Tris pH Research Articles

Electrochemical fingerprinting and phylogenetic analysis of Inula species

This study presents a groundbreaking approach in the chemotaxonomic classification of Inula species through electrochemical fingerprinting and multivariate data analysis. Focusing on 14 different Inula species, the research utilized differential pulse voltammetry to analyze leaf extracts in varying pH conditions (acetate pH 4.5, phosphate pH 7.0, and Tris pH 9.0). The electrochemical profiles generated were distinctive and informative, revealing specific oxidation peaks associated with phytochemical compounds. Principal component analysis (PCA) efficiently captured 91.0 % of the variance in these profiles, effectively distinguishing between species. Hierarchical cluster analysis further corroborated these findings, accurately classifying each species into distinct clusters. The method proved exceptionally precise in identifying unknown Inula samples, achieving a 100 % success rate when matched against a reference library. This approach significantly enhances the rapid identification and quality assurance of medicinal herbs, offering a simpler, faster alternative to traditional chemotaxonomic methods.

A rapid and efficient method using electroporation for releasing intracellular microcystin toxins from cultured and naturally occurring cyanobacterial cells in lake water

In cyanotoxin measurements, effective release of intracellular cyanotoxins through cell lysis is pivotal. The conventional method for cell lysis is repeated freeze-thaw (F-T), which has several disadvantages, including poor reproducibility since it is operator and equipment dependency and time-consuming. In this study, a rapid and sensitive method was developed using irreversible electroporation, reducing quantification time by over 6 h compared to F-T. Focusing on microcystins (MCs), we developed the most optimal electroporation medium (50 mM Tris (pH 7.0) with 0.5 % SDS) and determined the optimal intensity of electroporation using Microcystis culture. Microcystis cell rupture was validated by scanning electron microscopy. COMSOL simulations mirrored experimental conditions. Compared to F-T, this new method generated an average 13.7 % (6.7 ppb) more MCs from lake water samples (p ≥ 0.05). This innovation, surpassing the time-consuming F-T process, emerges as a valuable tool for timely decision-making in water safety advisory and cyanotoxin management in various settings.

Technical note: Stability of tris pH buffer in artificial seawater stored in bags

Abstract. Equimolal tris (2-amino-2-hydroxymethyl-propane-1,3-diol) buffer in artificial seawater is a well characterized and commonly used standard for oceanographic pH measurements. We evaluated the stability of tris pH when stored in purportedly gas-impermeable bags across a variety of experimental conditions, including bag type and storage in air vs. seawater over 300 d. Bench-top spectrophotometric pH analysis revealed that the pH of tris stored in bags decreased at a rate of 0.0058±0.0011 yr−1 (mean slope ±95 % confidence interval of slope). The upper and lower bounds of expected pH change at t=365 d, calculated using the averages and confidence intervals of slope and intercept of measured pH change vs. time data, were −0.0042 and −0.0076 from initial pH. Analyses of total dissolved inorganic carbon confirmed that a combination of CO2 infiltration and/or microbial respiration led to the observed decrease in pH. Eliminating the change in pH of bagged tris remains a goal, yet the rate of pH change is lower than many processes of interest and demonstrates the potential of bagged tris for sensor calibration and validation of autonomous in situ pH measurements.

Does and duration of exposure time dependent regulation of proteins in the urinary bladder of hamsters exposed to sodium arsenite

Does and duration of exposure time dependent regulation of proteins in the urinary bladder of hamsters exposed to sodium arsenite

Regulation of transgelin and GST-pi proteins in the tissues of hamsters exposed to sodium arsenite

Regulation of transgelin and GST-pi proteins in the tissues of hamsters exposed to sodium arsenite

Polyethyleneimine-modified resins effectively remove porcine circovirus and cellular prion protein

