Buffer For Use Research Articles

Metal-Organic Coordination Polymer for Delivery of a Subunit Broadly Acting Influenza Vaccine.

A zinc-carnosine (ZnCar) metal-organic coordination polymer was fabricated in biologically relevant N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid (HEPES) buffer for use as a vaccine platform. In vitro, ZnCar exhibited significantly less cytotoxicity than a well-established zeolitic imidazolate framework (ZIF-8). Adsorption of CpG on the ZnCar surface resulted in enhanced innate immune activation compared to soluble CpG. The model antigen ovalbumin (OVA) was encapsulated in ZnCar and exhibited acid-sensitive release in vitro. When injected intramuscularly on days 0 and 21 in C57BL/6 mice, OVA-specific serum total IgG and IgG1 were significantly greater in all groups with ZnCar and antigen compared to soluble controls. Th1-skewed IgG2c antibodies were significantly greater in OVA and CpG groups delivered with ZnCar for all time points, regardless of whether the antigen and adjuvant were co-formulated in one material or co-delivered in separate materials. When broadly acting Computationally Optimized Broadly Reactive Antigen (COBRA) P1 influenza hemagglutinin (HA) was ligated to ZnCar via its His-tag, significantly greater antibody levels were observed at all time points compared to soluble antigen and CpG. ZnCar-formulated antigen elicited increased peptide presentation to B3Z T cells in vitro and production of IL-2 after ex vivo antigen recall of splenocytes isolated from vaccinated mice. Overall, this work displays the formation of a zinc-carnosine metal-organic coordination polymer that can be applied as a platform for recombinant protein-based vaccines.

Scale-model test for disposal pit of high-level radioactive waste and theoretical evaluation of self-sealing of bentonite-based buffers

Bentonite is attracting greater attention in Japan and some other countries as a buffer for use in repositories of high-level radioactive waste (HLW). Bentonite-based buffers for HLW disposal are expected, because of their swelling deformation, to fill spaces between buffers and walls of disposal pits or between buffers and waste containers designated as overpack. Bentonite has a self-sealing capability. This study conducts scale-model tests simulating the relation between the buffer and interstitial space. It also investigates the validity of theoretical equations for swelling presented by Komine and Ogata (published in 2003 and 2004) to evaluate buffer self-sealing capabilities by comparing calculated and experimentally obtained results of scale-model tests. Results of the experimental work and calculations described herein highlight bentonite’s self-sealing capability and demonstrate the high applicability of these Komine and Ogata equations to quantify filling of interstitial spaces by bentonite-based buffer swelling.

Optical Buffering in a Bottle Microresonator on an Optical Fiber

Manufacturing highly efficient optical communications and computing devices requires designing dense integrated photonic circuits. Unfortunately, surface roughness of microscopic optical signaling devices results in light attenuation, decreasing the efficiency of the optical devices. A microscopic optical buffer would alleviate these issues; a potential solution is the Surface Nanoscale Axial Photonics (SNAP) platform. The SNAP platform operates because of the propagation of whispering gallery modes (WGMs) around the surface of an optical fiber. Because WGMs undergo slow axial propagation, they can be mathematically described by the one-dimensional Schrödinger equation. In this project, we calculate solutions to the effective wave equation to model the circulation of whispering gallery modes. We study the evolution of Gaussian-shaped wave pulses in a bottle microresonator on an optical fiber. By analyzing the propagation of WGMs within the bottle microresonator, we can examine the feasibility of creating a microscopic optical buffer for use in optical signal processing. In this project, we find analytical and numerical solutions to the effective wave equation, which is strikingly similar to the Schrödinger equation. We then use the numerical solutions to the effective wave equation to develop a model of the system in Mathematica.

A Systematic Investigation of the Best Buffers for Use in Screening by MALDI–Mass Spectrometry

Matrix-assisted laser desorption/ionization–mass spectrometry (MALDI-MS) offers a label-free alternative for the screening of biochemical targets in both 1536- and 6144-assay formats, as well as potentially providing increased sensitivity, reproducibility, and the simultaneous detection of multiple assay components within a specified m/z range. Ion suppression effects are one of the principal limitations reported for MS analysis. Within MALDI-MS screening, it has been identified that certain biochemical components incorporated into the assay (e.g., the buffers used to preserve the physiological conditions of the enzyme, salts, and other additives) induce suppression of the analyte ion signals monitored. This poorly understood phenomenon of ion suppression is a key reason the screening community has been reluctant to shift their investigations toward MS methods with reduced sample cleanup. Using acetylcholine as an assay substrate mimic, we have generated robust data to quantify the degree to which the most highly used components (base buffers, additional components, detergents, cell culture media, and other additives) within current screening assays are compatible with MALDI-MS. Here, the most suitable buffers and components, along with their identified optimal concentrations in terms of limiting ion suppression effects, are proposed for use in screening assays measured by MALDI-MS.

