Ivacaftor pharmacokinetics and lymphatic transport after enteral administration in rats.

Background: Ivacaftor is a modern drug used in the treatment of cystic fibrosis. It is highly lipophilic and exhibits a strong positive food effect. These characteristics can be potentially connected to a pronounced lymphatic transport after oral administration. Methods: A series of studies was conducted to describe the basic pharmacokinetic parameters of ivacaftor in jugular vein cannulated rats when dosed in two distinct formulations: an aqueous suspension and an oil solution. Additionally, an anesthetized mesenteric lymph duct cannulated rat model was studied to precisely assess the extent of lymphatic transport. Results: Mean ± SD ivacaftor oral bioavailability was 18.4 ± 3.2% and 16.2 ± 7.8%, respectively, when administered as an aqueous suspension and an oil solution. The relative contribution of the lymphatic transport to the overall bioavailability was 5.91 ± 1.61% and 4.35 ± 1.84%, respectively. Conclusion: Lymphatic transport plays only a minor role in the process of ivacaftor intestinal absorption, and other factors are, therefore, responsible for its pronounced positive food effect.

The potential of polyaniline-coated stationary phase in hydrophilic interaction liquid chromatography-based solid-phase extraction for glycopeptide enrichment.

Glycopeptide enrichment is a crucial step in glycoproteomic analysis, often achieved through solid-phase extraction (SPE) on polar stationary phases in hydrophilic interaction liquid chromatography (HILIC). This study explores the potential of polyaniline (PANI)-coated silica gel for enriching human immunoglobulin G (IgG). Experimental conditions were varied to assess their impact on glycopeptide enrichment efficiency, comparing PANI-cotton wool SPE with conventional cotton wool as SPE sorbents. Two formic acid concentrations (0.1% and 1%) in elution solvent were tested, revealing that higher concentrations led to earlier elution of studied glycopeptides, especially for sialylated glycopeptides. Substituting formic acid with acetic acid increased the interaction of neutral glycopeptides with the PANI-modified sorbent, while sialylated glycopeptides showed no significant change in enrichment efficiency. Acetonitrile concentration in the elution solvent (5%, 10%, and 20%) affected the enrichment efficiency with most glycopeptides eluting at the lowest acetonitrile concentration. The acetonitrile concentration in conditioning and washing solutions (65%, 75%, and 85%) played a crucial role; at 65% acetonitrile, glycopeptides were least retained on the stationary phase, and neutral glycopeptides were even detected in the flow-through fraction. This study shows the potential of in-house-prepared PANI-modified sorbents for SPE-HILIC glycopeptide enrichment, highlighting the crucial role of tuning experimental conditions in sample preparation to enhance enrichment efficiency and selectivity.

Antifungal triazoles affect key non-target metabolic pathways in Solanum lycopersicum L. plants.

Several 1,2,4-triazoles are widely used as systemic fungicides in agriculture because they inhibit fungal 14ɑ-demethylase. However, they can also act on many non-target plant enzymes, thereby affecting phytohormonal balance, free amino acid content, and adaptation to stress. In this study, tomato plants (Solanum lycopersicum L. var. 'Cherrola') were exposed to penconazole, tebuconazole, or their combination, either by foliar spraying or soil drenching, every week, as an ecotoxicological model. All triazole-exposed plants showed a higher content (1.7-8.8 ×) of total free amino acids than the control, especially free glutamine and asparagine were increased most likely in relation to the increase in active cytokinin metabolites 15 days after the first application. Conversely, the Trp content decreased in comparison with control (0.2-0.7 ×), suggesting depletion by auxin biosynthesis. Both triazole application methods slightly affected the antioxidant system (antioxidant enzyme activity, antioxidant capacity, and phenolic content) in tomato leaves. These results indicated that the tomato plants adapted to triazoles over time. Therefore, increasing the abscisic and chlorogenic acid content in triazole-exposed plants may promote resistance to abiotic stress.

Analysis of the Ibotenic Acid, Muscimol, and Ergosterol Content of an Amanita Muscaria Hydroalcoholic Extract with an Evaluation of Its Cytotoxic Effect against a Panel of Lung Cell Lines In Vitro.

