetric-fluorescent bimodality for non-invasive detection of urea in sweat

The concentration of urea in sweat serves as a valuable indicator of an individual's overall health. In this study, we present a novel hydrogel sensor (BAF-CPu), based on cellulose nanofiber and polyvinyl alcohol, designed to achieve non-invasive in situ and highly sensitive detection of urea in sweat by combining the dual-mode response of colorimetric and ratiometric fluorescence techniques. The bright red fluorescent gold‑copper bimetallic nanoclusters and green fluorescent fluorescein isothiocyanate-modified cellulose nanofibers endowed BAF-CPu with proportional fluorescence responsive properties. Under the catalytic action of urease, the hydrolysis of urea raises the pH, resulting in diminished red fluorescence along with enhanced green fluorescence, and the fluorescence color of BAF-CPu changes from red to green. Moreover, BAF-CPu hydrogel encapsulates pH-responsive bromothymol blue (BTB), which changes from yellow to blue in the presence of urea. Importantly, BAF-CPu absorbs sweat by adhering directly to the skin surface, avoiding the complicated sampling process and improving the maneuverability of the detection process. With both ratiometric fluorescence and colorimetric modes, BAF-CPu is not only able to detect sweat in situ, but also can reduce the interference of the complex sweat environment on the urea detection, and realize the high sensitivity detection of urea in sweat.

The multi‐factorial modes of action of urease in the pathogenesis of incontinence associated dermatitis

AbstractBackgroundIncontinence Associated Dermatitis (IAD) is a type of skin inflammation caused by chronic exposure to urine and/or faeces. Current treatment strategies involve creating a barrier between the skin and urine/faeces rather than targeting specific irritants. Urease expressing pathogens catalyse the conversion of urea, present in urine, into ammonia. The accumulation of ammonia causes an elevation in skin pH which is believed to activate faecal enzymes which damage skin, and opportunistic pathogens, which lead to secondary infections.ObjectivesTo develop a better, multi‐factorial model of IAD pathogenesis, including the effect of urease‐expressing bacteria on skin, mechanism of damage of urease and urease‐triggered activity of faecal enzymes and secondary pathogens. To study the effect of urease inhibition on preventing IAD skin damage.MethodsFive separate studies were made using ex vivo porcine skin and in vivo human skin models. Measurements of the change in skin barrier function were made using skin impedance, trans‐epidermal water loss (TEWL), stratum corneum moisture and pH. Skin was exposed to artificial urine, inoculated with various microbes, enzymes and chemicals to examine the influence of: 1) urease‐positive Proteus mirabilis 2) ammonia, 3) combination of P. mirabilis and a faecal enzyme, trypsin, 4) combination of P. mirabilis and opportunistic pathogens, Candida albicans and Staphylococcus aureus, 5) inhibition of urease using acetohydroxamic acid (AHA) on barrier function.ResultsThe urease‐mediated production of ammonia had two principal effects: it elevated skin pH and caused inflammation, leading to significant breakdown in skin (stratum corneum) barrier function. Urease was found to further increase the activity of faecal enzymes and opportunistic pathogens, due to elevated skin pH. The urease inhibitor, AHA, was shown to have significantly reduced damage to skin barrier function, measured as its electrical resistance.ConclusionsTargeted therapeutic strategies should be developed to prevent the manifestation of IAD, rather than creating a generic barrier between skin and urine/faeces. Urease has been identified as a crucial component in the manifestation of IAD, due to its role in the production of ammonia. Urease inhibition provides a promising therapeutic target to halt the progression of IAD.

