Gene Expression Plasticity Research Articles

Dietary and Endocrine Regulation of Endogenous Hydrogen Sulfide Production: Implications for Longevity.

Hydrogen sulfide (H2S) at the right concentration is associated with numerous health benefits in experimental organisms, ranging from protection from ischemia/reperfusion injury to life span extension. Given the considerable translation potential, two major strategies have emerged: supplementation of exogenous H2S and modulation of endogenous H2S metabolism. Recent Advances: Recently, it was reported that hepatic H2S production capacity is increased in two of the best-characterized mammalian models of life span extension, dietary restriction, and hypopituitary dwarfism, leading to new insights into dietary and hormonal regulation of endogenous H2S production together with broader changes in sulfur amino acid (SAA) metabolism with implications for DNA methylation and redox status. Here, we discuss the role of dietary SAAs and growth hormone (GH)/thyroid hormone (TH) signaling in regulation of endogenous H2S production largely via repression of H2S generating enzymes cystathionine γ-lyase (CGL) and cystathionine β-synthase (CBS) on the level of gene transcription, as well as reciprocal regulation of GH and TH signaling by H2S itself. We also discuss plasticity of CGL and CBS gene expression in response to environmental stimuli and the potential of the microbiome to impact overall H2S levels. The relative contribution of increased H2S to health span or lifespan benefits in models of extended longevity remains to be determined, as does the mechanism by which such benefits occur. Nonetheless, our ability to control H2S levels using exogenous H2S donors or by modifying the endogenous H2S production/consumption equilibrium has the potential to improve health and increase "shelf-life" across evolutionary boundaries, including our own. Antioxid. Redox Signal. 28, 1483-1502.

Plasticity of DNA methylation and gene expression under zinc deficiency in Arabidopsis roots.

DNA methylation is a heritable chromatin modification that maintains chromosome stability, regulates transposon silencing and appears to be involved in gene expression in response to environmental conditions. Environmental stress alters DNA methylation patterns that are correlated with gene expression differences. Here, genome-wide differential DNA methylation was identified upon prolonged zinc (Zn) deficiency, leading to hypo- and hypermethylated chromosomal regions. Preferential CpG methylation changes occurred in gene promoters and gene bodies, but did not overlap with transcriptional start sites. Methylation changes were also prominent in transposable elements. In contrast, non-CpG methylation differences were exclusively found in promoters of protein-coding genes and in transposable elements. Strongly Zn deficiency-induced genes and their promoters were mostly non-methylated, irrespective of Zn supply. Differential DNA methylation in the CpG and CHG, but not in the CHH context, was found close to a few up-regulated Zn deficiency genes. However, the transcriptional Zn deficiency response in roots appeared little correlated with associated DNA methylation changes in promoters or gene bodies. Furthermore, under Zn deficiency, developmental defects were identified in an Arabidopsis mutant lacking non-CpG methylation. The root methylome thus responds specifically to a micronutrient deficiency and is important for efficient Zn utilization at low availability, but the relationship of differential methylation and differentially expressed genes is surprisingly poor.

Plasticity and evolutionary divergence in gene expression associated with alternative habitat use in larvae of the European Fire Salamander.

Transcriptomes of organisms reveal differentiation associated with the use of different habitats. However, this leaves open how much of the observed differentiation can be attributed to genetic differences or to transcriptional plasticity. In this study, we disentangle causes of differential gene expression in larvae of the European fire salamander from the Kottenforst forest in Germany. Larvae inhabit permanent streams and ephemeral ponds and represent an example of a young evolutionary split associated with contrasting ecological conditions. We hypothesized that adaptation towards differences in water temperature plays a role because the thermal regime between stream and pond habitats differs notably. Tissue samples from tail fins of larvae were collected to study gene expression using microarrays. We found ample evidence for differentiation among larvae occupying different habitats in nature with 2,800 of 11,797 genes being differentially expressed. We then quantified transcriptional plasticity towards temperature and genetic differentiation based on controlled temperature laboratory experiments. Gene-by-environment interactions modelling revealed that 28% of the gene expression divergence observed among samples in nature could be attributed to plasticity related to water temperature. Expression patterns of only a small number of 101 genes were affected by the genotype. Our analysis demonstrates that effects of environmental factors must be taken into account to explain variation of gene expression in salamanders in nature. Notwithstanding, it provides first evidence that genetic factors determined gene expression divergence between pond and stream ecotypes and could be involved in adaptive evolution.

