The integration of reactive oxygen species (ROS) and calcium signalling in abiotic stress responses.

Reactive oxygen species (ROS) and calcium (Ca2+ ) signalling are interconnected in the perception and transmission of environmental signals that control plant growth, development and defence. The concept that systemically propagating Ca2+ and ROS waves function together with electric signals in directional cell-to-cell systemic signalling and even plant-to-plant communication, is now firmly imbedded in the literature. However, relatively few mechanistic details are available regarding the management of ROS and Ca2+ signals at the molecular level, or how synchronous and independent signalling might be achieved in different cellular compartments. This review discusses the proteins that may serve as nodes or connecting bridges between the different pathways during abiotic stress responses, highlighting the crosstalk between ROS and Ca2+ pathways in cell signalling. We consider putative molecular switches that connect these signalling pathways and the molecular machinery that achieves the synergistic operation of ROS and Ca2+ signals.

Signal transduction mechanisms in heme-based globin-coupled oxygen sensors with a focus on a histidine kinase (AfGcHK) and a diguanylate cyclase (YddV or EcDosC).

Heme is a vital cofactor of proteins with roles in oxygen transport (e.g. hemoglobin), storage (e.g. myoglobin), and activation (e.g. P450) as well as electron transfer (e.g. cytochromes) and many other functions. However, its structural and functional role in oxygen sensing proteins differs markedly from that in most other enzymes, where it serves as a catalytic or functional center. This minireview discusses the mechanism of signal transduction in two heme-based oxygen sensors: the histidine kinase AfGcHK and the diguanylate cyclase YddV (EcDosC), both of which feature a heme-binding domain containing a globin fold resembling that of hemoglobin and myoglobin.

Control of Excitation/Inhibition Balance in a Hippocampal Circuit by Calcium Sensor Protein Regulation of Presynaptic Calcium Channels.

Activity-dependent regulation controls the balance of synaptic excitation to inhibition in neural circuits, and disruption of this regulation impairs learning and memory and causes many neurological disorders. The molecular mechanisms underlying short-term synaptic plasticity are incompletely understood, and their role in inhibitory synapses remains uncertain. Here we show that regulation of voltage-gated calcium (Ca2+) channel type 2.1 (CaV2.1) by neuronal Ca2+ sensor (CaS) proteins controls synaptic plasticity and excitation/inhibition balance in a hippocampal circuit. Prevention of CaS protein regulation by introducing the IM-AA mutation in CaV2.1 channels in male and female mice impairs short-term synaptic facilitation at excitatory synapses of CA3 pyramidal neurons onto parvalbumin (PV)-expressing basket cells. In sharp contrast, the IM-AA mutation abolishes rapid synaptic depression in the inhibitory synapses of PV basket cells onto CA1 pyramidal neurons. These results show that CaS protein regulation of facilitation and inactivation of CaV2.1 channels controls the direction of short-term plasticity at these two synapses. Deletion of the CaS protein CaBP1/caldendrin also blocks rapid depression at PV-CA1 synapses, implicating its upregulation of inactivation of CaV2.1 channels in control of short-term synaptic plasticity at this inhibitory synapse. Studies of local-circuit function revealed reduced inhibition of CA1 pyramidal neurons by the disynaptic pathway from CA3 pyramidal cells via PV basket cells and greatly increased excitation/inhibition ratio of the direct excitatory input versus indirect inhibitory input from CA3 pyramidal neurons to CA1 pyramidal neurons. This striking defect in local-circuit function may contribute to the dramatic impairment of spatial learning and memory in IM-AA mice.SIGNIFICANCE STATEMENT Many forms of short-term synaptic plasticity in neuronal circuits rely on regulation of presynaptic voltage-gated Ca2+ (CaV) channels. Regulation of CaV2.1 channels by neuronal calcium sensor (CaS) proteins controls short-term synaptic plasticity. Here we demonstrate a direct link between regulation of CaV2.1 channels and short-term synaptic plasticity in native hippocampal excitatory and inhibitory synapses. We also identify CaBP1/caldendrin as the calcium sensor interacting with CaV2.1 channels to mediate rapid synaptic depression in the inhibitory hippocampal synapses of parvalbumin-expressing basket cells to CA1 pyramidal cells. Disruption of this regulation causes altered short-term plasticity and impaired balance of hippocampal excitatory to inhibitory circuits.

