Epigenetics and transgenerational inheritance

Epigenetic modifications can alter the function of genes. The epigenetics changes are caused by environmental effects, which lead to chemical modifications of the DNA or the chromatin. The mechanisms involve the influence of small interfering siRNAs on gene silencing. Epigenetic changes normally last only during the life‐time of an individual and are erased in embryos and eggs for a naive progeny. The genomes are reprogrammed and the chemical modifications removed to restart the next generation. However, there are mechanisms that allow the genome to escape from such a clearing effect so that modifications can be transmitted to one or more subsequent generations. In the germline of animal cells small RNAs, including piRNAs, have evolved which guarantee a higher degree of fidelity for transmission of genetic information, guarding especially against the detrimental effect caused by transposon activity. piRNA is essential for transposon silencing for survival of a species and protection of subsequent generations. Inactivation of piRNA results in abundant transposon activity and sperm infertility. The effect in humans has been described but is less distinct. Some stress‐induced epigenetic changes are transitory in mice and can be reversed by a change of environment or lifestyle.


Transgenerational inheritance
Inheritance of epigenetic modifications through several generations has recently attracted attention.Acquired properties have been described that can modify the genes by chemical reactions without altering the primary structure and sequence of the DNA.The epigenetic modifications can be transmitted in some cases to the next generation or even multiple subsequent generations.Such epigenetic changes and inheritance result in non-genetic transmission of acquired complex phenotypes from parents to offspring.This has been observed using epidemiological studies, mostly in animals, where environmental effects can be more easily controlled than in humans.
Epigenetic changes, which are accumulated during a lifetime, are mostly erased during development of eggs and sperm.The epigenetic changes include RNA modification, methylation of the DNA and chemical modifications of histones due to environmental influences.For transgenerational transmission the epigenetic changes have to affect the germline, which can then also affect somatic cells and development (Chen et al., 2016).Transport of RNA by extracellular vesicles through sperm may also affect the developing progeny (Sciamanna et al., 2019).
Environmental factors involved in transgenerational inheritance include temperature, nutrients, hunger, exposure to chemicals and toxins and endocrine factors.The inherited traits may be minor but some can also lead to more severe diseases and disorders.This may depend on dosage or other parameters of the environmental factors.
Inheritance of acquired characteristics has long been discussed in the context of Lamarckism (Heard & Martienssen, 2014).Now it may be necessary to extend Charles Darwin's concept of natural selection to incorporate epigenetic changes to organisms occurring during their lifetimes as a result of environmental conditions, and transmission of these to next generations (Fig. 1).

Major molecular mechanisms contribute to epigenetic inheritance
Methylation of DNA and modifications of histones such as methylation (H3K4me3), acetylation or other alterations.Transient chemical modifications of DNA are based on transfer of a methyl group onto the base cytosine C in CpG dinucleotides.The mechanism involves a DNA methyl transferase, which regulates gene expression by modulating the DNA including promoters and can affect recruited proteins involved in gene repression or transcription factors binding to DNA.This epigenetic effect can inactivate gene expression.Methylation influences development and characteristics of differentiated cells by altered gene expression.DNA methylation patterns can change due to environmental risk factors such as alcohol, drugs, toxins, diet, stress, or neural injury (Moore et al., 2013).Reprogramming is a protection of the progeny.
Other major epigenetic modifications include post-translational histone methylation or histone acetylation, which can change chromatin structure.Histone modifications can also affect transgenerational inheritance of genes.Inheritance of acquired properties has been described for many generations in C. elegans, but also in fruit fly Drosophila, in mammals and even in pluripotent stem cells (Fitz-James & Cavalli, 2022: Miska & Ferguson-Smith, 2016).Histones can be post-translationally modified by removal or addition of acetylation, by methylation, ubiquitination, or phosphorylation.Histone modifications have been shown to be transgenerational epigenetic signals in phenomena such as obesity (Li et al., 2021;Wan et al., 2022), while trauma or fear can lead to transgenerational DNA modifications, which can also be reversed during the animals's lifetime (Gapp et al., 2014).
RNA variants in epigenetic inheritance.Furthermore, non-coding RNAs, as well as coding small RNAs, are implicated in epigenetic transgenerational inheritance.Non-coding ncRNA or small-interfering siRNA, such as miRNAs, and small RNA strands interfere with transcription, and piRNAs and siRNAs act via RNA interference pathways (RNAi) (Heard & Martienssen, 2014).miRNAs cause trauma in mice, induced by early separation of newborn mice from their mothers, leading to altered siRNA in sperm (Gapp et al., 2014).In C. elegans survival after starvation can depend on RNAi pathways (Rechavi et al., 2014).ncRNA is the most abundant RNA expressed in mammalian genomes.Several small ncRNA-mediated gene-silencing pathways exist, which mainly defend their endogenous genomes against potentially deleterious foreign nucleic acids.Some ncRNAs, such as miRNAs, are changed in stressed organisms; they act on gene expression through the RISC pathway, which can alter the profile of expressed genes and lead to transgenerational inheritance of the altered gene expression.If the alteration is incompletely erased, it can be reproduced and replicated to successive generations.
Another possibility is that primary epigenetic signals are erased but faithfully reconstructed in the progeny based on secondary signals (Chen et al., 2016;Miska & Ferguson-Smith, 2016).These changes can be caused by exposure to toxins, hunger, diet, alcohol, or stress during pregnancy.
Viruses and transposable elements are a threat for genomes via their ability to integrate after excision, which can break the chromosomes, leading to a genotoxic event.ncRNA causes an epigenetic effect based on sequence-specific base-pairing with nascent RNA transcripts, including many RNA species, such as small RNAi, long ncRNAs (lncRNAs), and small tRNA fragments derived from tRNAs that are present in sperm.The various short RNAs can act as epigenetic factors affecting subsequent generations (Klastrup et al., 2019;Nilsson et al., 2018).Non-DNA sequence-based mechanisms are still highly controversial (Miska & Ferguson-Smith, 2016).