Influence of polymer modification of complex fertilizer on the efficiency of phosphorus and potassium use by winter wheat on the southern chernozem

In modern conditions of unprecedented challenges, an alternative to the technological degradation of fertilizer application systems can be the development of original forms of long-acting fertilizers. Despite significant progress in the adaptation of progressive systems of agrochemical impacts, the use of traditional forms of mineral fertilizers does not allow using the full potential of the introduced nutrients, since the processes of incomplete denitrification, immobilization and leaching (leaching) of the main macronutrients (NPK), along with the action of urease, lead to unproductive loss of nutrients in the soil. In addition to economic losses, there are negative environmental consequences – eutrophication of water bodies, emission of greenhouse gases. The studies were carried out in 2022 under production conditions in the Kursk district of the Stavropol Territory on the farm of “StavAgroCom” on southern carbonate chernozem. As a modifier of mineral fertilizers, one of the most common synthetic polymers, polyvinyl alcohol (PVA), was used. The polymer consumption rate was 3, 5 and 7 kg per 1 ton of fertilizer. Yield growth was positively correlated with the dose of polymer in the fertilizer. The analysis of soil properties was carried out by the method of principal components. The resulting clustering in the coordinates of the first two components (in total they explain 65% of the data variation) shows significant differences in soil properties under different experimental options. To assess the efficiency of the use of nutrients depending on the dose of the polymer, a differential indicator was proposed – the additional efficiency of the use of nutrients (E). It was estimated from the increase in yield relative to the zero dose of the polymer per unit reduction in the concentration of this element in the soil according to the proposed formula. It is shown that the polymer use in fertilizer gives a statistically significant increase in the efficiency of phosphorus and potassium use by winter wheat.

Biotransformation of urea in the water of water bodies

Introduction. The transformation of substances is an urgent problem of environmental hygiene. Urea (carbamide) is a product of human and animal life - enters water objects with household collector wastewater, surface runoff in areas that use urea as fertilizer, waste discharges from livestock complexes, and effluents of enterprises that produce carbamide. Two aspects of the problems associated with urea contamination of water bodies can be identified: the lack of a direct instrumental method for controlling urea and the lack of a comprehensive approach to controlling urea and its biotransformation products: ammonium, nitrites and nitrates. These two factors determined the relevance of the research. Materials and methods. The studies used the «Stayer» ionic chromatography system with anionic and cationic separation columns: Phenomenex Star-Ion A-300 100/4.6 USA; Shodex IC YS-50 150/4.6 Japan; when developing the urea analysis method, the «Aquilon» cationic column AQULINE C1P, 5µ,150/4.6 was used. The detection is conductometric and amperometric. The research objects were: surface, bottled, tap water, model water solutions of carbamide. As a urease source, the soil (sod-podzolic) from the Educational and Experimental Soil-Ecological Center of the Lomonosov Moscow State University “Chashnikovo” was used. Results. A new highly sensitive ion chromatographic method for determining urea in the water of various water bodies with a sensitivity of 5 mg/dm3 with direct sample input and using sample preparation - 0.5 mg/dm3 is proposed. The results of studies of model aqueous solutions of urea, surface water, tap water of Moscow, many bottled water samples on the content of urea and products of its biochemical transformation are presented. The presence of urea in the surface water and tap water of Moscow was revealed, and a correlation between the urea content and ammonium ions, nitrites and nitrates in the water was stated. Limitations of the study. The studies were carried out with waters whose total mineralization did not exceed 15 mg-eq/dm3 (water of river Don), and the urea content established in the water samples was not lower than 0.5 mg/dm3. Conclusion. When monitoring urea contamination of various water bodies, it is necessary to consider the products of its biotransformation under the action of urease, for as a result of the processes of urea biotransformation, a low-toxic substance can form more toxic products: nitrites, nitrates and ammonium. Applying the kinetic approach to study the processes of urea biotransformation in water under model conditions showed that the extreme type of kinetic curves corresponds to the formation of intermediate products - nitrites, and the increasing type corresponds to the formation and accumulation of final products - nitrates and ammonium ions.

Features and Applications of Urine Stabilization Methods: A Review

The recovery of nutrients from unconventional water such as domestic sewage is considered as a sustainable solution to environmental sanitation, personal hygiene, water, and food safety issues. Source separation of urine and the sequenced treatment techniques are the promising approaches to recover the resources from this unconventional water. However, in the storage of urine, urea is hydrolyzed under the action of urease, resulting in odor, precipitation and the loss of ammonia, which is a challenge to be overcome in the process of urine recycling. This review collates research related to urine stabilization, and aims to summarize the characteristics and applications of existing urine stabilization methods, such as acidification, alkalization, electrochemistry, inhibitors and etc. Overall, acidification and alkalization have higher dosage requirements and have an attentional impact on the environment; electrochemical technology is suitable for decentralized sanitation facilities, but the inhibition duration is short; inhibitors are the least effective and are usually used to regulate urease activities in soil environment rather than for urine stabilization. Choosing the appropriate approaches of urine stabilization should focus on the overall perspective of urine resource utilization, consider the separation method and recovery form, and combine it with the concentration technology.