Distinct mechanisms coordinate transcription and translation under carbon and nitrogen starvation in Escherichia coli.

Bacteria adapt to environmental stress by producing proteins that provide stress protection. However, stress can severely perturb the kinetics of gene expression, disrupting protein production. Here, we characterized how Escherichia coli mitigates such perturbations under nutrient stress through the kinetic coordination of transcription and translation. We observed that, when translation became limiting under nitrogen starvation, transcription elongation slowed accordingly. This slowdown was mediated by (p)ppGpp, the alarmone whose primary role is thought to be promoter regulation. This kinetic coordination by (p)ppGpp was critical for the robust synthesis of gene products. Surprisingly, under carbon starvation, (p)ppGpp was dispensable for robust synthesis. Characterization of the underlying kinetics revealed that under carbon starvation, transcription became limiting, and translation aided transcription elongation. This mechanism naturally coordinated transcription with translation, alleviating the need for (p)ppGpp as a mediator. These contrasting mechanisms for coordination resulted in the condition-dependent effects of (p)ppGpp on global protein synthesis and starvation survival. Our findings reveal a kinetic aspect of gene expression plasticity, establishing (p)ppGpp as a condition-dependent global effector of gene expression.

Nuclear organization mediates cancer-compromised genetic and epigenetic control.

Nuclear organization is functionally linked to genetic and epigenetic regulation of gene expression for biological control and is modified in cancer. Nuclear organization supports cell growth and phenotypic properties of normal and cancer cells by facilitating physiologically responsive interactions of chromosomes, genes and regulatory complexes at dynamic three-dimensional microenvironments. We will review nuclear structure/function relationships that include: 1. Epigenetic bookmarking of genes by phenotypic transcription factors to control fidelity and plasticity of gene expression as cells enter and exit mitosis; 2. Contributions of chromatin remodeling to breast cancer nuclear morphology, metabolism and effectiveness of chemotherapy; 3. Relationships between fidelity of nuclear organization and metastasis of breast cancer to bone; 4. Dynamic modifications of higher-order inter- and intra-chromosomal interactions in breast cancer cells; 5. Coordinate control of cell growth and phenotype by tissue-specific transcription factors; 6. Oncofetal epigenetic control by bivalent histone modifications that are functionally related to sustaining the stem cell phenotype; and 7. Noncoding RNA-mediated regulation in the onset and progression of breast cancer. The discovery of components to nuclear organization that are functionally related to cancer and compromise gene expression have the potential for translation to innovative cancer diagnosis and targeted therapy.

Similarities in temperature‐dependent gene expression plasticity across timescales in threespine stickleback (Gasterosteus aculeatus)

Phenotypic plasticity occurs at a variety of timescales, but little is known about the degree to which plastic responses at different timescales are associated with similar underlying molecular processes, which is critical for assessing the effects of plasticity on evolutionary trajectories. To address this issue, we identified differential gene expression in response to developmental temperature in the muscle transcriptome of adult threespine stickleback (Gasterosteus aculeatus) exposed to 12, 18 and 24°C until hatch and then held at 18°C for 9months and compared these results to differential gene expression in response to adult thermal acclimation in stickleback developed at 18°C and then acclimated to 5 and 25°C as adults. Adult thermal acclimation affected the expression of 7,940 and 7,015 genes in response to cold and warm acclimation, respectively, and 4,851 of these genes responded in both treatments. In contrast, the expression of only 33 and 29 genes was affected by cold and warm development, respectively. The majority of the genes affected by developmental temperature were also affected by adult acclimation temperature. Many genes that were differentially expressed as a result of adult acclimation were associated with previously identified temperature-dependent effects on DNA methylation patterns, suggesting a role of epigenetic mechanisms in regulating gene expression plasticity during acclimation. Taken together, these results demonstrate similarities between the persistent effects of developmental plasticity on gene expression and the effects of adult thermal acclimation, emphasizing the potential for mechanistic links between plasticity acting at these different life stages.

Hypothesis: The Psychedelic Ayahuasca Heals Traumatic Memories via a Sigma 1 Receptor-Mediated Epigenetic-Mnemonic Process.

