ABSTRACT
This study aimed to evaluate the impact of supplementing sodium bicarbonate or a commercial blend of buffering agents (BBA) comprising calcareous calcitic, magnesium oxide, calcareous algae, and sodium bicarbonate on the productive, behavioral and metabolic parameters of Holstein cows fed starchy diets. Over a 60-day trial period, thirty-six multiparous cows with an average milk yield of 38.84 ± 9.24 kg/day and 63.74 ± 18.63 days in milk (DIM), were randomly divided into two groups. The control group (n = 18) received a supplementation of 1.1% dry matter (DM) of sodium bicarbonate (Raudi®, Totalmix, Brazil), while the BBA group (n = 18) was administered with 0.5% DM of a blend of buffering agents (Equalizer®, Nutron/Cargill, Brazil). The mean values of ruminal pH (control 6.80 ± 0.06 and BBA 6.77 ± 0.06; P > 0.05) and volatile fatty acid (VFA) production (control: acetate 62.63 ± 1.29%, propionate 22.99 ± 1.07%, butyrate 14.30 ± 0.52%; BBA: acetate 63.07 ± 1.32%, propionate 23.47 ± 1.10%, butyrate 13.70 ± 0.57%), were similar (P > 0,05) between the two groups. The value of faecal pH was higher (P < 0.05) in the BBA group (6.25 ± 0.02) than the control group (6.12 ± 0.02). Animals treated with BBA exhibited lower (P < 0,05) dry matter intake (DMI) (24.75 ± 0.64 kg/day), higher feed efficiency (FE) (1.64 ± 0.03), and reduced feeding frequency (52.89 ± 3.73 n°/day) than the control group (DMI, 26.75 ± 0.62 kg/day; FE, 1.50 ± 0.03; feeding frequency, 66.07 ± 3.64 n°/day). Milk production remained similar across both groups (control, 39.11 ± 0.92 kg/day and BBA, 39.87 ± 0.92 kg/day; P > 0.05). Notably, the control group displayed a higher (P < 0,05) concentration of milk protein (1.21 ± 0.05 kg/day) than the BBA (1.18 ± 0.05 kg/day) group. The study concluded that both treatments effectively buffered the rumen and mitigated the risk of ruminal acidosis. Moreover, the higher faecal pH in the BBA-treated group suggests potential intestinal action attributable to the synergistic effects of diverse additives with buffering properties. Despite a reduced DMI, BBA-treated animals exhibited improved FE.
Subject(s)
Animal Feed , Diet , Lactation , Rumen , Animals , Cattle/physiology , Female , Lactation/drug effects , Diet/veterinary , Rumen/metabolism , Rumen/drug effects , Animal Feed/analysis , Dietary Supplements/analysis , Milk/chemistry , Buffers , Sodium Bicarbonate/administration & dosage , Sodium Bicarbonate/pharmacology , Fatty Acids, Volatile/metabolism , Fatty Acids, Volatile/analysis , Random Allocation , Hydrogen-Ion Concentration , Behavior, Animal/drug effects , Animal Nutritional Physiological Phenomena/drug effects , BrazilABSTRACT
Understanding cellular functions, particularly in their intricate complexity, can greatly benefit from the spatial mapping of diverse molecules through multitarget single-molecule localization microscopy (SMLM). Existing methodologies, primarily restricting the encoding dimensions to color and lifetime or requiring cyclic staining, often involve broad chromatic detection, specialized optical configurations, or sophisticated labeling techniques. Here, we propose a simple approach called buffer-exchange stochastic optical reconstruction microscopy (beSTORM), which introduces an additional dimension to differentiate between single molecules irrespective of their spectral properties. This method leverages the distinguishable photoblinking responses to distinct buffer conditions, offering a straightforward yet effective means of fluorophore discrimination. Through buffer exchanges, beSTORM achieves multitarget SMLM imaging with minimal crosstalk. Direct integration with expansion microscopy (ExM) demonstrates its capability to resolve up to six proteins at the molecular level within a single emission color without chromatic aberration. Overall, beSTORM presents a highly compatible imaging platform, promising significant advancements in highly multiplexed nanoscopy for exploring multiple targets in biological systems with nanoscale precision.
