Adult muscle stem cells, originally called satellite cells, are essential formuscle repair and regeneration throughout life. Besides a gradual loss of massand function, muscle aging is characterized by a decline in the repair capacity,which blunts muscle recovery after injury in elderly individuals. A major efforthas been dedicated in recent years to deciphering the causes of satellite celldysfunction in aging animals, with the ultimate goal of rejuvenating oldsatellite cells and improving muscle function in elderly people. This reviewfocuses on the recently identified network of cell-intrinsic and -extrinsicfactors and processes contributing to the decline of satellite cells in oldanimals. Some studies suggest that aging-related satellite-cell decay is mostlycaused by age-associated extrinsic environmental changes that could be reversedby a “youthful environment”. Others propose a central role forcell-intrinsic mechanisms, some of which are not reversed by environmentalchanges. We believe that these proposals, far from being antagonistic, arecomplementary and that both extrinsic and intrinsic factors contribute to musclestem cell dysfunction during aging-related regenerative decline. The lowregenerative potential of old satellite cells may reflect the accumulation ofdeleterious changes during the life of the cell; some of these changes may beinherent (intrinsic) while others result from the systemic and local environment(extrinsic). The present challenge is to rejuvenate aged satellite cells thathave undergone reversible changes to provide a possible approach to improvingmuscle repair in the elderly. The microenvironment of the SC includes interstitial cells (such asfibro/adipogenic progenitors [FAPs] and macrophages), blood vessels, extracellularmatrix proteins, and secreted factors 6. These components help to sustain the SC population in resting muscle andtheir regenerative capacity in response to muscle injury through as yet largelyunknown mechanisms. In healthy muscle, SCs are in a quiescent, non-proliferativestate but become activated and proliferate in response to muscle injury. A subset ofthe proliferating cells commits to differentiation and fuses with damaged fibers,while another subset of activated SCs self-renews and re-instates quiescence, thuspreserving a pool of stem cells for future regeneration 9. Balanced fate decisions are essential for maintaining the stem-cell pool andat the same time repairing muscle damage. Muscle regeneration is compromised byperturbations in aged muscle and muscular disease states that shift the equilibriumof SCs toward myogenic commitment or self-renewal 10.Muscle is one of the few tissues with the capacity to regenerate throughout most ofour life. This capacity is gradually lost and is minimal in advanced old age. Muscleregeneration relies on a heterogeneous population of adult stem cells, known assatellite cells (SCs), which reside in a niche between the muscle sarcolemma and thebasal lamina of each muscle fiber 14. Expression analysis of quiescent SCs distinguishes them from other SC fates 17, revealing a transcription profile that includes genes involved in theinhibition of proliferation and adhesion to the anatomical niche and others requiredfor the metabolic demands of quiescence 18. Interestingly, many silent genes in the quiescent SC are marked by“active chromatin”, indicating that they are in a“poised” state, primed for fast release from quiescence to theactivated state 20. Another crucial factor for maintaining SC quiescence is Notch signaling 23. Notch activation in quiescent SCs inhibits MyoD expression and induces Pax7expression, which further reduces MyoD protein stability 25. Thus, at least one role of Notch signaling is to prevent MyoD expression inthe quiescent state. Interestingly, the transcription factor Forkhead box protein O3(FoxO3), also required for quiescence re-entry during self-renewal, was recentlydemonstrated to induce Notch signaling by increasing the expression of Notchreceptors 26. Therefore, the FoxO3–Notch–Pax7–MyoD axis may be onepathway regulating the quiescent state (either its maintenance or reacquisitionduring regeneration). However, it is clear that the maintenance of SC quiescencerequires other, as-yet-uncharacterized epigenetic, transcriptional, andpost-transcriptional regulators.Quiescent SCs are characterized by the expression of several molecules, including thePaired box protein Pax7 (regarded as a definitive SC marker), and by the absence ofmuscle regulatory factors (MRFs) 33. These environmental signals induce the immediate expression of the myogenictranscription factors MyoD and Myf5, which control the transcriptional program ofactivated SCs 36. Transcriptome analysis of activated SCs reveals the upregulation of genesimplicated in cell-cycle progression, metabolic processes, and responses to theimmune system 20. Unlike the situation in quiescence, many genes expressed in activated SCsare associated with a repressive chromatin state that is possibly needed to restrictcommitment to the myogenic fate 42. A certain proportion of SCs must self-renew to preserve stem cells forfuture regeneration events. Like other adult stem cells, SCs can undergo symmetricor asymmetric cell division. One daughter cell of asymmetric cell division isdestined to self-renew and replenish the quiescent SC pool and the other todifferentiate. The mode of cell division is dependent on many parameters, includingdivision orientation, environmental signaling events, distribution of cellularcomponents, and expression patterns 44. It is becoming evident that SCs are heterogeneous and are composed ofsubpopulations with distinct gene expression profiles and different propensities forself-renewal and differentiation (reviewed in The activation of SCs is triggered by damage-associated molecular patterns (DAMPs),growth factors, and cytokines released by resident cells and infiltratinginflammatory cells in response to muscle injury 47. This decline is not the cause of sarcopenia but blunts muscle recovery afterinjury in elderly individuals. Recent studies have addressed the potentialinvolvement of SCs in sarcopenia, with distinct conclusions 49; however, this review focuses only on the altered functions of SCs during themuscle regeneration process with aging. The age-related regenerative decline of SCsis due to age-associated extrinsic/environmental changes as well ascell-intrinsic/autonomous changes  to gene promoters in aged cells,and SCs isolated from aged mice show elevated levels and altered distribution ofthe H3K27me3 repressive mark 20. These changes likely affect gene expression and contribute to thederegulation of signaling pathways necessary for an efficient regenerativeresponse, as described above. One pathway that is highly active in aged SCs isthe p38 mitogen-activated protein kinase (MAPK) (reviewed in 58). It remains unclear if high p38 MAPK activity in SCs is induced byintracellular signal transduction/transcriptional changes (intrinsic) or byextracellular ligands (extrinsic). High p38 MAPK activity is reported to reduceproliferative activity 59 and to decrease asymmetric cell divisions 60, ultimately reducing the number of self-renewed SCs. Self-renewal andregenerative capacity of “old SCs” is restored by exvivo treatment with a small-molecule p38 MAPK inhibitor 44. Another gene whose expression is affected by epigenetic changes isCdkn2a, which encodes the cell-cycle inhibitor p16INK4A, thought to drive cellular senescence 34. In young SCs, p16 INK4A is silenced by the PRC1-mediatedrepressive histone H2AK119Ub modification; H2AK119Ub is significantly reduced inSCs isolated from geriatric mice, resulting in p16 INK4A derepression 47. Increased p16 INK4A levels cause geriatric SCs to enter apre-senescent state. Interestingly, p38 MAPK may induce cellular senescence byactivating p16 INK4A61. A model could thus be drawn in which intrinsic p38 activity affects oldSCs in at least two ways: reducing asymmetric cell division and self-renewal andalso activating p16 INK4A expression, driving these cells to apre-senescent state.The age-associated functional defects observed in SCs may reflect alterations toepigenetic and transcriptional programs. Transcriptional changes could explainreduced antioxidant activity, changes in protein folding, reduced myogenicdifferentiation, and the tendency of these cells to adopt fibroblastic andadipogenic fates 63. STAT3 drives the expression of MyoD and commitment to myogenicdifferentiation, and its high activity therefore reduces SC self-renewal. Aswith p38 MAPK, transient pharmacological inhibition of STAT3 in aged miceincreases the population of proliferating SCs and improves muscle regeneration 63.The SCs of old mice also have elevated activity of the JAK–STAT pathway 64. SCs from geriatric mice are characterized by low baseline autophagy , resulting in accumulation of damaged proteins,dysfunctional mitochondria, and oxidative stress that lead to the senescentstate 64. Consistent with this, SC senescence in aging mice is driven by a declinein the level of oxidized cellular nicotinamide adenine dinucleotide (NAD+) thatimpairs mitochondrial activity. Treatment with the NAD+ precursor nicotinamideriboside rejuvenates SC function 65. It is presently unknown whether the block in autophagy and mitochondrialfunction is linked to the activation of p38 MAPK, and there is a need for moreresearch into the potential links between proteotoxicity and senescence in agingstem cells.Another cell-intrinsic change observed in old and geriatric SCs is unbalancedproteostasis (protein homeostasis) 66. In contrast to its detrimental role for SC quiescence maintenance, FGFsignaling plays an important role in SC proliferation in vitroand in vivo68, thus suggesting a possible dual role for some growth factors during theregeneration