Ayahuasca ingestion modulates brain activity, neurotransmission, gene expression and epigenetic regulation. N,N-Dimethyltryptamine (DMT, one of the alkaloids in Ayahuasca) activates sigma 1 receptor (SIGMAR1) and others. SIGMAR1 is a multi-faceted stress-responsive receptor which promotes cell survival, neuroprotection, neuroplasticity, and neuroimmunomodulation. Simultaneously, monoamine oxidase inhibitors (MAOIs) also present in Ayahuasca prevent the degradation of DMT. One peculiarity of SIGMAR1 activation and MAOI activity is the reversal of mnemonic deficits in pre-clinical models. Since traumatic memories in post-traumatic stress disorder (PTSD) are often characterised by “repression” and PTSD patients ingesting Ayahuasca report the retrieval of such memories, it cannot be excluded that DMT-mediated SIGMAR1 activation and the concomitant MAOIs effects during Ayahuasca ingestion might mediate such “anti-amnesic” process. Here I hypothesise that Ayahuasca, via hyperactivation of trauma and emotional memory-related centres, and via its concomitant SIGMAR1- and MAOIs- induced anti-amnesic effects, facilitates the retrieval of traumatic memories, in turn making them labile (destabilised). As Ayahuasca alkaloids enhance synaptic plasticity, increase neurogenesis and boost dopaminergic neurotransmission, and those processes are involved in memory reconsolidation and fear extinction, the fear response triggered by the memory can be reprogramed and/or extinguished. Subsequently, the memory is stored with this updated significance. To date, it is unclear if new memories replace, co-exist with or bypass old ones. Although the mechanisms involved in memory are still debated, they seem to require the involvement of cellular and molecular events, such as reorganisation of homo and heteroreceptor complexes at the synapse, synaptic plasticity, and epigenetic re-modulation of gene expression. Since SIGMAR1 mobilises synaptic receptor, boosts synaptic plasticity and modulates epigenetic processes, such effects might be involved in the reported healing of traumatic memories in PTSD patients. If this theory proves to be true, Ayahuasca could come to represent the only standing pharmacological treatment which targets traumatic memories in PTSD. Lastly, since SIGMAR1 activation triggers both epigenetic and immunomodulatory programmes, the mechanism here presented could help understanding and treating other conditions in which the cellular memory is dysregulated, such as cancer, diabetes, autoimmune and neurodegenerative pathologies and substance addiction.

Erratum: Non-adaptive plasticity potentiates rapid adaptive evolution of gene expression in nature.

This corrects the article DOI: 10.1038/nature15256.

Adolescent THC exposure in female rats leads to cognitive deficits through a mechanism involving chromatin modifications in the prefrontal cortex

Increasing cannabis consumption among adolescents, studies that link its early use with mental illnesses, and the political debate on cannabis legalization together call for an urgent need to study molecular underpinnings of adolescent brain vulnerability. The emerging role of epigenetic mechanisms in psychiatric diseases led us to hypothesize that epigenetic alterations could play a role in causes and subsequent development of the depressive/psychotic-like phenotype induced by adolescent, but not adult, Δ9-tetrahydrocannabinol (THC) exposure in female rats. We performed a time-course analysis of histone modifications, chromatin remodelling enzymes and gene expression in the prefrontal cortex of female rats after adolescent and adult THC exposure. We also administered a specific epigenetic drug (chaetocin) with THC to investigate its impact on THC-induced behavioural alterations. Adolescent THC exposure induced alterations of selective histone modifications (mainly H3K9me3), impacting the expression of genes closely associated with synaptic plasticity. Changes in both histone modifications and gene expression were more widespread and intense after adolescent treatment, suggesting specific adolescent susceptibility. Adolescent THC exposure significantly increased Suv39H1 levels, which could account for the enhanced H3K9me3. Pharmacological blockade of H3K9me3 during adolescent THC treatment prevented THC-induced cognitive deficits, suggesting the relevant role played by H3K9me3 in THC-induced effects. Only female rats were investigated, and the expression studies were limited to a specific subset of genes. Through a mechanism involving SUV39H1, THC modifies histone modifications and, thereby, expression of plasticity genes. This pathway appears to be relevant for the development of cognitive deficits.

Increased socially mediated plasticity in gene expression accompanies rapid adaptive evolution.