Subject(s)
Single Molecule Imaging , Single Molecule Imaging/methods , Buffers , Nanotechnology/methods , Fluorescent Dyes/chemistry , Microscopy, Fluorescence/methods , Microscopy/methodsABSTRACT
Amoeboid cells such as the protist Dictyostelium, human neutrophils, and the fungus B.d. chytrid move by extending pseudopods. The trajectories of cell movement depend on the size, rhythm, and direction of long series of pseudopods. These pseudopod properties are regulated by internal factors such as memory of previous directions and by external factors such as gradients of chemoattractants or electric currents. Here a simple method is described that defines the X, Y time coordinates of a pseudopod at the start and the end of the extension phase. The connection between the start and end of an extending pseudopod defines a vector, which is the input of different levels of analysis that defines cell movement. The primary information of the vector is its spatial length (pseudopod size), temporal length (extension time), extension rate (size divided by time), and direction. The second layer of information describes the sequence of two (or more) pseudopods: the direction of the second pseudopod relative to the direction of the first pseudopod, the start of the second pseudopod relative to the extension phase of the first pseudopod (the second starts while the first is still extending or after the first has stopped), and the alternating right/left extension of pseudopods. The third layer of information is provided by specific and detailed statistical analysis of these data and addresses question such as: is pseudopod extension in buffer in random direction or has the system internal directional memory, and how do shallow external electrical or chemical gradients bias the intrinsic pseudopod extension. The method is described for Dictyostelium, but has been used successfully for fast-moving neutrophils, slow-moving stem cells, and the fungus B.d. chytrid.
Subject(s)
Chemotaxis , Dictyostelium , Chemotaxis/physiology , Dictyostelium/physiology , Dictyostelium/cytology , Pseudopodia/physiology , Cell Movement/physiology , Humans , Buffers , Neutrophils/cytology , Neutrophils/physiologyABSTRACT
Near UV and visible light photodegradation can target therapeutic proteins during manufacturing and storage. While the underlying photodegradation pathways are frequently not well-understood, one important aspect of consideration is the formulation, specifically the formulation buffer. Citrate is a common buffer for biopharmaceutical formulations, which can complex with transition metals, such as Fe(III). In an aqueous solution, the exposure of such complexes to light leads to the formation of the carbon dioxide radical anion (â¢CO2-), a powerful reductant. However, few studies have characterized such processes in solid formulations. Here, we show that solid citrate formulations containing Fe(III) lead to the photochemical formation of â¢CO2-, identified through DMPO spin trapping and HPLC-MS/MS analysis. Factors such as buffers, the availability of oxygen, excipients, and manufacturing processes of solid formulations were evaluated for their effect on the formation of â¢CO2- and other radicals such as â¢OH.
Subject(s)
Anions , Carbon Dioxide , Ferric Compounds , Light , Photolysis , Ultraviolet Rays , Carbon Dioxide/chemistry , Anions/chemistry , Ferric Compounds/chemistry , Buffers , Chromatography, High Pressure Liquid/methods , Citric Acid/chemistry , Chemistry, Pharmaceutical/methods , Tandem Mass Spectrometry/methods , Excipients/chemistry , Free Radicals/chemistryABSTRACT
This study comprehensively evaluated the DNA/RNA Defend Pro (DRDP) sample collection buffer, designed to inactivate and stabilize patient samples. The primary objectives were to assess DRDP's efficacy in ensuring sample stability, facilitating extraction-free polymerase chain reaction (PCR), and ensuring compatibility with rapid antigen testing (RAT). Ninety-five diagnostic nasopharyngeal swab samples tested for influenza virus (influenza A), respiratory syncytial virus (RSV A), and/or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were 10-fold diluted with DRDP and anonymized. Initial characterization and retesting of these samples using cobas Liat confirmed 88 samples as positive, validating the presence of viral targets. Results from rapid antigen testing showed lower sensitivity compared to nucleic acid amplification testing (NAAT) but maintained perfect specificity, with 40 out of 88 positive samples by cobas Liat also testing positive for RAT. Direct RT-qPCR of DRDP-diluted samples demonstrated robust compatibility, with 72 out of 88 samples positive for cobas Liat also testing positive by direct RT-qPCR. Non-concordant results could be explained by the 200-fold lower input of extraction-free NAAT. Stability testing involved incubating 31 positive samples at 4 °C, 20 °C, and 37 °C for 7 days, with extraction-free NAAT. DRDP guaranteed viral RNA stability at all temperatures for influenza A, SARS-CoV-2, and RSV A, showing stability up to 7 days at 4 °C. In conclusion, DRDP is an effective stabilizing medium compatible with direct RT-qPCR and rapid antigen testing and shows great potential for optimizing diagnostic processes, particularly in resource-limited or time-sensitive scenarios.
