This requires a deeper understanding of the precise molecular and functional attributes of the DG granule cells in vivo as well as the DG cells derived in vitro. In this review we outline the neuroanatomical, molecular and cell-biological components of the granule cell differentiation pathway, including some growth- and transcription factors essential for their development. We summarize the functional characteristics of DG granule neurons, including the electrophysiological features of immature and mature granule cells and the axonal pathfinding characteristics of DG neurons. Additionally, we discuss landmark studies on the generation of dorsal telencephalic precursors from pluripotent stem cells (PSCs) as well as DG neuron differentiation in culture. Finally, we provide an outlook and comment critical aspects.The dentate gyrus (DG) receives highly processed information from the associative cortices functionally integrated in the trisynaptic hippocampal circuit, which contributes to the formation of new episodic memories and the spontaneous exploration of novel environments. Remarkably, the DG is the only brain region currently known to have high rates of neurogenesis in adults (Andersen et al.,  Key structures for DG development are: (1) the cortical hem (CH) and the anti-hem; (2) the radial glia; (3) the Cajal-Retzius cells (CR); and (4) the surrounding meninges. DG development is regulated by growth factor gradients which in turn activate transcription factor expression in the corresponding precursor cells and in the differentiating neurons. The DG originates in three proliferative matrices and the cellular events leading to its formation can be best described as subdivided into three developmental stages.Before thinking of producing dentate gyrus (DG) granule cells (or the entire DG itself) DG development begins with the expansion of the undifferentiated neuroepithelium sheet  of the early cortical primordium. During this phase, signal morphogens are secreted from two discrete telencephalic regions: the CH, a long and narrow region caudal and adjacent to the presumptive hippocampal neuroepithelium, and the more rostrally located anti-hem. The CH is considered one of the organizers , which encodes a protein essential for the migration of neurons along radial glia. The formation of the radial glial scaffold is triggered by a cascade of interactions involving Reln, Disabled1 (Dab) and Integrin beta1 ; these migrating precursors constitute the secondary proliferative matrix. This is a unique phenomenon: a migrating population of mitotically active progenitors at various differentiation stages. The migration is guided by a radial glial scaffold, which originates from the fimbrial region and leads to the pial side of the cortex and the HF. Additionally, a special kind of neurons, the CR cells, originating in the CH and migrating towards the HF, provide factors essential for DG precursor migration  accumulate in this region and form the tertiary proliferative matrix. Granule cells originate in all three matrices. CR cells surrounding the HF and the pial surface define the granule cell layer (GCL)’s well-known V-shaped progenitor and granule cells become gradually limited to the tertiary matrix. In the early postnatal phase, the subgranular zone  develops from the tertiary matrix Bayer, .in vivo causes newly differentiated neurons with shorter dendrites and simpler branching  maturation of hippocampal neurons is probably regulated by a genomic network mostly independent from external stimuli; this would explain the fact that the sequence of events leading to the functional  differentiation of hippocampal neurons is the same for neurons generated in embryonic and early postnatal brains and for neurons generated in the adult , encoding secreted modulators of WNT signaling, whose functions in the DG have not been analyzed yet.The CH secretes bone morphogenetic proteins (BMPs) and WNT protein ligands (as well as growth and differentiation factors (GDFs); see Sections “Fibroblast Growth Factors (FGFs)” and “Cytokine SDF1 Secreted by Cajal-Retzius cells”). The anti-hem secretes fibroblast growth factor 7 (FGF7), epidermal growth factor (EGF) and sonic hedgehog (SHH), as well as secreted frizzled-related protein 2  causes severe hippocampal abnormalities  quiescence  which in turn induces Smoothened (Smo), encoding a component of the SHH receptor complex, leads to abnormalities in regions of postnatal neurogenesis, e.g., a reduced amount of granule DG cells compared to other hippocampal regions , necessary for FGFs to exert their effects on the target cells is expressed in the entire hippocampus (CA and DG), from the neuronal precursors of the neuroepithelium to the postmitotic pyramidal cells and DG granule cells; mutants deficient in Fgfr1 have 30%–50% less granule cells in the DG , a protein secreted by the Cajal-Retzius neurons . The cThe interplay between WNT and BMP ligands and SHH establishes and sustains the DG stem cell niche  region , APC down-regulated 1 (Apcdd1), Rspo1-3 and Sfrp1 .We have mentioned above SHH , a secEmx2 is expressed within the diencephalon roof with a caudo-rostral gradient , neurogenin 2 (Neurog2) and T-box TF2 .Transcription factor gene Neurog2 is a bHLH transcription factor gene with proneural function expressed in the embryonic neuroepithelium. It promotes the neuronal fate in multipotent stem cells. Neurog2 activates the expression of several neuronal differentiation genes, e.g., the NeuroD family of transcription factors  is a bHLH proneural gene expressed with a dorso-ventral gradient in the cortical plate and tertiary matrix of the DG. The DG and cerebellum are completely missing in Neurod1 conditional knockouts  is a transcription factor gene expressed by the migration stream of newly generated granule cell precursors in the secondary matrix and also by mature granule cells in the tertiary matrix of the emerging DG  is necessary for NSCs to find their specific location in the emerging DG  is a transcription factor gene expressed in DG granule cells. Its function affects two essential developmental processes, i.e., the proliferation and the differentiation of granule cells. Since Bcl11b is expressed in postmitotic DG granule neurons, its influence on the proliferation compartment is probably effected through secretion of a factor acting on dividing cells of the proliferation matrix (positive feedback). A direct transcriptional target of BCL11B, the cell-cell adhesion protein desmoplakin (DSP), is essential for the proper differentiation of DG granule cells  knockout mice, DG precursor cell migration is disturbed so that they accumulate in the neuroepithelium (VZ). This results in the absence of DG granule cells  show a DG GCL partially absent, cytoarchitectonically disorganized  are also responsible for the proliferation of DG precursors in the first matrix , Couptf1 is positioned at the crossroads between precursor proliferation and migration, two cellular phenomena tightly linked in the DG  and Ccnd2 (a cyclin). While Tlx is dispensable for DG development, it is essential for adult neurogenesis from the subgranular zone (DG) and VZ-SVZ  while keeping specific cell culture conditions. Embryoid bodies (EBs) and single culture systems were used as starting point, and sometimes pre-differentiated cells were transplanted to brains i et al.  focused i et al. .By using an optimized serum-free suspension culture with addition of the WNT antagonist DKK1, FOXG1-positive telencephalic cells can be generated; these can be made to express typical dorsal telencephalic markers paired box 6 (PAX6) and EMX1 by the subsequent administration of WNT3a.Zbtb20 as well as Prox1 (DG granule cell marker), while the other expressed Zbtb20 in combination with glutamate ionotropic receptor kainate type subunit 4 , a marker of pyramidal cells of CA. However, these cells showed immature firing patterns and did not achieve the reconstruction of the primary hippocampal neuronal circuitry: specific CA1 pyramidal neurons could not be generated, and the formation of the DG or regionally specific hippocampal tissues could not be recapitulated , NOGGIN (BMP-antagonist), the ALK5 (TGFB pathway) inhibitor SB431542 and the SHH inhibitor cyclopamine. Then, WNT3a and brain-derived neurotrophic factor (BDNF) were added and co-cultures were initiated as single cell suspensions on hippocampal astrocytes. The resultant DG precursor-like cells were then transplanted into the hippocampus of brains in vivo, and their integration and maturation were followed. However, this study lacks detailed co-localization studies with DG markers, so that the final differentiation phenotypes of the DG-like cells cannot be assessed , capacitance and action potential waveform—as well as synaptic properties .One key neuronal parameter, the resting membrane potential (Vm), could in principle be used to assess the functional status of neurons. However, the Vm of immature DG granule cells has been variously reported in the literature. Among others found relatively depolarized Vm values (around −50 mV) in immature DG granule cells, and  to differentiate into particular neuronal and glial phenotypes has extended our knowledge about human brain development and maturation and will help in the future to understand certain neurological disorders. The generation of specific neuronal cell types and brain structures ex vivo will be an additional important step in brain research, to regulate the directed neuronal differentiation of crucial brain development events in stem cells and neuronal precursors.NSC research has advanced very fast in the last few years, in particular in the fields of in vitro from the cells of patients with hippocampal CNS disorders.For this reason, an exceptional opportunity for modeling neurological disorders and for drug design may be provided by the generation of DG granule neurons Harvesting central nervous system (CNS) tissue from living patients suffering brain disorders it is not possible. By using patient-derived neural induced PSCs (iPSCs), analysis of CNS disorders is now within reach of scientists and clinicians. Neural iPSCs are able to form functional neurons and glial cells; however, developing differentiation strategies for the generation of specific neurons and glia cells and their disease-relevant subtypes is still a significant challenge. A series of breakthroughs in the field of neural human PSCs (hPSC) differentiation during the past decade, has made it possible to use iPSCs as the basis of CNS disease models  as well as for the screening of appropriate therapeutic drugs. The immediate goal to this end is to develop and refine protocols for the reliable production of DG granule cells from ESC- or iPSC-derived cells. The best existing method for the generation of DG granule cells in vitro with EB and single cell culturing is still very inefficient , Gli3, Emx2 and Ascl1 