APP/PS1 mice; however, compared with the control mice, the HDI-treated mice exhibited upregulated divalent metal transporter 1 (DMT1) and ferroportin (Fpn) expression, and downregulated transferrin receptor (TFR) expression. Importantly, we confirmed that there were significantly fewer NeuN-positive neurons in both APP/PS1 and WT mice given HDI, than in the respective controls. Moreover, this iron-induced neuron loss may involve increased ROS and oxidative mitochondria dysfunction, decreased DNA repair, and exacerbated apoptosis and autophagy. Although HDI administration might trigger protective antioxidant, anti-apoptosis, and autophagy signaling, especially in pathological conditions, these data clearly indicate that chronic iron exposure results in neuronal loss due to apoptosis, autophagy, and ferroptosis, hence increasing the risk for developing AD.Although the causal relationship between Alzheimer’s disease (AD) and iron overload remains unclear, iron dyshomeostasis or improper transport mechanisms are speculated to lead to the accumulation of this neurotoxic metal in the hippocampal formation and other cerebral areas related to neurodegenerative diseases, resulting in the formation of reactive oxygen species (ROS) and, ultimately, cell death. In this study, exposure to high dietary iron (HDI) revealed no significant difference in the number of iron-positive cells and iron content in the cortex and hippocampal region between wild-type (WT) and  Alzheimer’s disease (AD) is the most common neurodegenerative disease, and is characterized by senile plaques (SPs) formed from β-amyloid (Aβ)  and neurStudies have implicated cellular overload of iron, a necessary cofactor in many metabolic processes in the central nervous system, along with iron-induced oxidative stress, in the development of AD ,5,6. ExcIn addition to Aβ, iron has been found to be relevant to the formation of NFTs in AD patient brains ,12. FurtAPP/PS1) mice and littermate C57BL/6J mice.Of course, pathological neuronal iron accumulation has been observed in AD brains ,20. IncrAPP/PS1 double Tg mouse model of AD after treatment with high iron in the drinking water. We detected factors related to neurotoxicity, apoptosis, autophagy, ferroptosis, oxidative stress, and DNA damage. Our results showed that a high level of iron-induced neuron death is caused by a mixture of factors in both the normal and pathological conditions.In this study, we evaluated the forms of neuron death and their mechanisms in WT mice and the APP/PS1 (APPswe/PSEN1dE9) double Tg mice and C57BL/6J (WT) mice were originally obtained from Jackson Laboratory . The mice were maintained in a controlled environment , with a standard diet and distilled water available ad libitum. For subsequent experiments, we intercrossed these mice to generate APP/PS1 and WT littermate mice. All experimental procedures using animals were designed to minimize suffering and the number of subjects used. These studies were conducted in accordance with the guidelines for the care and use of medical animals developed by the Ministry of Health of the People’s Republic of China (1998), and the ethical standards for laboratory animals in Northeastern University (#161031).APP/PS1 (AD) and APP/PS1 + Fe (AD + Fe) (n = 8 in each group). The high-iron groups were treated with 5 g/L ferric ammonium citrate (FAC)  for three months, and the control groups (C57BL/6J and APP/PS1) were given distilled water.We divided the 9-month-old male mice into four groups: C57BL/6J (WT), C57BL/6J + Fe (WT + Fe), Three months later, control- and FAC-treated mice were anesthetized with sodium pentobarbital  and then transcardially perfused with 0.9% saline. After the blood was completely perfused, the mouse brain was then immediately removed and cut in half on an icy plate along the sagittal plane. One half of the brain was retained in 4% paraformaldehyde for morphological evaluation, and the other half was frozen at −80 °C for the molecular biology experiments.4 [Fe (CN)6]  and 2% HCl was dropped on sections, followed by incubation at room temperature for 30 min. After being washed three times by 0.01 M PBS, the