High Concentrations Of Urea Research Articles

Silver-mediated base pairings: towards dynamic DNA nanostructures with enhanced chemical and thermal stability

The thermal and chemical fragility of DNA nanomaterials assembled by Watson–Crick (WC) pairing constrain the settings in which these materials can be used and how they can be functionalized. Here we investigate use of the silver cation, Ag+, as an agent for more robust, metal-mediated self-assembly, focusing on the simplest duplex building blocks that would be required for more elaborate Ag+–DNA nanostructures. Our studies of Ag+-induced assembly of non-complementary DNA oligomers employ strands of 2–24 bases, with varied base compositions, and use electrospray ionization mass spectrometry to determine product compositions. High yields of duplex products containing narrowly distributed numbers of Ag+ can be achieved by optimizing solution conditions. These Ag+-mediated duplexes are stable to at least 60 mM Mg2+, higher than is necessary for WC nanotechnology schemes such as tile assemblies and DNA origami, indicating that sequential stages of Ag+-mediated and WC-mediated assembly may be feasible. Circular dichroism spectroscopy suggests simple helical structures for Ag+-mediated duplexes with lengths to at least 20 base pairs, and further indicates that the structure of cytosine-rich duplexes is preserved at high urea concentrations. We therefore propose an approach towards dynamic DNA nanomaterials with enhanced thermal and chemical stability through designs that combine sturdy silver-mediated ‘frames’ with WC paired ‘pictures’.

Conformations of a Metastable SH3 Domain Characterized by smFRET and an Excluded-Volume Polymer Model

Conformational states of the metastable drkN SH3 domain were characterized using single-molecule fluorescence techniques. Under nondenaturing conditions, two Förster resonance energy transfer (FRET) populations were observed that corresponded to a folded and an unfolded state. FRET-estimated radii of gyration and hydrodynamic radii estimated by fluorescence correlation spectroscopy of the two coexisting conformations are in agreement with previous ensemble x-ray scattering and NMR measurements. Surprisingly, when exposed to high concentrations of urea and GdmCl denaturants, the protein still exhibits two distinct FRET populations. The dominant conformation is expanded, showing a low FRET efficiency, consistent with the expected behavior of a random chain with excluded volume. However, approximately one-third of the drkN SH3 conformations showed high, nearly 100%, FRET efficiency, which is shown to correspond to denaturation-induced looped conformations that remain stable on a timescale of at least 100 μs. These loops may contain interconverting conformations that are more globally collapsed, hairpin-like, or circular, giving rise to the observed heterogeneous broadening of this population. Although the underlying mechanism of chain looping remains elusive, FRET experiments in formamide and dimethyl sulfoxide suggest that interactions between hydrophobic groups in the distal regions may play a significant role in the formation of the looped state.

Folding PDZ2 Domain Using the Molecular Transfer Model.

A major challenge in molecular simulations is to describe denaturant-dependent folding of proteins in order to make direct comparisons with in vitro experiments. We use the molecular transfer model (MTM), currently the only method that accomplishes this goal, albeit phenomenologically, to quantitatively describe urea-dependent folding of the PDZ2 domain, a protein that is important in molecular recognition and signaling. Experiments show that urea-dependent unfolding rates of the PDZ2 domain exhibit a downward curvature at high urea concentrations ([C]’s), which has been interpreted as indicating the presence of a sparsely populated high energy intermediate. Simulations using the MTM and a coarse-grained self-organized polymer (SOP) representation of PDZ2 are used to show that the intermediate (IEQ), which has some native-like character, is present in equilibrium both in the presence and absence of urea. The free energy profiles as a function of the structural overlap order parameter show that there are t...

Delipidation of cytochrome c oxidase from Rhodobacter sphaeroides destabilizes its quaternary structure.

