Chemical aspects of hydrogen sulfide measurements in physiological samples.

BACKGROUND: Owing to recent discoveries of many hydrogen sulfide-mediated physiological processes, sulfide biology is in the focus of scientific research. However, the promiscuous chemical properties of sulfide pose complications for biological studies, which led to accumulation of controversial observations in the literature. SCOPE OF REVIEW: We intend to provide an overview of fundamental thermodynamic and kinetic features of sulfide redox- and coordination-chemical reactions and protonation equilibria in relation to its biological functions. In light of these chemical properties we review the strengths and limitations of the most commonly used sulfide detection methods and recently developed fluorescent probes. We also give a personal perspective on blood and tissue sulfide measurements based on proposed biomolecule-sulfide interactions and point out important chemical aspects of handling sulfide reagent solutions. MAJOR CONCLUSIONS: The diverse chemistries of sulfide detection methods resulted in orders of magnitude differences in measured physiological sulfide levels. Investigations that were aimed to dissect the underlying molecular reasons responsible for these controversies made the important recognition that there are large sulfide reserves in biological systems. These sulfide pools are tightly regulated in a dynamic manner and they are likely to play a major role in regulation of endogenous-sulfide-mediated biological functions and avoiding toxic side effects. GENERAL SIGNIFICANCE: Working with sulfide is challenging, because it requires considerable amounts of chemical knowledge to adequately handle reagent sulfide solutions and interpret biological observations. Therefore, we propose that a rigorous chemical approach could aid the reconciliation of the increasing number of controversies in sulfide biology. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Dissociation kinetics of open-chain and macrocyclic gadolinium(III)-aminopolycarboxylate complexes related to magnetic resonance imaging: catalytic effect of endogenous ligands.

The kinetics of the metal exchange reactions between open-chain Gd(DTPA)(2-) and Gd(DTPA-BMA), macrocyclic Gd(DOTA)(-) and Gd(HP-DO3A) complexes, and Cu(2+)  ions were investigated in the presence of endogenous citrate, phosphate, carbonate and histidinate ligands in the pH range 6-8 in NaCl (0.15 M) at 25 °C. The rates of the exchange reactions of Gd(DTPA)(2-) and Gd(DTPA-BMA) are independent of the Cu(2+) concentration in the presence of citrate and the reactions occur via the dissociation of Gd(3+)  complexes catalyzed by the citrate ions. The HCO(3)(-)/CO(3)(2-) and H(2)PO(4)(-) ions also catalyze the dissociation of complexes. The rates of the dissociation of Gd(DTPA-BMA), catalyzed by the endogenous ligands, are about two orders of magnitude higher than those of the Gd(DTPA)(2-). In fact near to physiological conditions the bicarbonate and carbonate ions show the largest catalytic effect, that significantly increase the dissociation rate of Gd(DTPA-BMA) and make the higher pH values (when the carbonate ion concentration is higher) a risk-factor for the dissociation of complexes in body fluids. The exchange reactions of Gd(DOTA)(-) and Gd(HP-DO3A) with Cu(2+) occur through the proton assisted dissociation of complexes in the pH range 3.5-5 and the endogenous ligands do not affect the dissociation rates of complexes. More insights into the interaction scheme between Gd(DTPA-BMA) and Gd(DTPA)(2-) and endogenous ligands have been obtained by acquiring the (13)C NMR spectra of the corresponding diamagnetic Y(III)-complexes, indicating the increase of the rates of the intramolecular rearrangements in the presence of carbonate and citrate ions. The herein reported results may have implications in the understanding of the etiology of nephrogenic systemic fibrosis, a rare disease that has been associated to the administration of Gd-containing agents to patients with impaired renal function.

Kinetics of the exchange reactions between Gd(DTPA)2-, Gd(BOPTA)2-, and Gd(DTPA-BMA) complexes, used as MRI contrast agents, and the triethylenetetraamine-hexaacetate ligand.