sections were stained with diaminobenzidine (DAB) dye solution  + 200 mL TrisHCL + 60 μL 30% H2O2) for 3 min, with observation under an optical microscope. The microscopic brown particles were considered positive staining. Images of stained sections were collected on a light microscope . After three repeated experiments, the images were quantified using ImageJ analysis software , and the area of positive staining was counted. Then, Graphpad prism software  was used for differential analysis. Subsequent staining results were analyzed using the same method.The frozen brain sections were hydrated with distilled water for 5 min. Then, a mixture of 2% KBrain tissue samples were accurately weighed with an analytical balance, and the samples were treated with 200 μL concentrated nitric acid at 98 °C for 15 min. After cooling to room temperature, the samples were diluted to 5 mL with 1% nitric acid. A graphite furnace atomic absorption spectrometer  was used to detect the iron level.The mouse brain was removed from 4% paraformaldehyde, dehydrated, embedded in paraffin, and finally cut into 5 μm sections. The coronal paraffin sections were then dewaxed and rehydrated, and treated with endogenous peroxidase inhibitor  for 10 min. L.A.B solution  was added for 20 min to retrieve the antigens. After each treatment, the sections were washed three times with 0.01 M PBS for 5 min each time, and blocked with goat serum for 30 min. Then, the sections were incubated with only one primary antibody overnight at 4 °C. The primary antibodies used for immunohistochemistry were rabbit anti-GFAP  and rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1) . Next, the sections were treated with the corresponding biotinylated secondary antibody  for 1 h at room temperature, and then treated with HRP-streptavidin for 30 min. The staining steps were identical to Perl’s-DAB iron staining. The samples were then immersed in distilled water to stop the reaction, and finally counterstained with hematoxylin. After these steps were completed, the sections were dehydrated and sealed. Images of the stained sections were collected using an optical microscope .The frozen brain sections were rehydrated and treated with L.A.B. solution  for 20 min. They were then blocked with goat serum for 30 min, and incubated overnight at 4 °C with both the mouse-anti-Aβ (1:400) and rabbit-anti-NeuN  antibodies. Subsequently, sections were incubated in DyLight 594-labeled goat anti-mouse IgG and 488-labeled goat anti-rabbit IgG  at room temperature for 1 h. Finally, the sections were labeled with 4′,6-diamidino-2-phenylindole (DAPI)  and sealed with anti-fluorescence quencher . The sections were observed and photographed using a confocal fluorescence microscope .The frozen brain sections were hydrated with distilled water for 5 min, incubated in Nissl staining solution  for 10 min at room temperature, washed twice with distilled water for a few seconds, and placed into 95% ethanol for 5 min, followed by 100% ethanol for 2 min. The sections were then cleared with xylene for 10 min and sealed. The images of the stained sections were collected with a light microscope .Mouse brain homogenates were lysed in RIPA buffer  containing protease inhibitor cocktail, and then centrifuged at 13,000 r/min for 25 min at 4 °C. The supernatants were collected, and the total protein levels were measured using an ultraviolet (UV) 1700 PharmaSpec ultraviolet spectrophotometer . Thirty micrograms of protein were loaded onto 10% SDS polyacrylamide gels, and transferred onto polyvinylidene fluoride (PVDF) membranes . After the membranes were blocked with 5% nonfat milk in TBS containing 0.1% Tween-20 for 1 h, the transferred PVDF membranes were probed overnight with the following antibodies: goat anti-AIF , rabbit anti-Bax , mouse anti-Bcl-2 , mouse anti-Beclin-1, rabbit anti-caspase 3 , rabbit anti-DMT1 , rabbit anti-Fpn , mouse anti-GFAP , rabbit anti-GPX4 , rabbit anti-Iba1 , rabbit anti-LC3 A/B , rabbit anti-MTH1 , rabbit anti-m-TOR , mouse anti-MUTYH , mouse anti-OGG1 , rabbit anti-P62 , rabbit anti-PARP1 , rabbit anti-p-m-TOR , rabbit anti-SOD1 , mouse anti-TFR, rabbit