Recent theory predicts that increased phenotypic plasticity can facilitate adaptation as traits respond to selection. When genetic adaptation alters the social environment, socially mediated plasticity could cause co-evolutionary feedback dynamics that increase adaptive potential. We tested this by asking whether neural gene expression in a recently arisen, adaptive morph of the field cricket Teleogryllus oceanicus is more responsive to the social environment than the ancestral morph. Silent males (flatwings) rapidly spread in a Hawaiian population subject to acoustically orienting parasitoids, changing the population's acoustic environment. Experimental altering crickets' acoustic environments during rearing revealed broad, plastic changes in gene expression. However, flatwing genotypes showed increased socially mediated plasticity, whereas normal-wing genotypes exhibited negligible expression plasticity. Increased plasticity in flatwing crickets suggests a coevolutionary process coupling socially flexible gene expression with the abrupt spread of flatwing. Our results support predictions that phenotypic plasticity should rapidly evolve to be more pronounced during early phases of adaptation.

Modularity Facilitates Flexible Tuning of Plastic and Evolutionary Gene Expression Responses during Early Divergence.

Gene expression changes have been recognized as important drivers of adaptation to changing environmental conditions. Little is known about the relative roles of plastic and evolutionary responses in complex gene expression networks during the early stages of divergence. Large gene expression data sets coupled with in silico methods for identifying coexpressed modules now enable systems genetics approaches also in nonmodel species for better understanding of gene expression responses during early divergence. Here, we combined gene coexpression analyses with population genetics to separate plastic and population (evolutionary) effects in expression networks using small salmonid populations as a model system. We show that plastic and population effects were highly variable among the six identified modules and that the plastic effects explained larger proportion of the total eigengene expression than population effects. A more detailed analysis of the population effects using a QST - FST comparison across 16,622 annotated transcripts revealed that gene expression followed neutral expectations within modules and at the global level. Furthermore, two modules showed enrichment for genes coding for early developmental traits that have been previously identified as important phenotypic traits in thermal responses in the same model system indicating that coexpression analysis can capture expression patterns underlying ecologically important traits. We suggest that module-specific responses may facilitate the flexible tuning of expression levels to local thermal conditions. Overall, our study indicates that plasticity and neutral evolution are the main drivers of gene expression variance in the early stages of thermal adaptation in this system.

Does a plant-eating insect's diet govern the evolution of insecticide resistance? Comparative tests of the pre-adaptation hypothesis.

According to the pre‐adaptation hypothesis, the evolution of insecticide resistance in plant‐eating insects co‐opts adaptations that initially evolved during chemical warfare with their host plants. Here, we used comparative statistics to test two predictions of this hypothesis: (i) Insects with more diverse diets should evolve resistance to more diverse insecticides. (ii) Feeding on host plants with strong or diverse qualitative chemical defenses should prime an insect lineage to evolve insecticide resistance. Both predictions are supported by our tests. What makes this especially noteworthy is that differences in the diets of plant‐eating insect species are typically ignored by the population genetic models we use to make predictions about insecticide resistance evolution. Those models surely capture some of the differences between host‐use generalists and specialists, for example, differences in population size and migration rates into treated fields, but they miss other potentially important differences, for example, differences in metabolic diversity and gene expression plasticity. Ignoring these differences could be costly.

Reconfigurable Analog Signal Processing by Living Cells.

Living cells are known for their capacity for versatile signal processing, particularly the ability to respond differently to the same stimuli using biochemical networks that integrate environmental signals and reconfigure their dynamic responses. However, the complexity of natural biological networks confounds the discovery of fundamental mechanisms behind versatile signaling. Here, we study one specific aspect of reconfigurable signal processing in which a minimal biological network integrates two signals, using one to reconfigure the network's transfer function with respect to the other, producing an emergent switch between induction and repression. In contrast to known mechanisms, the new mechanism reconfigures transfer functions through genetic networks without extensive protein-protein interactions. These results provide a novel explanation for the versatility of genetic programs, and suggest a new mechanism of signal integration that may govern flexibility and plasticity of gene expression.

Cofactors As Metabolic Sensors Driving Cell Adaptation in Physiology and Disease.