Design of nonlinear metamorphic buffer layers for lattice-mismatched InxGa1−xAs/GaAs (001) semiconductor devices

Equilibrium studies of metamorphic (partially relaxed) buffer layers are important in understanding the strain and misfit dislocation density configurations. The authors present a theoretical study of the equilibrium strain and misfit dislocation density profiles as well as appropriate design equations for nonlinearly graded (logarithmic) buffers for use in accommodating the lattice mismatch of heteroepitaxial InxGa1−xAs/GaAs (001) semiconductor devices. Minimum energy calculations show that the nonlinearly graded profile may be beneficial for the control of defect densities in lattice-mismatched devices because they have several characteristics which enhance the mobility and glide velocities of dislocations, thereby promoting longer misfit segments with relatively few threading arms. This study suggest that the use of nonlinear metamorphic buffer layers are beneficial because they contain (1) a misfit dislocation free zone (MDFZ) adjacent to the interface, which avoids dislocation pinning defects associated with substrate defects, (2) a misfit dislocation free zone near the surface, which reduces pinning interactions near the device layer which will be grown on top, and (3) a large built-in strain in the top MDFZ, which enhances the glide of dislocations to sweep out threading arms. In addition, the authors show that the use of nonlinear compositionally grading may be superior to linearly graded layers depending on the specific application of the heterostructure. Moreover, the use of a nonlinearity coefficient (deviation of the average lattice mismatch) enables comparison of nonlinearly graded metamorphic buffer layers to traditionally grown linearly graded heterostructures. The authors also present approximate design equations for the widths of the misfit dislocation free zones, the built-in strain, and peak misfit dislocation density for the general logarithmically graded semiconductor with diamond or zinc blende crystal structure and (001) orientation, and show that these design equations are in fair agreement with detailed numerical energy minimization calculations.

Universal buffers for use in biochemistry and biophysical experiments.

The use of buffers that mimic biological solutions is a foundation of biochemical and biophysical studies. However, buffering agents have both specific and nonspecific interactions with proteins. Buffer molecules can induce changes in conformational equilibria, dynamic behavior, and catalytic properties merely by their presence in solution. This effect is of concern because many of the standard experiments used to investigate protein structure and function involve changing solution conditions such as pH and/or temperature. In experiments in which pH is varied, it is common practice to switch buffering agents so that the pH is within the working range of the weak acid and conjugate base. If multiple buffers are used, it is not always possible to decouple buffer induced change from pH or temperature induced change. We have developed a series of mixed biological buffers for protein analysis that can be used across a broad pH range, are compatible with biologically relevant metal ions, and avoid complications that may arise from changing the small molecule composition of buffers when pH is used as an experimental variable.

Design of S-Graded Buffer Layers for Metamorphic ZnS y Se1−y /GaAs (001) Semiconductor Devices

We present design equations for error function (or “S-graded”) graded buffers for use in accommodating lattice mismatch of heteroepitaxial semiconductor devices. In an S-graded metamorphic buffer layer the composition and lattice mismatch profiles follow a normal cumulative distribution function. Minimum-energy calculations suggest that the S-graded profile may be beneficial for control of defect densities in lattice-mismatched devices because they have several characteristics which enhance the mobility and glide velocities of dislocations, thereby promoting long misfit segments with relatively few threading arms. First, there is a misfit-dislocation-free zone (MDFZ) adjacent to the interface, which avoids dislocation pinning defects associated with substrate defects. Second, there is another MDFZ near the surface, which reduces pinning interactions near the device layer which will be grown on top. Third, there is a large built-in strain in the top MDFZ, which enhances the glide of dislocations to sweep out threading arms. In this paper we present approximate design equations for the widths of the MDFZs, the built-in strain, and the peak misfit dislocation density for a general S-graded semiconductor with diamond or zincblende crystal structure and (001) orientation, and show that these design equations are in fair agreement with detailed numerical energy-minimization calculations for ZnS y Se1−y /GaAs (001) heterostructures.