Polyethyleneimine (PEI) possesses various molecular weights (MWs), structures, and virus capture capacities. However, whether PEI can capture porcine circovirus (PCV) and animal cell-derived prion protein (PrPC) that may contaminate source materials is unclear. Therefore, we conducted a feasibility study to assess the effectiveness of PEI in removing PCV and PrPC as a model of pathogenic prions. The removal performance of PCV was evaluated by quantitative PCR using PEIs with various MWs, structures, and ion exchange capacities in Tris (pH 7.5) and acetate (pH 5.5) buffers under neutral (pH 7.5) to acidic (pH 5.5) conditions. Removal performances of PrPC were also evaluated by western blotting using PEIs with various MWs and structures. Tris buffer did not affect the ability of PEI-modified resins to remove PCV, whereas acetate buffer affected removal performances, except those of PEI-10K-Br and PEI-70K-Br, which showed high ion-exchange capacities. PrPC was captured by PEIs with high MWs, especially PEI-70K-Br, which was the most effective. The results of this feasibility study suggested that PEI-modified resin could remove PCV and PrPC. PEI-70K-Br with an ion-exchange capacity of at least 0.3 meq/mL appears suitable as a PEI molecule for pathogen capture or removal of PCV or PrPC from biological materials.

Decorating sdAbs with Chelators: Effect of Conjugation on Biodistribution and Functionality

Single domain antibodies (sdAbs) have proven to be valuable probes for molecular imaging. In order to produce such probes, one strategy is the functionalization of the reactive amine side chain of lysines with a chelator, resulting in a mixture of compounds with a different degree of conjugation. In this study, we implemented anion exchange chromatography (AEX) to separate the different compounds or fractions that were further characterized and evaluated to study the impact of the conjugation degree on pharmacokinetic properties and functionality. Anti-HER2 and anti-MMR sdAbs were functionalized with NOTA or DTPA chelator. Anion exchange chromatography was performed using 0.02 mol/L Tris pH 7.5 as the first solvent and 0.25 M or 0.4 M NaCl (in case of NOTA chelator or DTPA chelator, respectively) as the second solvent applied as a gradient. The fractions were characterized via mass spectrometry (MS), surface plasmon resonance (SPR), and isoelectric focusing gel electrophoresis (IEF), while in vivo studies were performed after radiolabeling with either 68Ga (NOTA) or 111In (DTPA) to assess the impact of the conjugation degree on pharmacokinetics. AEX could successfully be applied to separate fractions of (chelator)n-anti-HER2 and (chelator)n-anti-MMR sdAb constructs. MS confirmed the identity of different peaks obtained in the separation process. SPR measurement suggests a small loss of affinity for (chelator)3-anti-sdAb, while IEF revealed a correlated decrease in isoelectric point (pI) with the number of conjugated chelators. Interestingly, both the reduction in affinity and in pI was stronger with the DTPA chelator than with NOTA for both sdAbs. In vivo data showed no significant differences in organ uptake for any construct, except for (DTPA)n-anti-MMR, which showed a significantly higher liver uptake for (DTPA)1-anti-MMR compared to (DTPA)2-anti-MMR and (DTPA)3-anti-MMR. For all constructs in general, high kidney uptake was observed, due to the typical renal clearance of sdAb-based tracers. The kidney uptake showed significant differences between fractions of a same construct and indicates that a higher conjugation degree improves kidney clearance. AEX allows the separation of sdAbs with a different degree of conjugation and provides the opportunity to further characterize individual fractions. The conjugation of a chelator to sdAbs can alter some properties of the tracers, such as pI; however, the impact on the general biodistribution profile and tumor targeting was minimal.

Mechanical Properties of the Composite Material consisting of β-TCP and Alginate-Di-Aldehyde-Gelatin Hydrogel and Its Degradation Behavior

This work aimed to determine the influence of two hydrogels (alginate, alginate-di-aldehyde (ADA)/gelatin) on the mechanical strength of microporous ceramics, which have been loaded with these hydrogels. For this purpose, the compressive strength was determined using a Zwick Z005 universal testing machine. In addition, the degradation behavior according to ISO EN 10993-14 in TRIS buffer pH 5.0 and pH 7.4 over 60 days was determined, and its effects on the compressive strength were investigated. The loading was carried out by means of a flow-chamber. The weight of the samples (manufacturer: Robert Mathys Foundation (RMS) and Curasan) in TRIS solutions pH 5 and pH 7 increased within 4 h (mean 48 ± 32 mg) and then remained constant over the experimental period of 60 days. The determination surface roughness showed a decrease in the value for the ceramics incubated in TRIS compared to the untreated ceramics. In addition, an increase in protein concentration in solution was determined for ADA gelatin-loaded ceramics. The macroporous Curasan ceramic exhibited a maximum failure load of 29 ± 9.0 N, whereas the value for the microporous RMS ceramic was 931 ± 223 N. Filling the RMS ceramic with ADA gelatin increased the maximum failure load to 1114 ± 300 N. The Curasan ceramics were too fragile for loading. The maximum failure load decreased for the RMS ceramics to 686.55 ± 170 N by incubation in TRIS pH 7.4 and 651 ± 287 N at pH 5.0.