The fungus Amanita muscaria is universally recognizable for its iconic appearance; it is also widely regarded as poisonous, inedible, and even deadly. In spite of that, there have been documented cases of use of A. muscaria-containing preparations against various diseases, including cancer, to no apparent ill effect. The search for compounds that can be used to treat cancer among various plants and fungi has been intensifying in recent years. In light of this, we describe an HPLC HILIC analytical method for the evaluation of the content of the anticancer compound ergosterol (ERG) and the neuroactive alkaloids ibotenic acid (IBO) and muscimol (MUS) that contribute significantly to the unpleasant physiological syndrome associated with A. muscaria consumption. A 'homemade' A. muscaria tincture made using 80-proof rye vodka as the solvent, an A. muscaria extract made with a standardized water-ethanol solution as the solvent, and fractions obtained from the second extract via liquid-liquid extraction with nonpolar solvents were analyzed. The study also presents the results of capillary zone electrophoresis with contactless conductivity detection and UHPLC-MS/MS analyses of the IBO and MUS content of the two native A. muscaria extracts and an evaluation of the standardized extract's cytotoxic effect against a small panel of lung cell cultures in vitro. Our results show that the standardized extract has a significant cytotoxic effect and does not contain the compounds of interest in any significant quantity.

Triazoles as a Potential Threat to the Nutritional Quality of Tomato Fruits.

Triazole fungicides can threaten plants as abiotic stressors but can also positively affect plant defense by inducing priming. Thus, plant yield is also both protected and endangered by triazoles that may influence several metabolic pathways during maturation processes, such as the biosynthesis of saccharides or secondary metabolites. Here, Solanum lycopersicum L. plants were exposed to foliar and soil applications of penconazole, tebuconazole, or their combination, and their resulting effect on tomato fruits was followed. The exposure to the equimolar mixture of both triazoles influenced the representation of free proteinogenic amino acids, especially Gln, Glu, Gly, Ile, Lys, Ser and Pro, saccharide content, and led to a significant increase in the contents of total phenolics and flavonoids as well as positive stimulation of the non-enzymatic antioxidant system. Among the identified secondary metabolites, the most abundant was naringenin, followed by chlorogenic acid in tomato peel. In turn, all triazole-treated groups showed a significantly lower content of rosmarinic acid in comparison with the control. Foliar application of penconazole affected the fruit more than other single triazole applications, showing a significant decrease in antioxidant capacity, the total content of secondary metabolites, and the activities of total membrane-bound peroxidases and ascorbate peroxidase.

Using surface plasmon resonance, capillary electrophoresis and diffusion-ordered NMR spectroscopy to study drug release kinetics.

Nanomedicines, including polymer nanocarriers with controlled drug release, are considered next-generation therapeutics with advanced therapeutic properties and reduced side effects. To develop safe and efficient nanomedicines, it is crucial to precisely determine the drug release kinetics. Herein, we present application of analytical methods, i.e., surface plasmon resonance biosensor technology (SPR), capillary electrophoresis, and 1H diffusion-ordered nuclear magnetic resonance spectroscopy, which were innovatively applied for drug release determination. The methods were optimised to quantify the pH-triggered release of three structurally different drugs from a polymer carrier. The suitability of these methods for drug release characterisation was evaluated and compared using several parameters including applicability for diverse samples, the biological relevance of the experimental setup, method complexity, and the analysis outcome. The SPR method was the most universal method for the evaluation of diverse drug molecule release allowing continuous observation in the flow-through setting and requiring a small amount of sample.

Casein as protein and hydrolysate: Biostimulant or nitrogen source for Nicotiana tabacum plants grown in vitro?

In contrast to inorganic nitrogen (N) assimilation, the role of organic N forms, such as proteins and peptides, as sources of N and their impact on plant metabolism remains unclear. Simultaneously, organic biostimulants are used as priming agents to improve plant defense response. Here, we analysed the metabolic response of tobacco plants grown in vitro with casein hydrolysate or protein. As the sole source of N, casein hydrolysate enabled tobacco growth, while protein casein was used only to a limited extent. Free amino acids were detected in the roots of tobacco plants grown with protein casein but not in the plants grown with no source of N. Combining hydrolysate with inorganic N had beneficial effects on growth, root N uptake and protein content. The metabolism of casein-supplemented plants shifted to aromatic (Trp), branched-chain (Ile, Leu, Val) and basic (Arg, His, Lys) amino acids, suggesting their preferential uptake and/or alterations in their metabolic pathways. Complementarily, proteomic analysis of tobacco roots identified peptidase C1A and peptidase S10 families as potential key players in casein degradation and response to N starvation. Moreover, amidases were significantly upregulated, most likely for their role in ammonia release and impact on auxin synthesis. In phytohormonal analysis, both forms of casein influenced phenylacetic acid and cytokinin contents, suggesting a root system response to scarce N availability. In turn, metabolomics highlighted the stimulation of some plant defense mechanisms under such growth conditions, that is, the high concentrations of secondary metabolites (e.g., ferulic acid) and heat shock proteins.