Gene silencing
Gene silencing by RNA interference (RNAi) was first discovered in plants, where double-stranded (ds)RNA reagents can bind to and promote degradation of the target RNA.RNAs can induce silencing via small interfering RNAs, (RNAi), 21−23 base pairs (bp) in length, which are trimmed from longer RNAs by the enzyme Dicer.The duplex binds to RISC -the RNA-induced silencing complex -which contains a helicase that unwinds the duplex.The resulting single-stranded RNAs can then bind to complimentary RNAs and cause cleavage or translational arrest.This results in gene silencing.RNAi has been widely used to characterize genes involved in  diseases including lipid metabolism and cancer (Moelling et al., 2017(Moelling et al., , 2019;;Leung & Whittaker, 2005).
Silencing performed by small interfering double-stranded RNAs (RNAi), such as siRNA or micro-RNAs, miRNAs, is a defence mechanism.It protects the genetic information of animal germ cells from modifications of the chromatin or inhibits elongation of the RNA-by-RNA polymerase by transcriptional gene silencing.In this way, mobile genetic elements are kept in check.A continuous supply of small RNAs is required for maintenance of the silencing state.This can be achieved by amplification of small RNAs by the RNA-dependent RNA polymerase.

PIWI
The enzyme PIWI, which is present in plants, plays a general role in gene silencing.Visible white patterns in dark blue petunia flower heads once decorated the cover of the journal Science in 2004 (Song et al., 2004).In the article, the crystal structural similarities between Reverse Transcriptase (RT)/RNase H and PAZ/PIWI were described.Additional retroviral tools such as integrase, the nucleocapsid as RNA-binding protein, and the special structure of the nucleotides (3 OH and 5 phosphate) are present in the silencing complex, suggesting an evolutionary relationship between gene silencing and retroviruses (Moelling et al., 2006).The silencing mechanism is based on RISC, the RNA-induced silencing complex, where an RNA moiety, the silencing RNA or small interfering RNA (siRNA), binds to a target mRNA region and leads to the cleavage of the target RNA, the actual silencing mechanism.The cleavage enzyme is the Argonaut protein with its PIWI moiety, a member of the RNase H family (Broecker & Moelling, 2019;Majorek et al., 2014).RNase H-folds are involved in prokaryotic CRISPR-Cas, eukaryotic V(D)J recombination, the interferon system, splicing, R-loop-mediated gene regulation, mononucleotide excision, DNA repair, genome stability, disease (AGS), and retrotransposon silencing.The RNase H family comprises 152 members that are among the most ancient and abundant protein-folds.
A prominent role in gene silencing has been attributed to the PAZ and PIWI enzymes which are orthologues of the RT/RNase H enzymes involved in retroviral replication.Orthologues of the retroviral RT and RNase H enzymes are named PAZ (Piwi Argonaut Zwille) and PIWI (P-element induced wimpy testis), with the latter being characterized by a conserved active site motif aspartate-aspartate-glutamate (DDG) (Moelling et al., 2006).The C-terminal domain of PIWI is structurally and functionally related to the RNase H domain.The N-terminal domain of PIWI has a nuclear localization signal (Song et al., 2004).
The similarity between the viral components and those of the cellular silencing mechanism is striking and suggests some evolutionary relationship -adaptation of viral information by the host leading to its antiviral defence by a mechanism designated as superinfection exclusion, and ultimately to a general defence (Casier et al., 2019;Moelling et al., 2006Moelling et al., , 2017)).This may represent just one example of how viral information can drive host cell evolution via the supply of novel genetic information -which finally becomes independent of an active viral infection as immunity of the host cell (Broecker & Moelling, 2019;Villarreal 2011).