Manufacturing Considerations for the Development of Lipid Nanoparticles Using Microfluidics.

In the recent of years, the use of lipid nanoparticles (LNPs) for RNA delivery has gained considerable attention, with a large number in the clinical pipeline as vaccine candidates or to treat a wide range of diseases. Microfluidics offers considerable advantages for their manufacture due to its scalability, reproducibility and fast preparation. Thus, in this study, we have evaluated operating and formulation parameters to be considered when developing LNPs. Among them, the flow rate ratio (FRR) and the total flow rate (TFR) have been shown to significantly influence the physicochemical characteristics of the produced particles. In particular, increasing the TFR or increasing the FRR decreased the particle size. The amino lipid choice (cationic—DOTAP and DDAB; ionisable—MC3), buffer choice (citrate buffer pH 6 or TRIS pH 7.4) and type of nucleic acid payload (PolyA, ssDNA or mRNA) have also been shown to have an impact on the characteristics of these LNPs. LNPs were shown to have a high (>90%) loading in all cases and were below 100 nm with a low polydispersity index (≤0.25). The results within this paper could be used as a guide for the development and scalable manufacture of LNP systems using microfluidics.

Host-Pathogen Interactions in Human Polyomavirus 7‒Associated Pruritic Skin Eruption

Host-Pathogen Interactions in Human Polyomavirus 7‒Associated Pruritic Skin Eruption

Biochemical and computational study of an alginate lyase produced by Pseudomonas aeruginosa strain S21.

Objective(s):Alginates play a key role in mucoid Pseudomonas aeruginosa colonization, biofilm formation, and driving out of cationic antibiotics. P. aeruginosa alginate lyase (AlgL) is a periplasmic enzyme that is necessary for alginate synthesis and secretion. It also has a role in depolymerization of alginates. Using AlgLs in cystic fibrosis patients along with antibiotics enhances bacterial killing and host healing. In this study, we investigated the different biochemical properties of a newly isolated AlgL from P. aeruginosa S21 to complete the databank of AlgLsMaterials and Methods:The enzyme was extracted from the periplasmic space of the bacteria by the heat shock method. Using the TBA method, the enzyme activity and biochemical properties were assessed. The mutability of P. aeruginosa S21 AlgL to increase its thermal stability was investigated. The most favorable mutations were studied computationally. The molecular dynamics simulation (MDS) package GROMACS was used for determining the effect of S34R mutation on enzyme’s thermal stability.Results:Data showed that this enzyme has the best activity at 37 °C and pH 7.5 and it can degrade mannuronate blocks, guluronate blocks, and sodium alginate. After 7 hr at 80 °C, 45% of the enzyme activity was retained. This enzyme needed 15 min to completely degrade accessible sodium alginate. Tris buffer, pH 8.5 and Britton-Robinson buffer, pH 7.0 were the preferable buffers for the enzyme activity. MDS of native and mutated enzymes showed desirable results.Conclusion: P. aeruginosa S21 AlgL can be used in medical and industrial applications to degrade alginates.

Identification Of A Novel Histone Derived Antimicrobial Peptide In Airway Surface Liquid.