Extraction, precipitation and characterization of urease from Vicia faba L.

This study aimed to extract urease enzyme from available plant source which was broad beans (Vicia faba L.) using aqueous solution in a ratio1:3(w:v) .The crude extract appeared enzyme activity 33.3 U/ml. Results of this study revealed the possibility to precipitate urease enzyme after extraction using different precipitation methods consisted of Acetone precipitation; Alcohol precipitation and Ammonium sulfate precipitation, these ways of urease precipitation gave enzyme activities 74.8; 43.6 and 82.2 U/ml respectively .The enzyme precipitation using 60% saturation ratio of Ammonium sulfate gave the maximum activity of urease precipitation among other precipitation methods. Enzyme characterization appeared the optimum pH of urease activity was 8.0 and gave enzyme activity 93.8U/ml, while the optimum pH of urease stability was 6.0; the enzyme maintains its activity. On the other side the optimum temperature of precipitated urease activity was 50°C and the enzyme activity reached 99.3U/ml while the optimal temperature of urease stability was 40°C ,the precipitated urease maintain 100% of activity. The effect of other factors on urease action were studied also, these are consisted of the storage time of enzyme ,urease maintain its activity for 8days and the activity begun to decline after that time; the effect of time reusability was also revealed that the enzyme could be used for six times and maintain its activity. The influence of different mineral salts on precipitated urease were also recorded ;these salts were CuSO4 , MgSO4, MgCl2,KCl,CaCl2and FeSo4, results appeared that magnesium and calcium salts were activator for the precipitated urease while copper ,potassium and ferrous salts were inhibitor of the precipitated urease. Urea concentration as an enzyme substrate were also examined and results appeared the optimal substrate concentration was 30mM; enzyme activity reached to125.2U/ml.

Homology Modeling, Molecular Docking and Molecular Dynamics Based Functional Insights into Rice Urease Bound to Urea

Urease (EC 3.5.1.5) is an important member of most popular amidohydrolases superfamily that is well known for catalyzes the hydrolysis of urea into ammonia and carbon dioxide. Urease protein exclusively found in a wide range of living organisms including plant, algae, bacteria, fungi and some invertebrates. In plants, urease play an important role of recapturing the nitrogen from urea. Despite its critical interplay in plants the structural and functional aspects of urease in O. sativa are still unresolved. In the present study, a three-dimensional structure of rice urease was deduced by using homology modelling based approach. Molecular dynamics simulations were performed to gain further insight into the molecular mechanism and mode of action of urease of rice. Further, the possible binding interactions of modeled structure of urease with urea were assessed by using a geometry-based molecular docking algorithm. The study reveals the role of Ser324, Ala329 and Val385 of rice urease enzyme in binding with the substrate urea. In conclusion, this study presents a 3D model of rice urease and helps understanding the molecular basis for the mechanism of urease interaction with substrate urea at atomic level.

Conformational change results in loss of enzymatic activity of jack bean urease on its interaction with silver nanoparticle.

Urease is an enzyme produced by microbes such as bacteria, yeast and fungi. Plants also produce this enzyme. Urease action splits urea into ammonia and carbamate. This action is having important implications in agro-chemical, medicinal and environment. Therefore there is always a constant search for new and novel compounds which could inhibit this enzyme. Here we have studied the interaction of jack bean urease (JBU) with silver nanoparticle to analyze the influence of the resultant protein corona formation on the catalytic property of JBU. Several techniques like UV-Vis, gel shift assay and CD spectroscopy have been used to characterize this interaction. Urease activity assay suggests that the protein corona formation inhibits the enzymatic action of JBU. The loss of enzymatic action could be either due to the nanoparticle blocking the active site of JBU or a conformational change in the protein. The CD spectra of JBU-AgNP complexes clearly revealed significant changes in the secondary structural composition of the JBU and this could be the reason for the loss of enzymatic activity of JBU. This study revealed an interesting observation, where the interaction of AgNP with JBU resulted destabilization of hexameric nature of JBU which is otherwise highly stable. The results of the present study could be useful in the development of nanoparticle based material for inhibiting the ureolytic activity of ureases in different fields.