Delipidation of detergent-solubilized cytochrome c oxidase isolated from Rhodobacter sphaeroides (Rbs-CcO) has no apparent structural and/or functional effect on the protein, however affects its resistance against thermal or chemical denaturation. Phospholipase A2 (PLA2) hydrolysis of phospholipids that are co-purified with the enzyme removes all but two tightly bound phosphatidylethanolamines. Replacement of the removed phospholipids with nonionic detergent decreases both thermal stability of the enzyme and its resilience against the effect of chemical denaturants such as urea. In contrast to nondelipidated Rbs-CcO, the enzymatic activity of PLA2-treated Rbs-CcO is substantially diminished after exposure to high (>4M) urea concentration at room temperature without an alteration of its secondary structure. Absorbance spectroscopy and sedimentation velocity experiments revealed a strong correlation between intact tertiary structure of heme regions and quaternary structure, respectively, and the enzymatic activity of the protein. We concluded that phospholipid environment of Rbs-CcO has the protective role for stability of its tertiary and quaternary structures.

Elevated urinary urea by high-protein diet could be one of the inducements of bladder disorders.

BackgroundPrevious work found that urea accumulation in urothelial cells caused by urea transporter B knockout led to DNA damage and apoptosis that contributed to the carcinogenesis. The purpose of this study is to explore the potential connection between high urinary urea concentration and the bladder disorders.MethodsA high protein diet rat model was conducted by feeding with 40 % protein diet. In-silico modeling and algorithm, based on the results of microarray and proteomics from the bladder urothelium, were used for the reconstruction of accurate cellular networks and the identification of novel master regulators in the high-protein diet rat model. Pathway and biological process enrichment analysis were used to characterize predicted targets of candidate mRNAs/proteins. The expression pattern of the most significant master regulators was evaluated by qPCR and immunohistochemistry.ResultsBased on the analysis of different expressed mRNAs/proteins, 15 significant ones (CRP, MCPT2, MCPT9, EPXH2, SERPING1, SRGN, CDKN1C, CDK6, CCNB1, PCNA, BAX, MAGEB16, SERPINE1, HSPA2, FOS) were highly identified and verified by qPCR and immunohistochemistry. They were involved in immune and inflammatory response, cell cycle arrest, apoptosis and pathways in cancer. These abnormally activated processes caused the bladder interstitial congestion and inflammatory infiltrates under the thinner urothelium, cell desquamation, cytoplasm vacuolization, nucleus swelling and malformation in the high-protein diet group.ConclusionsWe provided evidences that high urinary urea concentration caused by high-protein diet might be a potential carcinogenic factor in bladder.

Preparation and characterization of single phase, biphasic and triphasic calcium phosphate whisker-like fibers by homogenous precipitation using urea

Whisker-like calcium phosphate fibers were prepared by a homogenous precipitation method under refluxing conditions in solutions with a Ca/P molar ratio of 1.67 at 90°C and pH 3.0 using urea as additive. The precipitates were characterized by XRD, FTIR, SEM, and FE-SEM. The ICP spectroscopy and elemental analyzer were used to determine Ca/P ratio and carbonate content of products, respectively. The results indicate that the morphology and structural characteristics of the precipitates depend on the urea concentration and reaction time. Whisker-like biphasic monetite/hydroxyapatite (HA) fibers with a mean length of 60µm and a mean width of 1.0µm were obtained with the use of a low concentration of urea. Calcination of the biphasic calcium phosphate at 800°C led to the formation of a triphasic mixture of HA/whitlockite (β-TCP)/calcium pyrophosphate (CPP) with a whisker-like morphology. The use of a higher concentration of urea was caused to form a mixed morphology of spherulites/whisker-like fibers consisting of octacalcium phosphate (OCP) and HA phases. When the reaction time was increased to 10 days, both the above biphasic calcium phosphates transformed to a single phase HA which its morphology and growth pattern were similar to those of a whisker, according to FE-SEM images. The HA whiskers produced by a lower amount of urea had a lower carbonate content compared to those obtained using a higher urea concentration.

Molecular Dissection of the Homotrimeric Sliding Clamp of T4 Phage: Two Domains of a Subunit Display Asymmetric Characteristics.