Serum and lymph pharmacokinetics of nilotinib delivered by yeast glucan particles per os.

Nilotinib is a selective tyrosine-kinase inhibitor approved for the treatment of chronic myeloid leukemia. It is poorly soluble in aqueous media and has a low oral bioavailability. Nilotinib encapsulation into yeast glucan particles (GPs) was investigated in this work as a means of increasing bioavailability. The amorphization of nilotinib in GPs resulted in an increased dissolution rate, which was confirmed by in vitro experiments using biorelevant dissolution media. Simultaneously, GPs containing nilotinib were effectively taken up by macrophages, which was quantified in vitro on cell cultures. The overall oral bioavailability in a rat model was approximately 39 % for nilotinib delivered in a reference formulation (Tasigna) and was almost doubled when delivered in GPs. The contribution of glucan particles to the lymphatic transport of nilotinib was quantified. When delivered by GPs, cumulative nilotinib absorption via the lymphatic system increased by a factor of 10.8 compared to the reference, but still represented arelative bioavailability of only 1.12 %. The cumulative uptake of GPs in the lymph was found to be 0.54 mg after a single dose of 50 mg. Yeast glucan particles can therefore serve as a drug delivery vehicle with a dual function: dissolution rate enhancement by amorphization, and, to asmaller extent, lymphatic delivery due to macrophage uptake.

On-capillary fluorescent labeling of saccharides for capillary electrophoresis.

The feasibility of on-capillary derivatization of saccharides by aromatic amine-based fluorescent labeling agents was tested. To avoid the problematic evolution of gaseous hydrogen cyanide, the Schiff base reduction by sodium cyanoborohydride, as the second step of the standard reductive amination protocol, was omitted. Glucose was used as a model analyte and 7-amino-1,3-naphthalenedisulfonic acid as the labeling agent. Our experiments showed that the direct reaction of the saccharide with the labeling agent in 2.5-M acetic acid yields a labeled product that is sufficiently stable to be separated from the labeling agent in 20-mM phosphate buffer, pH 3.5, and detected using UV detection. The glucose and label zones were introduced separately into the capillary and mixed using a negative voltage. Mixing voltage, its duration, the concentration of acetic acid in the reaction zone, and the waiting time between mixing and separation were optimized. To show the applicability of the procedure to a broader range of analytes, a mixture of different types of saccharides, that is, xylose (pentose), fucose (hexose), glucose (hexose), N-acetylglucosamine (N-acetylaminosaccharide), and lactose (disaccharide), was subjected to derivatization and analysis under the optimal conditions. The linearity and repeatability of the process were evaluated as critical parameters for its analytical applications. Six-point calibration dependences in the 1-50 mM range showed excellent determination coefficients of 0.9992 or higher for all five saccharides tested. The repeatability of the labeled saccharide peak areas was between 2.2% and 4.3%.

Application of Oil-in-Water Cannabidiol Emulsion for the Treatment of Rheumatoid Arthritis.