The kinetics of ligand exchange reactions occurring between the Gd(DTPA), Gd(BOPTA), and Gd(DTPA-BMA) complexes, used as contrast agents in MRI, and the ligand TTHA, have been studied in the pH range 6.5-11.0 by measuring the water proton relaxation rates at 25 °C in 0.15 M NaCl. The rates of the reactions are directly proportional to the concentration of TTHA, indicating that the reactions take place with the direct attack of the H(i)TTHA((6-i)-) (i = 0, 1, 2 and 3) species on the Gd(3+) complexes, through the formation of ternary intermediates. The rates of the exchange reactions of the neutral Gd(DTPA-BMA) increase when the pH is increased from 6.5 to 9, because the less protonated H(i)TTHA((6-i)-) species can more efficiently attack the Gd(3+) complex. The rates of the exchange reactions of [Gd(DTPA)](2-) and [Gd(BOPTA)](2-) also increase from pH 8.5 to 11, but from 6.5 to 8.5 an unexpected decrease was observed in the reaction rates. The decrease has been interpreted by assuming the validity of general acid catalysis. The protons from the H(i)TTHA((6-i)-) species (i = 2 and 3) can be transferred to the coordinated DTPA or BOPTA in the ternary intermediates when the dissociation of the Gd(3+) complexes occurs faster. The kinetic inertness of Gd(DTPA), Gd(BOPTA), and Gd(DTPA-BMA) differs very considerably; the rates of the ligand exchange reactions of Gd(DTPA-BMA), thus the rates of its dissociation, are 2 to 3 orders of magnitude higher than those of Gd(DTPA) and Gd(BOPTA). The rates of the ligand exchange reactions increase with increasing concentration of the endogenous citrate, phosphate, or carbonate ions at a pH of 7.4, but the effect of citrate and phosphate is negligible at their physiological concentrations. The increase in the reaction rates at the physiological concentration of the carbonate ion is significant (20-60%), and the effect is the largest for the Gd(DTPA-BMA) complex.

Reliable identification and quantification of anabolic androgenic steroids in dietary supplements by using gas chromatography coupled to triple quadrupole mass spectrometry.

The aim of the present research was the identification and quantification of specific anabolic androgenic steroids (AASs) and other sterane structured compounds in dietary supplements (DSs). The adulteration of DSs by these compounds is of a particular concern in athletes, because it might lead to a positive doping result. The research was focused on the optimization of a highly sensitive and selective GC-based analytical strategy using triple quadrupole MS as detector. Chromatographic method and multiple reaction monitoring (MRM) transitions of 28 target compounds were optimized. Sample clean-up was carried out by using a solid phase extraction (SPE) procedure, while the derivatization of AASs was performed by using N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA). The method was validated, and the following parameters were investigated: linearity range, limit of detection, accuracy, and precision expressed in terms of intra-day precision. The calibration curves were evaluated by using regression model and resulting in a good determination coefficients (R2 ≥ 0.9912). The residuals were scattered randomly around zero. The limits of detection (LODs) were lower than 7.0 ng g-1 or ng ml-1 . The accuracy assessment was evaluated in different forms of DSs characterized by high sample-to-sample variability (liquid, powder, tablet, capsule, protein, and herbal-based). Intra-day assay precision was in all cases lower than 20%. The developed analytical method was successfully applied to the analysis of 67 commercially available dietary supplements. In five cases, one or more steroid-type compounds were found in the concentration of 5 ng g-1 -100 µg g-1 , which might result adverse analytical findings in athletes.

New and High Virulent Pathotypes of Sunflower Downy Mildew (Plasmopara halstedii) in Seven Countries in Europe.

Downy mildew of sunflower, caused by Plasmopara halstedii (Farl.) Berl. et de Toni, is a relevant disease of this crop. High virulent pathotypes have been identified in several countries, while there are few data on the spread of P. halstedii pathotypes in some important sunflower-growing areas of Europe. The goal of this study was to give up-to-date information on the pathotype structure of P. halstedii in Hungary and provide some actual data on the virulence phenotype of the pathogen for six European countries. Infected leaves of different sunflower hybrids and volunteers were collected in seven countries (Hungary, Bulgaria, Serbia, Turkey, Greece, Romania, and Italy) between 2012 and 2019. A universally accepted nomenclature was used with a standardized set of sunflower differential lines for pathotype characterization of isolates. The virulence pattern of the isolates was determined by a three-digit code (coded virulence formula, CVF). A total of 109 P. halstedii isolates were characterized. As a result of our survey, 18 new P. halstedii pathotypes were identified in Europe. Two out of the eighteen pathotypes were detected from the Asian part of Turkey. The detailed distribution of pathotypes in Hungary is also discussed.