Subject(s)
Nasopharynx , SARS-CoV-2 , Specimen Handling , Nasopharynx/virology , Humans , Specimen Handling/methods , SARS-CoV-2/isolation & purification , SARS-CoV-2/immunology , SARS-CoV-2/genetics , RNA, Viral/analysis , RNA, Viral/isolation & purification , RNA, Viral/genetics , Buffers , Influenza A virus/isolation & purification , Influenza A virus/immunology , Influenza A virus/genetics , Sensitivity and Specificity , COVID-19/diagnosis , COVID-19/virology , Antigens, Viral/analysis , Influenza, Human/diagnosis , Influenza, Human/virologyABSTRACT
I present the perspective that the divalent metalome and the metabolome can be modeled as a network of chelating interactions instead of separate entities. I review progress in understanding the complex cellular environment, in particular recent contributions to modeling metabolite-Mg2+ interactions. I then demonstrate a simple extension of these strategies based approximately on intracellular Escherichia coli concentrations. This model is composed of four divalent metal cations with a range of cellular concentrations and physical properties (Mg2+, Ca2+, Mn2+, and Zn2+), eight representative metabolites, and interaction constants. I applied this model to predict the speciation of divalent metal cations between free and metabolite-chelated species. This approach reveals potentially beneficial properties, including maintenance of free divalent metal cations at biologically relevant concentrations, buffering of free divalent metal cations, and enrichment of functional metabolite-chelated species. While currently limited by available interaction coefficients, this modeling strategy can be generalized to more complex systems. In summary, biochemists should consider the potential of cellular metabolites to form chelating interactions with divalent metal cations.
Subject(s)
Cations, Divalent , Escherichia coli , Cations, Divalent/metabolism , Cations, Divalent/chemistry , Escherichia coli/metabolism , Escherichia coli/genetics , Chelating Agents/chemistry , Chelating Agents/metabolism , Models, Biological , Metabolome , Magnesium/metabolism , Magnesium/chemistry , Buffers , Zinc/metabolism , Zinc/chemistryABSTRACT
PURPOSE: Chemical modifications in monoclonal antibodies can change hydrophobicity, charge heterogeneity as well as conformation, which eventually can impact their physical stability. In this study, the effect of the individual charge variants on physical stability and aggregation propensity in two different buffer conditions used during downstream purification was investigated. METHODS: The charge variants were separated using semi-preparative cation exchange chromatography and buffer exchanged in the two buffers with pH 6.0 and 3.8. Subsequently each variant was analysed for size heterogeneity using size exclusion chromatography and dynamic light scattering, conformational stability, colloidal stability, and aggregation behaviour under accelerated stability conditions. RESULTS: Size variants in each charge variant were similar in both pH conditions when analyzed without extended storage. However, conformational stability was lower at pH 3.8 than pH 6.0. All charge variants showed similar apparent melting temperature at pH 6.0. In contrast, at pH 3.8 variants A3, A5, B2, B3 and B4 display lower Tm, suggesting reduced conformational stability. Further, A2, A3 and A5 exhibit reduced colloidal stability at pH 3.8. In general, acidic variants are more prone to aggregation than basic variants. CONCLUSION: Typical industry practice today is to examine in-process intermediate stability with acidic species and basic species taken as a single category each. We suggest that perhaps stability evaluation needs to be performed at specie level as different acidic or basic species have different stability and this knowledge can be used for clever designing of the downstream process to achieve a stable product.