Transposons
In animal cells small RNAs, such as piRNAs, have a role in silencing in order to defend cells against transposons as well as viruses.Transposons are often designated as molecular parasites, indicating their dangerous properties and relationship to viruses.piRNAs, consisting of PIWI protein bound to small RNAs, are predominantly expressed in germline cells, are generally 24-32 nucleotides in length and are enriched in the germline.To reach high fidelity in transmitting genetic information to the next generation, some multi-cellular eukaryotes have evolved piRNA-based pathways to silence transposable elements in the germline.piRNA, the PIWI-interacting RNA, tames transposon expression in germ cells (Broecker & Moelling, 2015).Transposons are particularly active during embryogenesis in animals and in the germline.Primary piRNAs are amplified by a 'ping-pong' mechanism, which couples piRNA synthesis to target RNA silencing in a loop.This increases the number of antisense piRNA transcripts for silencing of target transposon transcripts (Czech & Hannon, 2016;Brennecke et al., 2008).Single-stranded RNA binds to the PIWI protein, leading to the PIWI-interacting RNA, piRNA.It preserves genome integrity by transposon silencing in germline cells since piRNAs are only found in male and female reproductive tissues (Ashe et al., 2012).piRNA complexes scan for and detect nascent transposon transcripts and induce transcriptional repression through the silencing mechanisms by sequence-specific small RNAs (Czech & Hannon, 2016).PiRNAs affect methyltransferases and methylation, which are required for silencing of transposons, designated as the 'guardians of the genomes' (Malone & Hannon, 2009).A decrease or absence of PIWI proteins is correlated with an increased expression of transposons, which have a high potential to cause deleterious effects in their host.Deletion in the piRNA pathway leads to infertility in D. melanogaster, and is embryonically lethal (Brennecke et al, 2007).piRNA complexes are mostly involved in post-transcriptional silencing of transposable elements and other repeat-derived transcripts, but can also be involved in the regulation of other genetic elements in germ line cells.
Long-term memory becomes independent of the piRNA trigger but remains dependent on the nuclear RNAi/chromatin pathway.A piRNA-mediated multigenerational epigenetic inheritance mechanism plays a major role in C. elegans nematodes where it can last for up to 20 generations.In contrast to germline cells, in somatic cells gene expression changes affect the next generations only (Ashe et al., 2012).
The role of PIWIs in genome stability in germline cells, controlling transposon mobility and uncontrolled transposition, may fit with the error catastrophe concept described by M. Eigen (Eigen, 2002).
Transposons move around as small mobile elements within the genome, designated as 'jumping genes' .Their movement can be considered as lock-in of virus-like elements within a cellular genome that lack envelope proteins.These are required for viral life cycles to leave a cell and infect a new one.Transposable elements can be described using computer Word program terminology as cut-and-paste, whereby DNA pieces are cut out and transferred to another location by integration.A more complex mechanism is copy-and-paste, whereby the DNA is transcribed to mRNA, then copied to a DNA transcript by the Reverse Transcriptase and reintegrated at another location.These elements, designated as retrotransposons, lead to gene duplication.They can also multiply within genomes.Integration of DNA fragments into a DNA genome is associated with gene disruption and is a potentially hazardous genotoxic event for the host DNA, leading to mutations and disruptions of genes that can result in the development of cancer (Moelling & Broecker, 2015;Moelling et al., 2017).
Transposon activity is especially dangerous in germ cells, in eggs and sperms, because it could affect the next generation.The protective mechanism against transposon activity is based on small RNAs which can 'tame' transposons by binding antisense, preventing their 'jumping' .
Small RNA was shown to influence gene expression in fruit fly embryos (Fabry et al, 2021), where certain transposons are highly active very early during development and are then controlled by small RNAs such as piRNAs.Normally, epigenetic reprogramming occurs in the germline and early embryos.It resets the DNA methylation pattern to the original state before the environmental insults.This mechanism guarantees a new beginning, a reset to a naive state free of dangerous residual changes that may have been caused by the environment.Sometimes the mechanism of reprogramming is incomplete and does not lead to fully pluripotent cells.This then results in transmission of epigenetic changes to the next generation.Transgenerational changes due to toxins such as DDT, hydrocarbons and plastic compounds, smoking, diabetes, high fat diet, hunger, stress, drought or heat promote transgenerational inherited diseases in various animal models such as flies, worms, rodents, zebrafish, and in plants and even in pluripotent stem cells (Fitz-James & Cavalli, 2022;Miska & Ferguson-Smith, 2016) Other RNA structures are thought to be involved in reprogramming.tRNAs and its halves are newly detected players in inheritance (Gapp & Miska, 2016;Klastrup et al., 2019).They recently gained attention in mature sperm and in a mammalian cell line after viral infection.A recent study reported the presence of tRNA fragments in mature sperm (Peng et al., 2012).Furthermore, tRNA fragments and their modifications were shown to be affected by altered parental diet (Klastrup et al., 2019).These studies suggested that small tRNAs (stRNAs) in sperm can be carriers of heritable information.Also, long RNA in sperm contributes to the transmission of the effects of previous environmental exposure (Gapp et al., 2020), and extracellular vesicles can transport RNA to sperm cells and affect the developing progeny (Sciamanna et al., 2019).
Other special structures may be involved in reprogramming of cells.These include R-loops, which form RNA-DNA hybrids in a three-stranded structure, where one of the two DNA strands is displaced by hybridization with a complementary RNA and looped out.R-loops represent 5% of the human genome and 8% of yeast genomes and have been described to be involved in reprogramming of cells (Rinaldi et al., 2021;Yan et al., 2020).RNA pseudoknot structures and G-tetrads have also been discussed in reprogramming (Rassoulzadegan et al., 2021).