stages. Other signaling molecules showing increased expression inthe aging niche include TGFβ and canonical Wnt, both implicated in thesuppression of SC stemness and in their transdifferentiation from a myogenic toa fibrogenic lineage 69. It is worth noting that the transdifferentiation of SCs into other celltypes, such as fibroblastic or adipogenic cells, may constitute ratherinfrequent events in aging or dystrophic muscle and in cell culture 72. Notch signaling, required to maintain the quiescent state, is reduced inthe aged niche, and the importance of Notch in maintaining regenerativepotential is demonstrated by the finding that Notch inhibition in young SCscauses regenerative defects while its activation in aged SCs restores theirregeneration capacity 75. Moreover, old myofibers express insufficient amounts of the Notch ligandDelta1, which is necessary to maintain SC quiescence 76. Recently, additional evidence on the requirement of SC–nicheinteractions for the maintenance of SC function and tissue repair capacity hasbeen provided 78. The expression of the cell surface receptor β1-integrin and theextracellular matrix (ECM) protein fibronectin is altered in old SCs and theirniche, respectively 80. Importantly, restoring their function rescues muscle regeneration in oldmice. How these various local signals interconnect awaits furtherinvestigation.SCs are affected by the local microenvironment (niche) as well as the systemiccirculation, both of which undergo aging-associated alterations. The expressionof several extracellular ligands increases during aging in the niche,compromising SC quiescence and reducing their regenerative potential. Niche FGFsignaling is elevated with aging owing to the release of FGF2 by myofibers anddecreased expression of Spry1, which encodes Sprouty1, an inhibitor of FGFsignaling. Genetic elimination of Spry1 in SCs promotes FGF signaling, resultingin loss of quiescence and a subsequent reduction in SC number. Spry1 eliminationduring adult muscle repair led to persistent ERK MAPK activation, which impairedthe self-renewal of a subset of SCs 85 and heterochronic parabiosis, wherein two mice are surgically joined suchthat they share the same circulatory system 89. Interestingly, joining young and aged mice improved the regenerativeresponse to muscle injury in the aged partner 74, indicating that young blood contains “rejuvenatingfactors”, and a major effort has been directed at identifying thesemolecules. One candidate is oxytocin, a hypothalamic hormone that declines withage in the blood and whose receptor is downregulated in SCs of aged mice 90. Administration of oxytocin to aged mice enhances SC proliferation anddifferentiation and improves overall regenerative potential after muscle injury 90. Another candidate is GDF11; however, its influence on SCs is debated.GDF11 is a member of the TGFβ family that shows structural and functionalhomology to myostatin 91. While one group observed its decline in the blood of aged animals andhumans and showed that administration of recombinant GDF11 to old mice improvedSC regeneration 89, another group reported that the levels of GDF11 increase with age andthat its administration to old mice has no beneficial effects and may evenworsen regeneration after muscle injury in young mice 92. More recent studies found no evidence that GDF11 rejuvenates old stemcells or extends lifespan in models of progeria and reported no improvement inmuscular dystrophy 96.The influence of the systemic circulation on SCs was demonstrated inheterochronic whole muscle transplant experiments 110. Because these cells may also experience age-related alterations, it islikely that the crosstalk between them and the SCs will be affected with agingand hence provoke consequences on the repair process. Similarly, changes in theinteractions between cells and the ECM during aging, by modifying tissuestiffness and topography, may alter SC regenerative functions 112.Distinct cell types residing in the niche or infiltrating the injured muscle havebeen shown to influence SC functions by releasing growth factors and cytokines,which may act at the distinct myogenic stages during the regeneration process.These cell types include FAPs and other resident progenitor cells, severalimmune cell types such as macrophages, eosinophils, and T lymphocytes, neurons,or endothelial cells exvivo-rejuvenated SCs into old animals (intrinsic effect). The simplestexplanation for these effects is the heterogeneous nature of SCs. Even in old age,the SC population includes a small percentage of functional SCs, with only limitedaccumulated damage that can be reversed still by extrinsic signaling factors or byex vivo pharmacological inhibition of stress pathways such asp38 MAPK or JAK/STAT3. It is thus likely that the success of biochemical or geneticstrategies applied to old SCs in transplantation experiments results from theproliferative amplification of a subset of highly regenerative cells. Alternatively,the health and fitness of old SCs could be increased by refueling “cleanup” activities such as autophagy (which declines with aging) to eliminatedamage, thus improving SC regenerative capacity after muscle injury and intransplantation procedures. Future interventions that could also be considered forcombating age-related muscle regenerative decline may utilize the restoration ofSC–niche interactions via the delivery of bioengineered molecules.The significant advances in the understanding of SC aging open up real possibilitiesfor improving SC regenerative potential as a possible treatment for aging anddiseased muscles. The emerging evidence indicates that the functional and numericalloss of SCs is a progressive process occurring throughout the lifetime of theorganism. The long-lived quiescent SC accumulates many lesions caused by loss ofhomeostasis, metabolic alterations, and the aging environment. Although this processis gradual, it is accelerated in advanced old age to the extent that SCs becomepractically non-functional owing to senescence or apoptosis. In this context,disputes about which factors, intrinsic or extrinsic, are more dominant in dictatingthe fate of old SCs seem misplaced, and it is likely that both make importantcontributions to SC functional decline with aging. A degree of success has beenobtained in restoring the regenerative capacity of old muscle with both parabiosisexperiments (extrinsic effect) and transplantation of 47 implies that any treatment to rejuvenate endogenous stem cells should beimplemented before this point of no return. It is also important to consider thelink between SC regenerative potential and quiescence. It is generally well acceptedthat the more quiescent a stem cell is, the more regenerative capacity it has. Ithas also become clear that somatic stem cell populations are heterogeneous, withcells showing differing levels of quiescence 113. Subpopulations of quiescent SCs with distinct regenerative capacities havebeen identified based on the differential expression of markers such as Pax7, CD34,Myf5, and M-Cadherin 117. Highly quiescent subpopulations probably change with aging to become lessquiescent and therefore of reduced regenerative capacity. SC heterogeneity shouldtherefore be further investigated, with the aim of deciphering the molecular basisof quiescence. Understanding the quiescent state will allow early intervention aimedat preserving the highly regenerative quiescent subpopulations throughout life.Likewise, strategies directed towards the expansion of relevant subpopulations ofresident progenitor cells in the SC niche may be envisioned for reversing theage-associated muscle regenerative loss. Another unresolved issue is the interactionamong the various events contributing to the loss of SC regenerative potential withaging. Research needs to focus on determining which events are causative and whichare consequential. For example, DNA damage may induce the loss of baseline autophagyflux in old SCs, or alternatively DNA damage may be the consequence of oxidativestress resulting from the loss of autophagy flux. Defining the hierarchy of eventsleading to SC deterioration will enable the targeting of upstream events in order toachieve more efficient rejuvenation of SCs. Last but not least, in a low-turnovertissue like muscle, much of the damage to the quiescent SC is the result of thegradual decline (aging) of the niche composition and the systemic system. Futureefforts to rejuvenate the regenerative potential of SCs should thus adopt a holisticview of the SC and its supportive environment.The accumulated evidence outlined in this review indicates a number of cleardirections for future research. The key finding that the SC pool enters a state ofirreversible senescence at a geriatric age INK4a47, STAT3 78, and p38 MAPK 59, augmentation of autophagic flux 64, NAD+ repletion 65, and the administration of rejuvenating hormones like oxytocin 90. While these approaches hold great promise, their translation from mouse tohuman will require significant technological advances to eliminate or minimize thepotentially broad side effects. Interestingly, SC activity has been found toincrease in response to simple lifestyle changes that modify cell metabolism, suchas adopting a low-calorie diet 118. Similarly, exercise has been shown to enhance SC numbers and function andhence promote better muscle regeneration in rodents 122. This serves as a reminder that we should consider not only sophisticatedmethods but also simple innovative approaches deriving from our understanding of thesystem.Current efforts to rejuvenate SCs in aged mice include genetic and pharmacologicalinhibition of p16 ECM, extracellular matrix; FAP, fibro/adipogenic progenitor; MAPK, mitogen-activatedprotein kinase; MRF, muscle regulatory factor; NAD+, nicotinamide adeninedinucleotide; SC, satellite cell.