Subject(s)
Antibodies, Monoclonal , Protein Stability , Antibodies, Monoclonal/chemistry , Hydrogen-Ion Concentration , Drug Stability , Protein Conformation , Protein Aggregates , Chromatography, Ion Exchange/methods , Hydrophobic and Hydrophilic Interactions , Chromatography, Gel , Colloids/chemistry , Biological Products/chemistry , Humans , BuffersABSTRACT
The phage lysin field has done nothing but grow in the last decades. As a result, many different research groups around the world are contributing to the field, often with certain methodological differences that pose a challenge to the interpretation and comparison of results. In this work, we present the case study of three Acinetobacter baumannii-targeting phage lysins (wild-type endolysin LysMK34 plus engineered lysins eLysMK34 and 1D10) plus one lysin with broad activity against Gram-positive bacteria (PlySs2) to provide exemplary evidence on the risks of generalization when using one of the most common lysin evaluation assays: the killing assay with resting cells. To that end, we performed killing assays with the aforementioned lysins using hypo-, iso- and hypertonic buffers plus human serum either as the reaction or the dilution medium in a systematic manner. Our findings stress the perils of creating hypotonic conditions or a hypotonic shock during a killing assay, suggesting that hypotonic buffers should be avoided as a test environment or as diluents before plating to avoid overestimation of the killing effect in the assayed conditions. As a conclusion, we suggest that the nature of both the incubation and the dilution buffers should be always clearly identified when reporting killing activity data, and that for experimental consistency the same incubation buffer should be used as a diluent for posterior serial dilution and plating unless explicitly required by the experimental design. In addition, the most appropriate buffer mimicking the final application must be chosen to obtain relevant results.
Subject(s)
Acinetobacter baumannii , Bacteriophages , Bacteriophages/chemistry , Bacteriophages/physiology , Bacteriophages/genetics , Acinetobacter baumannii/drug effects , Acinetobacter baumannii/virology , Osmolar Concentration , Microbial Viability/drug effects , Buffers , Humans , Viral Proteins/genetics , Viral Proteins/metabolism , Viral Proteins/chemistry , Endopeptidases/metabolism , Endopeptidases/chemistryABSTRACT
Biofouling on marine surfaces causes immense material and financial harm for maritime vessels and related marine industries. Previous reports have shown the effectiveness of amphiphilic coating systems based on poly(dimethylsiloxane) (PDMS) against such marine foulers. Recent studies on biofouling mechanisms have also demonstrated acidic microenvironments in biofilms and stronger adhesion at low-pH conditions. This report presents the design and utilization of amphiphilic polymer coatings with buffer functionalities as an active disruptor against four different marine foulers. Specifically, this study explores both neutral and zwitterionic buffer systems for marine coatings, offering insights into coating design. Overall, these buffer systems were found to improve foulant removal, and unexpectedly were the most effective against the diatom Navicula incerta.
Subject(s)
Biofilms , Biofouling , Diatoms , Dimethylpolysiloxanes , Biofouling/prevention & control , Diatoms/physiology , Dimethylpolysiloxanes/chemistry , Animals , Buffers , Surface Properties , Hydrogen-Ion ConcentrationABSTRACT
Polyamines (PAs) including putrescine (PUT), spermidine (SPD) and spermine (SPM) are small, versatile molecules with two or more positively charged amino groups. Despite their importance for almost all forms of life, their specific roles in molecular and cellular biology remain partly unknown. The molecular structures of PAs suggest two presumable biological functions: (i) as potential buffer systems and (ii) as interactants with poly-negatively charged molecules like nucleic acids. The present report focuses on the question, whether the molecular structures of PAs are essential for such functions, or whether other simple molecules like small peptides with closely spaced positively charged side chains might be suitable as well. Consequently, we created titration curves for PUT, SPD, and SPM, as well as for oligolysines like tri-, tetra-, and penta-lysine. None of the molecules provided substantial buffering capacity at physiological intracellular pH values. Apparently, the most important mechanism for intracellular pH homeostasis in neurons is not a buffer system but is provided by the actions of the sodium-hydrogen and the bicarbonate-chloride antiporters. In a similar approach we investigated the interaction with DNA by following the extinction at 260 nm when titrating DNA with the above molecules. Again, PUT and tri-lysine were not able to interact with herring sperm DNA, while SPD and SPM were. Obviously, the presence of several positively charged groups on its own is not sufficient for the interaction with nucleic acids. Instead, the precise spacing of these groups is necessary for biological activity.