The airway surface liquid (ASL), a protective layer secreted by the airway epithelium, represents the first line of defence against inhaled infectious material. It contains a complex array of secreted proteins and peptides that aid the neutralisation and removal of inhaled microbes and toxicants. The ASL also contains many proteases that can cleave proteins to generate further bioactive peptides. We therefore hypothesised that the airway ASL sustained an extensive peptidome containing novel bioactive peptides. We examined the ASL peptide profile of normal human bronchial epithelial cells transformed with BMI‐1 (NHBE‐BMI1) and cell‐lines Calu3 (submucosal adenocarcinoma) and H441 (Clara cell like adenocarcinoma). ASL was acquired by washing the apical surface of epithelial monolayers grown at air‐liquid interface with PBS (100μl) at 0, 24 and 120 hours after exposure to hyperglycaemic (25mM glucose) or normoglycaemic (5mM glucose and 20mM mannitol) basolateral medium. The ASL peptides < 10kDa, were subsequently isolated and analysed using the Q Exactive™ HF‐X Hybrid Quadrupole‐Orbitrap™ Mass Spectrometer. The resulting spectra of native peptides and their subsequent fragments were analysed using the pNovo De novo sequencing tool. Our preliminary results identified 4765 unique peptides (NHBE‐BMI1: 646, Calu3: 3150, H441: 1197). Of these, 37 peptides were common to all samples regardless of glycaemic state. A number of histone derived peptides were identified in all samples and cell lines. These peptides had a proline‐alanine rich N‐terminal region and a cationic C‐terminal, similar to antimicrobial peptides from histone H1 found in Atlantic salmon skin mucous. Synthetic peptides were produced by SPOT synthesis and the antimicrobial activity tested against luminescent Pseudomonas aeruginosa over 18 hours at 37°C in (100mM Tris (pH 7) and 20mM glucose). One of the crude peptides elicited a dose dependent inhibition of bacterial luminescence at 4.63±1.34 μM (n=3), indicating no bacteria survived. Truncated variants of this peptide containing the alanine‐proline rich N‐terminal sequence or the cationic C‐terminal sequence were produced to determine if a specific region of the peptide was required for antimicrobial activity. The N and the C terminal peptides had their antimicrobial activity significantly decreased, only reducing bacterial luminescence by 28.06±2.27% and 35.28±9.45% respectively compared to the full‐length peptide; 99.99±0.001% (all normalised to untreated bacterial luminescence, n=4, p<0.0001, ±SD). This indicates that both regions are required for antimicrobial activity. An immunoblot of concentrated native ASL peptides and cell lysates showed the presence of known histone protein epitopes in our peptide of interest. Furthermore, we identified this histone‐derived peptide in sputum samples from both Cystic Fibrosis and normal healthy patients. Our preliminary data indicates that this novel histone derived peptide may serve an antimicrobial function in the ASL.Support or Funding InformationMedical Research Council London Intercollegiate Doctoral Training Programme

Enhancement of hydroxyapatite dissolution through structure modification by Krypton ion irradiation

Hydroxyapatite (HA) synthesized by a wet chemical route was subjected to heavy ion irradiation, using 4 MeV Krypton ion (Kr17+) with ion fluence ranging from 1 × 1013 to 1 × 1015 ions/cm2. Glancing incidence X-ray diffraction (GIXRD) results confirmed the phase purity of irradiated HA with a moderate contraction in lattice parameters, and further indicated the irradiation-induced structural disorder, evidenced by broadening of the diffraction peaks. High-resolution transmission electron microscopy (HRTEM) observations indicated that the applied Kr irradiation induced significant damage in the hydroxyapatite lattice. Specifically, cavities were observed with their diameter and density varying with the irradiation fluences, while a radiation-induced crystalline-to-amorphous transition with increasing ion dose was identified. Raman and X-ray photoelectron spectroscopy (XPS) analysis further indicated the presence of irradiation-induced defects. Ion release from pristine and irradiated materials following immersion in Tris (pH 7.4, 37 ℃) buffer showed that dissolution in vitro was enhanced by irradiation, reaching a peak at 0.1dpa. We examined the effects of irradiation on the early stages of the mouse osteoblast-like cells (MC3T3-E) response. A cell counting kit-8 assay (CCK-8 test) was carried out to investigate the cytotoxicity of samples, and viable cells can be observed on the irradiated materials.