Stability, water exchange, and anion binding studies on lanthanide(III) complexes with a macrocyclic ligand based on 1,7-diaza-12-crown-4: extremely fast water exchange on the Gd3+ complex.

The picolinate-derivative ligand based on the 1,7-diaza-12-crown-4 platform (bp12c4(2-)) forms stable Ln(3+) complexes with stability constants increasing from the early to the middle lanthanides, then being relatively constant for the rest of the series (logK(LnL) = 16.81(0.06), 18.82(0.01), and 18.08(0.05) for Ln = La, Gd, and Yb, respectively). The complex formation is fast, allowing for direct potentiometric titrations to assess the stability constants. In the presence of Zn(2+), the dissociation of [Gd(bp12c4)](+) proceeds both via proton- and metal-assisted pathways, and in this respect, this system is intermediate between DTPA-type and macrocyclic, DOTA-type chelates, for which the dissociation is predominated by metal- or proton-assisted pathways, respectively. The Cu(2+) exchange shows an unexpected pH dependency, with the observed rate constants decreasing with increasing proton concentration. The rate of water exchange, assessed by (17)O NMR, is extremely high on the [Gd(bp12c4)(H(2)O)(q)](+) complex (k(ex)(298) = (2.20 +/- 0.15) x 10(8) s(-1)), and is in the same order of magnitude as for the Gd(3+) aqua ion (k(ex)(298) = 8.0 x 10(8) s(-1)). In aqueous solution, the [Gd(bp12c4)(H(2)O)(q)](+) complex is present in hydration equilibrium between nine-coordinate, monohydrated, and ten-coordinate, bishydrated species. We attribute the fast exchange to the hydration equilibrium and to the flexible nature of the inner coordination sphere. The large negative value of the activation entropy (DeltaS = -35 +/- 8 J mol(-1) K(-1)) points to an associative character for the water exchange and suggests that water exchange on the nine-coordinate, monohydrated species is predominant in the overall exchange. Relaxometric and luminescence measurements on the Gd(3+) and Eu(3+) analogues, respectively, indicate strong binding of endogenous anions such as citrate, hydrogencarbonate, or phosphate to [Ln(bp12c4)](+) complexes (K(aff) = 280 +/- 20 M(-1), 630 +/- 50 M(-1), and 250 +/- 20 M(-1), respectively). In the ternary complexes, the inner sphere water molecules are fully replaced by the corresponding anion. Anion binding is favored by the positive charge of the [Ln(bp12c4)](+) complexes and the adjacent position of the two inner sphere water molecules. To obtain information about the structure of the ternary complexes, the [Gd(bp12c4)(HCO(3))] and [Gd(bp12c4)(H(2)PO(4))] systems were investigated by means of density functional theory calculations (B3LYP model). They show that anion coordination provokes an important lengthening of the distances between the donor atoms and the lanthanide ion. The coordination of phosphate induces a more important distortion of the metal coordination environment than the coordination of hydrogencarbonate, in accordance with a higher binding constant for HCO(3)(-) and a more important steric demand of phosphate.

Measurements for Sulfide-Mediated Inhibition of Myeloperoxidase Activity.

Oxidative stress-alleviating and inflammation-mediatory functions of hydrogen sulfide were reported to be key features of its biological actions. However, the underlying molecular mechanisms of these biological observations are not fully understood. In conditions where sulfide was proposed to be protective against oxidative stress- or inflammation-induced tissue damage (e.g., reperfusion injury, atherosclerosis, vascular inflammation), the reactive oxidant-producing function of a key neutrophil enzyme, myeloperoxidase, was reported to be a protagonist on the detrimental side. We recently described favorable interactions between sulfide and myeloperoxidase and proposed that the potent inhibition of myeloperoxidase activities could contribute to sulfide's beneficial functions in a number of cardiovascular pathologies. Our chapter is dedicated to aid future studies and drug development endeavors in this area by providing methodological guidance on how to assess the inhibitory potential of sulfide on myeloperoxidase enzymatic activities in isolated protein systems, in neutrophil homogenates, and in live neutrophil preparations.