Biological pH buffers in IVF: help or hindrance to success

Minimizing environmental stress helps maintain cellular homeostasis and is a crucial component in optimizing embryo development in vitro and resulting ART success. One stressor of particular interest is pH. Biologic buffers, such as HEPES and MOPS, are valuable tools for stabilizing pH. The objective of this manuscript is to summarize efficacy and impact of various pH buffers used during IVF lab procedures Keyword searches were performed using Pubmed and Medline and relevant literature reviewed. Various pH buffers have been used with varying degrees of success for gamete and embryo processing in a variety of animal species, as well as in human. Though biologic buffers off a means to improve pH stability, not all buffers may be appropriate for use with gametes and embryos. Specific buffers may have undesired effects, and these may be buffer, species, cell type or concentration dependent. Continued research is needed to further refine and improve the use of biologic buffers for use in human ART.

Glucuronoxylomannan from Cryptococcus neoformans Down-regulates the Enzyme 6-Phosphofructo-1-kinase of Macrophages

The encapsulated yeast Cryptococcus neoformans is the causative agent of cryptococosis, an opportunistic life-threatening infection. C. neoformans is coated by a polysaccharide capsule mainly composed of glucuronoxylomannan (GXM). GXM is considered a key virulence factor of this pathogen. The present work aimed at evaluating the effects of GXM on the key glycolytic enzyme, 6-phosphofructo-1-kinase (PFK). GXM inhibited PFK activity in cultured murine macrophages in both dose- and time-dependent manners, which occurred in parallel to cell viability decrease. The polysaccharide also inhibited purified PFK, promoting a decrease on the enzyme affinity for its substrates. In macrophages GXM and PFK partially co-localized, suggesting that internalized polysaccharide directly may interact with this enzyme. The mechanism of PFK inhibition involved dissociation of tetramers into weakly active dimers, as revealed by fluorescence spectroscopy. Allosteric modulators of the enzyme able to stabilize its tetrameric conformation attenuated the inhibition promoted by GXM. Altogether, our results suggest that the mechanism of GXM-induced cell death involves the inhibition of the glycolytic flux.

Simultaneous Derivatization and Extraction of Primary Short-chain Aliphatic Amines Using an Ultrasonic-mixed Water-ionic Liquid Two-phase System for Liquid Chromatography

Here, we report on a procedure in which ultrasonic waves were used to mix a water-ionic liquid two-phase system to simultaneously derivatize amines with dansyl chloride and extract, and the resulting dansyl amines into an ionic liquid (IL) at room temperature within 15 min. The ILs used were room-temperature ionic liquids (RTILs), such as 1-alkyl-3-methylimidazolium hexafluorophosphate ([C(n)MIM][PF(6)], n = 4, 6, 8), which are insoluble in water, and can extract more than 88% of the total dansyl amines (Dns-amines) present in aqueous solution. The Dns-amines formed were separated by chromatography in 20 µL of RTIL in a mobile-phase gradient of acetonitrile-water flowing through an Agilent Zorbax SB-C18 column. The eluents were analyzed at 333 nm using a UV detector. The optimal analytical conditions were achieved by using the following: RTILs with a 4-carbon alkyl chain; a 7-mL water sample and 1 mL each of 5 mM dansyl chloride in acetone and 20 mM sodium tetraborate buffer for use with a fixed volume (1 mL) of IL; buffer pH of 10.4; duration of 15 min; and an acetonitrile-water ratio of 50:50 from zero time to 3 min, 80:20 from 4 to 10 min, and 50:50 from 11 to 16 min. In comparison to the single-phase hot-water (70°C) derivatization process reported earlier, the simultaneous process developed here showed a higher derivatization efficiency at the tested concentration. The linear range of analysis (0.14-7.14 mg/L) was determined, and the correlation coefficient (R) ranged from 0.9878 to 0.9980. Furthermore, the estimated percentage of recovery from amine-spiked river-water samples ranged from 76 to 108%. The method presented here is simple and fast, and does not involve the use of volatile organic extractants.

Establishment of a Protein Frequency Library and Its Application in the Reliable Identification of Specific Protein Interaction Partners

The reliable identification of protein interaction partners and how such interactions change in response to physiological or pathological perturbations is a key goal in most areas of cell biology. Stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry has been shown to provide a powerful strategy for characterizing protein complexes and identifying specific interactions. Here, we show how SILAC can be combined with computational methods drawn from the business intelligence field for multidimensional data analysis to improve the discrimination between specific and nonspecific protein associations and to analyze dynamic protein complexes. A strategy is shown for developing a protein frequency library (PFL) that improves on previous use of static “bead proteomes.” The PFL annotates the frequency of detection in co-immunoprecipitation and pulldown experiments for all proteins in the human proteome. It can provide a flexible and objective filter for discriminating between contaminants and specifically bound proteins and can be used to normalize data values and facilitate comparisons between data obtained in separate experiments. The PFL is a dynamic tool that can be filtered for specific experimental parameters to generate a customized library. It will be continuously updated as data from each new experiment are added to the library, thereby progressively enhancing its utility. The application of the PFL to pulldown experiments is especially helpful in identifying either lower abundance or less tightly bound specific components of protein complexes that are otherwise lost among the large, nonspecific background.