anti-xCT , and mouse anti-β-actin . The immunoblots were washed and treated with the appropriate species of horseradish peroxidase (HRP)-conjugated secondary antibody , and immunological complexes were visualized by enhanced chemiluminescence (ECL) kits  using the ChemiDoc XRS system and the accompanying Quantity One software . The immunoreactive bands were quantified using ImageJ analysis software.The brain tissue was placed in 0.1 M PBS solution at a 1:9 ratio of brain tissue (g) to PBS volume (mL), ultrasonically fragmented, and centrifuged at 1000 r/min for 10 min. The supernatant was removed, and the protein concentration was determined. The relevant indicators were then measured using commercial assay kits . In brief, the ROS levels were measured by adding 5 μL of supernatant and 195 μL of 2′,7′-dichlorofluorescein diacetate (DCFH-DA)  to a 96-well plate, which was incubated for 30 min at 37 °C in the dark. Fluorescence detection was then carried out using an excitation wavelength of 500 nm and an emission wavelength of 525 nm . The detection result was expressed by the fluorescence value/mg protein. Malondialdehyde (MDA) levels were assessed following the manufacturer’s instructions, and the samples were incubated at 95 °C for 40 min. The absorbance was measured at 532 nm to calculate the MDA levels, which were expressed as nmol per mL. Protein concentrations were measured by a BCA protein assay .p < 0.01, and statistically significant if p < 0.05.All results were obtained from three repeated experiments and presented as the mean ± SEM. The difference between groups was analyzed by the unpaired two-tailed Student t test for the two data sets, or the one-way analysis of variance (ANOVA) for the four data sets. The results were reported to be highly statistically significant if APP/PS1 mice (APP/PS1 mice after treatment with HDI (p > 0.05). Simultaneously, we used AAS to evaluate the iron content , divalent metal transporter 1 (DMT1), and Fpn—the only iron export protein of neurons . Nevertheless, the expression of DMT1 and Fpn was significantly increased after HDI treatment in both the WT and APP/PS1 mouse brains . These results suggested that exogenous iron might penetrate the blood–brain barrier (BBB) and enter into the central nervous system (CNS) of adult mice, to induce iron redistribution by regulating the expression and function of brain iron-transport-related proteins.To investigate the reason for the HDI-induced neurodegeneration in the mouse brain, we first examined the level of iron and iron-related transporter proteins. Perl’s-DAB iron staining showed that HDI increased the number of iron-positive cells in the cortex and hippocampal region in WT and PS1 mice A, howeve content C, and th neurons D,E. TFR APP/PS1 mice and WT littermate controls at 12 months of age. As shown in APP/PS1 mouse brains, relative to their respective controls . Meanwhile, the NeuN-positive neurons in the cortex and hippocampus of HDI-treated APP/PS1 mice were apparently reduced compared with HDI-treated WT mice, accompanied by an increase of Aβ-positive plaques . We then aimed to define the functional state of these neurons in the cortex and hippocampus by Nissl staining. In both tissues, Nissl body levels were decreased by HDI treatment in both APP/PS1 mice and WT littermate controls , suggesting that HDI treatment inhibits the activity of neurons. We also examined NeuN protein expression in the brain tissues of HDI-treated APP/PS1 mice and WT mice, and found that NeuN protein expression was markedly downregulated compared to that in the respective controls . This is consistent with the idea that chronic excess iron exposure might significantly undermine the structure and functions of specific neurons, ultimately causing neuronal loss.To characterize the effects of HDI on the neuropathology of AD, we assessed changes in Aβ plaques, neurons, glial cells, and associated marker proteins in the brains of APP/PS1 mouse brains after HDI . Simultaneously, in both the cortex and hippocampus of HDI-treated WT and APP/PS1 mice, immunohistochemistry staining demonstrated a sharp increase in GFAP-positive