Sliding clamp proteins are circular dimers or trimers that encircle DNA and serve as processivity factors during DNA replication. Their presence in all the three domains of life and in bacteriophages clearly indicates their high level of significance. T4 gp45, besides functioning as the DNA polymerase processivity factor, also moonlights as the late promoter transcription determinant. Here we report a detailed biophysical analysis of gp45. The chemical denaturation of gp45 probed by circular dichroism spectroscopy, tryptophan fluorescence anisotropy, and blue-native polyacrylamide gel electrophoresis suggests that the protein follows a three-state denaturation profile and displays an intermediate molten globule-like state. The three-state transition was found to be the result of the sequential unfolding of the two domains, the N-terminal domain (NTD) and the C-terminal domain (CTD), of gp45. The experiments involving Trp fluorescence quenching by acrylamide demonstrate that the CTD undergoes substantial changes in conformation during formation of the intermediate state. Further biophysical dissection of the individual domain reveals contrasting properties of the two domains. The NTD unfolds at low urea concentrations and is also susceptible to protease cleavage, whereas the CTD resists urea-mediated denaturation and is not amenable to protease digestion even at higher urea concentrations. These experiments allow us to conclude that the two domains of gp45 differ in their dynamics. While the CTD shows stability and rigidity, we find that the NTD is unstable and flexible. We believe that the asymmetric characteristics of the two domains and the interface they form hold significance in gp45 structure and function.

Atomistic insights into deep eutectic electrolytes: the influence of urea on the electrolyte salt LiTFSI in view of electrochemical applications.

The influence of urea on the conducting salt lithium bis-(trifluoromethanesulfonyl)-imide (LiTFSI) in terms of lithium ion coordination numbers and lithium ion transport properties is studied via atomistic molecular dynamics simulations. Our results indicate that the presence of urea favors the formation of a deep eutectic electrolyte with pronounced ion conductivities which can be explained by a competition between urea and TFSI in occupying the first coordination shell around lithium ions. All simulation findings verify that high urea concentrations lead to a significant increase of ionic diffusivities and an occurrence of relatively high lithium transference numbers in good agreement with experimental results. The outcomes of our study point at the possible application of deep eutectic electrolytes as ion conducting materials in lithium ion batteries.

Effect of glycine betaine on the hydrophobic interactions in the presence of denaturant: A molecular dynamics study

Hydrophobic interactions are very important in a variety of systems such as proteins. Osmoprotectant glycine betaine (GB) stabilizes proteins against denaturation and counteracts harsh conditions such as presence of high concentration of urea. Therefore, it is interesting to specifically investigate the effect of osmolytes on hydrophobic interactions to explore whether the latter plays any role in the counteraction of denaturing property of urea. In this work, molecular dynamics simulations have been performed on neopentane (NEP), a non-polar molecule, in aqueous GB, urea, and GB-urea mixture to understand the effect of GB on hydrophobic interactions in the presence of urea. The results were analyzed by calculating radial distribution function, potential of mean force, coordination number, and interaction energies. The results indicate that the presence of GB decreases hydrophobic interactions between urea and NEP. We also find that GB and urea molecules strongly interact with each other through H bonding and electrostatic interactions. However, GB and urea have negligible effect on hydrophobic interactions between the NEP molecules. This study provides insights into the role of hydrophobic interactions in osmolyte induced counteraction of harsh conditions.

Outbreak of Peracute Urea Toxicosis in Sheep Following the Consumption of New Packages of Grain Barley

 The objective of this report was to describe peracute urea poisoning deaths in 84 Ile de France and Merinos breed sheep due to following the consumption of new packages of grain barley on July 30, 2014 in Ivrindi, Balıkesir, Turkey. The owner of the sheep and a local veterinarian reported that in the farm there were in total 800 sheep, and 325 of animals were fed with new packages of grain barleys and 84 of which were observed dead after feeding in three-four hours. Clinical signs in all affected animals prior to death were uneasiness and ataxia, incoordination, grinding teeth, salivation, severe struggling and bellowing, abdominal bloat, lateral re-cumbence, convulsions, and deaths occurred in one and half hour after first clinical signs. The presences of white urea particles were intensive especially at the bottom of the packages of barley. Urea, crude protein, nitrate and nitrite analysis were performed in barley samples. Analysed barley samples had quite high urea concentration (8.76%) and crude protein (27.81%). Anamnesis, clinical signs and laboratory analysis indicated peracute urea poisoning in sheep. 
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Urea-induced binding between diclofenac sodium and bovine serum albumin: a spectroscopic insight.