Subject(s)
DNA , Peptides , Polyamines , RNA , Hydrogen-Ion Concentration , DNA/chemistry , DNA/metabolism , Buffers , RNA/chemistry , RNA/metabolism , Polyamines/chemistry , Polyamines/metabolism , Peptides/chemistry , Peptides/metabolism , Animals , Spermidine/chemistry , Spermidine/metabolism , Spermine/chemistry , Spermine/metabolism , Putrescine/chemistry , Putrescine/metabolismABSTRACT
The use of a ternary mobile-phase system comprising ammonium sulphate, sodium chloride, and phosphate buffer was explored to tune retention and enhance selectivity in hydrophobic interaction chromatography. The accuracy of the linear solvent-strength model to predict protein retention with the ternary mobile-phase system based on isocratic scouting runs is limited, as the extrapolated retention factor at aqueous buffer conditions (k0) cannot be reliably established. The Jandera retention model utilizing a salt concentration averaged retention factor (k¯0) in aqueous buffer for ternary systems overcomes this bottleneck. Gradient retention factors were derived based on isocratic scouting runs after numerical integration of the isocratic Jandera model, leading to retention-time prediction errors below 11 % for linear gradients. Furthermore, an analytical expression was formulated to predict HIC retention for both linear and segmented linear gradients, considering the linear solvent-strength (LSS) model within ternary salt systems, relying on a fixed k0. The approach involved conducting two gradient scouting runs for each of the two binary salt systems to determine model parameters. Retention-time prediction errors for linear gradients were below 12 % for lysozyme and 3 % for trypsinogen and α-chymotrypsinogen A. Finally, the analytical expression for a ternary mobile-phase system was used in combination with a genetic algorithm to tune the HIC selectivity. With an optimized segmented ternary gradient, a critical-pair separation for a mixture of 7 proteins was achieved within 15 min with retention-time prediction errors ranging between 0.7 and 15.7 %.
Subject(s)
Ammonium Sulfate , Hydrophobic and Hydrophilic Interactions , Muramidase , Muramidase/chemistry , Muramidase/analysis , Ammonium Sulfate/chemistry , Sodium Chloride/chemistry , Chromatography, Liquid/methods , Algorithms , Buffers , Phosphates/chemistry , Phosphates/analysis , Chymotrypsinogen/chemistry , Models, ChemicalABSTRACT
Light exposure during manufacturing, storage, and administration can lead to the photodegradation of therapeutic proteins. This photodegradation can be promoted by pharmaceutical buffers or impurities. Our laboratory has previously demonstrated that citrate-Fe(III) complexes generate the â¢CO2- radical anion when photoirradiated under near UV (λ = 320-400 nm) and visible light (λ = 400-800 nm) [Subelzu, N.; Schöneich, C. Mol. Pharmaceutics 2020, 17 (11), 4163-4179; Zhang, Y. Mol. Pharmaceutics 2022, 19 (11), 4026-4042]. Here, we evaluated the impact of citrate-Fe(III) on the photostability and degradation mechanisms of disulfide-containing proteins (bovine serum albumin (BSA) and NISTmAb) under pharmaceutically relevant conditions. We monitored and localized competitive disulfide reduction and protein oxidation by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis depending on the reaction conditions. These competitive pathways were affected by multiple factors, including light dose, Fe(III) concentration, protein concentration, the presence of oxygen, and light intensity.
Subject(s)
Antibodies, Monoclonal , Ferric Compounds , Light , Oxidation-Reduction , Serum Albumin, Bovine , Tandem Mass Spectrometry , Ultraviolet Rays , Serum Albumin, Bovine/chemistry , Tandem Mass Spectrometry/methods , Animals , Antibodies, Monoclonal/chemistry , Ferric Compounds/chemistry , Chromatography, High Pressure Liquid , Buffers , Photolysis , Cattle , Citric Acid/chemistry , Disulfides/chemistry , Iron/chemistryABSTRACT
BACKGROUND: Lipids such as phosphatidic acids (PAs) and cardiolipins (CLs) present strongly tailing peaks in reversed phase liquid chromatography, which entails low detectability. They are usually analyzed by hydrophilic interaction liquid chromatography (HILIC), which hampers high-throughput lipidomics. Thus, there is a great need for improved analytical methods in order to obtain a broader coverage of the lipidome in a single chromatographic method. We investigated the effect of ammonium bicarbonate (ABC) on peak asymmetry and detectability, in comparison with ammonium formate (AFO) on both a conventional BEH C18 column and an HST-CSH C18 column. RESULTS: The combination of 2.5 mM ABC buffer pH 8 with an HST-CSH C18 column produced significantly improved results, reducing the asymmetry factor at 10 % peak height of PA 16:0/18:1 from 8.4 to 1.6. Furthermore, on average, there was up to a 54-fold enhancement in the peak height of its [M - H]- ion compared to AFO and the BEH C18 column. We confirmed this beneficial effect on other strongly tailing lipids, with accessible phosphate moieties e.g., cardiolipins, phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, phosphorylated ceramide and phosphorylated sphingosine. Furthermore, we found an increased detectability of phospho- and sphingolipids up to 28 times in negative mode when using an HST-CSH C18 column. The method was successfully applied to mouse liver samples, where previously undetected endogenous phospholipids could be analyzed with improved chromatographic separation. SIGNIFICANCE: In conclusion, the use of 2.5 mM ABC substantially improved the peak shape of PAs and enhanced the detectability of the lipidome in negative mode on an RPLC-ESI-Q-TOF-MS system on both BEH C18 and HST-CSH C18 columns. This method provides a wider coverage of the lipidome with one single injection for future lipidomic applications in negative mode.