Clock gene predicted migration and breeding phenology of long-distance migratory barn swallows (Hirundo rustica) that were tracked year-round using light-level geolocators. Increasing methylation at Clock poly-Q was associated with earlier spring departure from the African wintering area, arrival date at the European breeding site, and breeding date. Higher methylation levels also predicted increased breeding success. Thus, we showed for the first time in any species that CpG methylation at a candidate gene may affect phenology and breeding performance. Methylation at Clock may be a candidate mechanism mediating phenological responses of migratory birds to ongoing climate change.Individuals often considerably differ in the timing of their life-cycle events, with major consequences for individual fitness, and, ultimately, for population dynamics. Phenological variation can arise from genetic effects but also from epigenetic modifications in DNA expression and translation. Here, we tested if CpG methylation at the poly-Q and 5′-UTR loci of the photoperiodic  Organisms fine-tune the timing of their life-cycle events (i.e. their phenology) according to circannual variation in ecological conditions12Clock genes, in particular, code for elements of a transcriptional-translational feedback loop Clock and Bmal1 products heterodimerize to produce a transcription factor, which acts both as a positive driver of the molecular oscillations and as an ‘output’ signal from the circadian oscillatorClock carboxyl-terminal polyglutamine repeat (poly-Q) domain, which serves as a transcriptional trans-activation domainPerception of day length (photoperiod) variation is a core mechanism that dictates the timing of seasonal behavioural and physiological changes of several organisms79Clock polymorphism predicts among-individual variation in breeding, migration and molt time schedules in some species151617181920Hirundo rustica), Clock exhibits low polymorphism at the poly-Q region16Clock poly-Q region showing delayed phenology1622Clock and other photoperiodic genes, can be small even in populations showing large phenological variation15Strong genetic determinism of phenological traits may evolve because of the advantages that strict endogenous control of circannual rhythmicity can afford in terms of appropriate timing of life history events24Epigenetic processes can reconcile small genetic variation in phenological genes with often large among-individuals variation in phenology, and thus also with the observed phenological responses of populations to climate changeClock gene31323334Clock genotype, expressed as number of the poly-Q repeats at the Clock region, because genetic polymorphism at poly-Q has been shown to predict phenological variation in barn swallows16Clock poly-Q region and on the relationships between genotype and migration phenology have been reported in a previous studyHere, for the first time we analyzed individual variation in major phenological events  in relation to methylation at two regions (poly-Q and 5′-UTR) of the photoperiodic Clock for seasonal breeding success. The link between methylation and breeding success could be an indirect one, being mediated by the cascading ‘carry-over’ effectsIn addition, we analyzed the consequences of methylation at 92 = 1.37, P = 0.173; 5′-UTR: males: 80.95% , females: 81.27% , t92 = 0.39, P = 0.696).Methylation at both loci did not significantly differ between males and females , females: 77.11% ; tMethylation levels at poly-Q recorded in two consecutive years  were positively correlated , whereas there was no significant correlation between years in methylation at the 5′-UTR . Hence, methylation levels at poly-Q at one life stage reflect methylation also at other life stages.Departure date from the wintering area was significantly earlier in females but not in males with higher levels of poly-Q methylation . ArrivalClock poly-Q on phenology were independent of the underlying individual genetic constitution at Clock poly-Q.In barn swallows, phenological traits including timing of spring migration and breeding, and molt schedules during wintering are predicted by genetic polymorphism at the poly-Q Clock regions1622per se or in combination with the effect of sex  but not for males . Methylation at 5′UTR did not predict breeding success , with no statistically significant evidence for sex-dependent effects . This re success .Phenological events occur in ordered sequences and each of them is therefore potentially influenced by timing of the preceding eventsClock poly-Q region can have a major impact in regulating timing of migration and breeding. Methylation at Clock poly-Q explained a very large fraction (>30%) of the variance in spring migration phenology, arrival dates at the breeding site and thus in seasonal breeding success, particularly in females. In males, the effects of methylation on spring migration phenology were considerably weaker than in females, and the consequences of methylation for seasonal breeding success seemed to be independent of timing of spring migration and breeding date. The sign of the relationship between methylation levels and dates of phenological events, with larger methylation levels being associated with earlier phenology, was consistent with the expectation since hypermethylation is expected to reduce the overall quantity of the CLOCK/BMAL1 transcription factor, thus reducing the transcriptional activation of the clock genes. Such reduction may possibly mimic the condition of small length of the poly-Q stretch, which is associated with earlier phenology of breeding, migration and molt1622Clock poly-Q, as demonstrated by analyses where we controlled statistically for the effect of length of the Clock poly-Q repeat. It should be noted that in the present study we measured Clock methylation in peripheral blood because for ethical reasons we could not sample other tissues (e.g. brain). While the present results should be considered under this caveat, methylation differences measured in blood have been suggested could have potential for being a good proxy of the changes occurring in other tissues, in particular at a central levelVariation in ecologically important traits can be generated by epigenetic modifications of the expression and function of genes even when genetic variation at those traits is small. Epigenetic processes may therefore be pivotal to our understanding of how organisms respond to environmental variation via phenotypic plasticityClock has a major role in controlling endogenous circadian rhythmicity12Clock can be entrained by local environmental factors thereby participating in the regulation of seasonality in behavioural and physiological traitsClock genotype has been shown to predict phenological variation in breeding, migration or plumage molt in several animals from diverse taxa15182223Clock and phenological variation at photoperiod-dependent activities, like migration in birds, is suggested by the observation that DNA methylation can affect circadian rhythmicity41Onchorhyncus mykiss)As a photoperiodic gene, Clock poly-Q in our study was apparent only among females, while methylation levels did not differ between the sexes. This result may suggest differentially larger phenological control by methylation at Clock in females. Thus, in females methylation at Clock could constrain early departure from the wintering grounds, thereby generating carry-over effects on timing of subsequent migration and arrival to the breeding site and, ultimately, affecting seasonal breeding successadaptive variation in timing of start of migration. Decisions on timing of migration may have to be tuned according to the ecological conditions experienced during wintering and their effects on individual physiological state or, for example, the progress of the single annual molt that barn swallows normally complete during winteringClock thereby allowing barn swallows to tune their migration phenology according to their physiological state. Hence, variation in methylation may serve as an adaptive, condition-dependent epigenetic control mechanism of individual timing of migration. Under the hypothesis that variation in methylation depends on ecological conditions during wintering, an ultimate, functional interpretation of the differentially larger effect of methylation at Clock poly-Q on phenology of females compared to males is that performance during spring migration and subsequent breeding performance of females is more dependent on the carry-over effects of condition during wintering as compared to males, thereby requiring stronger epigenetic controlThe association between migration phenology and methylation at The proximate mechanisms that cause variation in methylation levels between barn swallows and sex-dependence of the effects of methylation on phenology are open to speculation. Methylation of DNA has been shown to be affected by a host of environmental factors, ranging from nutrition to psychological stress and exposure to environmental pollution30Clock poly-Q, but path analysis suggested partly different causative links between methylation, spring migration and breeding phenology, and seasonal breeding success in either sex. In females, positive carry-over effects of methylation levels on breeding success were largely mediated by timing of spring migration and arrival to the breeding site, which depends on methylation and strongly predicts seasonal reproductive success mostly via an effect on number of breeding events per season. Indeed, early breeding translates into larger number of broods and thus reproductive output per breeding seasonClock poly-Q have consistent implications for seasonal reproductive in both sexes, but the mechanisms underpinning this relationship are different in either sex.Intriguingly, in both sexes seasonal breeding success was predicted by methylation levels at Epigenetic processes may boost the evolutionary potential of populations when faced with environmental challenges45464850The barn swallow is a small (ca. 20 g), long-distance migratory passerine bird5253We studied barn swallows at a total of 29 breeding colonies ( = farms) in southern Switzerland  and northern Italy 55Full details of the geolocator deployment procedures have been reported in related studies5555Light data recorded by light-level geolocators were used to obtain information on phenological variables as reported in a previous studyClock gene: one close to the ATG start-codon in the 5′-UTR and the second in the last exon of the gene where the poly-Q region is present (hereafter ‘poly-Q’ for brevity). As the complete genomic sequence of Clock in the barn swallow is not available, the two loci were identified as previously described in detailsUpon capture we collected a blood sample from all individuals by puncturing the brachial vein for methylation analysis in blood cells. Genomic DNA from clotted whole blood, previously rehydrated with an aqueous solution containing 20% sodium dodecyl sulphate (SDS) and 1 M Tris (pH 8.0), was extracted using the QIAamp DNA Mini kit 250 #51306). The analysis of methylation was conducted on two CG loci of the 6. The anet al.Clock methylation. Repeatability estimates (intra-class correlation coefficient) were 0.481  for Clock poly-Q exon and 0.413  for 5′-UTR.DNA methylation was quantified by bisulfite-PCR and pyrosequencingWe used Gaussian linear mixed models to analyze variation in phenological variables and seasonal number of eggs in relation to sex (fixed effect) and percentage methylation at either focal loci (continuous covariate). In the models, the interaction term between percentage methylation and sex was initially included, and then excluded when statistically non-significant. In all models, year (2 levels) and area (3 levels) were included as random factors. The effect of year or area were tested by likelihood ratio tests comparing the model including or, respectively, excluding each random effect at a time.Clock poly-Q and seasonal breeding success  the simple correlation coefficients between pairs of variables .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