Chromatin architectures and epigenetic fingerprint regulation are fundamental for genetically determined biological processes. Chemical modifications of the chromatin template sensitize the genome to intracellular metabolism changes to set up diverse functional adaptive states. Accumulated evidence suggests that the action of epigenetic modifiers is sensitive to changes in dietary components and cellular metabolism intermediates, linking nutrition and energy metabolism to gene expression plasticity. Histone posttranslational modifications create a code that acts as a metabolic sensor, translating changes in metabolism into stable gene expression patterns. These observations support the notion that epigenetic reprograming-linked energy input is connected to the etiology of metabolic diseases and cancer. In the present review, we introduce the role of epigenetic cofactors and their relation with nutrient intake and we question the links between epigenetic regulation and the development of metabolic diseases.

Epigenetic Regulation of Memory-Therapeutic Potential for Disorders

Background: Memory is a vital function which declines in different physiological and pathological conditions such as aging and neurodegenerative diseases. Research in the past has reported that memory formation and consolidation require the precise expression of synaptic plasticity genes. However, little is known about the regulation of these genes. Epigenetic modification is now a well established mechanism that regulates synaptic plasticity genes and neuronal functions including memory. Therefore, we have reviewed the epigenetic regulation of memory and its therapeutic potential for memory dysfunction during aging and neurological disorders.Method: Research reports and online contents relevant to epigenetic regulation of memory during physiological and pathological conditions have been compiled and discussed.Results: Epigenetic modifications include mainly DNA methylation and hydroxymethylation, histone acetylation and methylation which involve chromatin modifying enzymes. These epigenetic marks change during memory formation and impairment due to dementia, aging and neuro-degeneration. As the epigenetic modifications are reversible, they can be modulated by enzyme inhibitors leading to the recovery of memory.Conclusion: Epigenetic modifications could be exploited as a potential therapeutic target to recover memory disorders during aging and pathological conditions.

Phenotypic plasticity in gene expression and physiological response in red drum Sciaenops ocellatus exposed to a long-term freshwater environment

Red drum (Sciaenops ocellatus) is a euryhaline fish commonly found in the Gulf of Mexico and along the Atlantic coast of North America. Because of high commercial demand and its euryhaline characteristics, aquaculture of this species has diversified from marine to low-salinity aquaculture systems. In recent years, interest in the feasibility of producing red drum in inland freshwater systems has grown and this prompted us to investigate its osmoregulatory capacity after rearing for 8months in a freshwater aquaculture system. We compared the activities of several genes and enzymes involved in the osmoregulatory process in freshwater-acclimatized (FW) and seawater (SW) red drum. The gene expression profiles were variable: the expression of genes encoding Na+/K+-ATPase (NKA) and the cystic fibrosis transmembrane regulator (CFTR) was slightly higher in SW than FW fish, while phosphoenolpyruvate carboxykinase (PEPCK) and the glucocorticoid receptor messenger RNA (mRNA) levels were higher in FW red drum. The total plasma K concentration was 60.3% lower, and gill NKA activity was 63.5% lower in FW than in SW fish. PEPCK activity was twofold higher in FW than in SW red drum. Similarly, liver glycogen was 60% higher in FW fish. In summary, both gene expression and the enzyme activity data support the phenotypic plasticity of red drum and suggest that the limited capacity for ion homeostasis observed, in particular the low plasma K concentration, was due to the composition of freshwater and does not necessarily reflect a physiological inability to osmoregulate.

The potential role of genetic assimilation during maize domestication.

Domestication research has largely focused on identification of morphological and genetic differences between extant populations of crops and their wild relatives. Little attention has been paid to the potential effects of environment despite substantial known changes in climate from the time of domestication to modern day. In recent research, the exposure of teosinte (i.e., wild maize) to environments similar to the time of domestication, resulted in a plastic induction of domesticated phenotypes in teosinte. These results suggest that early agriculturalists may have selected for genetic mechanisms that cemented domestication phenotypes initially induced by a plastic response of teosinte to environment, a process known as genetic assimilation. To better understand this phenomenon and the potential role of environment in maize domestication, we examined differential gene expression in maize (Zea mays ssp. mays) and teosinte (Zea mays ssp. parviglumis) between past and present conditions. We identified a gene set of over 2000 loci showing a change in expression across environmental conditions in teosinte and invariance in maize. In fact, overall we observed both greater plasticity in gene expression and more substantial changes in co-expressionnal networks in teosinte across environments when compared to maize. While these results suggest genetic assimilation played at least some role in domestication, genes showing expression patterns consistent with assimilation are not significantly enriched for previously identified domestication candidates, indicating assimilation did not have a genome-wide effect.