astrocytes and Iba1-positive microglia, with thick processes and enlarged somata , indicating activation of astrocytes and microglia, respectively. Although previous morphological analysis has indicated that AD tissue is associated with activated microglia and Aβ plaques [As with neurons, glial cells are also involved in the regulation of iron homeostasis in the brain . The Wes plaques ,30, whetAPP/PS1 mouse brain. These results indicated that HDI could distinctly activate glial cells and increase Aβ-induced neuroinflammation in the APP/PS1 mouse brains, we performed a series of experiments to detect the oxidative stress index and endogenous antioxidants. As shown in APP/PS1 mouse brains, especially in the former. Glutamate-cystine transporter (xCT) is a cystine-glutamate antiporter. It transports extracellular cystine into cells, which is converted to cysteine for glutathione (GSH) synthesis [p < 0.05 or p < 0.01, respectively). Conversely, the expression levels of these antioxidants were significantly decreased in the APP/PS1 mouse brains after HDI treatment . These results suggested that the brain may trigger a compensatory response to iron-induced oxidative damage under normal physiological conditions, while the compensatory response is severely inhibited in conditions of Aβ accumulation.Iron is potentially toxic due to the generation of free radicals and ROS through the Fenton reaction. However, the human body is equipped with detoxifying mechanisms that alter redox regulation, and can even repair damage caused by reactive species . To examynthesis . Glutathynthesis . Superoxp < 0.01) and APP/PS1 (p < 0.01) mouse brains, suggesting that HDI may induce DNA damage, resulting in downstream events leading to neuron loss.Additionally, in order to detect iron-induced oxidative DNA damage, several DNA damage repair proteins, including mammalian MutY homologous DNA glycosidase (MUTYH), 8-hydroxyguanine DNA glycosidase (OGG1) and 8-hydroxy guanine nucleotidase (MTH1), were employed. After HDI treatment, the expression levels of MUTYH, OGG1, and MTH1 were significantly decreased in both the WT . Similar changes in apoptosis-associated proteins were seen in the brains of HDI-treated AD mice relative to their respective controls, although these changes were weaker .Iron overload and oxidative damage are known to drive neuronal cell death ,34. Therd brains ,36,37,38p < 0.01), whereas HDI treatment significantly decreased p-m-TOR expression in both groups (p < 0.01). Calpain, which is independent of m-TOR, participates in intracellular autophagy and apoptosis by cleaving various protein substrates [APP/PS1 mice than in the brains of the respective controls (p < 0.01). Next, the protein levels of Beclin-1, LC3A/B, and p62, which are reliable markers of autophagy [APP/PS1 mice (p < 0.01), whereas the levels of Beclin-1 and LC3A/B were significantly increased in the brains of both WT and APP/PS1 mice after HDI treatment .Given the prominent role of chronic iron exposure in AD, and the increased autophagy latency in the development of severe neuron loss caused by iron accumulation, we then investigated the contribution of autophagy to brain damage. m-TOR is an evolutionarily conserved PI3-kinase family member that plays a key role in integrating different biochemical and growth factor signals, and regulates the occurrence of autophagy . We founbstrates . Here, cutophagy , were prTaken together, our results suggest that excessive iron exposure plays a critical role in regulating neuron loss.Despite the evidence regarding the pathological accumulation of iron in AD brains ,43, onlyAPP/PS1 mice [p > 0.05) in brain iron levels in both WT and APP/PS1 mice; however, compared with the control mice, the HDI-treated mice showed upregulated expression of DMT1 and Fpn, and downregulated expression of TFR. Apparently, neurocytes can both inhibit ferric iron internalization into the cytosol via downregulating TFR, so that the internalized ferric iron is reduced to ferrous iron via upregulating DMT1, and encourage ferrous iron export by upregulating Fpn [APP/PS1 mice. Aβ has been shown to bind ferrous ions [APP/PS1 mice. These