should provide the starting cell source for the directed differentiation of DG granule cells. Guiding the differentiation of neurons with dorsal telencephalic phenotype through various embryonic stages towards the DG granule neuron phenotype will require a strict following of the key developmental events, starting with the sequential administration of appropriate growth factors and transcription factors, mimicking the differentiation of the DG neuroepithelium towards the different proliferation matrices and finally immature and mature granule cells. Major immediate challenges concern the scheduling and titration of these factors. In this context, it would be most interesting to analyze possible synergistic effects of BMP7 and WNT3a on the production of Emx2/Lef1-expressing IPCs and of the Prox1/NeuroD1/Calb2-expressing neurons that they generate during DG granule cell differentiation in vitro. IPC differentiation into DG granule cells is enhanced by expression of key transcription factors, particularly Emx2, Neurog2, Tbr2, NeuroD1, Lef1 and Prox1 (Hodge et al., Dorsal telencephalic precursor cells, generated in vitro, expression of the key dorsal telencephalic transcription factors needs to be activated in a sequence and time course analogous to those that occur during normal brain development (Li and Pleasure, in vitro production of DG granule cells are other neurons expressing similar combinations of transcription factors. For instance, the combination Emx2-Tbr2-NeuroD1-Lef1 is also expressed by pyramidal cells of the CA, and Emx2-Tbr2 are also expressed by CR cells. Ccnd2 and transcription factors like Nfix, Tlx and Ascl1, which play a major role in postnatal and adult granule cell neurogenesis, will have to be tested for their influence on the proliferation and maintenance of already generated granule cell precursors as well as differentiated granule cells.DG granule cell differentiation could also be forced by sequential and coordinated expression of the essential transcription factors. Obviously, when attempting to differentiate neuronal stem cells into granule cells in vivo and in vitro. Subsequently, conditional overexpression of transcription factors during the directed differentiation of PSCs towards DG granule precursor could be attempted. Lineage tracing of the different stages of granule cell development in combination with single cell expression profiling would be of importance to analyze the sequence of molecular events determining granule cell fate.Ultimately, single cell expression profiling of cells marked with stage-specific reporter constructs could be needed in order to molecularly define with precision the differentiation stages of granule cells in vitro is a major challenge, particularly for complex regions of the nervous system (Watanabe et al., Alternatively, the cellular engineering of organizers such as the CH could be attempted. However, the 3-D cultivation of neuronal tissues in vivo and in vitro, is essential to elaborate protocols generating viable, transplantable neuronal cells; consider for instance the problems posed by the axonal growth-inhibiting milieu of the adult nervous systems (Skutella and Nitsch, Finally, further characterization of the molecular guidance cues of DG granule cells, Growth-inhibition factors for re-growing axons and dendrites in the adult CNS post-lesion make this integration process difficult. Therefore, it is essential to support the outgrowth and navigation of neuronal processes and their integration in the existing entorhinal-DG-CA circuitry. This can be attempted by administering anti-myelin-associated factors (Xu L. et al., in vitro differentiated neurons identical to their natural models.Despite of all accomplishments in this field, a long way is still ahead for the generation of MH and SC drafted the article. GA-B and TS wrote the final version. PN drew the Figure.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.


A bud sport is a lateral shoot, inflorescence or single flower/fruit with a visibly different phenotype from the rest of the plant. The new phenotype is often caused by a stable somatic mutation in a single cell that is passed on to its clonal descendants and eventually populates part or all of a meristem. In many cases, a bud sport can be vegetatively propagated, thereby preserving the novel phenotype without sexual reproduction. Bud sports provide new characteristics while retaining the desirable qualities of the parent plant, which is why many bud sports have been developed into popular cultivars. We present an overview of the history of bud sports, the causes and methods of detecting somaclonal variation, and the types of mutant phenotypes that have arisen spontaneously. We focus on examples where the molecular or cytological changes causing the phenotype have been identified. Analysis of these sports has provided valuable insight into developmental processes, gene function and regulation, and in some cases has revealed new information about layer-specific roles of some genes. Examination of the molecular changes causing a phenotype and in some cases reversion back to the original state has contributed to our understanding of the mechanisms that drive genomic evolution. Careful analysis of unusual shoots on parent plants provides valuable insights into mechanisms driving genomic evolution. Sometimes, plants produce shoots, single flowers or fruit that are visibly and genetically different from the rest of the plant. These ‘bud sports’ are caused by stable mutations and usually retain the positive characteristics of the parent plant. They are useful for cultivating novel varieties, particularly in species with low genetic variability. Toshi Foster at the New Zealand Institute for Plant and Food Research in Palmerston North, New Zealand, and Maria Jose Aranzana at the Center for Research in Agricultural Genomics in Barcelona, Spain, reviewed current knowledge of bud sports and their genetic origins. Analyses of buds using next-generation sequencing is providing insights into the function and regulation of genes and mutations, with applications in agriculture and plant biology. Many perennial species have a long juvenile period and generation time, some are self-incompatible, and most are highly heterozygous, which means valuable qualities may be lost through sexual reproduction. For these reasons, many woody perennials are vegetatively propagated by cutting, grafting, and budding, which can preserve desirable genotypes over long periods of time. Indeed, there are examples of wine grapes that have been clonally propagated for centuries4. By 1936 there were at least 1664 known fruit tree bud sports, representing 32% of the plant patents issued by the U.S. Patent Office at that time5. More recently, Okie6 reported that more than 170 commercialized cultivars of peach and nectarine are derived from bud sport mutations.Occasionally, a lateral shoot, inflorescence or single flower/fruit is discovered with a visibly different phenotype from the rest of the plant. These are called bud sports and are often caused by a stable somatic mutation in a single cell that is passed on to its clonal descendants and eventually populates part or all of a meristem, enabling vegetative propagation of the new mutant. Although these are relatively rare events, sport mutations often provide valuable new characteristics while retaining the desirable qualities of the parent plant. Therefore, somatic mutation represents a mechanism to generate new genetic variability which is especially important for species with low levels of variation. Many economically important perennial cultivars are bud sportsChelidonium majus (celandine) plant found in a herb garden7. In 1644, gardener Pietro Nati noticed a shoot bearing unusual fruit (the “Bizarria” orange) growing from the graft junction of two types of citrus8. Charles Darwin was fascinated by “sporting plants” and published numerous reports of spontaneous mutants12. In his famous book, The Variation of Plants and Animals Under Domestication11, Darwin noted that “Many cases have been recorded of a whole plant, or a single branch, or bud suddenly producing flowers different from the proper type in colour, form, size, doubleness, or other character. Half the flower, or a smaller segment sometimes changes colour”. In some cases, the molecular or cytological change(s) causing the new phenotype have been identified, which has contributed to our knowledge of the mechanisms that lead to the formation of sports and provided novel information about gene function and regulation. This review will highlight some of these examples.The identification of bud sports relies on astute observation by the grower, breeder, or gardener. While these have likely been observed since humans have been cultivating plants, the earliest report of a bud sport was published by the botanist Gaspard Bauhin in 1598 and describes the unusual leaf phenotype of a 13. Cells in the tunica tend to divide anticlinally  such that they form one or more clonally distinct layers . Additionally, epigenetic variations such as DNA methylation, histone modification, chromatin remodelling, and RNA silencing can cause stable changes in gene expression, generating sports.24 estimated a mutation rate of 1.1% of SSR alleles between peach sports. More recently, high-throughput next-generation sequencing (NGS) technologies have been used to estimate variability between bud mutants, providing more comprehensive information. Whole-genome sequences of “Fuji” apple and four bud mutant cultivars were compared to identify small polymorphisms, which revealed an average rate of eight single-nucleotide polymorphisms (SNPs) and 1.2 indels per Mb over the genome25. In grape, Carrier et al.26 compared whole-genome sequences of Pinot noir and three of its clones and found 1.6 SNPs and 5.1 indels per Mb.The most frequent mutations caused by DNA polymerase errors are point mutations, tandem repeats, small insertions and deletions (indels), and base mismatches. Polymerase slippage is known to produce variability in simple sequence repeat (SSR) regions. For example, Aranzana and co-workers27 and does not require a homologous sequence to ligate the two DSB; it can result in small insertions or deletions of DNA at the break location and presumably contributes to microsatellite instability28. In contrast, the HR pathway requires an intact DNA molecule as template, such as the sister chromatid in a cell in S or G2 phases or the homologous chromosome. While recombination with the sister chromatid will not produce a mutation, recombination with the homologous chromosome may result in loss of heterozygosity or genome rearrangements30. Migliaro et al.31 characterized grape sports that were likely generated after independent DSBs and subsequent repair produced deletions ranging in size from a single bp to larger than Mbs along chromosome 2. More