Cryptococcusgattii urease as a virulence factor and the relevance of enzymatic activity in cryptococcosis pathogenesis.

Ureases (EC 3.5.1.5) are Ni(2+) -dependent metalloenzymes produced by plants, fungi and bacteria that hydrolyze urea to produce ammonia and CO2 . The insertion of nickel atoms into the apo-urease is better characterized in bacteria, and requires at least three accessory proteins: UreD, UreF, and UreG. Our group has demonstrated that ureases possess ureolytic activity-independent biological properties that could contribute to the pathogenicity of urease-producing microorganisms. The presence of urease in pathogenic bacteria strongly correlates with pathogenesis in some human diseases. Some medically important fungi also produce urease, including Cryptococcusneoformans and Cryptococcusgattii. C.gattii is an etiological agent of cryptococcosis, most often affecting immunocompetent individuals. The cryptococcal urease might play an important role in pathogenesis. It has been proposed that ammonia produced via urease action might damage the host endothelium, which would enable yeast transmigration towards the central nervous system. To analyze the role of urease as a virulence factor in C.gattii, we constructed knockout mutants for the structural urease-coding gene URE1 and for genes that code the accessory proteins Ure4 and Ure6. All knockout mutants showed reduced multiplication within macrophages. In intranasally infected mice, the ure1Δ (lacking urease protein) and ure4Δ (enzymatically inactive apo-urease) mutants caused reduced blood burdens and a delayed time of death, whereas the ure6Δ (enzymatically inactive apo-urease) mutant showed time and dose dependency with regard to fungal burden. Our results suggest that C.gattii urease plays an important role in virulence, in part possibly through enzyme activity-independent mechanism(s).

Cuban zeolite as ammonium carrier in urea-based fertilizer pellets: Photoacoustic-based sensor for monitoring N-ammonia losses by volatilization in aqueous solutions

The loss of mineral nitrogen by ammonia volatilization is one of the main drawbacks in modern agriculture. This work proposes the use of a quantum cascade laser-based photoacoustic sensor to continuously monitor ammonia emission rates from fertilizer pellets in aqueous solutions. Pellets were obtained mixing different contents (2.5, 20 and 40wt.%) of Cuban zeolite with urea. The pH of aqueous solutions was also monitored and its maximum value was 9.5 for pellets with 40wt.% of zeolite. Urease obtained from soybean seed extract was added to the solution. Ammonia emission rates were enhanced due to the catalytic action of urease on the urea hydrolysis. The use of Cuban zeolite (40wt.%) reduced the ammonia volatilization from the solutions by approximately 33%. The results give us important clues to reach a better understanding of the kinetics of ammonia release from pellets and the use of zeolite in agriculture as an innovative material to control the retention of mineral nitrogen into soils.

English

In agriculture high urease activity during urea fertilization causes substantial environmental and economical problems by releasing abnormally large amount of ammonia into the atmosphere which leads to plant damage as well as ammonia toxicity. All over the world, urea is the most widely applied nitrogen fertilizer. Due to the action of enzyme urease; urea nitrogen is lost as volatile ammonia. For efficient use of nitrogen fertilizer, urease inhibitor along with the urea fertilizer is one of the best promising strategies. Urease inhibitors also provide an insight in understanding the mechanism of enzyme catalyzed reaction, the role of various amino acids in catalytic activity present at the active site of enzyme and the importance of nickel to this metallo enzyme. By keeping it in view, the present study was designed to dock three urease inhibitors namely Hydroquinone (HQ), Phenyl Phosphorodiamate (PPD) and N-(n-butyl) Phosphorothiocic triamide (NBPT) against Hydroquinone glucosyltransferase using molecular docking approach. The 3D structure of Hydroquinone glucosyltransferase was predicted using homology modeling approach and quality of the structure was assured using Ramachandran plot. This study revealed important interactions among the urease inhibitors and Hydroquinone glucosyltransferase. Thus, it can be inferred that these inhibitors may serve as future anti toxic constituent against plant toxins.