Calcium sensor regulation of the CaV2.1 Ca2+ channel contributes to long-term potentiation and spatial learning.

Many forms of short-term synaptic plasticity rely on regulation of presynaptic voltage-gated Ca2+ type 2.1 (CaV2.1) channels. However, the contribution of regulation of CaV2.1 channels to other forms of neuroplasticity and to learning and memory are not known. Here we have studied mice with a mutation (IM-AA) that disrupts regulation of CaV2.1 channels by calmodulin and related calcium sensor proteins. Surprisingly, we find that long-term potentiation (LTP) of synaptic transmission at the Schaffer collateral-CA1 synapse in the hippocampus is substantially weakened, even though this form of synaptic plasticity is thought to be primarily generated postsynaptically. LTP in response to θ-burst stimulation and to 100-Hz tetanic stimulation is much reduced. However, a normal level of LTP can be generated by repetitive 100-Hz stimulation or by depolarization of the postsynaptic cell to prevent block of NMDA-specific glutamate receptors by Mg2+ The ratio of postsynaptic responses of NMDA-specific glutamate receptors to those of AMPA-specific glutamate receptors is decreased, but the postsynaptic current from activation of NMDA-specific glutamate receptors is progressively increased during trains of stimuli and exceeds WT by the end of 1-s trains. Strikingly, these impairments in long-term synaptic plasticity and the previously documented impairments in short-term synaptic plasticity in IM-AA mice are associated with pronounced deficits in spatial learning and memory in context-dependent fear conditioning and in the Barnes circular maze. Thus, regulation of CaV2.1 channels by calcium sensor proteins is required for normal short-term synaptic plasticity, LTP, and spatial learning and memory in mice.

Altered short-term synaptic plasticity and reduced muscle strength in mice with impaired regulation of presynaptic CaV2.1 Ca2+ channels.

Facilitation and inactivation of P/Q-type calcium (Ca(2+)) currents through the regulation of voltage-gated Ca(2+) (CaV) 2.1 channels by Ca(2+) sensor (CaS) proteins contributes to the facilitation and rapid depression of synaptic transmission in cultured neurons that transiently express CaV2.1 channels. To examine the modulation of endogenous CaV2.1 channels by CaS proteins in native synapses, we introduced a mutation (IM-AA) into the CaS protein-binding site in the C-terminal domain of CaV2.1 channels in mice, and tested synaptic facilitation and depression in neuromuscular junction synapses that use exclusively CaV2.1 channels for Ca(2+) entry that triggers synaptic transmission. Even though basal synaptic transmission was unaltered in the neuromuscular synapses in IM-AA mice, we found reduced short-term facilitation in response to paired stimuli at short interstimulus intervals in IM-AA synapses. In response to trains of action potentials, we found increased facilitation at lower frequencies (10-30 Hz) in IM-AA synapses accompanied by slowed synaptic depression, whereas synaptic facilitation was reduced at high stimulus frequencies (50-100 Hz) that would induce strong muscle contraction. As a consequence of altered regulation of CaV2.1 channels, the hindlimb tibialis anterior muscle in IM-AA mice exhibited reduced peak force in response to 50 Hz stimulation and increased muscle fatigue. The IM-AA mice also had impaired motor control, exercise capacity, and grip strength. Taken together, our results indicate that regulation of CaV2.1 channels by CaS proteins is essential for normal synaptic plasticity at the neuromuscular junction and for muscle strength, endurance, and motor coordination in mice in vivo.

Calcium sensor regulation of the CaV2.1 Ca2+ channel contributes to short-term synaptic plasticity in hippocampal neurons.