complex structural variations have been identified on three chromosomes of the grape sport Tempranillo blanco32, resembling a chromothripsis-like mechanism , which could have been generated by illegitimate re-joining of chromosome breaks in a unique event. Another example of structural variations that can alter gene expression and phenotype are copy number variants, which involve duplications or deletions of large segments of DNA34.Failures in the DNA replication process and damage caused by chemicals or radiation may produce DNA double-strand breaks (DSBs). Eukaryotic organisms have developed efficient mechanisms to repair DSBs through non-homologous end joining (NHEJ) and homologous recombination (HR) pathways. NHEJ is likely to occur more frequently than HR37. TEs can disrupt coding sequences, alter the expression of nearby genes and produce chromosome breakage leading to genome rearrangement and/or genome instability38. Retrotransposons are one class of TEs that are particularly disruptive because they replicate via reverse transcription of messenger RNAs and the duplicates integrate into other chromosomal locations. Related long terminal repeat (LTR) transposons are also sources of somatic variability as they can excise due to either homologous or illegitimate recombination between the terminal repeats, resulting in genomic loss or rearrangement. The activation and silencing of TEs is regulated by epigenetic mechanisms, therefore changes in such mechanisms may produce TE-driven somatic variability. Transcriptomic and epigenetic analyses have demonstrated variation in the transcription of genes associated with epigenetic mechanisms during bud dormancy release in fruit trees40, which may lead to the generation of sports. Stresses such as wounding, pruning, viral infection, and tissue culture are all known to induce the movement and/or activity of TEs42, as well as DNA damage43. McClintock44 first proposed that stress-induced activation of TEs could be a plant survival strategy to rapidly increase genotypic and phenotypic diversity in response to unfavourable conditions.TEs contribute to genome plasticity and cause the majority of somatic variation in plants45, while new strategies are proposed to identify TEs active during plant development46. For a more precise estimation of somatic variability rates, such methods should consider the layer specificity of some mutations as in Marroni et al47.In most cases, bud sport mutations are genetically identical to their parent except for the new mutation; comparison of the parent and sport genotypes provides an opportunity to identify the molecular lesion responsible for the new phenotype. Several molecular and sequencing methods accompanied by bioinformatics pipelines have been developed to detect novel somatic changes. NGS technologies, especially those producing long, single-molecule reads using nanopore sequencing, have proven reliable in the detection of TEs that are transpositionally active in plantsSports can arise from plants that are heterozygous for one or more loss-of-function mutation(s) that acquire independent new mutation(s) to the functional allele. Dominant gain-of-function mutations or a loss-of-function mutation to a haploinsufficient gene  can produce a different phenotype with a single mutation event.48. Many sports originate as chimeras, comprised of cells from two distinct genotypes20. Most of the bud sports identified affect the fruit, probably because they are easy to observe, and many ornamental sports have altered floral or leaf phenotypes. In the sections that follow, we will group sports by their altered phenotype.Lastly, not all bud sports are caused by genetic mutation. Hybridization between species and spontaneous changes in ploidy can introduce somatic variability leading to novel phenotypes49. Many ornamental plant cultivars originated as bud sports that change the appearance of flowers or inflorescences. Wild roses have a single whorl of five petals, whereas most cultivated roses (Rosa hybrida) have many petals. Dubois50 and co-workers demonstrated that the rose orthologue of AGAMOUS (RhAG) is expressed in whorls 3 and 4 of wild roses, consistent with its role as a C-function gene that has a key role in specifying stamen and carpel identity51. In double-flower roses, RhAG expression is restricted to a much smaller domain in the centre of the floral meristem and whorls of stamens are converted to petals. Analysis of sports that revert back to five petals also show expanded RhAG expression into whorl 350. The molecular lesion causing the misexpression of RhAG is unknown; identification of the mutation responsible may reveal information about the regulation of RhAG expression. The authors suggest that the boundary between A-function and C-function gene expression is very labile and might explain how double flower roses have arisen and been selected by humans more than twice.Humans have appreciated the beauty, colour and aromatic scent of flowers since at least 12,000 years ago52. A somatic variant of Carignan has a reiterated reproductive meristem (RRM) phenotype, leading to large, highly branched fruit clusters and delayed anthesis. The tendrils of RRM mutants are also more indeterminate than wild type, displaying multiple branches or even conversion to a leafy shoot. The RRM phenotype is caused by insertion of a TE into the promoter of VvTFL1A, a close homologue of Arabidopsis TERMINAL FLOWER1 (TFL)35. Genetic and genomic analyses demonstrate that the insertion of the transposon is associated with upregulation of VvTFL1A. This is consistent with previous studies showing that TFL genes control the length of developmental phases and maintain indeterminate inflorescence growth54.Somatic grape mutants with altered flower and inflorescence development produce some of the most conspicuous phenotypes and illustrate the developmental plasticity of the tendril52. Stamen and carpel development is abnormal in variants of Bouchalès and Mourvèdre, the latter being completely sterile. Although the molecular lesion(s) causing these phenotypes is unknown, the MADS box ABC genes would be prime candidates55.Mutants of Gamay, Morrastel, and Pinot initiate extra whorls of sepals and petals and are collectively known as multiple perianth whorlsRhododendron simsii hybrids) are colour sports56. Solid colour and variegated sports are likely L1 mutants because the new phenotype is not transmitted to progeny57. The variegated sports are likely to be transposon-mediated changes to pigment genes, although no direct evidence exists. The “picotee” phenotype is characterized by petals with a coloured centre and white margins. In broad-margined “picotee” mutants, the margin cells were found to be tetraploid and the coloured cells diploid, suggesting that positionally determined polyploidisation underlies this pattern. In general, coloured azalea sports were hypermethylated relative to their parent57. In carnation (Dianthus caryophyllus), the L1 layers showed very different patterns of methylation to L2 and L3 layers, and these patterns differed widely between sports and their parents58.More than half of commercial varieties of azalea  cultivar, Cheng61 and co-workers identified a small indel in RIANT, a gene encoding an anthocyanin transporter required for pigment accumulation. White flowers are homozygous for a 2-bp insertion which introduces a frameshift mutation and a premature stop codon. Red and pink flowers are heterozygous at the RIANT locus, with one non-functional allele and a second allele with either a 1-bp insertion or a 2-bp deletion that restores gene function. Periclinal, mericlinal, and sectorial chimeras with or without RIANT function produce white, pink, and red flowers 64 and almond (Prunus delcis)65. The pear sport is caused by genomic deletion of at least 4 kb that removes an S-RNase gene responsible for the SI reaction in the style. This mutation is only in the L1, providing evidence that the transmitting tissue in pear is L1-derived. In the self-compatible almond sport “Jeffries”, at least two mutations occurred, the deletion of one S haplotype and duplication of another, resulting in self-compatibility.Self-incompatibility (SI) is a genetic mechanism that prevents inbreeding in some flowering plants. In most plants in which this SI mechanism operates, SI is controlled by a single, multi-allelic Citrus reticulata) and seedless navel orange. The Zigui shatian pummelo (Citrus grandis Osbeck) sport produces self-pollinated fruit, but the seeds are sterile because of defective post-zygotic development66. The seedless mandarin sports Ougan67, Wuzishatangju68, and Huami Wugegonggan69 are caused by pollen abortion, blocked fertilization, and pollen sterility and embryo abortion, respectively.In most species, fruit and seed development are linked; however, there are examples where fruit development occurs in the absence of seed development. Other forms of SI occur after pollen germination and affect fertilization or embryo development, often leading to the development of a seedless fruit. Numerous seedless citrus sports have been identified, some of which have become popular cultivars such as satsuma mandarin  oxidase  and the ethylene receptor gene ETHYLENE INSENSITIVE 1 (ETR1) relative to their climacteric parent. A number of frameshift mutations were also identified in genes involved with sugar transport and ethylene biosynthesis. Similarly, early-ripening “Beni Shogun” apples show increased expression of ethylene synthesis and signal transduction genes74. Late-ripening Tardivo mandarin sports are less sensitive to ethylene and have decreased expression of ETR1 and ETR276. Transcriptomic and proteomic analyses of several late-maturing bud sports of sweet orange have revealed differential expression of genes involved with abscisic acid (ABA), ethylene, and jasmonic acid (JA) synthesis and signal transduction, as well as sugar metabolism and carotenoid biosynthesis79.Bud sports that ripen earlier or later than their parents have been identified in numerous species. While many aspects of fruit ripening are directly controlled by the hormone ethyleneVitis vinifera L.) for fresh fruit and wine making for over 10,000 years and from the second half of the 20th century clonal selection for wine grape breeding has been intensively used, so it is not surprising that many somatic variants have been identified that affect berry quality traits (mainly colour) and that these sports have been developed into cultivars. Grape berry colour is caused by the accumulation of anthocyanins in the berry skin (L1) and flesh (L2)80. Two tandemly repeated Myb regulatory genes, VvMybA1 and VvMybA2, regulate red berry colour83. Mutations and instability affecting this locus are the molecular basis for the majority of grape colour sports.Humans have been propagating grapevine , which requires exposure to cold to develop dark