results suggest that the brain has mechanisms to tightly control the level of iron entering the brain and neurocytes, but these mechanisms may become corrupt with age and/or chronic administration [Experimental iron overload using dietary supplementation with iron in the mouse is a well-established model ,45. RecePS1 mice , exposurting Fpn ,49. Thisous ions , suggeststration ,52.APP/PS1 mice [APP/PS1 mice, and a possible decrease in NeuN expression in 12-month-old WT and APP/PS1 mice, when compared to the respective controls. Using Western blot, we confirmed that the expression level of NeuN protein was significantly decreased by HDI treatment. Meanwhile, Nissl body levels were also decreased by HDI treatment in both APP/PS1 mice and WT littermate controls, indicating that the lower level of NeuN expression could be attributable to iron-induced neuron loss. However, iron overload in our model also impacted the gliocyte population, as we observed a significant increase in GFAP-positive astrocytes and Iba1-positive microglia. Our data are consistent with reports that iron accumulation and overload are associated with an increase in number, and greater activation of, microglia and astrocytes in aged brains, as well as in brains from individuals who had AD [Our previous work revealed that chronic iron exposure increases Aβ deposition, tau hyperphosphorylation, and synapse loss, leading to an exacerbation of cognitive dysfunction in 9-month-old PS1 mice ,53,54. Ao had AD ,57,58,59o had AD ,61, and o had AD . Therefoo had AD .APP/PS1 mouse brains. A variety of neurodegenerative diseases are associated with iron deposition and accompanied by brain tissue oxidative stress injury [APP/PS1 mouse brains, especially in the former. Concurrently, a decrease in the expression of the antioxidant enzymes xCT, GPX4, and SOD1 was associated with increased free radical generation in the APP/PS1 mouse brain after HDI treatment, whereas the expression levels of these antioxidants were significantly increased by HDI treatment in the WT mouse brain. These observations indicate that the iron alterations not only induce oxidative damage, but also trigger sufficient cellular compensation under normal physiological conditions [Next, we aimed to understand the mechanisms underlying the increase in neuron loss observed in HDI-treated WT and s injury . ROS levnditions , while tnditions ,66. Herenditions ,68.APP/PS1 mice. Although the ratio of Bcl-2/Bax was not significantly reduced by HDI treatment, we can still speculate that caspase-dependent apoptosis took place in our system. We then examined levels of autophagy markers  in 12-month-old WT and APP/PS1 mice with or without chronic iron exposure. We found that levels of p-m-TOR were dramatically decreased by HDI treatment, whereas the levels of calpain-1, Beclin-1, LC3A/B, and p62 were elevated. m-TOR is a variety of intracellular protein receptor that regulates the occurrence of autophagy [APP/PS1 mice. Collectively, these in vivo studies clearly indicate that chronic iron exposure results in neuronal loss due to apoptosis, autophagy, and ferroptosis, hence increasing the risk for the development of AD. Further studies are required to clarify whether ferroptosis is responsible for the iron-mediated cell death observed in the brains of WT mice.In fact, iron and ROS have been accepted as important mediators of cell death in many pathological processes that involve altered iron homeostasis. Studies have shown that iron overload in the AD brain can induce oxidative stress, leading to protein aggregation and neuron vulnerability ,70. Thusutophagy . The obsutophagy . Calpainutophagy , can parutophagy . Interesutophagy . Combineutophagy ,74. Furtutophagy , were siutophagy , these rutophagy ,76,77. Hutophagy . HoweverIn summary, we demonstrated that chronic high-dose iron exposure interferes with iron homeostasis and distribution, increases oxidative stress, exacerbates mitochondria dysfunction and DNA oxidative damage, and induces neuronal loss ; however, long-term high-dose iron administration following iron overload might trigger protective antioxidant, anti-apoptosis, and autophagy signaling. The present data, together with previous reports ,48,53,54