Short-term synaptic plasticity is induced by calcium (Ca(2+)) accumulating in presynaptic nerve terminals during repetitive action potentials. Regulation of voltage-gated CaV2.1 Ca(2+) channels by Ca(2+) sensor proteins induces facilitation of Ca(2+) currents and synaptic facilitation in cultured neurons expressing exogenous CaV2.1 channels. However, it is unknown whether this mechanism contributes to facilitation in native synapses. We introduced the IM-AA mutation into the IQ-like motif (IM) of the Ca(2+) sensor binding site. This mutation does not alter voltage dependence or kinetics of CaV2.1 currents, or frequency or amplitude of spontaneous miniature excitatory postsynaptic currents (mEPSCs); however, synaptic facilitation is completely blocked in excitatory glutamatergic synapses in hippocampal autaptic cultures. In acutely prepared hippocampal slices, frequency and amplitude of mEPSCs and amplitudes of evoked EPSCs are unaltered. In contrast, short-term synaptic facilitation in response to paired stimuli is reduced by ∼ 50%. In the presence of EGTA-AM to prevent global increases in free Ca(2+), the IM-AA mutation completely blocks short-term synaptic facilitation, indicating that synaptic facilitation by brief, local increases in Ca(2+) is dependent upon regulation of CaV2.1 channels by Ca(2+) sensor proteins. In response to trains of action potentials, synaptic facilitation is reduced in IM-AA synapses in initial stimuli, consistent with results of paired-pulse experiments; however, synaptic depression is also delayed, resulting in sustained increases in amplitudes of later EPSCs during trains of 10 stimuli at 10-20 Hz. Evidently, regulation of CaV2.1 channels by CaS proteins is required for normal short-term plasticity and normal encoding of information in native hippocampal synapses.

Imaging and analysis of genetically encoded calcium indicators linking neural circuits and behaviors.

Confirming the direct link between neural circuit activity and animal behavior has been a principal aim of neuroscience. The genetically encoded calcium indicator (GECI), which binds to calcium ions and emits fluorescence visualizing intracellular calcium concentration, enables detection of in vivo neuronal firing activity. Various GECIs have been developed and can be chosen for diverse purposes. These GECI-based signals can be acquired by several tools including two-photon microscopy and microendoscopy for precise or wide imaging at cellular to synaptic levels. In addition, the images from GECI signals can be analyzed with open source codes including constrained non-negative matrix factorization for endoscopy data (CNMF_E) and miniscope 1-photon-based calcium imaging signal extraction pipeline (MIN1PIPE), and considering parameters of the imaged brain regions (e.g., diameter or shape of soma or the resolution of recorded images), the real-time activity of each cell can be acquired and linked with animal behaviors. As a result, GECI signal analysis can be a powerful tool for revealing the functions of neuronal circuits related to specific behaviors.

Electrophoretic mobility shift in native gels indicates calcium-dependent structural changes of neuronal calcium sensor proteins.

In proteins of the neuronal calcium sensor (NCS) family, changes in structure as well as function are brought about by the binding of calcium. In this article, we demonstrate that these structural changes, solely due to calcium binding, can be assessed through electrophoresis in native gels. The results demonstrate that the NCS proteins undergo ligand-dependent conformational changes that are detectable in native gels as a gradual decrease in mobility with increasing calcium but not other tested divalent cations such as magnesium, strontium, and barium. Surprisingly, such a gradual change over the entire tested range is exhibited only by the NCS proteins but not by other tested calcium-binding proteins such as calmodulin and S100B, indicating that the change in mobility may be linked to a unique NCS family feature--the calcium-myristoyl switch. Even within the NCS family, the changes in mobility are characteristic of the protein, indicating that the technique is sensitive to the individual features of the protein. Thus, electrophoretic mobility on native gels provides a simple and elegant method to investigate calcium (small ligand)-induced structural changes at least in the superfamily of NCS proteins.

Control of catalysis in globin coupled adenylate cyclase by a globin-B domain.

The globin coupled heme containing adenylate cyclase from Leishmania major (HemAC-Lm) has two globin domains (globin-A and globin-B). Globin-B domain (210-360 amino acids) may guide the interaction between globin-A and adenylate cyclase domains for the regulation of catalysis. We investigated the role of globin-B domain in HemAC-Lm by constructing a series of mutants namely Δ209 (209 amino acids deleted), Δ360 (360 amino acids deleted), H161A, H311A and H311A-Δ209. Spectroscopic data suggest that the Δ209 and H311A-Δ209 proteins to be Fe(2+)-O2 form and apo form, respectively, indicating that His311 residue in the globin-B domain is crucial for heme binding in Δ209 protein. However, the H311A mutant is still of the Fe(2+)-O2 form whereas H161A mutant shows the apo form, indicating that only His161 residue in the globin-A domain is responsible for heme binding in full length enzyme. cAMP measurements suggest that the activities of Δ360 and Δ209 proteins were ∼10 and ∼1000 times lesser than full length enzyme, respectively, leading to the fact that globin-B domain inhibited catalysis rather than activation in absence of globin-A domain. These data suggest that the O2 bound globin-A domain in HemAC-Lm allows the best cooperation of the catalytic domain interactions to generate optimum cAMP.