Subject(s)
Bicarbonates , Animals , Mice , Buffers , Bicarbonates/chemistry , Lipids/chemistry , Chromatography, Reverse-Phase/methods , Surface Properties , Lipidomics/methods , Mice, Inbred C57BL , Hydrophobic and Hydrophilic Interactions , Phosphatidic Acids/chemistry , Liver/chemistryABSTRACT
The lipase from Prunus dulcis almonds was inactivated under different conditions. At pH 5 and 9, enzyme stability remained similar under the different studied buffers. However, when the inactivation was performed at pH 7, there were some clear differences on enzyme stability depending on the buffer used. The enzyme was more stable in Gly than when Tris was employed for inactivation. Then, the enzyme was immobilized on methacrylate beads coated with octadecyl groups at pH 7 in the presence of Gly, Tris, phosphate and HEPES. Its activity was assayed versus triacetin and S-methyl mandelate. The biocatalyst prepared in phosphate was more active versus S-methyl mandelate, while the other ones were more active versus triacetin. The immobilized enzyme stability at pH 7 depends on the buffer used for enzyme immobilization. The buffer used in the inactivation and the substrate used determined the activity. For example, glycine was the buffer that promoted the lowest or the highest stabilities depending on the substrate used to quantify the activities.
Subject(s)
Enzyme Stability , Enzymes, Immobilized , Lipase , Prunus dulcis , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Lipase/chemistry , Lipase/metabolism , Prunus dulcis/chemistry , Prunus dulcis/enzymology , Buffers , Hydrogen-Ion Concentration , Triacetin/chemistry , Triacetin/metabolism , Glycine/chemistry , Glycine/metabolism , Tromethamine/chemistry , Biocatalysis , Substrate Specificity , Phosphates/chemistry , Phosphates/metabolism , HEPES/chemistryABSTRACT
Background: Carbonic anhydrase VI (CA VI) is crucial in regulating oral pH and predicting susceptibility to dental caries. The hypothesis posits that caries activity may alter the CA VI function, diminishing its capacity to regulate pH effectively and potentially exacerbating cariogenic challenges. This 1-year cohort study sought to investigate the enzymatic activity of salivary CA VI and buffering capacity following a 20% sucrose rinse in 4 to 6.5-year-old children. Method: This research involved 46 volunteers categorized into three groups based on their caries status after follow-up: caries-free (CFee), arrested caries (CArrested), and caries active (CActive). Children underwent visible biofilm examination and saliva collection for salivary flow rate, buffering capacity, and CA VI analyses before and after a 20% sucrose rinse. Results: A reduction in the buffering capacity was observed after sucrose rinse in all groups. The CA VI activity decreased significantly in CFee and CArrested groups after sucrose rinse, although it did not change in the CActive group. An improvement in the buffering capacity and salivary flow rate was found at follow-up when compared with the baseline. After 1-year follow-up, buffering capacity and salivary flow rate increased in all groups, whilst the CA VI activity reduced only in CFree and CArrested children. Conclusion: Sucrose rinse universally reduces the salivary buffering capacity, while caries activity may disrupt CA VI activity response during a cariogenic challenge. After a year, increased salivary flow enhances buffering capacity but not CA VI activity in caries-active children.