Aberrant Function of the C-Terminal Tail of HIST1H1E Accelerates Cellular Senescence and Causes Premature Aging

Histones mediate dynamic packaging of nuclear DNA in chromatin, a process that is precisely controlled to guarantee efficient compaction of the genome and proper chromosomal segregation during cell division and to accomplish DNA replication, transcription, and repair. Due to the important structural and regulatory roles played by histones, it is not surprising that histone functional dysregulation or aberrant levels of histones can have severe consequences for multiple cellular processes and ultimately might affect development or contribute to cell transformation. Recently, germline frameshift mutations involving the C-terminal tail of HIST1H1E, which is a widely expressed member of the linker histone family and facilitates higher-order chromatin folding, have been causally linked to an as-yet poorly defined syndrome that includes intellectual disability. We report that these mutations result in stable proteins that reside in the nucleus, bind to chromatin, disrupt proper compaction of DNA, and are associated with a specific methylation pattern. Cells expressing these mutant proteins have a dramatically reduced proliferation rate and competence, hardly enter into the S phase, and undergo accelerated senescence. Remarkably, clinical assessment of a relatively large cohort of subjects sharing these mutations revealed a premature aging phenotype as a previously unrecognized feature of the disorder. Our findings identify a direct link between aberrant chromatin remodeling, cellular senescence, and accelerated aging.

BSA-based sample clean-up columns for ochratoxin A determination in wine: Method development and validation

Analytical chromatographic techniques for mycotoxins control are well established, but they often depend on costly immunoaffinity sample clean-up. Serum albumins, particularly that from bovine origin (BSA), have stable binding affinity towards some mycotoxins, and can be cheaper alternative receptors for sample clean-up due to their wide availability. Thus, this work used BSA immobilized in agarose beads as a novel solid-phase extraction method for quantification of ochratoxin A (OTA) in wine. Constructed BSA-agarose columns could extract OTA efficiently from red wine after its dilution (4-fold) in 0.1 M Tris pH 8.0. The method was linear (R2 = 0.9999) in the OTA concentration range studied (0.05 to 3.0 μg L−1), with recovery rates above 98%. It also showed low detection (0.017 μg L−1) and quantification (0.051 μg L−1) limits. The efficacy of the BSA-based method was further validated by direct comparison with commercial immunoaffinity columns. Portuguese wines analyzed by both methods had agreeing results.

<i>In Vitro</i> Resorbability of 3D Printed Hydroxyapatite in Two Different pH Buffered Solutions

Resorability of 3D printed hydroxyapatite (3DP HA) in deionized water solution which was buffered with succinic acid-NaOH (pH 5.5) and Tris(hydroxymethyl aminomethane) (pH 7.4) for 1, 7, 14 and 28 days was carried out. Weight change and release of calcium (Ca) and phosphorus (P) were used to evaluate the sample resorption. It was found that the weight of samples soaking in both pH 5.5 and 7.4 solutions decreased with increasing soaking times, but the degree of decrease was greater at pH 5.5 than at pH 7.4. ICP-OES results showed that the release of Ca and P in both pH solutions increased with immersing times. The amount of Ca and P released at pH 5.5 was higher than at pH 7.4. Phase composition of the samples and the microstructure of the sample were characterized using XRD and SEM respectively. XRD analysis showed that hydroxyapatite (HA) and octacalcium phosphate (OCP) phases were found at the center of all samples, but the intensity of OCP peaks tended to decrease with increasing times. Only HA was found on the sample surface after immersion in both pH solutions at all soaking periods. After immersion, newly formed crystals were seen both at the center and/or on the surface of samples. These results suggested that pH could influence the resorption of the samples and also the formation of new calcium phosphate crystals.

An air-assisted flow-gating interface for capillary electrophoresis.

A new kind of flow gating interface (FGI) has been designed for online connection of CE with flow-through analytical techniques. The sample is injected into the separation capillary from a space from which the BGE was forced out by compressed air. A drop of sample solution with a volume of 75 nL is formed between the outlet of the delivery capillary supplying the solution from the flow-through apparatus and the entrance to the CE capillary; the sample is hydrodynamically injected into the CE capillary from this drop. The sample is not mixed with the surrounding BGE solution during injection. The functioning of the proposed FGI is fully automated and the individual steps of the injection process are controlled by a computer. The injection sequence lasts several seconds and thus permits performance of rapid sequential analyses of the collected sample. FGI was tested for the separation of equimolar 50 μM mixture of the inorganic cations K+ , Ba2+ , Na+ , Mg2+ , and Li+ in 50 mM acetic acid/20 mM Tris (pH 4.5) as BGE. The obtained RSD values for the migration times varied in the range 0.7-1.0% and the values for the peak area were 0.7-1.4%; RSD were determined for ten repeated measurements.