Introduction: Rheumatoid arthritis (RA) is a chronic autoimmune disease with unknown cause. It mainly affects joints and, without proper treatment, negatively impacts their movement, causes painful deformities, and reduces the patients' quality of life. Current treatment options consist of various types of disease-modifying antirheumatic drugs (DMARDs), however 20-30% of patients are partially resistant to them. Therefore, development of new drugs is necessary. Possible option are compounds exhibiting their action via endocannabinoid system, which plays an important role in pain and inflammation modulation. One such compound - cannabidiol (CBD) has already been shown to attenuate synovitis in animal model of RA in in vivo studies. However, it has low bioavailability due to its low water solubility and lipophilicity. This issue can be addressed by preparation of a lipid containing formulation targeting lymphatic system, another route of absorption in the body. Materials and Methods: CBD-containing emulsion was prepared by high-shear homogenization and its droplet size distribution was analysed by optical microscopy. The relative oral bioavailability compared to oil solution as well as total availability of CBD were assessed in a cross-over study in rats and absorption of CBD via lymphatic system was observed. The effect of CBD on the animal model of RA was determined. Results: Compared to oil solution, the emulsion exhibited higher absolute oral bioavailability. Significant lymphatic transport of CBD was observed in all formulations and the concentrations in lymph were calculated. The therapeutic effect of CBD on RA was confirmed as an improvement in clinical symptoms as well as morphological signs of disease activity were observed during the study. Conclusion: In this work, we prepared a simple stable emulsion formulation, determined the pharmacokinetic parameters of CBD and calculated its absolute bioavailability in rats. Moreover, we successfully tested the pharmaceutical application of such a formulation and demonstrated the positive effect of CBD in an animal model of RA.

Pharmaceutical Product Characterization and Manufacturability of Surfactant-Enriched Oil Marbles with Abiraterone Acetate.

The present study investigates the physicochemical properties and stability of a novel lipid-based formulation-surfactant-enriched oil marbles containing abiraterone acetate. While the biopharmaceutical performance of this formulation has been reported recently, this study aims to fill the gap between a promising in vivo performance and industrial applicability. A series of techniques were employed to assess the solid-state characteristics of oil marble cores along with their physicochemical properties upon stability testing. The chemical stability of abiraterone acetate in the formulation was also investigated. The core of the formulation was found to be stable both physically and chemically over 12 months of storage. The in vitro performance of stressed samples was evaluated using a dissolution experiment. The formulation has successfully self-emulsified upon incubation in bio-relevant media, resulting in a fast and complete API release. An important issue connected with the excipient used as a covering material of oil marbles has been identified. The seemingly insignificant water sorption caused agglomeration of the oil marbles and consequently compromised the dissolution rate in some of the stressed samples. Replacing HPMC with lactose as a covering material resulted in more favorable properties upon storage. Overall, it has been shown that oil marbles are an industrially applicable concept of the solidified lipid-based formulation.

Human ultrarare genetic disorders of sulfur metabolism demonstrate redundancies in H2S homeostasis.

Regulation of H2S homeostasis in humans is poorly understood. Therefore, we assessed the importance of individual enzymes in synthesis and catabolism of H2S by studying patients with respective genetic defects. We analyzed sulfur compounds (including bioavailable sulfide) in 37 untreated or insufficiently treated patients with seven ultrarare enzyme deficiencies and compared them to 63 controls. Surprisingly, we observed that patients with severe deficiency in cystathionine ß-synthase (CBS) or cystathionine γ-lyase (CSE) - the enzymes primarily responsible for H2S synthesis - exhibited increased and normal levels of bioavailable sulfide, respectively. However, an approximately 21-fold increase of urinary homolanthionine in CBS deficiency strongly suggests that lacking CBS activity is compensated for by an increase in CSE-dependent H2S synthesis from accumulating homocysteine, which suggests a control of H2S homeostasis in vivo. In deficiency of sulfide:quinone oxidoreductase - the first enzyme in mitochondrial H2S oxidation - we found normal H2S concentrations in a symptomatic patient and his asymptomatic sibling, and elevated levels in an asymptomatic sibling, challenging the requirement for this enzyme in catabolizing H2S under physiological conditions. Patients with ethylmalonic encephalopathy and sulfite oxidase/molybdenum cofactor deficiencies exhibited massive accumulation of thiosulfate and sulfite with formation of large amounts of S-sulfocysteine and S-sulfohomocysteine, increased renal losses of sulfur compounds and concomitant strong reduction in plasma total cysteine. Our results demonstrate the value of a comprehensive assessment of sulfur compounds in severe disorders of homocysteine/cysteine metabolism and provide evidence for redundancy and compensatory mechanisms in the maintenance of H2S homeostasis.

Monosaccharide profiling of glycoproteins by capillary electrophoresis with contactless conductivity detection.