Specificity of the Metalloregulator CueR for Monovalent Metal Ions: Possible Functional Role of a Coordinated Thiol?

Metal-ion-responsive transcriptional regulators within the MerR family effectively discriminate between mono- and divalent metal ions. Herein we address the origin of the specificity of the CueR protein for monovalent metal ions. Several spectroscopic techniques were employed to study Ag(I) , Zn(II) , and Hg(II) binding to model systems encompassing the metal-ion-binding loop of CueR from E. coli and V. cholerae. In the presence of Ag(I) , a conserved cysteine residue displays a pKa  value for deprotonation of the thiol that is close to the physiological pH value. This property is only observed with the monovalent metal ion. Quantum chemically optimized structures of the CueR metal site with Cys 112 protonated demonstrate that the conserved Ser 77 backbone carbonyl oxygen atom from the other monomer of the homodimer is "pulled" towards the metal site. A common allosteric mechanism of the metalloregulatory members of the MerR family is proposed. For CueR, the mechanism relies on the protonation of Cys 112.

Illuminating the early signaling pathway of a fungal light-oxygen-voltage photoreceptor.

Circadian clocks are molecular timekeepers encountered in a wide variety of organisms, which allow to adapt the cell's metabolism and behavior to the daily and seasonal periods. Their function is regulated by light-sensing proteins, among which Vivid, a light-oxygen-voltage (LOV) sensitive domain of the fungus Neurospora crassa, constitutes one of the most prominent examples. Although the major photochemical and structural changes during the photocycle of this photosensor have been elucidated through experimental means, its signal transduction pathway is still poorly resolved at the molecular level. In this article, we show through molecular dynamics simulation that the primary steps after adduct formation involve a switch of Gln182 in vicinity of the chromophore FAD (flavin-adenine-dinucleotide), followed by a coupling between the Iß- and Hß-strands through H-bond formation between Gln182 and Asn161 as well as subsequent weakening of the H-bonding interaction between the Iß- and Aß-strands. These processes then induce a reorientation of the Aß-Bß-loop with respect to the protein core as well as a simultaneous contraction of the partially unfolded α-helix onto the α-Aß-linker at the Ncap. Finally, we demonstrate through additional dimer simulations that the light-induced conformational changes, observed in the monomeric case, play a decisive role in controlling the dimerization tendency of Vivid with its partner domains and that the light-state homodimer shows a much larger affinity for aggregation than the dark state.

Optical oxygen micro- and nanosensors for plant applications.

Pioneered by Clark's microelectrode more than half a century ago, there has been substantial interest in developing new, miniaturized optical methods to detect molecular oxygen inside cells. While extensively used for animal tissue measurements, applications of intracellular optical oxygen biosensors are still scarce in plant science. A critical aspect is the strong autofluorescence of the green plant tissue that interferes with optical signals of commonly used oxygen probes. A recently developed dual-frequency phase modulation technique can overcome this limitation, offering new perspectives for plant research. This review gives an overview on the latest optical sensing techniques and methods based on phosphorescence quenching in diverse tissues and discusses the potential pitfalls for applications in plants. The most promising oxygen sensitive probes are reviewed plus different oxygen sensing structures ranging from micro-optodes to soluble nanoparticles. Moreover, the applicability of using heterologously expressed oxygen binding proteins and fluorescent proteins to determine changes in the cellular oxygen concentration are discussed as potential non-invasive cellular oxygen reporters.

A catalogue of signal molecules that interact with sensor kinases, chemoreceptors and transcriptional regulators.

Bacteria have evolved many different signal transduction systems that sense signals and generate a variety of responses. Generally, most abundant are transcriptional regulators, sensor histidine kinases and chemoreceptors. Typically, these systems recognize their signal molecules with dedicated ligand-binding domains (LBDs), which, in turn, generate a molecular stimulus that modulates the activity of the output module. There are an enormous number of different LBDs that recognize a similarly diverse set of signals. To give a global perspective of the signals that interact with transcriptional regulators, sensor kinases and chemoreceptors, we manually retrieved information on the protein-ligand interaction from about 1,200 publications and 3D structures. The resulting 811 proteins were classified according to the Pfam family into 127 groups. These data permit a delineation of the signal profiles of individual LBD families as well as distinguishing between families that recognize signals in a promiscuous manner and those that possess a well-defined ligand range. A major bottleneck in the field is the fact that the signal input of many signaling systems is unknown. The signal repertoire reported here will help the scientific community design experimental strategies to identify the signaling molecules for uncharacterised sensor proteins.