Reactive oxygen species (ROS) are by-products of normal cell activity. They are produced in many cellular compartments and play a major role in signaling pathways. Overproduction of ROS is associated with the development of various human diseases , inflammation, and aging. Tumors continuously generate ROS at increased levels that have a dual role in their development. Oxidative stress can promote tumor initiation, progression, and resistance to therapy through DNA damage, leading to the accumulation of mutations and genome instability, as well as reprogramming cell metabolism and signaling. On the contrary, elevated ROS levels can induce tumor cell death. This review covers the current data on the mechanisms of ROS generation and existing antioxidant systems balancing the redox state in mammalian cells that can also be related to tumors. Reactive oxygen species (ROS) are formed as natural by-products of normal cell activity and participate in cellular signaling . The incTumor cells are characterized by a high level of ROS. ROS overproduction can result from changes in many processes, such as oxidative phosphorylation (OXPHOS), transition metal ions, oxidase activity, protein folding, thymidine, and polyamine catabolism –7. ROS cROS have a dual role in cancer development; on one hand, they can promote molecular genetic alterations that are necessary for tumor initiation, growth, and progression, as well as acquisition of treatment resistance . On the In this review, we discuss the main sources of ROS production in animal cells and the antioxidant defense systems that could be implicated in the redox state of cancer cells (to a significant or less significant extent).2) is reduced to water in the electron transport chain. The superoxide radical (O2•−) is produced at a number of sites in the mitochondria, including complex I (sites IQ and IF), complex III (site IIIQo), glycerol 3-phosphate dehydrogenase, Q oxidoreductase, pyruvate dehydrogenase, and 2-oxoglutarate dehydrogenase [2O2) in the MM, while Cu- and Zn-SOD convert the superoxide radical in the IMS or cytosol [2O2 in the MM can further be converted by mitochondrial aconitase to a hydroxyl radical (•OH) via a Fenton reaction [2O2 as by-products [b5 reductase [Mitochondria are a prime source of endogenous ROS due to its main role in oxidative ATP production, in which molecular oxygen  [Mitochondria are protected from ROS by multiple defense systems and antioxidants: glutathione peroxidases (GPXs), thioredoxin peroxidases (TRXPs), superoxide dismutases (SODs), peroxiredoxins (PRDXs), glutathione (GSH), thioredoxin 2 (TRX2), glutaredoxin 2 (GRX2), cytochrome c oxidase (complex IV), coenzyme Q, ascorbic acid, tocopherol, vitamin E, and carotene –26. Moretissues) , 28.Mitochondrion-generated ROS were widely shown to be implicated in various human pathologies, including inflammation, cancer, mitochondrial and neurodegenerative diseases, diabetes, chronic diseases, and aging , 29–32. 2O2 is decomposed with the participation of metal ions, such as iron (Fe), copper (Cu), zinc (Zn), and aluminum (Al), leading to the production of hydroxyl radical (•OH) and hydroxyl anion (OH−) [One of the major mechanisms of metal carcinogenicity is the ability of transition metal ions to induce oxidative stress . Fenton on (OH−) . Other con (OH−) . The hydon (OH−) .β-oxidation and α-oxidation as well as metabolism of purines, polyamines, amino acids, glyoxylate, reactive oxygen and nitrogen species (RNS), transition metal ions, and others [2O2, superoxide radical (O2•−), hydroxyl radical (•OH), nitric oxide (NO•), and peroxynitrite (ONOO−) [Peroxisomes have multiple functions in living cells, including fatty acid d others , 40. Per (ONOO−) .2O2-producing and H2O2-degrading organelle [2O2 [2O2 is released as a by-product during the normal catalytic activity of many peroxisomal enzymes, and it can also be generated by the spontaneous or enzymatic dismutation of the superoxide radical [2O2 can generate hydroxyl radicals (•OH) via a Fenton reaction [The peroxisome was first described