Transgenerational inheritance in various species
One of the best model systems for multigenerational inheritance is the nematode C. elegans, which has a short generation time of 3 days, and was the model in which RNAi interference was detected (Fire et al., 1998).siRNA pathways, RNA-dependent RNA polymerases for generation of secondary siRNA generations, and piRNAs contribute to gene silencing.The RNAi studies on silencing were performed by feeding the worms with bacteria expressing double-stranded RNA to induce silencing.It occurred in more than 60% of the animals for at least 4 generations.Drosophila and vertebrates have a more complex piRNA system, which includes the ping-pong amplification loop (Czech & Hannon, 2016).

Kit-gene in mice
Epigenetic changes with transgenerational transmission have been reported in detail in a mouse model (Rassoulzadegan, et al., 2006).Modification of the mouse kit gene can cause characteristic white spots in the tail that J Physiol 602.11 also appear in the progeny of heterozygotes.The kit gene codes for a receptor tyrosine kinase and was described as a retroviral oncogene by A. Ullrich (Yarden et al., 1987).Offspring maintain the white spots characteristic of Kit mutant animals in spite of a homozygous wild-type genotype.The modified phenotype results from a decrease in kit messenger RNA levels with the accumulation of non-polyadenylated RNA molecules of abnormal sizes.Sustained transcriptional activity at the postmeiotic stages -at which time the gene is normally silent -leads to the accumulation of RNA in sperm cells.
Microinjection of the RNA into fertilized eggs either of total RNA from heterozygotes or of kit-specific micro-RNAs induced a heritable white tail phenotype.These results show the mode of epigenetic inheritance in mice mediated by RNA and microRNA in the sperm.The white spot disease occurs in humans where it is still unresolved and may be due to epigenetic inheritance.It is correlated with melanocyte migration (Rassoulzadegan, et al., 2006).The white spot in the hair of Indira Gandhi was attributed to this disorder designated as piebaldism.
Several animal models have demonstrated that transgenerational transmission of metabolic disorders can originate from environmental factors.They can be based on maternal and paternal environmental factors.They include diet, toxins, neuronal stress and others.Indeed, sperm RNA from testis of obese mice recapitulated the metabolic phenotype of the parent in the offspring (Grandjean et al., 2015;Fitz-James & Cavalli, 2022).