Subject(s)
Carbonic Anhydrases , Dental Caries , Saliva , Sucrose , Child , Child, Preschool , Female , Humans , Male , Buffers , Carbonic Anhydrases/metabolism , Hydrogen-Ion Concentration , Longitudinal Studies , Saliva/enzymology , Saliva/chemistry , Sucrose/pharmacology , Enzyme Activation/drug effectsABSTRACT
OBJECTIVE: Saliva serves multiple important functions crucial for maintaining a healthy oral and systemic environment. Among them, the pH buffering effect, which is primarily mediated by bicarbonate ions, helps maintain oral homeostasis by neutralizing acidity from ingested foods. Therefore, higher buffering capacity, reflecting the ability to neutralize oral acidity, may influence taste sensitivity, especially for sour taste since it involves sensing H+ ions. This study aims to explore the relationship between salivary buffering capacity and taste sensitivities to the five basic tastes in healthy adult humans. DESIGN: Eighty seven healthy adult students participated in this study. Resting saliva volume was measured using the spitting method. The liquid colorimetric test was used to assess salivary buffering capacity. The whole-mouth taste testing method was employed to determine the recognition threshold for each tastant (NaCl, sucrose, citric acid, quinine-HCl, monosodium glutamate). RESULTS: Taste recognition thresholds for sour taste as well as sweet, salty, and bitter tastes showed no correlation with salivary buffering capacity. Interestingly, a negative relationship was observed between recognition threshold for umami taste and salivary buffering capacity. Furthermore, a positive correlation between salivary buffering capacity and resting saliva volume was observed. CONCLUSIONS: Salivary buffering capacity primarily influences sensitivity to umami taste, but not sour and other tastes.
Subject(s)
Buffers , Hydrogen-Ion Concentration , Saliva , Taste Perception , Taste Threshold , Adult , Female , Humans , Male , Young Adult , Citric Acid , Colorimetry , East Asian People , Healthy Volunteers , Japan , Saliva/chemistry , Saliva/metabolism , Taste/physiology , Taste Perception/physiology , Taste Threshold/physiologyABSTRACT
The choice of correct pH buffer is crucial in chemical studies modeling biological processes involving Cu2+ ions. Popular buffers for physiological pH are known to form Cu(II) complexes, but their impact on kinetics of Cu(II) complexation has not been considered. We performed a stopped-flow kinetic study of Cu2+ ion interactions with four popular buffers (phosphate, Tris, HEPES, and MOPS) and two buffers considered as nonbinding (MES and PIPPS). Next, we studied their effects on the rate of Cu2+ reaction with Gly-Gly-His (GGH), a tripeptide modeling physiological Cu(II) sites, which we studied previously at conditions presumably excluding the buffer interference [Kotuniak, R.; Angew. Chem., Int. Ed. 2020, 59, 11234-11239]. We observed that (i) all tested pH 7.4 buffers formed Cu(II) complexes within the stopped-flow instrument dead time; (ii) Cu(II)-peptide complexes were formed via ternary complexes with the buffers; (iii) nevertheless, Good buffers affected the observed rate of Cu(II)-GGH complex formation only slightly; (iv) Tris was a competitive inhibitor of Cu(II)-GGH complexation; while (v) phosphate was a reaction catalyst. This is particularly important as phosphate is a biological buffer.
Subject(s)
Copper , Copper/chemistry , Buffers , Hydrogen-Ion Concentration , Kinetics , Coordination Complexes/chemistry , Peptides/chemistry , Oligopeptides/chemistry , Ions/chemistryABSTRACT
The structural stability and therapeutic activity of Stem Bromelain (BM) have been explored by unravelling the interaction of stem BM in presence of two different types of anionic surfactants namely, bile salts, NaC and NaDC and the conventional anionic surfactants, SDDS and SDBS, below, at and above the critical micelle concentration (cmc) in aqueous phosphate buffer of pH 7. Different physicochemical parameters like, surface excess (Γcmc), minimum area of surfactants at air water interface (Amin) etc. are calculated from tensiometry both in absence and presence of BM. Several inflection points (C1, C2 and C3) have been found in tensiometry profile of surfactants in presence of BM due to the conformational change of BM assisted by surfactants. Similar observation also found in isothermal titration calorimetry (ITC) profiles where the enthalpy of micellization (ΔH0obs) of surfactants in absence and presence of BM have calculated. Further, steady state absorption and fluorescence spectra monitoring the tryptophan (Trp) emission of free BM and in presence of all the surfactants at three different temperatures (288.15 K, 298.15 K, and 308.15 K) reveal the nature of fluorescence quenching of BM in presence of bile salts/surfactants. Time resolved fluorescence studies at room temperature also support to determine the several quenching parameters. The binding constant (Kb) of BM with all the surfactants and free energy of binding (∆G0 of bile salts/surfactants with BM at different temperatures have been calculated exploiting steady state fluorescence technique. It is observed that, the binding of NaC with BM is greater as compared to other surfactants while Stern-Volmer quenching constant (KSV) is found greater in presence of SDBS as compared with others which supports the surface tension and ITC data with the fact that surface activity of surfactant(s) is decreasing with the binding of the surfactants at the core or binding pocket of BM. Circular Dichroism (CD) study shows the stability of secondary structure of BM in presence of NaC and NaDC below C3, while BM lost its structural stability even at very low surfactant concentration of SDDS and SDBS which also supports the more involvement of bile salts in binding rather than surfactants. The molecular docking studies have also been substantiated for better understanding the several experimental investigations interaction of BM with the bile salts/surfactants.