During neural development, neural precursors transition from a proliferative state within their germinal niches to a migratory state as they relocate to their final laminar positions. Transitions across these states are coupled with dynamic alterations in cellular polarity. This key feature can be seen throughout the developing vertebrate brain, in which neural stem cells give rise to multipolar or unpolarized transit-amplifying progenitors. These transit-amplifying progenitors then expand to give rise to mature neuronal lineages that become polarized as they initiate radial migration to their final laminar positions. The conventional understanding of the cellular polarity regulatory program has revolved around signaling cascades and transcriptional networks. In this review, we discuss recent discoveries concerning the role of the Siah2 ubiquitin ligase in initiating neuronal polarity during cerebellar development. Given the unique features of Siah ubiquitin ligases, we highlight some of the key substrates that play important roles in cellular polarity and propose a function for the Siah ubiquitin proteasome pathway in mediating a post-translational regulatory network to control the onset of polarization. A salient feature of neurogenesis is the expansion of neural precursors within their germinal niches. This expansion is followed by cell cycle exit, cellular differentiation, and migration of the cells to their distant sites of function . In the Drosophila seven in absentia (SINA) protein was first identified as being required for compound eye development (Drosophila Egf receptor (DER) homolog, except that the R7 photoreceptor specification requires an additional burst of Ras–MAPK activity, which is triggered by another receptor tyrosine kinase, sevenless (sev) . SINA also regulates sensory organ precursor cell fate in a Ras-dependent manner by reducing Notch signaling  . Sev is ss (sev) . Sev or ss (sev) . A recesignaling . Subsequfication . SINA-infication . Finallyfication . Thus, DSiah1a, Siah1b, and Siah2, whereas humans have only two such genes, designated SIAH1 and SIAH2 . The RING domain and zinc-finger motifs are highly conserved between Sina and Siah . The combined loss of Siah1a and Siah2 leads to embryonic and neonatal lethality  exit  exit , we predFigures 1C,D). Most notably, in the hypoxic response pathway, Siah targets the prolyl hydroxylases Phd1 and Phd3 for degradation under hypoxic conditions, leading to the stabilization of the transcription factor hypoxia-inducible factor 1a (Hif1a) and the subsequent transcription of key hypoxia-response genes  differentiation . The CGNFigure 2A). Siah2 was the first E3 ubiquitin ligase found to interact with the Pard6 component of the Pard complex via a two-hybrid screen . Taken together, these results show that Siah2 regulates CGN migration through several aspects, including cellular adhesion and the cytoskeletal elements that propel migration.Live-cell imaging first indicated that drebrin might be involved in CGN cytoskeletal interplay, as the protein is cyclically transferred from the neuronal soma to the proximal leading process in migration. Lattice light-sheet microscopy, with its superior signal-to-noise ratio and spatial resolution, showed drebrin to be localized to two sub-plasma membrane f-actin-containing collars parallel to the long axis of the CGN leading process that were undetectable at lower resolution. Further examination of the relation of drebrin to plasma membrane, f-actin, and microtubules with super-resolution structured illumination imaging (with approximately 100-nm resolution) showed that the CGN proximal leading process possesses a central core of microtubules and that a layer of drebrin and f-actin intervenes between the plasma membrane and microtubules. These results were surprising, as previous migration models posited that microtubules directly interfaced with the membrane in migrating neurons . FunctioA major goal in developmental biology is to identify polarity-signaling components that will yield insights into the mechanisms controlling tissue morphogenesis. Given the key role of Siah2 in directly regulating cell polarity to control the movement of CGNs to their final laminae, we recently reviewed previous computational screens  and noteDrosophila Numb, an endocytic adaptor protein. In the developing cortex, Numb and Numbl accumulate at the apical end-feet of RGCs, where they co-localize with cadherins, conferring polarity on RGCs (numb and numbl genes affects the cadherin-based adherens junction and disrupts RGC polarity, leading to defects in cortical lamination defects and the generation of neuronal lineages. Given that both Numb and Pard3 have pro-adhesive activities Siah LOF enhancement of RGC junctions could be expected in the developing cerebral cortex of Siah mutant mice. Recently, the tumor suppressor Aspp2 was shown to regulate cell polarity by directly interacting with Pard3 (Numb was revealed to be an interacting partner of Siah1 in a yeast two-hybrid screen . Interac on RGCs . Conditith Pard3 . This inth Pard3 . StructuSiah regulation of polarity may extend beyond the adhesion turnover events described above to direct regulation of signaling events. Consistent with this, Pard3 interacts with Dab2, an endocytic adaptor that regulates the endocytosis of certain extracellular receptors and influences the trafficking of cell-signaling components . In a reAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tissue engineered scaffolds for adipose restoration/repair has significantly evolved in recent years. Patients requiring soft tissue reconstruction, caused by defects or pathology, require biomaterials that will restore void volume with new functional tissue. The gold standard of autologous fat grafting (AFG) is not a reliable option. This review focuses on the latest therapeutic strategies for the treatment of adipose tissue defects using biomolecule formulations and delivery, and specifically engineered biomaterials. Additionally, the clinical need for reliable off-the-shelf therapies, animal models, and challenges facing current technologies are discussed. Adipose tissue is distributed throughout the body and is categorized by location such as subcutaneous , intraabdominal  and other sites . Adipose tissue plays an essential role in the protection of underlying structures, providing insulation, contributing function, and imparting a normal human appearance. This appearance, and even function, can be compromised by disease, trauma, tumor resection, or congenital defects of the subcutaneous adipose tissue. These pathologies and defects of the adipose tissue remain a reconstructive challenge for plastic surgeons and clinicians. According to the American Society of Plastic Surgeons, the most common reconstructive procedures are breast construction, burn care, cleft lip/palate repair, tumor removal, and treatment from dog bites . In partTwo major reconstruction options for breast adipose tissue deficits in the clinic include implant or flap surgery using the patient’s own skin, fat, and/or muscle. Currently, flap surgeries are not without morbidity. Patients receiving autologous reconstruction are at high risk for specific complications including wound infection, flap/prosthesis failure, and reoperation. Other general complications of major surgery such as deep vein thrombosis, blood transfusions, pneumonia, and pulmonary embolism can lead to prolonged length of stay in the hospital . Patientin vitro cultured adipocytes via injection . Assuming the patient has adipose tissue to spare, AFG harvests the patient’s own fat, typically from the abdomen or thighs, via liposuction and deposits the tissue into the defect site with syringe injections. AFG is usually minimally invasive and carries several advantages in comparison to implant- or flap-based soft tissue reconstruction. With minimal scarring from the defect site and no foreign body reaction, the patient recovery time is less than 48 h. However, while the general outcomes for these procedures are positive in the short-term, the long-term results can be unpredictable due to post-graft resorption rates reaching as high as 90% . Anothernjection . Unfortunjection .The field of adipose tissue engineering evolved to address the clinical need discussed earlier. The following sections describe how current therapies insufficiently restore body contour. This review focuses on the wide range of biomaterials for resolving adipose tissue defects and the most current cutting edge therapeutic strategies, specifically encapsulated biomolecules. This comprehensive overview guides the rationale for the need of a reliable off-the-shelf biomaterial that will mostly likely contain a scaffold-encapsulation therapy combination to restore and repair the adipose defect site.in vitro/in vivo. Several methods of encapsulation have been used to localize the delivery of the therapeutic agents. Table 1 contains a comprehensive list of therapeutic agents, encapsulation strategies used, and the expected biological responses from their use. The effects of these therapeutic agents on preadipocyte differentiation and adipose tissue have been described further in illustration . FGF-2 is a member of fibroblast growth factors and most widely used angiogenic factor to stimulate blood vessel infiltration and subsequent de novo adipogenesis  , fibroblogenesis . Insulinogenesis . IGF-1 aogenesis . Vasculaogenesis . Pioglitogenesis . It is bogenesis . Dexametogenesis . Direct Figure 2, many plastic surgeons have adopted the “lipo-structure technique” or Coleman technique for the microinjection of fat particles to minimize fat graft loss. Using a Coleman microcannula, light negative pressure is created by drawing the plunger of a 10-ml syringe connected to a 3-mm cannula when introduced in the subcutaneous cavity per a small incision. The cannula is moved through the adipose section manually, loosening the tissue, and collecting the fat into the syringe. For processing, the collected fat tissue is transferred to 10 mL tubes for centrifugation to separate into four layers: (a) oily fraction; (b) aqueous fraction; (c) a cell pellet; and (d) the purified fat flanked by the oily and aqueous fractions  with saline or M2 macrophages under the scalps of C57BL/6 mice. After 3 months, M2 supplemented grafts resulted in significantly higher mean volumes and exhibited an increase in vascular density compared to controls. New therapies which include both biomolecules and cells are now being studied to increase viability within AFG and consistently enhance retention. For example, Phipps et. al proposed the use of M2 macrophages to improve fat graft retention via proangiogenic factors such as VEGF and basic fibroblast growth factor-2 (FGF-2) . AdditioResearchers have utilized biomaterials to encapsulate growth factors and pharmaceuticals aiming to develop clinically relevant tissue engineering therapies . VEGF waRubin et al. utilized PLGA MS to encapsulate two adipogenic factors, dexamethasone (Dex) and insulin, as a method to stimulate adipogenesis in ASCs. Results showed that ASCs treated with dexamethasone and insulin microspheres contained a significantly higher number of adipocytes compared to control groups . More reIn another study, 2. When SVF or ASC cells are mixed with the fat graft, the strategy is known as cell-assisted lipotransfer (CAL). Recently, Additional proposed therapies for improved retention and tissue restoration in AFG are the use of the stromal vascular fraction (SVF) and/or ASCs. SVF can be acquired during enzymatic collagenase digestion of excised human fat . Clinicaex vivo expanded ASCs , consists of multiple repeating disaccharide units of N-acetyl-D-glucosamine and ocedures .in vivo (®11) pre-adipocyte seeded scaffold implanted in subcutaneous pockets of human volunteers, using a 1 cm median sub-umbilical skin incision, up to 16 weeks, has been reported  to induce neovascularization and adipogenesis in a rat vascular pedicle model of adipose tissue engineering   . Vessel /ml dose .In vitro studies using a spinner flask showed ASCs in culture attach and seed into microbeads at a high capacity. The ability to