red fruit. The blood orange was first documented in Italy in 164692 and was propagated clonally and sexually for centuries. A second blood orange sport arose in China in the late 1800s or earlier93. Both sports are caused by insertion of a retroelement near Ruby, a Myb gene that regulates anthocyanin production in fruit94. Cold induces retroelement transcription which activates transcription of Ruby. Many of the blood oranges found throughout Europe are derived from recombination between the LTRs that were maintained as periclinal chimeras94.One of the most conspicuous fruit sports is the blood orange  and a hybrid of Asian and European pear (Pyrus pyrifolia and P. communis), respectively. Both are associated with decreased methylation of promoter regions of orthologous genes MdMYB1 and PyMYB1096. Conversely, “Blondee”, a yellow sport of a red parent, is associated with demethylation of the MdMYB10 promoter97. Some apple cultivars can produce fruit with both solid and striped pigmentation. The regions of red stripes are associated with increased expression of MdMYB10 and decreased methylation in the MdMYB10 promoter98.Epigenetic changes to Prunus persica) arose from at least three independent loss-of-function mutations to the carotenoid cleavage dioxygenase4 (PpCCD4) gene100. These lesions are caused by an SNP introducing a premature stop, a dinucleotide (CT) insertion in a microsatellite region creating a frameshift mutation causing a premature stop, or the insertion of a retrotransposon into an intron. Genotyping yellow and white cultivars and somatic revertants demonstrated that yellow flesh peaches have two non-functional PpCCD4 alleles, whereas the white progenitors and revertants have at least one functional PpCCD4 allele, which is sufficient to block carotenoid accumulation in the flesh. Two white bud sport mutants, revertants from yellow cultivars, were used to validate the causal relationship between allele and phenotype. Both white sports have a yellow suture (an L1-derived tissue), indicating that these are periclinal chimeras comprised of a PpCCD4-deficient L1 and a revertant L2 that has restored one functional PpCCD4 allele100. In one of the sports, the insertion of another CT in the mutated microsatellite region restored the reading frame and removed the stop codon99. In the other sport, deletion of the retrotransposon led to restoration of gene function100.Yellow flesh peaches , and grapefruit104 (Citrus paradisi Macf.) have greatly increased accumulation of lycopene in the fruit. The genetic lesions causing the increase in lycopene accumulation have not been identified; however, one report showed that a cluster of six candidate genes exhibited gene dosage variation and decreased transcription between the sport Hong Anliu and its parent105. A yellow citrus sport Pinalate has yellow fruit with high levels of carotenes and decreased ABA content106. Three citrus sports with brown skin have been reported to be caused by defective chlorophyll degradation107, altered carotenoid accumulation108, and defective synthesis or accumulation of β-citraurin108.Pink or red sports in orange110. Interestingly, giant fruit sports in apple111 and pear112 both showed fruit-specific increases to DNA content and cell size, suggesting that fruit cell size is under separate regulation to other parts of the plant. Another giant pear sport had no change in ploidy, but showed increased expression of an actin-related protein that is involved with regulating cell proliferation in Arabidiopsis114.After fruit colour, the most obvious type of sport mutant is one that has a different fruit size or shape. Final fruit size results from cell division and enlargement, and many fruit size sports are clearly affected in one of these processes. Increased DNA content caused by endoreduplication or chimeric polyploidization often results in larger cells and increased lateral organ and/or fruit sizeDiospyros kaki Thumb.) that also increases sugar content in fruit and causes a more compact tree architecture115. Application of exogenous cytokinin restores normal fruit size116, suggesting that the mutant gene involves cytokinin biosynthesis or signal transduction. The progenitor of TTN is non-aploid (2n = 9×) and has produced many sports, raising the possibility that recent increases in ploidy have led to increased transposon movement and/or chromosomal rearrangements.“Totsutanenashi” (TTN) is a small fruit sport in Japanese persimmon (fleshless berry (flb) mutation disrupts cell division and differentiation in the mesocarp of the fruit, resulting in a 10-fold reduction in berry weight117. Observations that the flb phenotype was unstable and that some progeny obtained from sexual propagation showed a new phenotype that failed to set fruit led to the discovery that the original flb mutant is an L2 chimera118. The flb mutation is caused by the insertion of a TE into the promoter of VvPI, resulting in ectopic expression of the grape homologue of Arabidopsis PISTILATA120. Analysis of plants that carry the mutation in the L1, L2, or in both layers demonstrate the differential effects of ectopic VvPI expression in each layer. VvPI expression in the L2 blocks flesh development by preventing mesocarp cells from differentiating, while VvPI expression in the L1 and L2 disrupts carpel development at fruit set119. This is an excellent example of how the study of a sport mutant has provided new insights into the layer-specific role of PI in floral and fleshy fruit development. Interestingly, both the TTN and flb L2 sports have increased sugar content and decreased phenolic compounds, which suggest a potential connection between fruit size and biochemical properties117.In grape, the 121. Genotypic analysis indicates that nectarines were introduced or arose in Europe multiple times, probably as bud sports122. Genomic data from five peach/nectarine accessions indicated that the insertion of a LTR retroelement in the coding sequence of PpeMYB25 is the likely cause of the hairless nectarine phenotype123. In Arabidopsis, loss-of-function mutations to the closely related MYB gene, GLABRA1, result in hairless leaves124. Nectarine sports of peach are frequently observed in orchards and some have been commercialized.Nectarines were first identified in China over 2000 years ago125. Analysis of the flat fruit indicates they have fewer cells in the vertical axis due to earlier cessation of cell division relative to round fruit126. The flat fruit phenotype has been associated with a 10 kb deletion that removes the first 693 bp of a leucine-rich receptor-like kinase (LRR-RLK) gene (PRUPE.6G281100/ppa025511m), orthologous to the Arabidopsis BRASSINOSTEROID INSENSITIVE1 ASSOCIATED KINASE (BAK1) group125. BAK1-like genes regulate cell division and meristem size in Arabidopsis128, rice130, and maize131. A bud sport mutant reverting from flat to round , which may itself be a bud sport mutant that originated in China. Individuals heterozygous for S have flat fruit, while those homozygous for S show early abortion of the fruit133. Occasionally, leaf sectors lacking the hairy phenotype appear on Pinot Meunier suggesting that it is a periclinal chimera with a mutant L1133. Indeed, plants regenerated from L1 or L2 layers indicate L1-derived plants are hairy, while L2-derived plants are hairless2. More striking is the dwarfed phenotype of L1-derived plants, caused by very short internodes  oxygenase was upregulated 14-fold over “McIntosh” in young axillary buds. Overexpression of this gene (MdCo31) in Arabidopsis resulted in very short inflorescences due to reduced internode lengths137. Members of this gene family are involved with the biosynthesis of ethylene, flavonoids, gibberellins, and defence against downy mildew. Several studies have used RNAseq to identify differentially expressed genes (DEGs) between columnar and standard trees139. Many of the DEG from shoot meristem tissue are involved with hormone metabolism and signalling140. Genes involved with lignin and terpene biosynthesis, and pathogen/pest attack response were highly upregulated in “Wijcik” leaves36. Two other genes near the insertion are upregulated in “Wijcik”. One encodes a helix-loop-helix transcription factor and the other Downy Mildew Resistant 6 (DMR6), a regulator of defence genes.In the early 1960s, apple grower Anthony Wijcik noticed an abnormal shoot on a “McIntosh” tree in his orchardSome annual crop plants are clonally propagated for a variety of reasons. Clonal propagation preserves the genotype and phenotype of the parent plant, which could be lost in sexual propagation. Prevention of sexual reproduction helps avoid inbreeding depression which is common in outcrossing species. In varieties selected for seedlessness, such as banana, sexual propagation may not be possible.Solanum tuberosum) cultivars are bud sports that alter the pigmentation of the tuber including the leading French fry processing cultivar “Russet Burbank“145. Many coloured potato sports are periclinal chimeras that show a range of pigmentation from white to purple145. Careful analysis of colouration patterns in these sports and their sexual offspring revealed that the L1 gives rise to most of the tuber skin except for a small patch below each eye, which is L2 derived143. The “Kostroma mutant” does not affect the tuber, but shows pronounced dissection of leaf blade and flower corolla146. Spontaneous mutations giving rise to coloured tubers and sectorial chimeras have also been developed into popular cultivars of sweetpotato (Ipomoea batatas)147.A large number of potato (Manihot esculenta Crantz) is an important staple crop in Sub-Saharan Africa, the tropics, sub-tropics, and South Pacific islands. Cassava sports with altered leaf morphology and increased vigour are associated with spontaneous changes in ploidy, probably caused by the fertilization of unreduced gametes150. Many of the crops grown in Oceania originated from a very limited number of introduced genotypes and have been mostly or exclusively vegetatively propagated. Somaclonal variation has been a valuable source of phenotypic diversity for farmers growing taro (Colocasia esculenta Schott) and yam 151, bananas and plantains (Musa spp.)153, and kava (Piper methysticum Forst f.)154.Cassava (Bud sport mutants introduce new genetic variability, which is crucial for species that lack variability or cannot be sexually reproduced. Somatic mutations can also provide valuable new characteristics while retaining the desirable qualities of the parent plant, which is why many popular cultivars have originated from sports. In some cases, the analysis of sports has revealed new information about layer-specific roles of some genes or has provided valuable insight into gene function during specific developmental processes. As sequencing technologies improve, sports also provide a means to examine mechanisms that drive genomic evolution.