Cloning, purification and preliminary crystallographic analysis of the complex of Helicobacter pylori α-carbonic anhydrase with acetazolamide.

Helicobacter pylori infection of the stomach can lead to severe gastroduodenal diseases such as gastritis, peptic ulcers and gastric cancers. Periplasmic H. pylori α-carbonic anhydrase (HpαCA) is essential for the acclimatization of the bacterium to the acidity of the stomach. Through the action of urease and carbonic anhydrases, the H. pylori periplasmic pH is maintained at around 6 in an environment with a pH as low as 2, which in turn facilitates the maintenance of a cytoplasmic pH close to neutral, allowing growth in the gastric niche. Crystals of HpαCA in complex with the inhibitor acetazolamide have been grown by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitating agent. The crystals have the symmetry of space group P2(1)2(1)2(1), with unit-cell parameters a = 37.0, b = 82.4, c = 150.8 Å. An X-ray diffraction data set was collected from a single crystal to 1.7 Å resolution. Calculation of the self-rotation function using this data and molecular replacement showed that the asymmetric unit contains an HpαCA dimer.

Enzymatic generation of oil-displacing compositions under conditions of low-temperature viscous-oil reservoirs

The enzymatic hydrolysis of urea contained in an oil-displacing composition by the action of plant urease has been studied. Optimal ratios of urea to natural urease-containing sources have been determined for the intense hydrolysis accompanied by the release of CO2, which reduces the viscosity of crude oil, and ammonia, which forms an alkaline buffer system, thereby increasing the detergent properties of the composition. The effect of hydrolysis products on the rheological characteristics of heavy crude from the Usa oil-field has been examined. The recovery of viscous crude oil from a model reservoir using the oil-displacing composition and urease has been investigated.

An Electrochemical Enzyme Immunochip Based on Capacitance Measurement for the Detection of IgG

This study describes the development of an electrochemical array immunochip for the detection of IgG. Interdigitated immunochip platforms were fabricated by sputtering gold on a glass wafer by using MEMS process and then were coated with Eudragit S100, an enteric polymer, forming an insulating layer over the working area of immunochips. The breakdown of the polymer layer was exemplified by the catalytic action of urease which, in the presence of urea, caused an alkaline pH change. This subsequently caused an increase of the double layer capacitance of the underlying electrode. Used in conjunction with a competitive immunoassay format, this allowed the ratio of initial to final electrode capacitance to be directly linked with the concentration of analyte, i.e. IgG. Responses to IgG could be detected at IgG concentration as low as 250 ngmL �1 and showed good linearity up to IgG concentration as high as 20 µgmL �1 .

Volatilização de amônia do solo após a aplicação de ureia convencional ou com inibidor de urease