Lanthanide complexes based on a 1,7-diaza-12-crown-4 platform containing picolinate pendants: a new structural entry for the design of magnetic resonance imaging contrast agents.

We have synthesized a new macrocyclic ligand, N,N'-Bis[(6-carboxy-2-pyridyl)methyl]-1,7-diaza-12-crown-4 (H 2bp12c4), designed for complexation of lanthanide ions in aqueous solution. The X-ray crystal structure of the Gd (III) complex shows that the metal ion is directly bound to the eight donor atoms of the bp12c4 ligand, the ninth coordination site being occupied by an oxygen atom of a carboxylate group of a neighboring [Gd(bp12c4)] (+) unit, while the structure of the Lu (III) analogue shows the metal ion being only eight-coordinate. The hydration numbers obtained from luminescence lifetime measurements in aqueous solution of the Eu (III) and Tb (III) complexes suggest an equilibrium in aqueous solution between a dihydrated ( q = 2), ten-coordinate and a monohydrated ( q = 1), nine-coordinate species. This has been confirmed by a variable temperature UV-vis spectrophotometric study on the Eu (III) complex. The structure of the complexes in solution has been investigated by (1)H and (13)C NMR spectroscopy, as well as by theoretical calculations performed at the DFT (B3LYP) level. The results indicate that the change in hydration number occurring around the middle of the lanthanide series is accompanied by a change in the conformation adopted by the complexes in solution [Delta(lambdalambdalambdalambda) for q = 2 and Lambda(deltalambdadeltalambda) for q = 1]. The structure calculated for the Yb (III) complex (Lambda(deltalambdadeltalambda)) is in good agreement with the experimental structure in solution, as demonstrated by the analysis of the Yb (III)-induced paramagnetic (1)H shifts.

Arthropods dataset from different genetically modified maize events and associated controls.

Arthropods from four genetically modified (GM) maize hybrids (coleopteran resistant, coleopteran and lepidopteran resistant, lepidopteran resistant+herbicide tolerant and coleopteran resistant and herbicide tolerant) and non-GM varieties were sampled during a two-year field assessment. A total number of 363 555 arthropod individuals were collected. This represents the most comprehensive arthropod dataset from GM maize, and together with weed data, is reasonable to determine functional groups of arthropods and interactions between species. Trophic groups identified from both phytophagous and predatory arthropods were previously considered non-target organisms on which possible detrimental effects of Bacillus thuringiensis (Bt) toxins may have been directly (phytophagous species) or indirectly (predators) detected. The high number of individuals and species and their dynamics through the maize growing season can predict that interactions are highly correlational, and can thus be considered a useful tool to assess potential deleterious effects of Bt toxins on non-target organisms, serving to develop biosafety risk hypotheses for invertebrates exposed to GM maize plants.

Hydrogen Sulfide Abrogates Hemoglobin-Lipid Interaction in Atherosclerotic Lesion.

The infiltration of red blood cells into atheromatous plaques is implicated in atherogenesis. Inside the lesion, hemoglobin (Hb) is oxidized to ferri- and ferrylHb which exhibit prooxidant and proinflammatory activities. Cystathione gamma-lyase- (CSE-) derived H2S has been suggested to possess various antiatherogenic actions. Expression of CSE was upregulated predominantly in macrophages, foam cells, and myofibroblasts of human atherosclerotic lesions derived from carotid artery specimens of patients. A similar pattern was observed in aortic lesions of apolipoprotein E-deficient mice on high-fat diet. We identified several triggers for inducing CSE expression in macrophages and vascular smooth muscle cells including heme, ferrylHb, plaque lipids, oxidized low-density lipoprotein, tumor necrosis factor-α, and interleukin-1ß. In the interplay between hemoglobin and atheroma lipids, H2S significantly mitigated oxidation of Hb preventing the formation of ferrylHb derivatives, therefore providing a novel function as a heme-redox-intermediate-scavenging antioxidant. By inhibiting Hb-lipid interactions, sulfide lowered oxidized Hb-mediated induction of adhesion molecules in endothelium and disruption of endothelial integrity. Exogenous H2S inhibited heme and Hb-mediated lipid oxidation of human atheroma-derived lipid and human complicated lesion. Our study suggests that the CSE/H2S system represents an atheroprotective pathway for removing or limiting the formation of oxidized Hb and lipid derivatives in the atherosclerotic plaque.