Human females have a unique duration of post-reproductive longevity, during which sex-specific mechanisms ma influence later-life mechanisms of neuronal resilience and vulnerability. The maintenance of energy metabolism, through the oxidative phosphorylation (OXPHOS) apparatus, is essential for brain health. Given the known association between reproductive period (years from menarche to menopause) and cognitive aging, we examined the hypothesis that cumulative estrogen exposure across the lifetime may be associated with differential methylation of genes in the OXPHOS pathway.Using DNA methylation patterns in the post-mortem dorsolateral prefrontal cortex (DLPFC) of 426 women prospectively followed until death in the Religious Orders Study and Rush Memory and Aging Project, we examined the relationship between reproductive period (subtracting age at menarche from age at menopause) and DNA methylation of a published set of autosomal OXPHOS genes previously implicated in stroke susceptibility. We then performed an unsupervised analysis of methylation levels across the Hallmark pathways from the Molecular Signatures Database.E2F, MYC and MTORC1 signaling, fatty acid metabolism and DNA repair.We observed a strong association between reproductive period and DNA methylation status across OXPHOS CpGs. We replicated this association between reproductive period and DNA methylation in a much larger set of OXPHOS genes in our unsupervised analysis. Here, reproductive period also showed associations with methylation in genes related to This study provides evidence from both a supervised and unsupervised analyses, that lifetime cumulative endogenous steroid exposures may play a role in maintenance of post-menopausal cellular balance, including in brain tissue. The maintenance of energy metabolism through the oxidative phosphorylation (OXPHOS) apparatus is essential for brain health –5. UnderIn an aging population, sex-specific mechanisms  may To examine the hypothesis that methylation of genes in the OXPHOS pathway is associated with lifetime estrogen exposure, we leveraged a rich data set of 426 female dorsolateral prefrontal cortex samples, from two well-phenotyped longitudinal cohorts.Religious Orders Study (ROS) and the Rush Memory and Aging Project (MAP)[We examined DNA methylation patterns in the post-mortem dorsolateral prefrontal cortex (DLPFC) of 456 women who were enrolled in 2 prospectively followed cohorts maintained by investigators by the Rush Alzheimer’s Disease Center in Chicago, IL: the ect (MAP), 22. TheAs in previous manuscripts, we analyzed the ROS and MAP cohorts jointly since they were designed to be combined, are collected by a single investigative team, and a large set of phenotypes collected are identical in both studies. All aspects of these studies were approved by the Institutional Review Boards of Rush University Medical Center and Partners Healthcare. More detailed information regarding the two cohorts can be found in previously published literature , 22.At baseline, participants were asked about current and past estrogen-based hormone therapy (HT) use (ever vs. never), age at which they started and stopped taking estrogen, age at menarche and menopause , and whether menopause had occurred naturally or been induced surgically. We excluded women with other types of menopause. Current HT use was verified by inventory of prescription bottles that participants brought to the interview, with an agreement of 93%. As previously, data from 10 women were excluded because they provided highly unlikely ages at menopause (<20 or >60 years and 4 were excluded due to unlikely ages at menarche (>30) . ReproduWe used a set of 95 autosomal genes encoding proteins directly involved in the OXPHOS respiratory chain, selected based on published criteria from a chemical dissection of mitochondrial function, and previously associated with risk of ischemic stroke and intracranial hemorrhage in a large stroke consortium [Our methods for obtaining methylation measures at 415,848 discrete CpG dinucleotides in ROS/MAP subjects have been previously described . BrieflyPreviously described quality checks of the data included (1) using the detection p-value criteria recommended by Illumina (i.e. < 0.01) to ensure the use of good quality probes, and (2) removal of probes strongly predicted by Illumina and observed by our team to cross-hybridize with the sex chromosomes based on sequence alignment or of autosomal, polymorphic CpGs where methylation level of these CpGs could be affected by a subject’s genotype (i.e. in which a Single Nucleotide Polymorphic site (SNP) with a minor allele frequency (MAF) ≥0.01 exists within 10 base pairs upstream or downstream of the CpG site. Finally, we removed subjects with poor quality individual data, according to Principal Component Analysis (PCA) , and toGene expression data exists for 335 subjects of the 426 subjects with DNA methylation data. Gene expression was measured using FPKM values obtained from Illumina HiSeq RNA sequencing as previously described .For our analyses, we used the β-values reported by the Illumina platform for each probe as the methylation level measurement for the targeted CpG site, as previously described . These βIn order to examine the association between cumulative estrogen exposure and OXPHOS gene methylation we first used a series of linear models to test the association of each probe with reproductive period (age of menopause minus age of menarche) adjusting for age at death, study (ROS or MAP), experimental batch, bisulfite conversion efficiency, the proportion of neuronal cells in the sample, race, cIn order to examine the links between OXPHOS gene expression and reproductive span, as with the methylation data, linear models were first used to assess the relationship between all 23,205 genes with average FPKM greater than 1 and reproductive span accounting for the same confounders as in the methylation models. A Wilcoxon-rank-sum test was then used to test if the OXPHOS genes were more associated with reproductive span than all other genes. To assess if OXPHOS probes were correlated with gene expression, for each gene the average correlation between the gene and all its annotated probes were calculated.Table 1. The mean (SD) menopausal age for women with natural menopause was 48.2 (5.4) and for surgical menopause was 42.3 (7.1). We found a large diversity in the duration of reproductive period, defined as age at last period minus age at first period, a marker of long-term estradiol exposure; this ranged from 8 to 48 years (median = 35). Methylation levels from prefrontal cortex samples were measured using an Illumina infinium HumanMethylation450K BeadChip as previously described [The 426 women who were part of one of two prospective cohorts of aging and dementia the Religious Orders Study and the Rush Memory and Aging Project (ROSMAP), were free of known dementia at study enrollment and followed annually until brain donation at death. Their demographic characteristics at the time of autopsy are summarized in escribed , 28, 29.In our first approach, we performed hypothesis-driven analyses starting with the list of 95 OXPHOS genes previously shown to be associated with neurovascular diseases. We examined the association between reproductive period and DNA methylation in the 1344 CpGs annotated by Illumina as being in the TSS, 5’ or 3’ UTR or gene body of the 95 genes [−4; adjusted for age at death, study (ROS or MAP), experimental batch, bisulfite conversion efficiency, estimated proportion of neuronal cells in the sample, race, cigarette pack-years smoked, menopause type and hormone therapy use). This association was primarily driven by positive relationships between methylation of OXPHOS CpGs and reproductive period . Table 2 shows the OXPHOS CpGs associated with reproductive period with a nominal p-value less than 0.01, as well as according to more stringent FDR. The most differentially methylated OXPHOS probe lies in the transcription start site of NDUFS8.We tested our primary hypothesis that reproductive period was assocated with methylation changes in OXPHOS genes using two complementary approaches. NDUFS8 are depicted in Fig 1.As an illustration, the methylation patterns of probes within ±20kb of −6, n = 300) and surgical  menopause. HT use did not mitigate these associations.Supporting a causal association between hormonal exposures and methylation changes, we noted an association between reproductive period and OXPHOS gene methylation levels in subjects with both natural  among 10 pathways that were differentially methylated with an FDR of 0.00012 (Table 3).e (MSigDB) to findThe Hallmark oxidative phosphorylation pathway largely overlaps with the set of 95 OXPHOS genes [The ROSMAP cohort has gene expression data for 335 of the women in our analyses . Here, tUsing both a hypothesis-driven and an unsupervised pathway analysis approach, we found that longer reproductive period was significantly associated with subsequent levels of methylation across a set of OXPHOS genes, genes known to be associated with stroke, a major cause of worldwide mortality and neurological morbidity.It is known that sex influences epigenetic modifications, including DNA methylation, in the blood and across many tissues, potentiThis study had three important limitations. First, given their complexity and potential for inactivation and dosing effects, the sex chromosomes were not included in the current methylation analyses; however, OXPHOS genes are found primarily on autosomes. Second, we used only an estimate of the proportion of neuronal cells in the sample, and methylation levels across other cell types might have been confounders. Third, the lack of association between reproductive exposures and RNA expression suggests that other variables in the postmenopausal period may play a stronger role in regulating expression. Finally, it is important to note that the study did not evaluate for other possible modifiers of DNA methylation, such as smoking or medications.Human females are unique among mammals in the duration of post-reproductive longevity. Previous investigations of sex-specific mechanisms leading to neurological resilience have uncovered an association between reproductive period, i.e. a woman’s period of maximal exposure to levels of estradiol and other gonadal hormones, and cognitive decline in old age. Here, we advanced these findings mechanistically with reporting a significant association of reproductive period with methylation levels across genes in the OXPHOS pathway. This provides some mechanistic suggestion that early and mid-life hormonal exposures may influence risk of late-life neurological morbidity and mortality via epigenetic regulation of energy metabolism.S1 FigA plot demonstrating the ranks of each of the 1,344 OXPHOS probes with respect to all 420,132 included probes. Each black vertical line represents an OXPHOS probe. A centered cumulative sum is also plotted as a blue line. Tracking the ranks of probes from left to right, when the blue line increases the number of OXPHOS probes seen is increasing more than expected and vice versa for when the blue line decreases.(PNG)Click here for additional data file.