Saccharides form one of the major constituents of biological macromolecules in living organisms. Many biological processes including protein folding, stability, immune response and receptor activation are regulated by glycosylation. In this work, we optimized a capillary electrophoresis method with capacitively coupled contactless conductivity detection for the separation of eight monosaccharides commonly found in glycoproteins, namely D-glucose, D-galactose, D-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-fucose, N-acetylneuraminic acid, and D-xylose. A highly alkaline solution of 50 mM sodium hydroxide, 22.5 mM disodium phosphate, and 0.2 mM CTAB (pH 12.4) was used as a background electrolyte in a 10 µm id capillary. To achieve baseline separation of all analytes, a counter-directional pressure of -270 kPa was applied during the separation. The limits of detection of our method were below 7 µg/ml (i.e., 1.5 pg or 1 mg/g protein) and the limits of quantification were below 22 µg/ml (i.e., 5 pg or 3 mg/g protein). As a proof of concept of our methodology, we performed an analysis of monosaccharides released from fetuin glycoprotein by acid hydrolysis. The results show that, when combined with an appropriate pre-concentration technique, the developed method can be used as a monosaccharide profiling tool in glycoproteomics and complement the routinely used LC-MS/MS analysis.

Liposomes: preparation and characterization with a special focus on the application of capillary electrophoresis.

Liposomes are nowadays a matter of tremendous interest. Due to their amphiphilic character, various substances with different properties can be incorporated into them and they are especially suitable as a model system for controlled transport of bioactive substances and drugs to the final destination in the body; for example, COVID-19 vaccines use liposomes as a carrier of mRNA. Liposomes mimicking composition of various biological membranes can be prepared with a proper choice of the lipids used, which proved to be important tool in the early drug development. This review deals with commonly used methods for the preparation and characterization of liposomes which is essential for their later use. The alternative capillary electrophoresis methods for physico-chemical characterization such as determination of membrane permeability of liposome, its size and charge, and encapsulation efficiency are included. Two different layouts using liposomes to yield more efficient separation of various analytes are also presented, capillary electrochromatography, and liposomal electrokinetic chromatography.

Seed Protection of Solanum lycopersicum with Pythium oligandrum against Alternaria brassicicola and Verticillium albo-atrum.

Pythium oligandrum, strain M1, is a soil oomycete successfully used as a biological control agent (BCA), protecting plants against fungal, yeast, and oomycete pathogens through mycoparasitism and elicitor-dependent plant priming. The not yet described Pythium strains, X42 and 00X48, have shown potential as BCAs given the high activity of their secreted proteases, endoglycosidases, and tryptamine. Here, Solanum lycopersicum L. cv. Micro-Tom seeds were coated with Pythium strains, and seedlings were exposed to fungal pathogens, either Alternaria brassicicola or Verticillium albo-atrum. The effects of both infection and seed-coating on plant metabolism were assessed by determining the activity and isoforms of antioxidant enzymes and endoglycosidases and the content of tryptamine, amino acids, and heat shock proteins. Dual culture competition testing and microscopy analysis confirmed mycoparasitism in all three Pythium strains. In turn, seed treatment significantly increased the total free amino acid content, changing their abundance in both non-infected and infected plants. In response to pathogens, plant Hsp70 and Hsp90 isoform levels also varied among Pythium strains, most likely as a strategy for priming the plant against infection. Overall, our results show in vitro mycoparasitism between Pythium strains and fungal pathogens and in planta involvement of heat shock proteins in priming.

LEGO-Lipophosphonoxins: A Novel Approach in Designing Membrane Targeting Antimicrobials.

The alarming rise of bacterial antibiotic resistance requires the development of new compounds. Such compounds, lipophosphonoxins (LPPOs), were previously reported to be active against numerous bacterial species, but serum albumins abolished their activity. Here we describe the synthesis and evaluation of novel antibacterial compounds termed LEGO-LPPOs, loosely based on LPPOs, consisting of a central linker module with two attached connector modules on either side. The connector modules are then decorated with polar and hydrophobic modules. We performed an extensive structure-activity relationship study by varying the length of the linker and hydrophobic modules. The best compounds were active against both Gram-negative and Gram-positive species including multiresistant strains and persisters. LEGO-LPPOs act by first depleting the membrane potential and then creating pores in the cytoplasmic membrane. Importantly, their efficacy is not affected by the presence of serum albumins. Low cytotoxicity and low propensity for resistance development demonstrate their potential for therapeutic use.