A comparison of Activating Solutions with Hatchery Water in Artificial Insemination of Rainbow Trout (Oncorhynchus mykiss)

In the propagation of rainbow trout, the available water supply used for incubation of fertilized egg is generally used for also spermatozoa being activated to reach eggs. The aim of the present study was to assess comparisons of the effects of two lab-made activating solutions and hatchery water on progressive sperm motility percentage (%), duration of progressive sperm motility (s), and fertilization success in artificial insemination of rainbow trout. For this purpose, an activating solution (A1) containing 60 mM NaHCO3, 50 mM Tris pH=9.0 and another activating solution (A2) containing 20 mM Tris, 30 mM glycine, 125 mM NaCl, pH=9.0, and also hatchery water (HW) were used for activation of spermatozoa and fertilization. The average motility percentages of samples activated by HW, A1 and A2 were observed >90% with no significant differences, while the durations of progressive motility were found to be significantly different as 22.5±0.7 s, 30.0±1.4 s and 30.5±0.7 s respectively. The lowest average fertilization rate (64.6±1.4 %) was obtained using HW, while those values were 89.4±5.1 and 91.3±0.6 % using A1 and A2 respectively. Consequently, both motility durations and fertilization rates obtained by using A1 and A2 were significantly higher than those values of obtained by HW.

Selective release of overexpressed recombinant proteins from E. coli cells facilitates one-step chromatographic purification of peptide-tagged green fluorescent protein variants

The need for fast and convenient protein purification is enormous, not only on the industrial scale for the production of therapeutics but also in life science research. Most protein purification strategies require multiple steps, which are time consuming, cost and resource intensive. Selective release of the intracellular target product can considerably facilitate downstream processing by minimizing the HCP concentration before subsequent purification processes. Here, we present a simple method to selectively release soluble overexpressed proteins from E. coli with minimal laboratory equipment requirements. This method is based on the creation of pores in the cell envelope by a single freeze-thaw cycle. Proteins of various sizes were released by several 50 mM Tris pH 9.5 buffer washes with high yields. Consequently, the need for detergents and other additives were circumvented. Green fluorescent protein (GFP), was tagged with peptides of various charges and polarity. Releasing these GFP variants with our method enabled the successful one-step chromatographic purification on a quaternary amine resin despite the different isoelectric points of the target proteins. Up to 90% of the target protein elution peak during anion exchange chromatography was of >95% purity. This optimized process is more productive compared to multi-step purification procedures currently available in literature.

Geranylgeranylacetone induction of HSP90α exerts cryoprotective effect on Acipenser sinensis sperm

Heat Shock Protein 90 (HSP90) is a fertility-associated protein, the expression of which positively correlates with sperm quality in many species. Geranylgeranylacetone (GGA) is reported to induce expression of HSP90. The present study aimed to investigate whether GGA induced expression of HSP90 in Acipenser sinensis sperm to exert a cryoprotective effect. Sperm from five male A. sinensis was combined with extender containing 20 mmol/L tris pH = 8.1, 10% v/v methanol, 2–5 mmol/L KCl, 15 mmol/L lactose, and 15 mmol/L trehalose, with GGA at 0, 14, 67, 135, 673, 1346, or 6731 μmol/L. After cryopreservation and thawing, the percentage of motile spermatozoa, spermatozoon curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), acrosome integrity, and membrane integrity, as well as fertility were evaluated. Sperm quality increased with the increase of GGA to 673 μmol/L, but decreased at higher concentrations. Expression levels of HSP90α were detected by Western blot in sperm frozen with GGA at 673 μmol/L (highest obtained sperm quality), 6731 μmol/L (highest GGA concentration), and a control without GGA. The expression of HSP90α increased with the increase in GGA, with lowest expression observed in the control. GGA was found to induce increase of HSP90α, and this increase was associated with higher quality cryopreserved sperm at concentrations ≤673 μmol/L. This research suggests a viable technique to increase the quality of cryopreserved A. sinensis sperm by adding GGA to induce expression of HSP90α.