In vitro cytotoxicity of four different buffers for use in peritoneal dialysis.

Various buffers can be used in fluids for peritoneal dialysis (PD). Lactate is the most frequently used buffer, but bicarbonate and pyruvate have been suggested as more biocompatible alternatives. In the past, acetate was used as a buffer in PD fluids, but was abandoned after being linked with sclerosing peritonitis and loss of ultrafiltration. When a new buffer for PD fluids is introduced, one of its most important characteristics is that it must be non-toxic, i.e. that it does not influence fundamental cellular functions. The aim of this study was to investigate the basal cytotoxicity of bicarbonate, acetate, lactate and pyruvate at neutral pH. As target cells, we used cultured mouse fibroblast-like L-929 cells, a well-known cell line approved by the authorities for regulatory use, and primary human mesothelial cells, which are the cells that line the peritoneal cavity and are exposed to the PD fluid in vivo. Pyruvate was more cytotoxic than lactate and bicarbonate, and no significant difference in cytotoxicity was found between lactate and bicarbonate. The human mesothelial cells were more sensitive to exposure to pyruvate than the L-929 fibroblast-like cells, but less sensitive to exposure to pure PD fluids. Thus, we recommend that both types of cell are used for the evaluation of the biocompatibility of PD fluids.

A Mutant Form of Vascular Endothelial Growth Factor (VEGF) That Lacks VEGF Receptor-2 Activation Retains the Ability to Induce Vascular Permeability

Vascular endothelial growth factor (VEGF) is a major mediator of vasculogenesis and angiogenesis both during development and in pathological conditions. VEGF has a variety of effects on vascular endothelium, including the ability to stimulate endothelial cell mitogenesis, and the potent induction of vascular permeability. These activities are at least in part mediated by binding to two high affinity receptors, VEGFR-1 and VEGFR-2. In this study we have made mutations of mouse VEGF in order to define the regions that are required for VEGFR-2-mediated functions. Development of a bioassay, which responds only to signals generated by cross-linking of VEGFR-2, has allowed evaluation of these mutants for their ability to activate VEGFR-2. One mutant (VEGF0), which had amino acids 83-89 of VEGF substituted with the analogous region of the related placenta growth factor, demonstrated significantly reduced VEGFR-2 binding compared with wild type VEGF, indicating that this region was required for VEGF-VEGFR-2 interaction. Intriguingly, when this mutant was evaluated in a Miles assay for its ability to induce vascular permeability, no difference was found when compared with wild type VEGF. In addition we have shown that the VEGF homology domain of the structurally related growth factor VEGF-D is capable of binding to and activating VEGFR-2 but has no vascular permeability activity, indicating that VEGFR-2 binding does not correlate with permeability activity for all VEGF family members. These data suggest different mechanisms for VEGF-mediated mitogenesis and vascular permeability and raise the possibility of an alternative receptor mediating vascular permeability.

Effect of pH on the stability of methacholine chloride in solution

Methacholine chloride bronchoprovocation challenges are performed for the diagnosis and investigation of hyperreactive airways. Over the last 20 yrs various formulations and pH values for the preparation of solutions of methacholine have been described. To determine the stability of methacholine chloride solutions prepared in a variety of buffers with differing pH values and under varying storage temperatures, we measured methacholine concentrations at intervals from 1 to 5 weeks. It was found that methacholine chloride solutions rapidly decompose if the pH is greater than 6 and that decomposition is more rapid as the pH is raised; solutions at pH 9, i.e. bicarbonate buffer, and stored at 27°C have degradation up to 36% after only one week. Solutions of the same pH but prepared in different buffers can have both varied rates of deterioration and different absolute amounts of methacholine hydrolysed, e.g. solutions prepared in pH 9 borate buffer and stored at 27°C have up to 60% degradation after 1 week. Solutions prepared in saline are stable probably because methacholine solutions are weakly acidic. The results emphasise the importance of preparing methacholine chloride in the proper buffers for use in the accurate assessment of airway responsiveness.