Inherited postnatal trauma in mice
In mice, non-Mendelian epigenetic inheritance via micro-RNAs or RNA transcript fragments in sperm appears to be the basis of transgenerational signals responsible for paternal transmission.Information transfer across generations has been shown for RNA after traumatic stress.A postnatal trauma was studied using a mouse model in which newborn mice were separated from their mothers.Such a traumatic stress early in life could be attributed to sncRNAs with altered mouse miRNA expression.
Injection of sperm RNAs from traumatized males into fertilized wild-type oocytes reproduced the behavioural and metabolic alterations in the resulting offspring (Gapp et al., 2014).This argues for a possible inheritance mechanism.Mainly miRNA and piRNA were affected.Exposure to chronic stress for 6 weeks during puberty or adulthood alters a pool of miRNA in sperm.Pathways for inheritance of epigenetic changes may differ if acquired after birth from those obtained during embryogenesis.There is a window with higher or lower susceptibilities to trauma and epigenetic transmission.The transmission of stress by sperm RNA needs further studies.Female rats who experienced poor maternal care as a result of separation become poor mothers themselves.Thus, antisocial stress in baby rats can lead to aggression in adulthood (Gapp et al., 2014).However, some of the stress-induced traits can be reversed in mammals by subsequent normal experiences (Gapp et al., 2014).This would give some hope for humans who may have undergone trauma experience but could be cured by later normal life.

Dutch Hunger Winter study -intergenerational inheritance
The inheritance of epigenetic marks in the immediate next generation is not a genuine transgenerational inheritance; it referred to as intergenerational inheritance.In females environmental effects can influence not only the F1 generation but also the F2 generation due to exposure of the germline in the womb; thus there is a direct toxic effect on the F2 generation.In subsequent generations the signatures disappear because there is no direct exposure to the environmental factors any more.Such studies in humans come mostly from retrospective analyses including the Dutch famine where an increased risk of mortality and diseases such as diabetes were observed in a subsequent generation.In the winter of 1944 refugees in Holland including pregnant women received limited food supply.The Dutch Hunger Winter studied in 2010 has shown that food restriction in utero had an influence on the metabolism and cardiovascular health of the offspring and resulted in age-associated decline of cognitive functions.Some babies had normal birth weights but later developed higher rates of obesity.The results show that there is a critical window during development, during which a starvation diet influences the subsequent health of the offspring.The factors involved such as hormones, oxygen, maternal illness or others are not known.However, the methylation status of many genes and gene expression were altered (Schulz, 2010).Although this study does not constitute a transgenerational inheritance study (an additional generation needs to be tested), the analysis clearly shows the nutritional environmental effect on the germline of an embryo as well as directly on the embryo.
The paternal effects of grandfathers on their offspring have also been studied and described.Early-life parental exposure to smoke or obesity can influence the health, phenotypes and general well-being of the offspring generation, with dosage and timing of exposure of environmental effectors in relation to the stage of development being important (Nilsson et al., 2018;Pembrey, 2010).