Mechanisms of Synaptic Vesicle Exo- and Endocytosis.

Within 1 millisecond of action potential arrival at presynaptic terminals voltage-gated Ca2+ channels open. The Ca2+ channels are linked to synaptic vesicles which are tethered by active zone proteins. Ca2+ entrance into the active zone triggers: (1) the fusion of the vesicle and exocytosis, (2) the replenishment of the active zone with vesicles for incoming exocytosis, and (3) various types of endocytosis for vesicle reuse, dependent on the pattern of firing. These time-dependent vesicle dynamics are controlled by presynaptic Ca2+ sensor proteins, regulating active zone scaffold proteins, fusion machinery proteins, motor proteins, endocytic proteins, several enzymes, and even Ca2+ channels, following the decay of Ca2+ concentration after the action potential. Here, I summarize the Ca2+-dependent protein controls of synchronous and asynchronous vesicle release, rapid replenishment of the active zone, endocytosis, and short-term plasticity within 100 msec after the action potential. Furthermore, I discuss the contribution of active zone proteins to presynaptic plasticity and to homeostatic readjustment during and after intense activity, in addition to activity-dependent endocytosis.

Moonlighting Proteins: The Case of the Hexokinases.

Moonlighting proteins are defined as proteins with two or more functions that are unrelated and independent to each other, so that inactivation of one of them should not affect the second one and vice versa. Intriguingly, all the glycolytic enzymes are described as moonlighting proteins in some organisms. Hexokinase (HXK) is a critical enzyme in the glycolytic pathway and displays a wide range of functions in different organisms such as fungi, parasites, mammals, and plants. This review discusses HXKs moonlighting functions in depth since they have a profound impact on the responses to nutritional, environmental, and disease challenges. HXKs' activities can be as diverse as performing metabolic activities, as a gene repressor complexing with other proteins, as protein kinase, as immune receptor and regulating processes like autophagy, programmed cell death or immune system responses. However, most of those functions are particular for some organisms while the most common moonlighting HXK function in several kingdoms is being a glucose sensor. In this review, we also analyze how different regulation mechanisms cause HXK to change its subcellular localization, oligomeric or conformational state, the response to substrate and product concentration, and its interactions with membrane, proteins, or RNA, all of which might impact the HXK moonlighting functions.

Preharvest Spray Hexanal Formulation Enhances Postharvest Quality in 'Honeycrisp' Apples by Regulating Phospholipase D and Calcium Sensor Proteins Genes.

'Honeycrisp' (Malus domestica Borkh.), a premium applecultivar, is highly susceptible to bitter pit and decline in quality during long-term storage. In order to enhance the quality, an aqueous composition containing hexanal was applied as a preharvest spray. The effects of hexanal were assessed on the treated fruit and compared with HarvistaTM (a sprayable 1-Methylcyclopropene based commercial formulation) applied and control fruit under both cold (2.5 °C; four months) and cold after room temperature storage (20 °C; 14 days) conditions. Color, firmness, and total soluble solids (TSS) did not show a significant change in response to any treatment at harvest, while abscisic acid (ABA) significantly reduced and tryptophan increased in response to hexanal, compared to HarvistaTM and control. The treatment effects on quality traits were observed during storage. Both hexanal and HarvistaTM sprayed apples had higher TSS under both cold and room temperature storage. In addition, both sprays enhanced firmness at room temperature storage. However, the effects of sprays on other quality traits showed a different pattern. Apples sprayed with hexanal had lower phospholipase D enzyme (PLD) activity, lower incidence of bitter pit, and decreased expression of MdPLDα1 compared to HarvistaTM and control. On the other hand, HarvistaTM treated fruit produced lower ethylene. Both sprays decreased the expression of MdPLDα4, MdCaM2, MdCaM4 and MdCML18 genes. Generally, PLD alpha has a direct role in promoting fruit senescence, whereas the calcium senor proteins (CaM/CMLs) may involve in fruit ripening process via calcium and ethylene interactions. Therefore, improved postharvest qualities, including the lower incidence of bitter pit in hexanal treated 'Honeycrisp', may be associated with lower membrane damage due to lower PLD enzyme activity and decreased expression of MdPLDα1 and MdPLDα4 genes throughout the storage period.