We investigated the interaction of diclofenac sodium (Dic.Na) with bovine serum albumin (BSA) in the absence and presence of urea using different spectroscopic techniques. A fluorescence quenching study revealed that the Stern-Volmer quenching constant decreases in the presence of urea, decreasing further at higher urea concentrations. The binding constant and number of binding sites were also evaluated for the BSA-Dic.Na interaction system in the absence and presence of urea using a modified Stern-Volmer equation. The binding constant is greater at high urea concentrations, as shown by the fluorescence results. In addition, for the BSA-Dic.Na interaction system, a static quenching mechanism was observed, which was further confirmed using time-resolved fluorescence spectroscopy. UV-vis spectroscopy provided information about the formation of a complex between BSA and Dic.Na. Circular dichroism was carried out to explain the conformational changes in BSA induced by Dic.Na in the absence and presence of urea. The presence of urea reduced the α-helical content of BSA as the Dic.Na concentration varied. The distance r between the donor (BSA) and acceptor (Dic.Na) was also obtained in the absence and presence of urea, using fluorescence resonance energy transfer. Copyright © 2015 John Wiley & Sons, Ltd.

Promoting protein self-association in non-glycosylated Thermomyces lanuginosus lipase based on crystal lattice contacts

We have used the crystal structure of Thermomyces lanuginosus lipase (TlL) to identify and strengthen potential protein–protein interaction sites in solution. As wildtype we used a deglycosylated mutant of TlL (N33Q). We designed a number of TlL mutants to promote interactions via interfaces detected in the crystal-lattice structure, through strengthening of hydrophobic, polar or electrostatic contacts or truncation of sterically blocking residues. We identify a mutant predicted to lead to increased interfacial hydrophobic contacts (N92F) that shows markedly increased self-association properties on native gradient gels. While wildtype TlL mainly forms monomer and <5% dimers, N92F forms stable trimers and dimers according to Size-Exclusion Chromatography and Small-Angle X-ray Scattering. These oligomers account for ~25% of the population and their enzymatic activity is comparable to that of the monomer. Self-association stabilizes TlL against thermal denaturation. Furthermore, the trimer is stable to dilution and requires high concentrations (>2M) of urea to dissociate. We conclude that crystal lattice contacts are a good starting point for design strategies to promote protein self-association.

Hypervolemic hypernatremia is the most common type of hypernatremia in the intensive care unit.

A high incidence of hypervolemic hypernatremia has been described in patients recovering from acute kidney injury (AKI) in intensive care units. However, this has been limited to only a few cases. One hundred fifty adult patients recovering from AKI in the intensive care unit of a single institution during a 6-year period, who developed hypernatremia during the course of their illness, were investigated. Serum and urine electrolytes, osmolality, urea nitrogen and creatinine were measured. The weights of these patients at the time of hypernatremia development and at presentation to the hospital were also measured. Even though the hypernatremia was mild in most patients (146-160 mEq/L), the average rise in serum sodium concentration was 14.5 ± 7.1 mEq/L. Of the 34 patients who had all urinary studies available, the average urine osmolality was 436 ± 128 mmol/kg of which 172 ± 54 mmol/L was contributed by sodium, potassium and their accompanying anion. Another 204 ± 96 mmol/L was accounted for by urea and creatinine (mainly urea). Almost all the patients had hypervolemia as evidenced by the presence of edema and an average weight gain of more than 9 ± 11 kg between the time of presentation and the onset of hypernatremia despite likely having lost muscle mass from being in the intensive care unit for several days. The weight data were available in 54 patients, and only eight of these patients had lost weight at the time of the development of hypernatremia. Hypervolemic hypernatremia is by far the most common cause of hypernatremia in patients in the intensive care unit. Even though the patients are in negative fluid balance at the time of the development of the hypernatremia, earlier saline administration has caused massive volume overload despite the ongoing losses. Post-AKI diuresis in the face of inability to maximally concentrate the urine because of renal failure often leads to mainly mild elevations in serum sodium concentration. The urine solute is mainly urea because of the often high serum urea concentrations with little electrolytes being present in the urine.

Factors impacting unbound vancomycin concentrations in different patient populations.