proliferate and differentiate was also confirmed with clear potential in culture up to 49 and 21 days, respectively  have been evaluated as an injectable cell delivery scaffold for ASCs . In vitrectively . An asseineering .While the majority of studies with collagen consisted of bovine collagen, salmon collagen has also been evaluated as a scaffold for its ligand protein interactions. This protein biomaterial was prepared with and without pioglitazone, a known inducer of adipogenesis via PPAR-γ . Using aWhile scaffold performance of collagen and HA has been analyzed individually, the combined form of these natural biomaterials have implications on the future use of multi-component scaffold. Collagen  and HA (derived from bovine vitreous humor) has been crosslinked to develop a three-dimensional scaffold that encourages adipose tissue development . CollageGelatin, a partially degraded product of collagen, contains amino acid sequences, which can enhance cell attachment . GelatinL-glutamic acid) to mimic natural ECM (L-glutamic acid)/CS scaffolds retained 90% volume and Oil red O staining indicated adipose tissue formation within the implant (Chitosan (CS) is another biodegradable natural material currently being used in tissue engineering applications ,b. The bural ECM . A porou implant . Minimal implant . Test rein vitro ASC survival and migration over a 10-day period. Subcutaneous injections of silk scaffolds in Sprague-Dawley rats demonstrated significant degradation after 90 days with new tissue formation, and cell-seeding ASCs into the silk scaffold may have contributed to new adipose tissue growth in vivo.Silk is another widely examined biomaterial for soft tissue repair. Silk is a naturally occurring biocompatible protein with tunable mechanical strength, adjustable degradation rate, and low inflammatory and immunogenic responses . In refein vitro and in vivo. The use of ASCs for in vivo injection studies showed significance on several examples. More comparison studies of natural biomaterials containing ASCs with different therapeutic agents in vivo would be immensely revealing to see if a specific grouping is ideal.Overall, the use of natural-biomaterial based therapies has led to considerably positive results both in vivo due to their surface topologies and ligand landscapes  beta 1, basic fibroblast growth factor, placenta growth factor and insulin-like growth factor 1 affecting angiogenesis have been identified in adipose tissues . Decelluin vitro  were combined with human DAT to study adipose tissue engineering implanted subcutaneously in nude rats for up to 8 weeks compared to human fat graft and hASC injection. Vessel density confirmed by IHC staining showed that human DAT-ASCs grafts at 4 and 8 weeks contained less vessels compared to the fresh human fat grafts. H&E staining also confirmed remodeling of fresh graft and surrounding tissue resembling new adipose formation . In a ceAs a strategy to increase clinical relevance, Han et al. assessed allogenic rat ASC-seeded DAT scaffolds implanted subcutaneously in an immunocompetent Wistar rat model. Cells were seeded 72 h prior to implantation . CompareSpecific ECM and cellular parameters can be adjusted to influence cellular behavior. For example, the effect of DAT particle size and cell density on ASC proliferation and differentiation was assessed in a composite methacrylate chondroitin sulfate (MSC)-DAT hydrogel. At a cellular density of 2500 cells/μl of MSC-DAT hydrogel, proliferation of human ASCs was significantly higher than using DAT of large particle size (∼280 μm) compared to the small particle size group (∼40 μm). However, at an ASC concentration of 5000 cells/μl of hydrogel, the small particle size group displayed a significantly outperformed all groups with the greatest amount of adipogenesis, identified by glycerol-3-phosphate dehydrogenase activity and laminin production .In an attempt to enhance fat graft volume retention, FGF-2 was loaded into an injectable decellularized adipose matrix (mouse-derived tissue) to be investigated as a potential soft-tissue replacement for reconstructive surgery . A 12-weThe use of decellularized ECM from tissues other than adipose have also been examined for soft tissue repair. For example, PLGA microspheres containing FGF-2 were combined with decellularized small intestinal submucosa (SIS) particulates and ASCs in an athymic mouse subcutaneous model for adipose tissue growth . InitialA variety of animals has been used in adipose tissue research, each serving a specific purpose. Athymic mice are frequently used to assess volume retention of human fat grafts under various experimental conditions (microspheres) as well as the growth/differentiation of human ASCs in scaffolds within a living system. Athymic mice provide the advantage of physiological conditions for a multi-week study without having to match cell-to-animal species. Substantial information can be obtained from these studies, but it is distant from clinical application in terms of size, function, and immune health.The use of an immunocompetent animal such as the Wistar rat is also common. The use of allogenic cells, autologous fat, and multi-component scaffolds have been explored and evaluated in the Wistar rat model. Materials are normally injected as a bolus subcutaneously with studies lasting up to 12 weeks. An animal model with a healthy immune system allow investigators to fully consider material degradation and immune responses related to adipose tissue formation. Adipose volume retention has been reported in this animal model at 12 weeks , providiIt would be useful to identify an animal model that reflects the nature of an adipose defect where subcutaneous tissue is removed and a potential scaffold is implanted to demonstrate inherent tissue regeneration. Some animals that could be appropriate for this model would be the rabbit, guinea pig, or pig. These animal models would also be appropriate to assess different size defects to identify any size limitations for a potential regenerative scaffold. Once a desirable biomaterial has been established, investigators must be able to approximately quantify the amount of tissue formation per concentration of drug and/or biological factors within scaffold of choice on top of the optimal cell concentration. These results and parameters are critical for translation of these biotechnologies as well as their appropriate application to an array of unique adipose defect cases in the clinic.The investigation of various therapies for sustained adipose tissue regeneration has advanced significantly since AFG was introduced by CM summarized the literature, wrote the review, drew the figure, and constructed the table. CI summarized literature related to biomolecules for adipose tissue repair. CY and KM provided critical comments on content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Induced pluripotent stem cell-derived neural stem cells (iNSCs) have significant potential as an autologous, multifunctional cell therapy for stroke, which is the primary cause of long term disability in the United States and the second leading cause of death worldwide. Here we show that iNSC transplantation improves recovery through neuroprotective, regenerative, and cell replacement mechanisms in a novel ischemic pig stroke model. Longitudinal multiparametric magnetic resonance imaging (MRI) following iNSC therapy demonstrated reduced changes in white matter integrity, cerebral blood perfusion, and brain metabolism in the infarcted tissue. The observed tissue level recovery strongly correlated with decreased immune response, enhanced neuronal protection, and increased neurogenesis. iNSCs differentiated into neurons and oligodendrocytes with indication of long term integration. The robust recovery response to iNSC therapy in a translational pig stroke model with increased predictive potential strongly supports that iNSCs may be the critically needed therapeutic for human stroke patients. Due to the epidemic magnitude of stroke, hundreds of clinical trials have been performed to assess therapeutics that mitigate the sequela of stroke injury2. Unfortunately, these treatments have all failed in clinical trials with the exception of tissue plasminogen activator (tPA), the only Food and Drug Administration (FDA) approved agent for treating stroke. Despite the benefits of tPA, <5% of patients receive this treatment due to its narrow therapeutic window and its effectiveness in only ischemic stroke4. The mechanism of tPA and the majority of other tested therapeutics focus on limiting ischemic damage by promoting reperfusion of brain tissue or acting as neuroprotectants. However, their functions have no direct restorative or regenerative action that would enhance replacement of brain tissue leading to improved functional recovery5. An assessment of the litany of failed clinical trials by the Stem Cell Emerging Paradigm in Stroke (STEPS) Consortium resulted in identifying key therapeutic development criteria including 1) the development of a regenerative cell therapy that will not only protect cells from ischemic injury but also replace lost and damaged tissues and 2) testing of novel therapeutics in translational animal models that would be more reflective of human pathology with improved predictability7. Recent breakthroughs enabling the robust generation of neural stem cells from autologous sources and the development of a robust pig stroke model make it possible to address both of these major criteria10.Stroke is the leading cause of long-term disability in the United States and the second leading cause of death worldwide12. These studies have demonstrated that upon transplantation, iNSCs have a dual mode of therapeutic action: 1) serving as a cell replacement therapy by differentiating into neurons and glia that integrate into surrounding tissues and 2) producing neuroprotective and regenerative growth factors that stabilize and promote healing of damaged parenchyma16. In rodent stroke models, transplanted iNSCs reduce microglia activation, glial scar formation, promote angiogenesis, reduce tissue atrophy, and differentiate to mature neuronal cell types that engraft into the host brain17. However, iNSCs were tested in a rodent brain which, when compared to the human, possesses many cerebral anatomical and physiological differences; therefore, these results may not be directly translatable to clinical applications. Compared to the rodent, the pig brain has greater anatomical and physiological similarities to humans with respect to gray to white matter composition, blood flow, gyral patterning, metabolism, and size - key factors that directly affect injury evolution, tissue recovery and treatment development25. Therefore, the use of the pig model to develop stroke therapies, particularly iNSC based therapy, could bridge the gap between preclinical rodent models and human clinical trials.Recent pioneering research has shown that induced pluripotent stem cells (iPSCs) can be generated from a patient’s own somatic cells. These iPSCs have similar plasticity to embryonic stem cells and are able to differentiate into neural stem cells (iNSC) which are used to effectively treat ischemic stroke in rodent modelsIn this study, we report for the first time that intraparenchymal transplantation of human iNSCs improves tissue recovery in the stroke-damaged brain in a pig model. Non-invasive longitudinal magnetic resonance imaging (MRI) of stroked animals provided evidence of recovery in brain metabolism, white matter integrity, and cerebral blood perfusion after iNSC therapy. Histologic assessment showed that iNSC therapy also resulted in neural protection, decreased microglial activation, and stimulated endogenous neurogenesis. Transplanted iNSCs survived for 12 weeks in the pig brain and differentiated into neuronal and glial cells. These results demonstrate that iNSCs have significant potential as a regenerative and cell replacement therapy in a robust translational large animal model, providing further insight into the potential of these cells as a therapeutic for patients.in vitro resulted in robust generation of Tuj1+/MAP2+ neurons , a measure of white matter integrity, in the ipsilateral hemisphere were compared to the contralateral hemisphere with calculated changes closer to 0 being more similar to normal tissue Fig. . FA valuThe levels of neurochemicals N-acetylaspartate , creatine , and choline  are indicative of brain metabolism and can be longitudinally measured by MRS to assess tissue damage and the effect of iNSC treatment Fig. . MR spec27. CBV was found to be significantly (p < 0.05) increased in iNSC treated animals at 12 weeks post-transplant compared to 24 hours post-stroke , which was not observed in non-treated animals  was performed to determine if iNSCs could normalize blood perfusion to the brain after stroke. Color-coded cerebral perfusion maps taken at 12 weeks post-transplant suggest increased cerebral blood flow at the area of the infarct in iNSC treated pigs compared to non-treated pigs Fig. . Cerebraals Fig. . This da2 vs. 278 ± 9 cells/mm2, respectively). However, there was no significant difference in the number of NeuN+ neurons between iNSC treated and normal control animals  at the lesion border, indicating that iNSC treatment leads to neuronal protection at the lesion site post-stroke  decline in the number of NeuN+ neurons in non-treated animals at the lesion border compared to normal control animals  before forming migratory chains directly adjacent to the vSVZ, deemed the abventricular SVZ (aSVZ)29. From the aSVZ, neuroblasts migrate toward the site of neural injury. We wanted to determine if neuroblasts in our pig MCAO model demonstrated this same migratory pattern, and if iNSC treatment promoted neuroblast migratory behavior from the SVZ. 12 weeks post-transplant, there was a significant (p < 0.05) increase in DCX+ neuroblasts at the level of the vSVZ in non-treated animals compared to normal control animals . Furthermore, there was a significant (p < 0.01) increase of DCX+ neuroblasts in the vSVZ in iNSC treated animals compared to non-treated animals  . However, there was a significant (p < 0.01) increase in DCX+ neuroblasts in the aSVZ in iNSC treated animals compared to non-treated animals  . However, we found significant (p < 0.05) increase in the amount of DCX+ neuroblasts at the lesion border in iNSC treated animals compared to normal control animals   have the ability to differentiate into immature neuronal cells, or neuroblasts, which migrate toward the site of injury replacing damaged and lost cells  and have a neuroprotective effect through the release of neurotrophic factorsly) Fig. . When asly) Fig. . Last, wly) Fig. . These rTransplanted human iNSC differentiation in the post-stroke pig brain was assessed by immunohistochemistry Fig. . Transpl31. We performed quantitative RT-PCR to determine if iNSC treatment resulted in changes in gene expression related to tissue recovery  that iNSC cell therapy leads to significant tissue recovery and cell replacement. This tissue level recovery strongly correlated with decreased immune (microglia) response and increased neuroblast migration, demonstrating that the iNSC recovery mechanism is both neuroprotective and regenerative. In addition, iNSCs demonstrated long term integration with robust cell differentiation into both neurons and oligodendrocytes. These results demonstrate that iNSCs are a multi-modal therapeutic with neuroprotective, regenerative, and cell replacement capabilities.The potential to perform autologous or allogeneic iNSC transplantation to replace and protect brain tissue has significant implications for the treatment of stroke, with promising rodent studies demonstrating drastic improvements in recovery. However, the enthusiasm of the preclinical success of iNSC therapy has been tempered with the hundreds of failed clinical trials of therapeutics previously developed in rodents and a need for a deeper understanding of the underlying recovery mechanisms32. Neurogenesis after NSC transplantation has been well documented in rodent stroke models. These studies showed that hESC- and fetal-derived NSC treatment enhances cell proliferation and migration in the SVZ, DG, and striatum, and a subset of these cells co-label with the neuroblast marker DCX and the mature neuron marker Fox3, indicating endogenous neurogenesis mediated by NSC transplantation may contribute to neuronal replacement in the stroke-damaged brain39. In the current study we provide evidence that iNSCs exhibit similar neurogenic behavior in the stroke-damaged pig brain. To our knowledge, we are the first to provide evidence of how stroke and iNSC therapy affects neurogenic activity in the SVZ in a gyrencephalic species. Stroke alone stimulated neurogenesis at the vSVZ, but iNSC treatment augmented the neurogenic activity at the vSVZ and was important for increased neuroblast migration to the aSVZ and lesion border, likely through enhanced trophic factor signaling mediated by transplanted iNSCs. Indeed, BDNF and NOG, the gene encoding Noggin, expression was upregulated in brain tissue of iNSC treated animals and have both been shown to stimulate neurogenesis from progenitor cells in the SVZ41. These results support a strong paracrine-mediated endogenous neurogenesis role of the transplanted iNSCs.Interestingly, we found that iNSC treatment promotes neurogenesis and the migration of DCX+ neuroblasts from the SVZ to the lesion border. Previous reports have shown that in addition to the SVZ proper (vSVZ), pigs possess an extensive aSVZ containing neuroblast chains extending laterally similar to human infants and other mammalian species which migrate toward the lesion after neural injuryet al. demonstrated TH+ neuronal protection in the substantia nigra one month post-iNSC transplantation, an effect attributed to unknown iNSC-derived trophic factors acting on host substantia nigra afferents14. In the current study, we found increased expression of the neurotrophic factors BDNF, GDNF, and NTF3 in the brain tissue of iNSC treated animals, which have been shown to protect neurons from mechanisms related to ischemic insult44. Furthermore, we found increased expression of the gene encoding neurite branching mediator Rtn4 in iNSC treated brain tissue, which further indicates that iNSCs may foster neural repair mechanisms after stroke.We found a higher number of neurons at the lesion border in iNSC treated animals compared to non-treated animals 12 weeks post-transplantation. Due to the limited number of identified engrafted iNSCs and migratory endogenous neuroblasts, it is unlikely that neuronal replacement would have such a profound effect on neuronal number by this time point. Therefore, we hypothesize that the observed increase in neurons at these sites is probably due to neuronal protection by iNSC trophic factor signaling. Polentes 47. Microglia, the resident macrophages of the brain, proliferate and migrate to the lesion where they produce cytotoxic substances that exacerbate brain injury after an ischemic event48. Clinical studies have shown that the pharmacologic amelioration of microglia activity improves outcome after stroke50. Our data indicates that iNSC treatment significantly reduced the number of microglia at the lesion border by 12 weeks post-transplant, thereby diminishing pro-inflammatory signaling. Indeed, we found a significant decrease in the expression of pro-inflammatory cytokines CSF2, IL-1β, and IL-6 in the brain tissue of iNSC treated animals52. Similar to our findings, Huang et al. and Chang et al. documented reduced microglia activity in rodent stroke models after fetal-derived and iPSC-derived NSC treatment, respectively30. However, we found no iNSC-mediated effects on astrogliosis, another hallmark of cerebral secondary injury, in our model. Similarly, Oki et al. found that iNSC transplantation did not influence the number of activated astrocytes after stroke13. Although reduced astrogliosis after iNSC therapy has been documented in other rodent models of stroke, it is possible that the cell line tested in this study, or other therapeutic factors such as cell dose or treatment window, did not possess the required signaling capabilities to mitigate astrogliosis in the pig brain after stroke17.Neuroinflammation is a hallmark of stroke pathology and is a component of the secondary injury cascade in both human and preclinical animal models17. Other groups have demonstrated astrocytic differentiation by iNSCs; however, we are the first to show evidence of robust oligodendrocyte differentiation by iNSCs after transplantation in a preclinical large animal stroke model56. We hypothesize that this finding may be attributed to the environmental signals inherent to a gyrencephalic brain with abundant white matter; external cues specifically mediated in white matter compartments may induce iNSCs to differentiate toward an oligodendrocyte fate57. Future studies are needed to elucidate the different signaling mechanisms needed to induce differentiation to region-specific phenotypes after iNSC transplantation in vivo.We demonstrate that our transplanted iNSCs differentiate into both neuronal and glial phenotypes by 12 weeks post-transplantation. The majority of the transplanted cells co-localized with the mature neuron marker NeuN while a smaller subset co-localized with the oligodendrocyte maker Olig2. Our data correlates with previous reports in rodents showing that transplanted iNSC preferentially take on a neuronal phenotype3. In the present study, stroke-induced deficits in CBV were significantly increased by iNSC therapy by 12 weeks post-transplantation. Previous studies have shown that NSC transplantation promotes the expression of the angiogenesis factor VEGF in endogenous tissues and augments angiogenic activity at the lesion border in rodent stroke models, resulting in improved perfusion59. In the present study, we observed a significant increase in the expression of the angiogenesis marker Ang1 12 weeks after iNSC treatment, which may in part explain the observed improvement in cerebral perfusion after iNSC treatment. Previous reports show that transplantation of the fetal NSC cell line CTX0E03, which highly expresses the Ang1 protein, increases the amount of microvessels in the stroked rat brain. A recent Phase I clinical trial testing CTX0E03 (PISCES) noted functional improvement after treatment60. Thus, the expression of Ang1 coupled with improved cerebral perfusion in our model suggests increased cerebral microvessel density in the peri-lesional area, which could improve functional recovery. Future studies are needed to further assess the mechanism of this improvement in cerebral vascular performance post-stroke.Cerebral blood perfusion status is believed to be the most important factor in the clinical management of stroke because restoration of regional perfusion provides substantial help in salvaging endangered ischemic brain tissue20. Previous studies in rodent stroke models aimed to recapitulate white matter injury demonstrate that stem cell treatment facilitates white matter reorganization, enhances connectivity, and improves FA through increases in myelination, axonal sprouting, and white matter bundle thickness in the ischemic boundary66. The use of the rodent model in these studies possesses certain limitations as the rodent brain contains only 14% white matter, while the human brain contains >60%; therefore white matter repair may be achieved more easily in the rodent than the human20. Furthermore, the PISCES trial demonstrated that NSC treatment increased FA value in white matter, a similar result observed in this study60. MRS results demonstrated recovery in neurometabolites NAA, Cr, and Cho in iNSC treated animals, complementing our histological findings that iNSC treatment enhances neuronal survival and promotes tissue recovery. Previous studies in rodents demonstrate increased NAA abundance in the stroked hemisphere mediated by adult stem cell trophic factor signaling correlated with improved functional outcome68. These results suggest notable enhancement in white matter structural and total brain metabolic recovery due to iNSC therapy.We also demonstrate that iNSC therapy leads to more similar FA values, an indicator of white