Quantitative Determination of Ca2+-binding to Ca2+-sensor Proteins by Isothermal Titration Calorimetry.

Diverse and complex molecular recognitions are central elements of signal transduction cascades. The strength and nature of these interaction modes can be determined by different experimental approaches. Among those, Isothermal titration calorimetry (ITC) offers certain advantages by providing binding constants and thermodynamic parameters from titration series without a need to label or immobilize one or more interaction partners. Furthermore, second messenger homeostasis involving Ca2+-ions requires in particular knowledge about stoichiometries and affinities of Ca2+-binding to Ca2+-sensor proteins or Ca2+-dependent regulators, which can be obtained by employing ITC. We used ITC to measure these parameters for a set of neuronal Ca2+-sensor proteins operating in photoreceptor cells. Here, we present a step wise protocol to (a) measure Ca2+ interaction with the Ca2+-sensor guanylate cyclase-activating protein 1, (b) to design an ITC experiment and prepare samples, (c) to remove Ca2+ nearly completely from Ca2+ binding proteins without using a chelating agent like EGTA.

Mapping Calcium-Sensitive Regions in GCAPs by Site-Specific Fluorescence Labelling.

Signal transduction processes that are under control of changes in cytoplasmic Ca2+-concentration involve Ca2+-sensor proteins, which often undergo pronounced conformational transitions triggered by Ca2+. Consequences of conformational changes can be the structural rearrangement of single amino acids, exposition of small patches of several amino acids, or the movement of whole protein regions or domains. Furthermore, these conformational changes can lead to the exposure or movement of posttranslationally attached acyl groups. These processes could then control the function of target proteins, for example, by Ca2+-dependent protein-protein interaction. Fluorescence spectroscopy allows for mapping these Ca2+-sensitive regions but needs site-specific fluorescence labelling. We describe the application of a new group of diaminoterephthalate-derived fluorescence probes targeting either cysteines in guanylate cyclase-activating proteins, named GCAPs, or azide moieties in covalently attached acyl groups. By monitoring Ca2+-dependent changes in fluorescence emission, we identify Ca2+-sensitive protein regions in GCAPs and correlate conformational changes to protein function.

Determination of Hydrodynamic Radius of Proteins by Size Exclusion Chromatography.

Size exclusion chromatography (SEC) or gel filtration is a hydrodynamic technique that separates molecules in solution as a function of their size and shape. In the case of proteins, the hydrodynamic value that can be experimentally derived is the Stokes radius (Rs), which is the radius of a sphere with the same hydrodynamic properties (i.e., frictional coefficient) as the biomolecule. Determination of Rs by SEC has been widely used to monitor conformational changes induced by the binding of calcium (Ca2+) to many Ca2+-sensor proteins. For this class of proteins, SEC separation is based not just on the variation in protein size following Ca2+ binding, but likely arises from changes in the hydration shell structure. This protocol aims to describe a gel filtration experiment on a prepacked column using a Fast Protein Liquid Chromatography (FPLC) system to determine the Rs of proteins with some indications that are specific for Ca2+ sensor proteins.

Isothermal Titration Calorimetry to Determine Apparent Dissociation Constants (K d) and Stoichiometry of Interaction (n) of C-di-GMP Binding Proteins.

Isothermal titration calorimetry (ITC) is a commonly used biophysical technique that enables the quantitative characterization of intermolecular interactions in solution. Based on enthalpy changes (ΔH) upon titration of the binding partner (e.g., a small-molecule ligand such as c-di-GMP) to the molecule of interest (e.g., a receptor protein), the resulting binding isotherms provide information on the equilibrium association/dissociation constants (K a, K d) and stoichiometry of binding (n), as well as on changes in the Gibbs free energy (ΔG) and entropy (ΔS) along the interaction. Here we present ITC experiments used for the characterization of c-di-GMP binding proteins and discuss advantages and potential caveats in the interpretation of results.