TP53 and LKB1 and hyperactivation of the mTOR pathway and of oncogenes such as MYC, RAS, and AKT have been shown to fuel nucleotide synthesis in tumor cells. The molecular mechanisms by which these signaling hubs influence metabolism, especially the metabolic pathways for nucleotide synthesis, continue to emerge. Here, we focus on the current understanding of the molecular mechanisms by which oncogenes and tumor suppressors modulate nucleotide synthesis in cancer cells and, based on these insights, discuss potential strategies to target cancer cell proliferation.Cancer cells exhibit a dynamic metabolic landscape and require a sufficient supply of nucleotides and other macromolecules to grow and proliferate. To meet the metabolic requirements for cell growth, cancer cells must stimulate de novo nucleotide synthesis to obtain adequate nucleotide pools to support nucleic acid and protein synthesis along with energy preservation, signaling activity, glycosylation mechanisms, and cytoskeletal function. Both oncogenes and tumor suppressors have recently been identified as key molecular determinants for de novo nucleotide synthesis that contribute to the maintenance of homeostasis and the proliferation of cancer cells. Inactivation of tumor suppressors such as  Signaling systems allow cells to sense their internal and external surroundings in an integrated manner and generate harmonized responses comprising processes such as growth, proliferation, differentiation, and survival. The development of cancer involves consecutive alterations encompassing signaling, metabolic and genetic modifications that that empower cells to escape self-regulating mechanisms that generally eliminate the survival of abnormally proliferating cells. Although cancer cells are heterogeneous in origin and cell type, most cancer cells share features called hallmarks . Some chMechanisms for proper cell division require the preservation of nucleotide pools used for DNA and RNA synthesis. Nucleotides can be produced through salvage pathways, via the recycling of existing nucleosides and nucleobases, or through the de novo synthesis pathways, using amino acids and small molecules to build the purine and pyrimidine rings. Unlike nonproliferating cells, proliferating cells such as immune cells and cancer cells are predisposed to use the de novo nucleotide synthesis pathways ,5. The mPurine synthesis differs from pyrimidine synthesis in that all reactions occur in the cytosol, and the purine ring is directly built onto the activated ribose, PRPP. The purine ring is synthesized from various substrates, including glutamine, glycine, bicarbonate, and N10-formyl-tetrahydrofolate (THF). After a 10-step reaction, inosine monophosphate (IMP) is produced and converted into guanosine monophosphate (GMP) (via the enzymes inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate synthetase (GMPS)) or adenosine monophosphate (AMP) (via metabolic reactions involving adenylosuccinate synthase (ADSS) and adenylosuccinate lyase (ADSL)) B. Once fIt is well recognized that cancer cells rewire their metabolism to enhance de novo nucleotide synthesis in order to grow and proliferate; however, the molecular events by which oncogenes or tumor suppressors modulate these metabolic pathways are not fully elucidated. Here, we systematically review the literature defining the influence of signaling pathways on nucleotide metabolism by first focusing on the short-term molecular features involving posttranslational modifications, then on the long-term processes comprising transcriptional mechanisms and finally on the reemergence of nucleotide metabolism as a new targetable weakness for cancer therapy.Altered expression and activity of metabolic enzymes, including enzymes involved in nucleotide synthesis, are regulated by oncogenes and tumor suppressor genes . To examAs mentioned above, de novo nucleotide synthesis is activated in proliferating cells in response to the enhanced demand for nucleotides to support RNA and DNA synthesis. Recently, several studies have indicated that de novo nucleotide synthesis required for cancer cell proliferation is directly regulated by oncogenes and tumor suppressors. An increasing number of oncogenes  and tumor suppressors ) and the primary metabolic sensor (mTOR complex 1 (mTORC1)) described below are being implicated in the acute regulation of nucleotide metabolism ,9,10,11.Mechanisms supporting cell growth and anabolic metabolism are coordinated by the mechanistic target of rapamycin (mTOR) . mTORC1 mTORC1 is a signaling and metabolic hub that senses nutrients and energy as well as other growth signals to regulate increases or decreases in anabolic and catabolic processes ,14,15,16A study published by Graves and colleagues connected ERK signaling to CAD activity. ERK induces phosphorylation of CAD on Thr456 in the carbamoyl phosphate synthetase 2 (CPS2) domain, which leads to a decrease in the affinity of CAD for the allosteric inhibitor uridine triphosphate (UTP) and a concomitant increase in the binding of the allosteric activator PRPP to the CPS2 domain ,25. Two Additionally, Carrey et al.,  revealedThe small GTPase RAS integrates growth signals through the activation of the receptor tyrosine kinase by growth hormones and is therefore involved in the control of cell growth, differentiation and survival . This grMultiple lines of evidence indicate that ERK activation leads to long-term stimulation of de novo pyrimidine and purine synthesis through the regulation of the transcription factor c-MYC ,34. It iTo control cell growth and survival, the PI3K/Akt pathway integrates environmental signals, notably growth factor signaling. Saha et al., (2014) demonstrated that upon various growth cues, Akt binds to transketolase (TKT), a key enzyme in the nonoxidative PPP  and direPRPP is synthesized by the enzyme phosphoribosylpyrophosphate synthetase (PRPS1/2/L1) by a one-step transfer of the β,γ-diphosphoryl group of adenosine triphosphate (ATP) to the C-1 hydroxyl group of α-d-ribose 5-phosphate. The availability of PRPP can be modulated by the allosteric binding of a purine nucleotide (ADP) to PRPS, which modulates nucleotide production in mammalian cells . RecentlGlycolysis produces ATP and metabolite intermediates that can be immediately used for anabolic metabolism . Pyruvat+ provides a pool of reducing equivalents for the synthesis of both nucleotides and lipids. Moreover, NADPH is essential for counterbalancing the high levels of reactive oxygen species (ROS) generated by increased metabolic activity [+. Akt directly phosphorylates and stimulates NAD kinase (NADK) in mammalian cells, thereby increasing the availability of cellular NADPH, which is required to mitigate stress from high ROS levels and sustain NADPH-dependent anabolic production of nucleotides and lipids in proliferating cells [The oxidative branch of the PPP producing NADPH from NADPactivity . A recenng cells ,55,56 , the activation of specific metabolic conduits and, more importantly, the synthesis of macromolecules such as proteins, lipids and nucleic acids from metabolic precursors ,58. HerePrps2 mRNA translation through a specialized cis-acting regulatory element and directs an increase in nucleotide biosynthesis. In addition, MYC controls the expression of genes associated with the uptake and catabolism of glutamine required for de novo nucleotide synthesis and other metabolic processes [MYC was first identified as a cellular homolog of v-myc, a retroviral gene that was found to induce tumorigenesis in chicken cells ,60. The rocesses . Hyperacrocesses ,72. For rocesses  demonstrUnderstanding the complexity of MYC-mediated metabolic rewiring in cancers, along with the ways in which MYC cooperates with other signaling drivers such as the mTORC1 and RAS/ERK pathways, can provide translational prospects for cancer therapy.MYC is activated downstream of the RAS-ERK pathway ,75. K-RADownstream of K-RAS, the oncogenic activation of BRAF, which encodes a serine-threonine kinase, also maintains nucleotide synthesis through MYC activation. BRAF mutations are common in various human cancers; the point mutation V600E accounts for 80% of all BRAF mutations, especially in melanoma . BRAF prLiver kinase B1 (LKB1) is a serine/threonine kinase inactivated in a range of cancers. Interestingly, inactivation of LKB1 is often accompanied by mutations in the RAS-RAF pathway in human cancers . A studyThe tumor suppressor protein 53 (TP53 or p53) is a transcription factor that regulates the expression of genes with diverse cellular functions. p53 is a major tumor suppressor found to be mutated in more than 50% of human cancers . AccumulUnlike de novo pyrimidine synthesis, de novo purine synthesis is stimulated by mTORC1 signaling in a more delayed manner via the regulation of the transcription factors MYC, sterol regulatory element binding protein (SREBP) and activating transcription factor 4 (ATF4), which induce the expression of specific metabolic enzymes required for de novo synthesis of purines. For example, ATF4 downstream of mTORC1 signaling, stimulates gene expression of the serine/glycine synthesis pathway and the mitochondrial tetrahydrofolate cycle, which produces the one-carbon formyl units necessary for purine ring assembly in the cytosol  Figure . More spThe proper execution of the regulated nucleotide synthesis program downstream of mTORC1 needs substantial input of nutrients and energy. Therefore, the activation state of mTORC1 is robustly influenced by the availability of building blocks  for the enhanced synthesis of macromolecules  ,93,94,95Upstream of mTORC1, activation of the PI3K/Akt pathway has been shown to stimulate nucleotide synthesis. Interestingly, loss of the tumor suppressor PTEN, a lipid phosphatase that dephosphorylates phosphatidylinositol -triphosphate (PIP3) to phosphatidylinositol -bisphosphate (PIP2), results in the activation of AKT and mTORC1 signaling and thus the promotion of cell growth and proliferation and the inhibition of apoptosis ,98. PTENYAP1 depletion resulted in reduced organ growth, and this phenotype was partially rescued by providing exogenous nucleosides [The Hippo signaling pathway regulates organ size and tissue growth in response to surrounding signals . These eleosides . The metAll of these findings highlight the existence of lines of communication connecting tumor suppressors, oncogenes and nucleotide synthesis in cancers. However, understanding