Effects of genetically modified maize events expressing Cry34Ab1, Cry35Ab1, Cry1F, and CP4 EPSPS proteins on arthropod complex food webs.

Four genetically modified (GM) maize (Zea mays L.) hybrids (coleopteran resistant, coleopteran and lepidopteran resistant, lepidopteran resistant and herbicide tolerant, coleopteran and herbicide tolerant) and its non-GM control maize stands were tested to compare the functional diversity of arthropods and to determine whether genetic modifications alter the structure of arthropods food webs. A total number of 399,239 arthropod individuals were used for analyses. The trophic groups' number and the links between them indicated that neither the higher magnitude of Bt toxins (included resistance against insect, and against both insects and glyphosate) nor the extra glyphosate treatment changed the structure of food webs. However, differences in the average trophic links/trophic groups were detected between GM and non-GM food webs for herbivore groups and plants. Also, differences in characteristic path lengths between GM and non-GM food webs for herbivores were observed. Food webs parameterized based on 2-year in-field assessments, and their properties can be considered a useful and simple tool to evaluate the effects of Bt toxins on non-target organisms.

Interactions of hydrogen sulfide with myeloperoxidase.

BACKGROUND AND PURPOSE: The actions of hydrogen sulfide in human physiology have been extensively studied and, although it is an essential mediator of many biological functions, the underlying molecular mechanisms of its actions are ill-defined. To elucidate the roles of sulfide in inflammation, we have investigated its interactions with human myeloperoxidase (MPO), a major contributor to inflammatory oxidative stress. EXPERIMENTAL APPROACH: The interactions of sulfide and MPO were investigated using electron paramagnetic resonance, electronic circular dichroism, UV-vis and stopped-flow spectroscopies. KEY RESULTS: We found favourable reactions between sulfide and the native-ferric enzyme as well as the MPO redox intermediates, ferrous MPO, compound I and compound II. Sulfide was a potent reversible inhibitor of MPO enzymic activity with an IC50 of 1 µM. In addition, the measured second-order rate constants for the reactions of sulfide with compound I [k = (1.1 ± 0.06) × 10(6) M(-1) s(-1)] and compound II [k = (2.0 ± 0.03) × 10(5) M(-1) s(-1)] suggest that sulfide is a potential substrate for MPO in vivo. CONCLUSION AND IMPLICATIONS: Endogenous levels of sulfide are likely to inhibit the activity of circulating and endothelium-bound MPO. The fully reversible inhibition suggests a mediatory role of sulfide on the oxidant-producing function of the enzyme. Furthermore, the efficient HOCl oxidation of sulfide to give polysulfides (recently recognized as important components of sulfide biology) together with MPO-catalysed sulfide oxidation and the lack of interaction between MPO and sulfide oxidation products, predict a modulatory role of MPO in sulfide signalling.

Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods.

BACKGROUND: Exosomes are emerging targets for biomedical research. However, suitable methods for the isolation of blood plasma-derived exosomes without impurities have not yet been described. AIM: Therefore, we investigated the efficiency and purity of exosomes isolated with potentially suitable methods; differential ultracentrifugation (UC) and size exclusion chromatography (SEC). METHODS AND RESULTS: Exosomes were isolated from rat and human blood plasma by various UC and SEC conditions. Efficiency was investigated at serial UC of the supernatant, while in case of SEC by comparing the content of exosomal markers of various fractions. Purity was assessed based on the presence of albumin. We found that the diameter of the majority of isolated particles fell into the size range of exosomes, however, albumin was also present in the preparations, when 1h UC at 4°C was applied. Furthermore, with this method only a minor fraction of total exosomes could be isolated from blood as deduced from the constant amount of exosomal markers CD63 and TSG101 detected after serial UC of rat blood plasma samples. By using UC for longer time or with shorter sedimentation distance at 4°C, or UC performed at 37°C, exosomal yield increased, but albumin impurity was still observed in the isolates, as assessed by transmission electron microscopy, dynamic light scattering and immunoblotting against CD63, TSG101 and albumin. Efficiency and purity were not different in case of using further diluted samples. By using SEC with different columns, we have found that although a minor fraction of exosomes can be isolated without significant albumin content on Sepharose CL-4B or Sephacryl S-400 columns, but not on Sepharose 2B columns, the majority of exosomes co-eluted with albumin. CONCLUSION: Here we show that it is feasible to isolate exosomes from blood plasma by SEC without significant albumin contamination albeit with low vesicle yield.