Volatilização de NH3 é a principal reação que diminui a eficiência de utilização pelas plantas do N proveniente da ureia, quando ela é aplicada sobre a superfície do solo. A fim de minimizar essa perda, produtos têm sido misturados à ureia para inibir temporariamente a ação da urease. Este trabalho objetivou avaliar alternativas de aplicação de um fertilizante com inibidor de urease, visando a diminuir a volatilização de NH3 relativamente à ureia convencional, em algumas condições ambientais e de solo. Foram desenvolvidos quatro experimentos, todos em condições de laboratório, em 2007 e 2008, em Cambissolo Húmico. Os tratamentos variaram em cada estudo e incluíram combinações de níveis de pH do solo (natural; 5,5; 6,3; e 6,8), umidade do solo (5, 10 ou 20 % de água) e temperaturas ambientais (18 e 35 ºC), além de estados físicos (sólido ou líquido) e de métodos de aplicação dos fertilizantes (na superfície ou incorporado ao solo). As unidades experimentais foram constituídas por bandejas plásticas (23 x 51 x 17 cm) com 12 kg de solo, numa espessura de 15 cm, sobre as quais foram instaladas câmaras coletoras de NH3. A amônia volatilizada foi determinada em várias épocas, nos primeiros 28 dias após a aplicação dos fertilizantes. O pico de volatilização diária de NH3 ocorreu sempre na primeira semana depois da adição dos fertilizantes ao solo, e aconteceu dois a três dias mais tarde para a ureia com inibidor de urease, em relação à ureia convencional. A volatilização de NH3 nem sempre foi maior para a ureia convencional em comparação ao fertilizante contendo inibidor de urease, tampouco para o estado líquido em relação ao granulado. A volatilização de NH3 aumentou com a elevação do pH, da temperatura e da dose aplicada de N e foi menor nos extremos de umidade (solo com 5 % ou com 20 % de água). Para os fertilizantes aplicados sobre a superfície do solo, a taxa máxima de perda diária foi correspondente a 14 kg ha-1 de N, e a perda total acumulada variou de 2 a 50 % do N aplicado, dependendo principalmente do estado físico em que o fertilizante foi aplicado, da umidade do solo e da temperatura ambiente. A incorporação dos fertilizantes amídicos ao solo foi a maneira mais eficaz de minimizar as perdas de N por volatilização.

Entrapment of live microbial cells in electropolymerized polyaniline and their use as urea biosensor

The lyophilized biomass of bacterium Brevibacterium ammoniagenes was immobilized in polystyrene sulphonate–polyaniline (PSS–PANI) conducting polymer on a Pt twin wire electrode by potentiostatic electropolymerization. The bacterial cells retained their viability as well as urease activity under entrapped state, as confirmed with bacterial live–dead fluorescent assay and enzymatic assays. The entrapped cells were visualized using scanning electron microscope. The immobilized cells were used as a source of unpurified urease to develop a conductometric urea biosensor. The catalytic action of urease in the sensor released ammonia, thereby causing an increase in the pH of the microenvironment. The pH dependant change in the resistivity of the polymer was used as the basis of sensing mechanism. The sensor response was linear over a range of 0–75mM urea with a sensitivity of 0.125mM−1. The sensor could be reused for 12–15 independent measurements and was quite stable in dry as well as buffered storage condition at 4°C for at least 7 days.

Modification of jack bean urease thiols by thiosulphinates contained in garlic extract: DTNB titration studies

The reactivity of the thiol groups in urease with thiosulphinate contained in garlic extract was spectroscopically characterised. The enzyme was incubated with the garlic extract and the reaction progress curves were recorded in the presence of thiol-selective reagent 5,5′-dithiobis(2-nitrobenzoic acid). Simultaneously the enzyme residual activity was also determined. The process was studied in 50 mM phosphate buffer, at pH 7.8 and ambient temperature. It was found that thiosulphinates act as time- and concentration-dependent inactivators of urease. The observed decrease of the enzyme activity corresponds to the number of the urease thiols modified by thiosulphinates. The modification of half of all the urease thiols (18 of 36) causes a slight (only 8–10%) decrease of the catalytic activity. The modification of the remaining 18 thiols results in significant disturbance of urease action until complete loss of the catalytic function occurs. This provides the evidence that Cys 592, the critical residue for urease activity, belongs to the enzyme thiols, which are less reactive and more resistant to chemical modification than the other thiols.

Expression of the urease gene of Agaricus bisporus: a tool for studying fruit body formation and post-harvest development

Fruit body initials of Agaricus bisporus contain high levels of urea, which decrease in the following developmental stages until stage 4 (harvest) when urea levels increase again. At storage, the high urea content may affect the quality of the mushroom, i.e. by the formation of ammonia from urea through the action of urease (EC 3.5.1.5). Despite the abundance of urea in the edible mushroom A. bisporus, little is known about its physiological role. The urease gene of A. bisporus and its promoter region were identified and cloned. The coding part of the genomic DNA was interrupted by nine introns as confirmed by cDNA analysis. The first full homobasidiomycete urease protein sequence obtained comprised 838 amino acids (molecular mass 90,694 Da, pI 5.8). An alignment with fungal, plant and bacterial ureases revealed a high conservation. The expression of the urease gene, measured by Northern analyses, was studied both during normal development of fruit bodies and during post-harvest senescence. Expression in normal development was significantly up-regulated in developmental stages 5 and 6. During post-harvest senescence, the expression of urease was mainly observed in the stipe tissue; expression decreased on the first day and remained at a basal level through the remaining sampling period.