A technique for the rapid removal of proteins from cell extracts containing polysaccharides

The contaminating proteins in microbial cell extracts containing desired polysaccharides, can be rapidly precipitated by adjusting the pH of the solution. In the present research, 90% of the contaminating proteins in the alkali extract of Mucor rouxii were eliminated by lowering the pH, through addition of an organic acid. The acid for pH adjustment was chosen so that it formed a readymade buffer for use in subsequent chromatographic steps for recovery of the polysaccharide. The pH adjustment procedure thus offers a rapid and easily scaleable method for protein removal from cell extracts containing polysaccharides.

‘Organic phase buffers’ control biocatalyst activity independent of initial aqueous pH

Combinations of triisooctylamine with its hydrochloride, or of triphenylacetic acid with its Na + salt, can function as buffers for use during biocatalysis in organic media. They can control the pH of an adjacent aqueous phase, even though both forms of each buffer remain in the organic phase. With 0.1 M aqueous NaCl, the mid-point pH values obtained with the two buffer systems are around pH 4.5 and 7.0, respectively. The activity of an immobilized subtilisin Carlsberg shows a strong dependence on the ratio of the two forms of the triphenylacetic acid buffer system. Without the buffer, the rate shows the normal dependence on the pH of the aqueous solution before drying; however, this is almost eliminated if the buffer is used. The amine buffer system can similarly affect the activity of an immobilized Rhizomucor miehei lipase.

Determination of fluoride in zinc concentrates, processing products and solutions with an ion-specific electrode

Determination of fluoride in hydrometallurgical zinc-plant processing products and solutions with a fluoride-ion electrode is described. Various types of buffers were tested, to find a suitable common buffer for use in analysis of all types of substances generated at the zinc plant. The method involves decomposition of samples either by fusion with sodium hydroxide or by leaching with perchloric acid to bring fluoride into solution. The fluoride concentration is measured directly with a fluoride-specific electrode. In standard addition tests 100% recovery was obtained when a citric acid/sodium nitrate buffer was used. Validation tests gave satisfactory results.

Synthesis of a new acrylamido buffer (acryloylhistamine) for isoelectric focusing in immobilized pH gradients and its analysis by capillary zone electrophoresis

By reacting acryloyl chloride with histamine a weakly basic acrylamido buffer for use in isoelectric focusing in immobilized pH gradients, acryloylhistamine [2-(4-imidazolyl)ethylamine-2-acrylamide], with a p K of 7.0 (25°C) was synthesized. Even though this compound has the same p K value as the commercially available p K 7.0 Immobiline, it has some interesting features: (a) it is strongly resistant to hydrolysis, in contrast to the p K 7.0 and 6.2 species; (b) it is hydrophilic; and (c) owing to its heteroaromatic ring, its amino groups are resistant to oxidation. The above properties (degradation kinetics, hydrophilicity, proneness to oxidation) were established in a quantitative manner by capillary zone electrophoresis analysis.

Selection of pH buffers for use in conductimetric microbiological assays.

Many metabolic activities of micro-organisms lead to changes in the pH value of cultures and consequently pH buffer compounds are potentially a major source of conductivity changes in cultures. To maximize changes in conductivity associated with microbial growth the pH buffer-associated changes should occur in a direction that reinforces those due to other metabolic activities. In agreement with this, studies with Escherichia coli showed that fermentation of glucose and aerobic growth on L-alanine yielded greater changes in the conductivity of media containing Tris(hydroxymethyl)aminomethane or L-histidine buffers than in a medium containing phosphate buffer, whereas aerobic growth on glucose or succinate yielded greater changes with phosphate buffer than with Tris(hydroxymethyl)aminomethane or L-histidine buffers. Criteria for the selection of appropriate pH buffer compounds are presented.

Inactivation of human alpha 1-proteinase inhibitor by cigarette smoke: effect of smoke phase and buffer.

In order to resolve a discrepancy in the literature, we have examined the in vitro inactivation of human alpha 1-proteinase inhibitor by direct exposures either to whole cigarette smoke or to filtered (i.e., gas-phase) smoke. Wyss and coworkers (2) reported that whole smoke does not inactivate the protein, whereas we reported that gas-phase smoke does. We now find that direct exposure to gas-phase cigarette smoke causes a slightly greater inactivation of the protein than does direct exposure to whole cigarette smoke, confirming our earlier suggestion that whole smoke is less oxidizing than is gas-phase smoke. This difference, however, does not explain the dramatic difference between our previous findings and those of Wyss and coworkers (2). The explanation for the discrepancy lies in the nature of the buffers used. Wyss and coworkers used Tris buffer and the use of Tris quenches the inactivation process almost completely. Our experiments used phosphate buffer. We suggest that Tris is an unsuitable buffer for use in experiments that probe the effects of cigarette smoke.