as an Hrganelle . Despitelle [2O2 , 44. H2O radical . H2O2 careaction .2•−) in both the matrix and membrane. In the matrix, two enzymes are responsible for O2•− generation, xanthine oxidoreductase (XOR), and urate oxidase (UO) [2•− and H2O2. However, UO expression was not detected in humans and some primates indicating that uric acid is a terminal compound of purine metabolism [•) [• can also be produced from L-arginine, in a reaction catalyzed by nitric oxide synthase (NOS) [2•− with NO• results in a highly reactive compound called peroxynitrite (ONOO−) [Peroxisomes produce superoxide radicals (Oase (UO) , 48. XORtabolism . The othtabolism . Moreovelism [•) . NO• canse (NOS) . The rea (ONOO−) .Several antioxidant systems regulate ROS levels in peroxisomes and defend cells from oxidative damage. These include a number of enzymes, such as CAT, superoxide dismutases, peroxiredoxins, glutathione S-transferases (GST), and epoxide hydrolase 2 (EPHX2), as well as nonenzymatic low-molecular weight antioxidants, which are reviewed in detail elsewhere , 45, 50.Changes in redox homeostasis contribute to cancer development and progression . The perThe endoplasmic reticulum (ER) has many general cellular functions, such as protein folding, synthesis, transport, and posttranslational modifications, as well as lipid metabolism and calcium storage . Alterat2O2 [2O2 and oxidized glutathione causes ER stress. Furthermore, the ratio between GSH and GSSG is an essential marker of the redox status in the ER lumen. Moreover, GSH was proposed as a potential protection mechanism from ER-associated ROS damage [Oxidative protein folding gives rise to a highly oxidative environment in the ER lumen . Protein2O2 , 71. EROS damage . PDI andS damage . ExpressS damage , 75 whilS damage –78. ERO1S damage . ElevateS damage . In the 2O2 generation through interconnections with NOX1 and NOX4, which belong to the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase protein family [phox complex in the ER membrane and is involved in releasing H2O2 in the ER lumen [5b [2O2 [PDI is also involved in Hn family , 82. In ER lumen . One morumen [b5 . MMO cat[b5 [2O2 , 86. Inc[b5 [2O2 , 88.Thymidine phosphorylase (TP) is a rate-limiting enzyme in thymidine catabolism that catalyzes the reversible conversion of thymidine to thymine and 2-deoxy-D-ribose-1-phosphate (DR1P) . TP is uThe natural polyamines (PAs), putrescine, spermidine, and spermine, are involved in multiple basic cellular functions, including growth, proliferation, differentiation, apoptosis, migration, and protection from stresses. They are involved in protein posttranslational modifications, regulation of ion channels, maintenance of nucleic acid, and protein structure and stability, as well as cell-cell communications . PA cont2O2 release. Spermidine and spermine can also be converted to prior polyamines, with the help of spermidine/spermine-N1-acetyltransferase (SSAT) and acetylpolyamine oxidase (APAO/PAOX). N-Acetyl-3-aminopropanaldehyde and H2O2 are produced as by-products of these reactions [2O2, ammonia, and 4-aminobutanal. However, as putrescine is present in relatively low amounts in most mammalian tissues, this reaction does not generate significant amounts of ROS [In mammals, putrescine is the first polyamine of the PA catabolism. It is synthesized with the participation of ornithine decarboxylase (ODC). Putrescine is further converted to higher polyamines, spermidine and spermine, a reaction catalyzed by spermidine synthase (SPDS) and spermine synthase (SPMS), respectively. S-Adenosylmethionine decarboxylase (AdoMetDC) supplies the aminopropyl groups in these reactions. The activities of ODC and AdoMetDC are considered to be a rate-limiting factors of PA biosynthesis. The other branch of PA catabolism involves the interconversion cycles where spermine is degraded to spermidine and spermidine to putrescine with the generation of toxic-reactive aldehydes and ROS. Spermine oxidase (SMO) catalyzes the conversion of spermine to spermidine, which is accompanied by 3-aminopropanal and Heactions , 96. Adds of ROS .SMOX and SAT1, which encode the SMO and SSAT