Liquid chromatography and capillary electrophoresis in glycomic and glycoproteomic analysis.

Glycosylation is one of the most significant and abundant post-translational modifications in cells. Glycomic and glycoproteomic analyses involve the characterization of oligosaccharides (glycans) conjugated to proteins. Glycomic and glycoproteomic analysis is highly challenging because of the large diversity of structures, low abundance, site-specific heterogeneity, and poor ionization efficiency of glycans and glycopeptides in mass spectrometry (MS). MS is a key tool for characterization of glycans and glycopeptides. However, MS alone does not always provide full structural and quantitative information for many reasons, and thus MS is combined with some separation technique. This review focuses on the role of separation techniques used in glycomic and glycoproteomic analyses, liquid chromatography and capillary electrophoresis. The most important separation conditions and results are presented and discussed.

Prediction of Intact N-Glycopeptide Retention Time Windows in Hydrophilic Interaction Liquid Chromatography.

Analysis of protein glycosylation is challenging due to micro- and macro-heterogeneity of the attached glycans. Hydrophilic interaction liquid chromatography (HILIC) is a mode of choice for separation of intact glycopeptides, which are inadequately resolved by reversed phase chromatography. In this work, we propose an easy-to-use model to predict retention time windows of glycopeptides in HILIC. We constructed this model based on the parameters derived from chromatographic separation of six differently glycosylated peptides obtained from tryptic digests of three plasma proteins: haptoglobin, hemopexin, and sex hormone-binding globulin. We calculated relative retention times of different glycoforms attached to the same peptide to the bi-antennary form and showed that the character of the peptide moiety did not significantly change the relative retention time differences between the glycoforms. To challenge the model, we assessed chromatographic behavior of fetuin glycopeptides experimentally, and their retention times all fell within the calculated retention time windows, which suggests that the retention time window prediction model in HILIC is sufficiently accurate. Relative retention time windows provide complementary information to mass spectrometric data, and we consider them useful for reliable determination of protein glycosylation in a site-specific manner.

Explaining dissolution properties of rivaroxaban cocrystals.

The aim of this study was to improve rivaroxaban water-solubility by cocrystal preparation and to understand this process. The screening with water-soluble coformers was performed via both mechanochemical and solution-mediated techniques. Two cocrystals of rivaroxaban with malonic acid and oxalic acid were prepared, and the structure of the cocrystal with oxalic acid was solved. Both cocrystals exhibit improved dissolution properties. The mechanism of the supersaturation maintenance was studied by in-situ Raman spectroscopy. The transformation into rivaroxaban dihydrate was identified as the critical step in the improved dissolution properties of both cocrystals. Moreover, the transformation kinetics and solubilization effects of the coformers were identified as responsible for the differences in the dissolution behavior of the cocrystals. In-vivo experiments proved that the use of cocrystal instead of form I of free API helped to increase the bioavailability ofrivaroxaban.

Liquid crystals purity assay using nonaqueous capillary electrokinetic chromatography.

A method for purity control of newly synthesized lactic acid-based liquid crystals has been developed. The electrokinetic chromatography proved to be suitable for the separation of these electroneutral substances from their impurities. The separations were performed in an acidic acetonitrile-based background electrolyte (BGE) with a pseudostationary phase formed by a cationic surfactant. During the optimization step, appropriate concentrations of cetyltrimethylammonium bromide, acetic acid, and water were seeked. In the optimized method, separations were carried out in acetonitrile with 1-mol/L acetic acid, 80-mmol/L cetyltrimethylammonium bromide, and 6% (v/v) water. Interesting positive effects of a small water content in the BGE on electroosmotic flow and resolution of liquid crystal substances from their impurities were observed and discussed. Samples of five liquid crystal substances, both pure and containing impurities from synthesis, were analyzed. The identification of analytes was based on a comparison of relative migration times related to the migration time of mesityl oxide. For all five samples, impurities were separated from the liquid crystals and the method thus showed its viability. To the best of our knowledge, this method is used for the first time for the purity control of newly synthesized liquid crystals. This method can be used to confirm or complement the results obtained by commonly used high-performance liquid chromatography and supercritical fluid chromatography methods. Furthermore, the electrokinetic chromatography method requires very small amounts of sample, solvents, and buffer constituents. Overall, its operational costs are significantly lower.