Involvement of the HP0165-HP0166 Two-Component System in Expression of Some Acidic-pH-Upregulated Genes of Helicobacter pylori

About 200 genes of the gastric pathogen Helicobacter pylori increase expression at medium pHs of 6.2, 5.5, and 4.5, an increase that is abolished or much reduced by the buffering action of urease. Genes up-regulated by a low pH include the two-component system HP0165-HP0166, suggesting a role in the regulation of some of the pH-sensitive genes. To identify targets of HP0165-HP0166, the promoter regions of genes up-regulated by a low pH were grouped based on sequence similarity. Probes for promoter sequences representing each group were subjected to electrophoretic mobility shift assays (EMSA) with recombinant HP0166-His(6) or a mutated response regulator, HP0166-D52N-His(6), that can specifically determine the role of phosphorylation of HP0166 in binding (including a control EMSA with in-vitro-phosphorylated HP0166-His(6)). Nineteen of 45 promoter-regulatory regions were found to interact with HP0166-His(6). Seven promoters for genes encoding alpha-carbonic anhydrase, omp11, fecD, lpp20, hypA, and two with unknown function (pHP1397-1396 and pHP0654-0675) were clustered in gene group A, which may respond to changes in the periplasmic pH at a constant cytoplasmic pH and showed phosphorylation-dependent binding in EMSA with HP0166-D52N-His(6). Twelve promoters were clustered in groups B and C whose up-regulation likely also depends on a reduction of the cytoplasmic pH at a medium pH of 5.5 or 4.5. Most of the target promoters in groups B and C showed phosphorylation-dependent binding with HP0166-D52N-His(6), but promoters for ompR (pHP0166-0162), pHP0682-0681, and pHP1288-1289 showed phosphorylation-independent binding. These findings, combined with DNase I footprinting, suggest that HP0165-0166 is an acid-responsive signaling system affecting the expression of pH-sensitive genes. Regulation of these genes responds either to a decrease in the periplasmic pH alone (HP0165 dependent) or also to a decrease in the cytoplasmic pH (HP0165 independent).

Characterization and optimization of experimental variables within a reproducible bladder encrustation model and in vitro evaluation of the efficacy of urease inhibitors for the prevention of medical device‐related encrustation

This study presents a reproducible, cost-effective in vitro encrustation model and, furthermore, describes the effects of components of the artificial urine and the presence of agents that modify the action of urease on encrustation on commercially available ureteral stents. The encrustation model involved the use of small-volume reactors (700 mL) containing artificial urine and employing an orbital incubator (at 37 degrees C) to ensure controlled stirring. The artificial urine contained sources of calcium and magnesium (both as chlorides), albumin and urease. Alteration of the ratio (% w/w) of calcium salt to magnesium salt affected the mass of encrustation, with the greatest encrustation noted whenever magnesium was excluded from the artificial urine. Increasing the concentration of albumin, designed to mimic the presence of protein in urine, significantly decreased the mass of both calcium and magnesium encrustation until a plateau was observed. Finally, exclusion of urease from the artificial urine significantly reduced encrustation due to the indirect effects of this enzyme on pH. Inclusion of the urease inhibitor, acetohydroxamic acid, or urease substrates (methylurea or ethylurea) into the artificial medium markedly reduced encrustation on ureteral stents. In conclusion, this study has described the design of a reproducible, cost-effective in vitro encrustation model. Encrustation was markedly reduced on biomaterials by the inclusion of agents that modify the action of urease. These agents may, therefore, offer a novel clinical approach to the control of encrustation on urological medical devices.