Subject(s)
Bromelains , Micelles , Molecular Docking Simulation , Surface-Active Agents , Thermodynamics , Bromelains/chemistry , Bromelains/metabolism , Surface-Active Agents/chemistry , Hydrogen-Ion Concentration , Anions/chemistry , Spectrometry, Fluorescence , BuffersABSTRACT
Free Calcium ions in the cytosol are essential for many physiological and physical functions. The free calcium ions are commonly regarded as a second messenger, are an essential part of brain communication. Numerous physiological activities, such as calcium buffering and calcium ion channel flow, etc. influence the cytosolic calcium concentration. In light of the above, the primary goal of this study is to develop a model of calcium distribution in neuron cells when a Voltage-Gated Calcium Channel and Sodium Calcium Exchanger are present. As we know, decreased buffer levels and increased calcium activity in the Voltage-Gated Calcium Channel and Sodium Calcium Exchanger lead to Alzheimer's disease. Due to these changes, the calcium diffusion in that location becomes disrupted and impacted by Alzheimer's disease. The model has been constructed by considering key factors like buffers and ER fluxes when Voltage-Gated Calcium Channels and Sodium Calcium Exchangers are present. Based on the physiological conditions of the parameters, appropriate boundary conditions have been constructed in the fuzzy environment. This model is considered a fuzzy boundary value problem with the source term and initial boundary conditions are modeled by triangular fuzzy functions. In this, paper we observed the approximate solution of the mathematical model which was investigated by the fuzzy undetermined coefficient method. The solution has been performed through MATLAB and numerical results have been computed using simulation. The observation made that the proper operation of the Voltage-Gated Calcium Channel and Sodium Calcium Exchanger is critical for maintaining the delicate equilibrium of calcium ions, which regulates vital cellular activities. Dysregulation of Voltage-Gated Calcium Channel and Sodium Calcium Exchanger activity has been linked to neurodegenerative illnesses like Alzheimer's disease.
Subject(s)
Calcium Channels , Calcium , Endoplasmic Reticulum , Neurons , Sodium-Calcium Exchanger , Calcium/metabolism , Neurons/metabolism , Sodium-Calcium Exchanger/metabolism , Calcium Channels/metabolism , Endoplasmic Reticulum/metabolism , Buffers , Humans , Fuzzy Logic , Models, BiologicalABSTRACT
The cross talk between calcium (Ca2+), IP3 and buffer dynamics regulate various mechanisms in hepatocyte cells. The study of independent systems of calcium, IP3, and buffer signaling provides limited information about cell dynamics. In the current study, coupled reaction-diffusion equations are used to design a cross-talk model for IP3, buffer, and calcium dynamics in a hepatocyte cell. The one-way feedback of calcium, buffer, and IP3 in ATP production, ATP degradation, and NADH production rate is incorporated into the model. Numerical simulation has been done using the Finite Element Method (FEM) along the spatial direction and the Crank-Nicolson (C-N) method along the temporal direction. The numerical results are analysed to determine the effects of alterations in processes of cross-talking dynamics of IP3, buffer, and calcium on ATP and NADH production and degradation rate of ATP in a hepatocyte cell under normal and obesity conditions. The comparative analysis of these findings unveils notable distinctions induced by obesity in calcium dynamics, ATP and NADH synthesis, and ATP degradation kinetics.