Agouti mouse model
In the Agouti mouse model, variation in the coat colour was investigated, focusing on the role of nutrition and environmental factors on the mouse (Dolinoy et al., 2006) and gene expression.The wild-type murine Agouti gene in mice codes for a hair cycle-specific promoter and results in a brown phenotype.An inserted retrovirus-related intracisternal A particle, IAP, a retrotransposon, can integrate upstream of the Agouti gene and function as cryptic promoter.The gene is transcribed not only in hair follicles but also in other cells.This leads to yellow fur and also to the onset of obesity, diabetes and early tumorigenesis in adult mice (Broecker & Moelling, 2019;Dolinoy, 2008).
The degree of methylation within the long terminal repeat (LTR) of the IAP affects the DNA and thereby leads to a wide range of colours.Unmethylated LTR leads to yellow fur, while methylated LTR leads to a brown colour, a pseudo-agouti phenotype.Hypomethylated transposable element promoters lead to high expression and the yellow colour.The number of IAPs in these mice reaches about one thousand copies.As has been shown for the human genome, there are about 40% transposable elements and 9% are retrotransposons (Lander et al., 2001).
Agouti mice were analysed for the effects of maternal exposure to the chemical Bisphenol A (BPA), which is present in many polycarbonate baby bottles.BPA exposure of the mother shifted the coat colour towards yellow.This was attributed to demethylation of many CpG sites inside the IAP.The Bisphenol A-induced hypomethylation was abolished by supplementing the mother's food with folic acid or vitamin B12.This is one of the best understood models showing that a simple change in diet can protect against environmental toxins and prevent disease (Broecker & Moelling, 2019b;Dolinoy, 2008;Goldberg et al., 2007).

Open questions
Is there a new RNA-associated 'memory' in sperm?The importance of small RNAs is enormous and the number of non-coding RNAs such as tRNAs, fragments thereof, miRNAs, siRNAs, and long non-coding RNAs and associated RNA modifications is increasing (Mattick & Amarai, 2022).Sperm RNA studies now offer a completely unexpected new window onto transgenerational inheritance (Chen et al., 2016).RNA in vesicles transmitted to sperm also needs more intensive studies (Sciamanna et al., 2019).Most surprising is the role of tRNAs and fragments thereof because this extends their contribution to life far beyond ribosomes and mechanisms of protein synthesis (Rassoulzadegan & Cuzin, 2018).Other important regulators and 'guardians' of gene expression are the circular RNAs (circRNA) (Hansen et al., 2013), which are products of splicing.One can speculate about their structure as non-coding circular closed RNA loops that look like a relative of ribozymes.circRNAs control gene expression; they have been designated as 'sponges' and function as decoys of miRNAs and transcriptional regulators (Hansen et al., 2013).Another surprise came with the discovery of the importance of catalytic RNAs, the ribozymes that function as the catalytic element within ribosomes, summarized in the slogan 'ribosomes are ribozymes' (Cech, 2000).Not one of the hundred proteins that constitute ribosomes is the active enzymes for peptide bond formation and polypeptide synthesis; this function is performed by an RNA enzyme, a ribozyme.Early during evolution even before the genetic code arose, these RNAs may have established their dominance and superior properties to other entities, since they were not replaced but survived through 3.8 Bio of years of evolution -proof of their fitness.There is a known tendency for RNAs to evolve towards proteins such as splicing RNAs, which are thought to have evolved to enzymes such as the RNases H described here (Zimmerly & Semper, 2015).The dependence of DNA polymerases on RNA primers points to another form of dominance of RNA over DNA.
A new dimension to transmission of non-genetic information came from sperm RNA, from transgenerational inheritance of RNA from stressed mouse-fathers to their offspring.What kind of RNA is involved?Size separation of the total RNA indicated that long RNA (>200 nucleotides) affected food uptake, whereas shorter RNA (<200 nucleotides) manifested itself as behavioural despair in the swim test (Zhang et al., 2019).Thus, an open question is how the specific RNA fraction of the sperm RNA results in different phenotypes -obesity and fear!This raises the question about the type of coding mechanism present in sperm RNA.To our present knowledge non-coding RNA has structural information but no coding information.The interactions of RNAs with other RNA or DNA and other epigenetic factors remains unknown.How sperm RNA-encoded information is decoded in early embryos to control offspring phenotypes also remains unclear.Is there a 'sperm RNA memory' and how is it deciphered?This is not only interesting scientifically but may also be important for future applications and precision medicine, and may help to control intergenerational transmission of obesity and type 2 diabetes mellitus susceptibility (Zhang et al., 2019).In humans it remains unknown to what extent sperm RNA can be directly amplified and how it can affect next generations by transgenerational effects.

Figure 1 .
Figure 1.Transgenerational inheritance can be mediated by non-coding RNA, such as tRNA fragments, transmitted through sperm A complex of non-coding RNA and the enzyme PIWI is formed, which, after amplification via the 'ping-pong' mechanism, influences gene expression by modification of histones or DNA, including promoters (P).

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Physiol 602.11