whether these signaling and metabolic routes could be used to define potential therapeutic avenues to target specific cancers requires further studies.The examination of the distinct metabolic dependencies of many tumors provided potential metabolic targets for exploiting nucleotide metabolism, some of which are being evaluated in preclinical models and clinical trials. Here, we discuss past and current developments in nucleotide metabolism research that have identified metabolic targets and highlighted features that might be exploited to improve cancer therapy.The primary medical success of antifolates led to the production of an entire class of drugs known as antimetabolites. Antimetabolites are small molecules that are chemically similar to nucleotide metabolites, but often competitively inhibit the activity of enzymes involved in nucleotide synthesis. Sidney Farber was the first to discover that aminopterin could induce disease remission of some cancers . AminoptRecent studies have revisited the use of nucleotide synthesis inhibitors to target tumor cell progression. Targeting cancer metabolism through the inhibition of DHODH has recently received much consideration . Steady-Interestingly, DHODH is the only enzyme in the de novo pyrimidine synthesis pathway that is localized in mitochondria and requires oxidative phosphorylation (OXPHOS) to function correctly . FurtherGiven that mTORC1 is a master regulator of cellular metabolism and induces metabolic stimulation of de novo purine and pyrimidine synthesis ,36 in caPurine dependency was further observed in acute lymphoblastic leukemia and glioblastoma . In addiThe control of cellular metabolism by growth factor-initiated signaling pathways is frequently deregulated in cancer. These oncogenic alterations promote increased nutrient uptake and anabolic metabolism to assemble macromolecules such as the proteins, lipids and nucleic acids indispensable for cell growth. These signaling pathways impact nucleotide metabolism, but the molecular links between these networks and the nucleotide synthesis pathways continue to be explored. The direct roles of mTOR and MYC in the regulation of nucleotide synthesis are currently recognized to be prominent across different types of tumors. Recent studies targeting DHODH or IMPDH reveal metabolic vulnerabilities potentially exploitable in cancer treatment. However, the use of brequinar (a DHODH inhibitor) as a single agent has not yet yielded significant clinical results . One potAdditionally, the cell proliferation rates of normal proliferating cells are often higher than those of cancer cells, and the involuntarily obliteration of normal proliferating cells with antimetabolite chemotherapy can cause serious side effects in patients. Despite these toxicities, antimetabolites are often approved in many modern chemotherapy regimens since they increase the patient survival rate and, in some cases, help eradicate the disease; however, the specificity of these therapies needs to be improved. The signaling and metabolic features of different types of cancers should be considered in the context of cancer therapies, but should be mechanistically investigated to determine effective future treatments specifically targeting cancer cell metabolism influenced by the dysregulation of particular signaling pathways.


Pesticide use is increasing in low- and middle-income countries (LMICs) including Costa Rica. This increase poses health risks to farm owners, farm workers, and communities living near agricultural farms.We aimed to examine the health effects associated with occupational pesticide exposure in farm owners and workers from conventional and organic smallholder farms in Costa Rica.We conducted a cross-sectional study involving 300 owners and workers from organic and conventional horticultural smallholder farms in Zarcero County, Costa Rica. During the baseline study visit, we administered a structured, tablet-based questionnaire to collect data on sociodemographic characteristics, pesticide exposure, and health conditions  and administered a neurobehavioral test battery ; we measured blood pressure, anthropometry , and erythrocytic acetylcholinesterase activity and also collected urine samples. In addition, a functional neuroimaging assessment using near-infrared spectroscopy was conducted with a subset of 50 study participants. During the follow-up study visit (~2-4 weeks after the baseline), we administered participants a short questionnaire on recent pesticide exposure and farming practices and collected hair, toenail, and urine samples. Urine samples will be analyzed for various pesticide metabolites, whereas toenails and hair will be analyzed for manganese (Mn), a biomarker of exposure to Mn-containing fungicides. Self-reported pesticide exposure data will be used to develop exposure intensity scores using an exposure algorithm. Furthermore, exposure-outcome associations will be examined using linear and logistic mixed-effects regression models.Fieldwork for our study was conducted between May 2016 and August 2016. In total, 113 farm owners and 187 workers from 9 organic and 83 conventional horticultural smallholder farms were enrolled. Data analyses are ongoing and expected to be published between 2019 and 2020.This study is one of the first to examine differences in health effects due to pesticide exposure between farm owners and workers from organic and conventional smallholder farms in an LMIC. We expect that this study will provide critical data on farming practices, exposure pathways, and how occupational exposure to pesticides may affect farm owners and workers’ health. Finally, we hope that this study will allow us to identify strategies to reduce pesticide exposure in farm owners and workers and will potentially lay the groundwork for a future longitudinal study of health outcomes in farm owners and workers exposed to pesticides.DERR1-10.2196/10914 Pesticides are extensively used in agriculture and for control of vector-borne diseases across the globe ,2. CurrePesticide applicators (either farm owners or workers) in smallholder farms are often exposed to these chemicals at different stages of the process  ,10. TherThe characterization of pesticide exposures in LMICs is challenging due to the short half-lives of most of these chemicals in the human body, limited availability of biomarkers of exposure, and lack of epidemiological data -20. As hSeveral studies have examined the health effects of occupational pesticide exposure in tropical settings -29. In aWe conducted a cross-sectional study of 300 farm owners and workers with repeated exposure assessment at two time points (to study the variability of pesticide exposure over time) between May 2016 and August 2016 . The specific objectives of the project are as follows :To assess occupational pesticide exposure in owners and workers of conventional and organic farms, using self-reported pesticide use data and biomarkers of pesticide exposure.To evaluate the association of occupational pesticide exposure  with self-reported symptoms of acute intoxication in the last 12 months.To examine the association of occupational pesticide exposure with self-reported respiratory and allergic outcomes in the last 12 months.To evaluate the association between occupational pesticide exposure and cardiometabolic effects, such as adiposity and high blood pressure.To assess the association of occupational exposure to organophosphates and carbamates with erythrocytic acetylcholinesterase (AChE) activity.To assess the association of occupational pesticide exposure with neurobehavioral outcomes, such as working memory, visual perception, and fine motor function.To examine the association of occupational pesticide exposure with changes in brain activity, assessed using functional near-infrared spectroscopy (fNIRS).This study is part of the Pesticide Use in Tropical Settings (PESTROP) Project, which aims to deepen our understanding of the environmental, health, and regulatory dimensions of pesticide use in conventional and organic agriculture in LMICs. The design of our research was partly informed by a study conducted in the same study area between 2014 and 2016 . This stAll study materials and procedures were approved by the human subjects committee of the Universidad Nacional in Costa Rica (UNA-CECUNA-ACUE-04-2016) and Ethical Board of the Ethikkommission Nordwest- und Zentralschweiz in Switzerland (EKNZ-UBE 2016-00771). Written informed consent was obtained from all study participants at enrollment. Study results will be communicated back to participants and stakeholders at restitution workshops .2 of horticultural farms) [The study was conducted in the Tapezco river catchment in the Zarcero County, Costa Rica . This ril farms)  and has l farms) ; chlorotl farms) .Conventional farms in the study area were identified using random global positioning system (GPS) points generated on the basis of smallholder land use data from 2015  . After aAfter organic and conventional farms in the study area were identified, farm owners were briefly informed about the study aims and procedures  as follows: “Station 1 ” included the administration of the informed consent and a structured questionnaire to collect data on sociodemographic characteristics, occupational history, pesticide exposure at work and at home, medical history including respiratory and allergic outcomes in the last 12 months, and a blood pressure measurement; “Station 2 ” included the administration of a neurobehavioral test battery  and a checklist of acute pesticide intoxication symptoms in the last 12 months; “Station 3 ” included measurements of the erythrocytic AChE activity, anthropometry , and blood pressure (second measurement) and urine sample collection; and “Station 4 ” included the fNIRS assessment (only completed by a subset of study participants).Participants were visited twice during the study duration, either at the farms where they worked or at their homes . The basThe follow-up study visits  were conducted by two trained research assistants and included the administration of a short questionnaire on recent pesticide exposure  and farming practices (administered only to farm owners) and collection of toenail and hair samples and a second urine specimen.All study instruments were pilot-tested in 10 farmers. Study protocols were administered by trained research assistants in Spanish, and data were entered directly