matter integrity, and brain metabolism in the ipsilateral hemisphere compared to the contralateral hemisphere by 12 weeks post-transplantation. Disruption of the myelinated and unmyelinated axon bundles present in white matter impairs communication between neurons in gray matter regions, often leading to motor dysfunction, neurobehavioral syndromes, and cognitive impairmentIn conclusion, we show for the first time in a translational large animal pig stroke model that iNSC therapy improves recovery through a combination of neuroprotective, regenerative, and cell replacement mechanisms resulting in improved white matter integrity, cerebral perfusion, and brain metabolism at the tissue level. These macro-level tissue changes in recovery were mechanistically supported with cellular level changes with iNSC treatment leading to reduced immune response, enhanced neurogenesis, and increased neuron survival. Finally, iNSCs survive long-term and differentiate predominantly into neurons. With the increased predictive potential of testing iNSC therapy in a gyrencephalic pig stroke model, these findings support that iNSC therapy may serve as a robust therapeutic in human patients and are deserving of further study.TM hNSC BC1, GlobalStem®, Rockville, MD; hereafter “iNSC”) were used for transplantation into the pig brain. Details for iNSC culture, flow cytometry, immunocytochemistry, and in vitro differentiation can be found in Supplementary Methods.Human induced pluripotent stem cell-derived neural stem cells  iNSCs were labeled with the lipophilic tracer 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide  for 9. Details can be found in Supplementary Methods.All experimental protocols performed in this study were reviewed, approved, and complied with the guidelines established by the University of Georgia Institutional Animal Care and Use Committee. Eight commercially bred, male castrated Landrace pigs aged 6 months and weighing approximately 180 lbs were obtained from the University of Georgia Swine Farm two weeks prior to surgery. The pig stroke model was prepared based on the procedure described previously and permanent middle cerebral artery occlusion (MCAO) was performed in all animals®, 20 mL, IV) was administered at a rate of 2 mL/second. Researchers were blinded to treatment group assignment when performing quantification analysis. Details can be found in Supplementary Methods.Magnetic resonance imaging (MRI) was performed 24 hours post-stroke and 1, 4, and 12 weeks post-transplantation on a Siemens 1.5 Tesla MRI system . Under the same anesthesia protocol utilized for surgery, MRI of the cranium was performed using an 8-channel head coil with the animal positioned in dorsal recumbency. Standard multiplanar MR brain imaging series at the coronal plane were acquired, including T1-weighted, T2-weighted, T2-weighted fluid attenuated inversion recovery (FLAIR), diffusion-weighted imaging (DWI), diffusion tensor imaging (DTI), dynamic susceptibility contrast perfusion-weighted imaging (PWI), and magnetic resonance spectroscopy (MRS). For PWI, the contrast agent  groups and were anesthetized under the same protocol utilized for the surgery. The surgery site was then reopened for iNSC transplantation into the brain parenchyma. Transplantation surgeries were performed utilizing a large animal stereotaxic frame (David Kopf Instruments) with modifications to meet our needs with pigs. The frame is equipped with a mounted Quintessential stereotaxic injector  to regulate cell injection location and parameters. Previous studies in our laboratory have shown that the use of immunosuppressive drugs did not improve iNSC survival in the pig brain (unpublished data); therefore, the use of immunosuppression was omitted in this study. The iNSC transplantation portion of this study was carried out in a single-blinded manner in which the researchers were aware of treatment group assignment. Details can be found in Supplementary Methods.Twelve weeks after MCAO surgery, all eight pigs were sedated utilizing the pre-operative drug protocol and immediately euthanized by intravenous pentobarbital overdose (1 mL/4.5 kg). Brains were removed and coronally sectioned using a brain slicer specific to pig . Details for tissue processing can be found in Supplementary Methods.Brain tissue samples were cryosectioned (Leica CM1950) at a thickness of 10 μm and collected on gelatin-coated microscopy slides for immunofluorescence analysis. Primary antibodies used were human nuclear antigen  neuronal nuclei , oligodendrocyte lineage transcription factor 2 , and glial fibrillary acidic protein . Details can be found in Supplementary Methods.Formalin-fixed whole coronal sections were routinely processed, embedded in paraffin, and stained with antibodies specific to NeuN , doublecortin , GFAP , and Iba1 . Four healthy pigs of the same age that received no MCAO surgery, iNSC injection, or vehicle injection were sacrificed and brains were processed as described above to serve as normal control animals. Researchers were blinded to treatment group assignment when performing quantification analysis. Details can be found in Supplementary Methods.Details can be found in Supplementary Methods.All quantitative data was analyzed with SAS version 9.3  and statistical significances between groups were determined by two-way analysis of variance and post-hoc Tukey’s Pair-Wise comparisons. Treatments where p-values were ≤0.05 were considered to be significantly different.The datasets generated during the current study are available from the corresponding author on a reasonable request.Supplementary Methods


Proterozoic rivers flowed through barren landscapes, and lacked interactions with macroscopic organisms. It is widely held that, in the absence of vegetation, fluvial systems featured barely entrenched channels that promptly widened over floodplains during floods. This hypothesis has never been tested because of an enduring lack of Precambrian fluvial-channel morphometric data. Here we show, through remote sensing and outcrop sedimentology, that deep rivers were developed in the Proterozoic, and that morphometric parameters for large fluvial channels might have remained within a narrow range over almost 2 billion years. Our data set comprises fluvial-channel forms deposited a few tens to thousands of kilometres from their headwaters, likely the record of basin- to craton-scale systems. Large Proterozoic channel forms present width:thickness ranges matching those of Phanerozoic counterparts, suggesting closer parallels between their fluvial dynamics. This outcome may better inform analyses of extraterrestrial planetary surfaces and related comparisons with pre-vegetation Earth landscapes. It has been previously assumed that deep river channels could not have developed in the Proterozoic due to lack of vegetation. Here, the authors present remote sensing and outcrop data to show that large scale and deeply channelled river networks did exist in the Proterozoic despite the absence of vegetation. The interpretation of ancient sedimentary environments relies on comparisons between their rock records and the dynamics of modern sedimentary landscapes. Pre-Ordovician terrestrial realms were devoid of macroscopic life forms136810Here we demonstrate that during a significant portion of the Proterozoic, large-scale fluvial systems had channels that shared morphometric characteristics with those of post-vegetation and modern rivers, irrespective of their planform or position within sedimentary basins. Our results disprove the widespread inference that all ancient unvegetated rivers were wide and shallow. This hypothesis is supported by a data set of width and thickness values from 156 fully preserved channel forms ranging in age from 1.9 to 1.0 Ga, once part of both proximal and distal drainage networks . Analysi12The recognition of channel forms is critical in fluvial sedimentology, and relies on the detection of prominent erosional scours overlain by sediment fills related to either stages of active discharge or channel abandonment. Active-channel fills typically contain gravelly and sandy deposits pointing to bedload traction by waterflows, while abandoned-channel fills consist of finer-grained, sandy and muddy deposits settled in slough waters after flow diversion to another conduit12Our data set includes active fixed-channel forms, active mobile-channel forms and channel-abandonment intervals that belonged to fluvial systems capable of developing alluvial cross-sections several hundred metres to a few kilometres wide  and 3, s15Our data set  is compi9232426923The aspect ratios (width:thickness) of observed channel forms are shown in logarithmic plots in W and T represent width and thickness of channel forms, respectively, and ±29 indicates the data set's s.d. (R2=0.6162). Since, by definition, a channel form must have values of W and T greater than zero, the linear regression includes the W=0, T=0 point  and the lack of sheeted channel fills dismiss the hypothesis that channel deposits aggraded within broad, shallow, highly mobile stream-beds. The linear correlation derived from our data set (W=100 T/3±29 T) provides a first-order approximation of the width of deep Proterozoic channel forms that are only partially preserved or exposed.Our evaluation of fluvial channel form and hydrodynamics in Proterozoic terrestrial landscapes stems from a novel approach based on remote-sensing observations of channel fills from extensive outcrop belts, at such scales that their morphometric attributes could not be fully appreciated by means of conventional ground surveys. Previous attempts to reconstruct Precambrian fluvial-channel geometries were based on outcrops a few hundred metres wideOur data set comprises the products of large fluvial systems originating in extensive and long-lived orogenic belts . It is a22324uniquely characterized by wide, shallow, frequently shifting channels of high aspect ratio, and thus we contend that parallels commonly drawn between Proterozoic fluvial systems and modern ephemeral rivers are not warranted. The perennial regime inferred from the observed channel deposits is related to stable patterns of discharge relative to sediment supply. Sediment production and delivery might have been relatively steady in the Proterozoic, with bedrock relief exposed to vigorous physical and acidic chemical weathering47919222324It is still widely accepted that the early Palaeozoic rise of vascular land plants contributed to a significant diversification of fluvial planform stylesA number of Precambrian fluvial units are exposed over vast and still underexplored intracratonic basins worldwide, and we expect that further advances gained from their study will expand the data set discussed here. Future remote-sensing analyses may adjust the morphometric parameters established here and will provide additional information on Archean to early Palaeoproterozoic fluvial-channel forms to further test hypotheses of evolving or invariant channel morphometry through deep time. Our data provide evidence that current hypotheses and conceptual models on the evolution of fluvial systems through deep geological time need revision. Hence, we call for a critical reassessment of the environmental controls on fluvial-channel patterns and morphometry. Once systematic, highly resolved morphometric compilations on alluvial channels from other planetary bodiesThis study is based on a quantitative data set of width and thickness for Proterozoic channel forms  identifiThe authors declare that all data supporting the finding of this research are available within the paper and its How to cite this article: Ielpi, A. et al. Morphometric convergence between Proterozoic and post-vegetation rivers. Nat. Commun.8, 15250 doi: 10.1038/ncomms15250 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary FiguresGeometric data for the Proterozoic channel forms analysed in this study.Geometric data, compiled from literature, for the Ordovician-Pleistocene channel forms analysed in this study.Geometric data, compiled from literature, for the modern perennial river cross-sections analysed in this study.