Short-term effects of different genetically modified maize varieties on arthropod food web properties: an experimental field assessment.

There is concern that genetically modified (GM) plants may have adverse affects on the arthropod biodiversity comprising agricultural landscapes. The present study report on a two year field experimental test of whether four different genotypic lines, some are novel with no previous field tests, of GM maize hybrids alter the structure of arthropod food webs that they harbour, relative to non-GM maize (control) that is widely used in agriculture. The different GM genotypes produced either Bt toxins, conferred glyphosate tolerance or a combination of the two traits. Quantitative food web analysis, based on short-term assessment assigning a total of 243,896 arthropod individuals collected from the treatments to their positions in food webs, revealed that complex and stable food webs persisted in each maize treatment. Moreover, food web structure remained relatively unchanged by the GM-genotype. The results suggest that at least in short-term period these particular GM maize genotypes will not have adverse effects on arthropod biota of agricultural landscapes.

Polysulfides link H2S to protein thiol oxidation.

AIMS: Hydrogen sulfide (H2S) is suggested to act as a gaseous signaling molecule in a variety of physiological processes. Its molecular mechanism of action was proposed to involve protein S-sulfhydration, that is, conversion of cysteinyl thiolates (Cys-S(-)) to persulfides (Cys-S-S(-)). A central and unresolved question is how H2S-that is, a molecule with sulfur in its lowest possible oxidation state (-2)-can lead to oxidative thiol modifications. RESULTS: Using the lipid phosphatase PTEN as a model protein, we find that the "H2S donor" sodium hydrosulfide (NaHS) leads to very rapid reversible oxidation of the enzyme in vitro. We identify polysulfides formed in NaHS solutions as the oxidizing species, and present evidence that sulfane sulfur is added to the active site cysteine. Polysulfide-mediated oxidation of PTEN was induced by all "H2S donors" tested, including sodium sulfide (Na2S), gaseous H2S, and morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate (GYY4137). Moreover, we show that polysulfides formed in H2S solutions readily modify PTEN inside intact cells. INNOVATION: Our results shed light on the previously unresolved question of how H2S leads to protein thiol oxidation, and suggest that polysulfides formed in solutions of H2S mediate this process. CONCLUSION: This study suggests that the effects that have been attributed to H2S in previous reports may in fact have been mediated by polysulfides. It also supports the notion that sulfane sulfur rather than sulfide is the actual in vivo agent of H2S signaling.

Development of an early-tier laboratory bioassay for assessing the impact of orally-active insecticidal compounds on larvae of Coccinella septempunctata (Coleoptera: Coccinellidae).

Early-tier studies are the initial step in the environmental risk assessment of genetically engineered plants on nontarget arthropods. They are conducted in the laboratory where surrogate species are exposed to higher concentrations of the arthropod-active compound than those expected to occur in the field. Thus, early-tier tests provide robust data and allow to make general conclusions about the susceptibility of the surrogate to the test substance. We have developed an early-tier test for assessing the toxicity of orally-active insecticidal compounds to larvae of the ladybird beetle Coccinella septempunctata L. (Coleoptera: Coccinellidae). Using potassium arsenate and the protease inhibitor E-64 as model compounds, we validated the bioassay set-up for C. septempunctata. Sucrose solution containing the test compound was offered to larvae for 24 h on the first day of each of its four larval instars. Subsequently, larvae were fed ad libitum with Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) eggs. Both compounds negatively affected C. septempunctata larval survival and development, and adult weight, indicating that the bioassay setup was able to detect dietary effects of insecticidal substances on the ladybird. Power analyses revealed that sample sizes of 20 or 45 per treatment are sufficient to detect 50 or 20% differences between the control and treatment groups, respectively, for the various measurement endpoints.