Gene expression stasis and plasticity following migration into a foreign environment.

Selection against migrants is key to maintaining genetic differences between populations linked by dispersal. However, migrants may mitigate fitness costs by proactively choosing among available habitats, or by phenotypic plasticity. We previously reported that a reciprocal transplant of lake and stream stickleback (Gasterosteus aculeatus) found little support for divergent selection. Here, we revisit that experiment to test whether phenotypic plasticity in gene expression may have helped migrants adjust to unfamiliar habitats. We measured gene expression profiles in stickleback via TagSeq and tested whether migrants between lake and stream habitats exhibited a plastic response to their new environment that allowed them to converge on the expression profile of adapted natives. We report extensive gene expression differences between genetically divergent lake and stream stickleback, despite gene flow. But for many genes, expression was highly plastic. Fish transplanted into the adjoining habitat partially converged on the expression profile typical of natives from their new habitat. This suggests that expression plasticity may soften the impact of migration. Nonetheless, lake and stream fish differed in survival rates and parasite infection rates in our study, implying that expression plasticity is not fast or extensive enough to fully homogenize fish performance.

Genetic accommodation in the wild: evolution of gene expression plasticity during character displacement.

Ecological character displacement is considered crucial in promoting diversification, yet relatively little is known of its underlying mechanisms. We examined whether evolutionary shifts in gene expression plasticity ('genetic accommodation') mediate character displacement in spadefoot toads. Where Spea bombifrons and S. multiplicata occur separately in allopatry (the ancestral condition), each produces alternative, diet-induced, larval ecomorphs: omnivores, which eat detritus, and carnivores, which specialize on shrimp. By contrast, where these two species occur together in sympatry (the derived condition), selection to minimize competition for detritus has caused S. bombifrons to become nearly fixed for producing only carnivores, suggesting that character displacement might have arisen through an extreme form of genetic accommodation ('genetic assimilation') in which plasticity is lost. Here, we asked whether we could infer a signature of this process in regulatory changes of specific genes. In particular, we investigated whether genes that are normally expressed more highly in one morph ('biased' genes) have evolved reduced plasticity in expression levels among S. bombifrons from sympatry compared to S. bombifrons from allopatry. We reared individuals from sympatry vs. allopatry on detritus or shrimp and measured the reaction norms of nine biased genes. Although different genes displayed different patterns of gene regulatory evolution, the combined gene expression profiles revealed that sympatric individuals had indeed lost the diet-induced gene expression plasticity present in allopatric individuals. Our data therefore provide one of the few examples from natural populations in which genetic accommodation/assimilation can be traced to regulatory changes of specific genes. Such genetic accommodation might mediate character displacement in many systems.

Contrasting gene expression programs correspond with predator-induced phenotypic plasticity within and across generations in Daphnia.

Research has shown that a change in environmental conditions can alter the expression of traits during development (i.e., "within-generation phenotypic plasticity") as well as induce heritable phenotypic responses that persist for multiple generations (i.e., "transgenerational plasticity", TGP). It has long been assumed that shifts in gene expression are tightly linked to observed trait responses at the phenotypic level. Yet, the manner in which organisms couple within- and TGP at the molecular level is unclear. Here we tested the influence of fish predator chemical cues on patterns of gene expression within- and across generations using a clone of Daphnia ambigua that is known to exhibit strong TGP but weak within-generation plasticity. Daphnia were reared in the presence of predator cues in generation 1, and shifts in gene expression were tracked across two additional asexual experimental generations that lacked exposure to predator cues. Initial exposure to predator cues in generation 1 was linked to ~50 responsive genes, but such shifts were 3-4× larger in later generations. Differentially expressed genes included those involved in reproduction, exoskeleton structure and digestion; major shifts in expression of genes encoding ribosomal proteins were also identified. Furthermore, shifts within the first-generation and transgenerational shifts in gene expression were largely distinct in terms of the genes that were differentially expressed. Such results argue that the gene expression programmes involved in within- vs. transgeneration plasticity are fundamentally different. Our study provides new key insights into the plasticity of gene expression and how it relates to phenotypic plasticity in nature.