Potatoes represent an important staple food crop across the planet. Yet, to maintain tuber quality and extend availability, there is a necessity to store tubers for long periods often using industrial-scale facilities. In this context, preserving potato quality is pivotal for the seed, fresh and processing sectors. The industry has always innovated and invested in improved post-harvest storage. However, the pace of technological change has and will continue to increase. For instance, more stringent legislation and changing consumer attitudes have driven renewed interest in creating alternative or complementary post-harvest treatments to traditional chemically reliant sprout suppression and disease control. Herein, the current knowledge on biochemical factors governing dormancy, the use of chlorpropham (CIPC) as well as existing and chemical alternatives, and the effects of pre- and post-harvest factors to assure potato tuber quality is reviewed. Additionally, the role of genomics as a future approach to potato quality improvement is discussed. Critically, and through a more industry targeted research, a better mechanistic understanding of how the pre-harvest environment influences tuber quality and the factors which govern dormancy transition should lead to a paradigm shift in how sustainable storage can be achieved. Solanum tuberosum) have been cultivated for more than 6000 years. Currently, potato is the fourth most important crop produced crop worldwide with an annual production of ca. 382 MT. Europe and Asia are the biggest producers with a share of 40.7% each, followed by America and Africa  . Potatoes provide an excellent source of nutrients and vitamins, but year-round availability depends on industrial-scale storage, especially in countries which rely on an annual crop. In the United Kingdom, approximately half of the total harvested tubers are stored for up to 11 months (Potato tubers (1 months . Sub-opt1 months . The Uni1 months . This st1 months .N-(3-chlorophenyl) carbamate (chlorpropham or CIPC) residues (mainly for ware potatoes destined for processing), control of sweetening processes, and ensuring tuber marketability , auxins, cytokinins (CKs), gibberellins (GAs), ethylene, and strigolactones (SLs)] and other compounds (viz. carbohydrates and organic acids) are believed to orchestrate the onset and further development of dormancy break  and endogenous signals . The relcy break .-1) has been reported to break endodormancy following short-term treatments (Endogenous ethylene is required at the earliest stage of dormancy initiation (endodormancy induction) ; howevereatments , but alseatments . Howevereatments . These fThe role of ABA is better understood. It is well known that a sustained synthesis and action of ABA is required for dormancy induction and maintenance . That saIt has also been demonstrated that, CKs and GAs are required for the reactivation of meristematic activity and sprout growth . Just prOptimum length of dormancy differs depending on cultivars and final usage of potato tubers. Thus, longer dormancy and delayed sprouting (at a desired time) would be best for ware potatoes storage, while accelerated sprouting would be preferable for seed potatoes. As reviewed by Suppression of sprout growth in potato tubers represents a crucial step to manage potato quality during storage. Sprouting can be inhibited by the application of chemical sprout suppressants and by controlling environmental conditions, e.g., cold storage, tuned humidity and regulated gas composition conditions. Due to its high efficacy, CIPC is the world’s most utilized sprout suppressant chemical; it inhibits meristematic cell division, delaying sprout development. Nevertheless, concerns about CIPC usage have increased following studies which described toxic and carcinogenic properties of CIPC and its metabolites . However-1 for processing potatoes and 24 g ton-1 for fresh market tubers) must be done through ‘active recirculation’ of storage air by fans to optimize CIPC application  have been found to suppress sprout length and sprout incidence in a range of potato cultivars when applied at first indication of sprouting  derived from caraway seed, can inhibit sprout growth for up to a year; however, the number of applications required may make this solution uncompetitive when compared to CIPC (-1) needs more investigation to elucidate whether it is environmentally safe  is another factor which strongly impacts quality; it triggers both maturation of the tuber periderm and stolon release, and in seed potato production it can also control tuber size. To manage all these variables a multifactorial approach is recommended to mitigate side effects which may affect quality . In this context, After harvest, tuber quality management aims to delay dormancy break and limit weight loss and sweetening of potatoes. Senescent sweetening is a natural process that occurs as a result of tuber aging; it is irreversible and involves cellular breakdown. Following cellular breakdown, structural and non-structural carbohydrates are depolymerized by hydrolytic enzymes. To delay this process, correct storage conditions are crucial. Cold storage is commonly used to control sprouting, yet temperature management depends on the intended market: tubers for the fresh market can be stored at temperatures below 7°C while tubers destined for the processing market need higher temperature (8–13°C) to preserve frying quality. Quality loss is also caused by ‘cold-induced sweetening’ when sucrose hydrolysis leads to reducing sugars accumulation; although it can be partially reversed by temperature reconditioning . Cold-in2, another storage extension gas, on frying quality is less clear. Studies on processing potato varieties showed negative effects on fry color when ethylene and CO2 were applied together. Despite this, cultivar, gas concentration and timing, and seasonality strongly affect responses of tubers to CO2 treatment . A recent study has shown that this type of sweetening can be prevented with a single application (24 h) of 1-methylcyclopropene (1-MCP) prior to early and late ethylene supplementation . The impreatment .Vinv, in potato tubers resulted in cold induced-sweetening inhibition and high quality processed tubers . Due to the complex genetic heterogeneity of modern potato cultivars, conventional potato breeding requires approximately 10 years for the phenotypic selection cycle: from crossing to variety release . The incd tubers . Other sd tubers . Despited tubers . The comThe release of the potato genome , improveAgrobacterium tumefaciens with plant viruses. This combination showed very promising results when Agrobacterium was coupled with DNA virus Geminivirus replicon (GVR), which allows a larger carrying capacity compared to RNA viruses. Potato plants modified with this technique exhibited reduced herbicide susceptibility  will improve tuber quality. These combined efforts will benefit the development of new cultivars with improved features and provide guidelines for more sustainable agricultural techniques and storage strategies. At the same time, alternative pre- and post-harvest technologies have to be embraced and further implemented by the potato industry. Through a more industry targeted research, the combination of genomics, pre- and post-harvest technologies will aid the preservation, enhancement, and viability of future tuber quality.In order to assure future potato tuber quality, industry and academic communities have to work together while considering consumers preferences. Deploying molecular and improved phenotyping techniques to increase the knowledge of mechanisms which mediate physiological responses during pre-harvest production, post-harvest storage and processing (MA: Paper writing (35%). RT: Paper writing (35%). SL: Paper writing (10%). AB: paper writing (10%). LT: Overall supervision (10%).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objective: Aggressive periodontitis (AgP) is frequently associated with the presence of Aggregatibacter actinomycetemcomitans, a Gram-negative pathogen, which expresses leukotoxin (Ltx) as a virulence factor. Ltx is known to disturb neutrophil function and to activate these cells. This study was aimed at investigating the ability of Ltx to alter the chemotactic behaviour of neutrophils, the most abundant leukocyte in periodontitis.Methods: Neutrophils from healthy blood donors were isolated and stimulated or primed with low concentrations of Ltx . Neutrophil migration in response to Ltx gradients and to the biofilm-derived chemotactic agent fMLP after Ltx-priming was monitored by real-time video microscopy using an Insall chamber.Results: Although Ltx alone enhanced neutrophil movement at all concentrations, low directional accuracy was observed compared to the positive control (fMLP). Ltx priming led to directional movement towards fMLP with significantly enhanced speed. In case of 10 ng/mL Ltx, however, this chemotactic response towards fMLP appeared to be delayed.Conclusion: The results indicate that Ltx induces non-directional neutrophil movement, whilst enhancing migration towards fMLP. Clincially, this may lead to the presence of a higher number of activated neutrophils in the periodontal tissues, potentially causing more pronounced host tissue damage.