The unbound drug hypothesis states that only unbound drug concentrations are active and available for clearance, and highly variable results regarding unbound vancomycin fractions have been reported in the literature. We have determined the unbound vancomycin fractions in four different patient groups by a liquid chromatography tandem mass spectrometry (LC-MS/MS) method and identified factors that modulate vancomycin binding. We have further developed and validated a prediction model to estimate unbound vancomycin concentrations. Vancomycin (unbound and total) concentrations were measured in 90 patients in four different hospital wards (hematology [n = 33 samples], intensive care unit [ICU] [n = 51], orthopedics [n = 44], and pediatrics [age range, 6 months to 14 years; n = 18]) by a validated LC-MS/MS method. Multiple linear mixed model analysis was performed to identify patient variables that were predictive of unbound vancomycin fractions and concentrations. The variables included in the model were patient age, ward, number of coadministered drugs with high protein binding, kidney function (estimated glomerular filtration rate [determined by Chronic Kidney Disease Epidemiology Collaboration formula]), alpha-1-acid glycoprotein, albumin, total bilirubin, IgA, IgM, urea, and total vancomycin concentrations. In the pediatric cohort, the median unbound vancomycin fraction was 81.3% (range, 61.9 to 95.9%), which was significantly higher (P < 0.01) than the unbound fraction found in the three adult patient cohorts (hematology, 60.6% [48.7 to 90.6%]; ICU, 61.7% [47.0 to 87.6%]; orthopedics, 56.4% [45.9 to 78.0%]). The strongest significant predictor of the unbound vancomycin concentration was the total drug concentration, completed by albumin in the pediatric cohort and albumin and IgA in the adult cohorts. Validation of our model was performed with data from 13 adult patients. A mean difference of 0.3 mg/liter (95% confidence interval [CI], -1.3 to 0.7 mg/liter; R(2) = 0.99 [95% CI, 0.95 to 0.99]) between measured and calculated unbound vancomycin concentrations demonstrated that the predictive performance of our model was favorable. Unbound vancomycin fractions vary significantly between pediatric and adult patients. We developed a formula to estimate the unbound fraction derived from total vancomycin, albumin, and IgA concentrations in adult patients.

The Folding process of Human Profilin-1, a novel protein associated with familial amyotrophic lateral sclerosis.

Human profilin-1 is a novel protein associated with a recently discovered form of familial amyotrophic lateral sclerosis. This urges the characterization of possible conformational states, different from the fully folded state, potentially able to initiate self-assembly. Under native conditions, profilin-1 is monomeric and possesses a well-defined secondary and tertiary structure. When incubated at low pH or with high urea concentrations, profilin-1 remains monomeric but populates unfolded states exhibiting larger hydrodynamic radius and disordered structure, as assessed by dynamic light scattering, far-UV circular dichroism and intrinsic fluorescence. Refolding from the urea-unfolded state was studied at equilibrium and in real-time using a stopped-flow apparatus. The results obtained with intrinsic fluorescence and circular dichroism indicate a single phase without significant changes of the corresponding signals before the major refolding transition. However, such a transition is preceded by a burst phase with an observed increase of ANS fluorescence, which indicates the conversion into a transiently populated collapsed state possessing solvent-exposed hydrophobic clusters. Kinetic analysis reveals that such state has a conformational stability comparable to that of the fully unfolded state. To our knowledge, profilin-1 is the first example of an amyloid-related protein where folding occurs in the absence of thermodynamically stable partially folded states.

C-phycocyanin from Arthrospira maxima LJGR1: production, extraction and protection

C-phycocyanin (C-PC) overproduced by Arthrospira maxima LJGR1 under high urea and salt concentrations was extracted by different methods and evaluated as an antioxidant activity. A. maxima was grown in flat plate photobioreactors, using modified Zarrouk medium with 624 mg L-1 urea and 15 g L-1 NaCl to produce 11 mg C-PC L-1d-1. Agitation, sonication at 35 and 45°C and enzymatic lysis of the cell wall were tried. The C-PC yields, purity and inhibition of the oxidant radicals (DPPH &amp; ABTS) obtained by the best extraction methods were 215 mg C-PC g-1, 0.74, 92% and 29%; when agitation and 221, 0.75, 87% and 27% using sonication at 45°C. Chia mucilage and amaranth flour coatings best preserved C-PC antioxidant activity from temperature variations and up to six days shelf life at 20°C. An activation energy of 160 kJ mol-1 has been obtained with chia mucilage, and the least color degradation rate (r∆E*) of 4.2 d-1 with amaranth flour.