into tablets  via an entry mask using Open Data Kit . QuestioWe based our sample size calculation on the difference in the erythrocytic AChE activity between organic and conventional farm owners and workers . With a Information on sociodemographic variables and occupational exposure to pesticides was collected using structured questionnaires . During . Specimens were stored at 4°C until the end of the fieldwork day. Then, samples were aliquoted in 15-mL plastic test tubes  and stored at −20°C until their shipment at 4°C to Lund University, Sweden, for analysis.Spot urine samples were collected during the baseline and follow-up visits in plastic containers of 100 mL Urine samples will be analyzed for multiple pesticide metabolites including, but not limited to, ethylenethiourea ; propylenethiourea ; 3,5,6-trichloropyridinol ; 3-phenoxybenzoic acid ; and hydroxy pyrimethanil (a metabolite of pyrimethanil). These pesticides and their metabolites were selected because they are among the most commonly used in Zarcero  and for Hair samples (~20-30 strands) were collected from the occipital region, within 2 mm from the scalp, using stainless-steel scissors during the follow-up study visit. The samples were then stored at room temperature in sterile plastic bags until their shipment to the Federal University of Bahia, Brazil.3 spectroscopic-grade acid in a microwave digestion oven . The digested material will be diluted to 10 mL with ultrapure water. Hair samples, certified reference material , and reagent blanks will be analyzed using electrothermal atomic absorption spectrometry with Zeeman background correction [Hair specimens will be analyzed for manganese (Mn), which is contained in ethylene bisdithiocarbamate fungicides, such as mancozeb. Briefly, the nearest centimeter scalp  of hair will be sonicated for 20 min in 0.5% Triton, rinsed 5 times with ultrapure water, sonicated for 10 min in 1-N nitric acid, rinsed once with 1-N nitric acid, and then rinsed 5 times with ultrapure water . Approxirrection . The anaToenail samples were collected during the follow-up study visit. Participants were asked to cut their toenails with clean stainless-steel nail clippers and put them inside of a sterile plastic bag. Toenail specimens were stored at room temperature until their shipment to Federal University of Bahia, Brazil.Toenail samples will be analyzed for Mn using the same procedure described above for hair samples. Briefly, nails will be washed in a Triton X-100 solution, put in acetone, repeatedly rinsed with ultrapure water, and then dried in an oven. Later, the dried nails will be digested with spectroscopic-grade acid in a microwave digestion oven , diluted to 10 mL with ultrapure water, and analyzed electrothermal atomic absorption spectrometry with Zeeman background correction ,43. All Participants were administered a checklist of symptoms of acute organophosphate and carbamate poisoning  during the 12-month period before the baseline study visit. This checklist has been previously used in studies of Latin American farm workers ,44.A short version of the European Community Respiratory Health Study II questionnaire  was admiStudy participants were administered the following eight neurobehavioral tests: Animals and words with initial letter “F”  ; Digit SThese tests were selected on the basis of previous studies of Latin American populations exposed to pesticides ,29, admiWe used fNIRS  to assess the cortical function associated with pesticide exposure in a random subsample of 50 study participants; a detailed description of our study methods can be found elsewhere . SpecifiStation 1 ” and “Station 3 ” and about 1.5 hours apart from each other) during the baseline study visit using an automatic sphygmomanometer (Advantage 6021N). In addition, they measured participants’ height (cm) and weight (kg) using a portable stadiometer and a digital scale . Waist circumference (cm) was measured using a tape measure Hoechstmass .Trained research assistants measured systolic and diastolic blood pressure at two different time points . Briefly, a small lancet (size 30) was used to collect a small sample of 10 µm from the tip of the index finger of each study participant and placed into a capillary tube. Blood samples were analyzed on site for the erythrocytic AChE activity and hemoglobin levels using the same collection instrument .We will explore differences in self-reported pesticide exposures and health outcomes  in farm workers from conventional and organic farms. Cumulative lifetime pesticide exposure, exposure during the last 12 months, and during the last week will be estimated using exposure intensity scores derived from a semiquantitative exposure algorithm  . ExposurAssociations of pesticide exposure with health outcomes of interest will be examined using both exposure scores and biomarkers of exposure. More specifically, we will fit linear or logistic (depending on the outcome) mixed-effects regression models  to explore the association of pesticide exposure with the following: (1) self-reported symptoms of acute pesticide poisoning in the last 12 months; (2) self-reported symptoms of respiratory and allergic symptoms and outcomes; (3) neurobehavioral outcomes and brain activity; (4) cardiometabolic effects ; and (5) erythrocytic AChE activity. These mixed-effects models will allow us to take into account the correlation between and within farms as well as between and within farm workers. Furthermore, we will fit generalized additive models to examine the nonlinearity of the exposure-outcome associations.a priori in our regression models. Furthermore, we will assess other potential confounders by adding them, one at a time, to the final models (models with a priori covariates). Additional covariates will be possibly included in the final models if they materially changed the magnitude of one or more exposure coefficients (>10%). Missing values (<10%) for covariates will be imputed by randomly selecting a value from the dataset or using multiple imputation techniques [We will identify potential confounders and known predictors of the health outcomes of interest  using directed acyclic graphs and will include them chniques . StatistThe fieldwork for this study was conducted between May 2016 and August 2016. A total of 300 participants, including 113 farm owners and 187 workers from nine organic (48 participants) and 83 conventional (252 participants) horticultural smallholder farms from Zarcero County, Costa Rica, were enrolled. We had a 6.3% (281/300) loss to follow-up of study participants between the baseline and follow-up study visits (conducted ~2-4 weeks apart). During the study implementation, we observed that farms were, on average, smaller than we expected (about three owners or workers per farm) and that there were not as many organic farms in the study area as anticipated (only 10 farms out of the 25 that we expected). Interviews to farm owners and key community actors conducted during this study revealed that many organic farm owners had recently started using synthetic pesticides due to the increasing costs of growing organic produce and getting certified as organic producers. Given the limited number of organic farms in the study area, we decided to include all farm owners and workers from organic farms located in the study area or within 5 km from it. In addition, to reach the targeted sample of 300 participants, we had to enroll more conventional farm owners and workers than what we had anticipated. Data analyses are ongoing and expected to be published between 2019 and 2021.This study has several limitations. First, its cross-sectional design will prevent us from identifying causal associations of pesticide exposure with health effects of interest. Second, we will not be able to exclude the possibility that there is recall or information bias, especially when relying on self-reported exposure to pesticides. In our study population, this bias may have worsened under the following conditions: (1) most study participants had a relatively low educational level; (2) pesticide use varied by crop and season; and (3) some farm owners did not communicate the specific pesticides that were used in their farms by their employees. Therefore, the validation of self-reported pesticide exposure data against biomarkers of exposure is crucial. Third, given that we had to enroll more conventional farm owners and workers than what we expected, and this could potentially threaten the internal validity of our study, we will assess participants’ pesticide exposures using exposure intensity scores and biomarker concentrations in addition to their farming practices. We will also run analyses using only data from the conventional farms to evaluate the effects of the miscalculated sample size on our exposure-outcome associations. Fourth, we observed some significant differences between owners and workers from conventional and organic farms  that could potentially confound the exposure-outcome associations. Hence, it was important to also collect detailed information on confounding factors and predictors of the outcomes of interest.The limitations of this study are offset by notable strengths, including (1) the quantification of pesticide metabolites and Mn concentrations in different biological matrices, which will allow us to validate the exposure information collected via questionnaires ; (2) the comparison of workers and owners from conventional and organic farms using comprehensive questionnaires on occupational pesticide exposure; and (3) the assessment of health outcomes using internationally standardized tests that will allow for direct comparison of the results from this study to those from studies of other populations.This study is one of the first studies to examine the health effects of exposure to a wide range of pesticides on Latin American workers from conventional and organic farms. In addition, this is one the first epidemiological studies to examine the association of pesticide exposure with brain cortical activity in farm workers. We expect that this study will provide critical data on how occupational exposure to common pesticides may affect farm owners’ and workers’ health. Finally, we hope that this study will allow us to identify strategies to reduce pesticide exposure in farm workers and will lay the groundwork for a future longitudinal study of health outcomes in farm owners and workers exposed to pesticides.