Enhancement of the solubility and stability of d-amino acid oxidase by fusion to an elastin like polypeptide

An elastin-like polypeptide (ELP) was fused to d-amino acid oxidases (DAAO). ELP–DAAO exhibited a better solubility in aqueous solutions than DAAO, and its enzymatic activity is about 1.6 times that of DAAO. The stability of the proteins was investigated by interacting with urea at various concentrations. The circular dichroism and fluorescence spectra were measured. The results demonstrated that that ELP–DAAO exhibited a much better stability than DAAO, and ELP–DAAO has retained the α-helix content with a high percentage even at a high urea concentration. The results of this work have demonstrated that the ELP tag can be utilized to purify DAAO, in the meantime the solubility and stability of the enzyme are improved.

Characterization of the Release of Urea from Coated Fertilizer by Fourier Transform Infrared Spectroscopy with Attenuated Total Reflectance

The development of a simple and cost-effective method for the determination of the release of coated urea has significant implications. Fourier-transform infrared spectroscopy with attenuated total reflectance was employed to determine the release of urea through univariate and multivariate calibration. The results indicated that univariate calibration did not accurately predict the release of urea, whereas partial least squares based on multivariate calibration performed significantly better. Partial least squares had the highest accuracy when the band located at 1420–1520 per centimeter was employed as the input. Moreover, the accuracy was further improved when segmented partial least squares models were developed at low and high urea concentrations. Unsegmented and segmented partial least squares models were employed, and release values were comparable to those measured by colorimetry. This work demonstrated the use of infrared spectroscopy and partial least squares to characterize the release of coated urea.

Thermodynamics and structure of aqueous electrolyte solutions of glycine at different temperatures

The enthalpies of dissolution of glycine in aqueous electrolyte solutions are measured by calorimetry at temperatures of 288 K, 298 K, and 313 K in the molality range from 0.25 mol/kg to 6 mol/kg NaCl and to 4.5 mol/kg KCl. The enthalpy and heat capacity parameters are determined for the pair and triple interactions of glycine with electrolyte in water. Special (singular) points are discovered on the concentration dependences of the heats of dissolution of glycine in aqueous electrolyte solutions and in urea solutions at different temperatures. At these points, such a structurally sensitive property as the entropy of dissolution is temperature independent and the partial heat capacity of glycine is equal to that of the crystalline amino acid. The position of the singular point on the concentration axis is identical for NaCl and KCl solutions; in the case of urea solutions, the singular point is achieved at a much higher concentration of urea.

Electrochemical incineration of the antibiotic ciprofloxacin in sulfate medium and synthetic urine matrix

The degradation of 100 mL of 0.245 mM of the antibiotic ciprofloxacin in 0.05 M Na2SO4 at pH 3.0 has been studied by electrochemical oxidation with electrogenerated H2O2 (EO-H2O2), electro-Fenton (EF), UVA photoelectro-Fenton (PEF) and solar PEF (SPEF). Electrolyses were performed with a stirred tank reactor using either a boron-doped diamond (BDD) or Pt anode and an air-diffusion cathode. In EF, PEF and SPEF, ciprofloxacin was rapidly removed due to its oxidation with •OH formed from Fenton's reaction between added Fe2+ and H2O2 generated at the cathode. The larger electrochemical incineration of the antibiotic was achieved by SPEF with BDD with 95% mineralization thanks to the additional attack by hydroxyl radicals formed from water oxidation at the BDD anode surface and the photolysis of final Fe(III)-oxalate and Fe(III)-oxamate species from sunlight. Up to 10 primary intermediates and 11 hydroxylated derivatives were identified by LC-MS, allowing the proposal of a reaction sequence for ciprofloxacin mineralization. A different behavior was found when the same antibiotic concentration was oxidized in a synthetic urine matrix with high urea content and a mixture of PO43−, SO42− and Cl− ions. Since Fenton's reaction was inhibited in this medium, only EO and EO-H2O2 processes were useful for mineralization, being the organics mainly degraded by HClO formed from Cl− oxidation. The EO process with a BDD/stainless steel cell was found to be the most powerful treatment for the urine solution, yielding 96% ciprofloxacin removal and 98% mineralization after 360 min of electrolysis at optimum values of pH 3.0 and current density of 66.6 mA cm−2. The evolution of released inorganic ions was followed by ion chromatography.