enzymes, respectively, was shown to be interconnected with infection, inflammation, and high risk of cancer. Several studies have reported that bacterial cytotoxins upregulate the expression of SMOX. The Helicobacter pylori virulence factor, cytotoxin-associated gene A (CagA) protein, promotes an increase in the SMOX mRNA level and enzyme activity in both human and animal gastric epithelial cells. This results in a significant increase in extra- and intracellular H2O2, leading to DNA damage and apoptosis, which can further contribute to inflammation and carcinogenesis [H. pylori infection induces overexpression of SMO and oxidative DNA damage and is associated with a high risk of gastric dysplasia and adenocarcinoma [H. pylori [H. pylori-induced upregulation of SMOX.Increased PA catabolism can lead to an elevated level of ROS and oxidative stress. Overexpression of ogenesis , 99. Usiarcinoma . Further. pylori . In the Bacteroides fragilis (ETBF) infection results in chronic inflammation and can promote colorectal carcinogenesis [B. fragilis toxin (BFT) has also been reported to increase SMOX expression leading to ROS generation and DNA damage in colonic epithelial cells [SMOX overexpression could be caused by the activation of the transcription factor C/EBPβ, which is involved in the regulation of inflammation and immunity, rather than the ETBF infection [SMOX expression and chronic inflammation was also observed in several precancerous conditions such as prostatic intraepithelial neoplasia (PIN) and chronic hepatitis [Enterotoxigenic ogenesis . B. fragal cells . A studynfection . The assepatitis –107. Intepatitis . This caOn the contrary, an antioxidant role of polyamines has been proposed. Multiple protection mechanisms from oxidative damage with PA participation were reported: direct ROS scavenging , 110, ch2O2. DAO activity in mammals varies and was found to be tissue specific. As such, high DAO activity was found in the placenta, kidneys, lungs, small intestine, and liver [Diamine oxidase (DAO) is a copper-containing amine oxidase that catalyzes the oxidation of polyamines, such as histamine, putrescine, spermidine, and to a lesser extent spermine , 117. Alnd liver , 119. Mond liver –122 and nd liver . Increasnd liver –126. Mornd liver , 128.2O2 as a by-product [Acetylpolyamine oxidase (APAO/PAOX) is related to flavin adenine dinucleotide- (FAD-) containing enzymes and catalyzes the oxidation of both spermidine and spermine in peroxisomes. Depending on the substrate, N1-acetylspermine or N1-acetylspermidine, APAO produces spermidine or putrescine, respectively, generating H-product . The pre-product . Inducti-product .2O2, and 3-aminopropanal [SMOX was shown in prostate and colorectal cancers [SMOX overexpression followed by downstream oxidative damage, chronic inflammation, and carcinogenesis is often induced by infections (discussed above).Spermine oxidase (SMO) is a FAD-dependent oxidase that acts directly on spermine generating spermidine, Hpropanal , 133. Unpropanal . SMO actpropanal . Elevate cancers , 105. Fu2O2 and O2•− through NADH oxidation [•) generating peroxynitrite (ONOO−). NO•, in turn, is produced by NOS activity or even by XOR under hypoxic conditions [Xanthine oxidoreductase (XOR) is a molybdenum iron-sulfur flavin hydroxylase that exists in two forms, xanthine dehydrogenase (XDH) and xanthine oxidase (XOD). XDH can be converted to XOD either irreversibly by proteolysis or reversibly by modification of cysteine residues , 137. Thxidation . Neverthxidation . XOR-relnditions . The assnditions . Decreasnditions . XOR actnditions –146. Thi2O2 either for direct oxidation of substrates or as a donor of oxygen atoms [2O2 and superoxide radicals are produced during the CYP monooxygenase cycle; the former can be further decomposed to hydroxyl radicals (•OH) in the presence of ferrous iron via a Fenton reaction [b5, oxygen concentration, oligomerization, and so on [Cytochrome P450 (CYP) oxidase is part of the microsomal electron transport system. It belongs to the CYP superfamily of integral membrane proteins that catalyze the oxidation of numerous organic substrates, accompanied by the reduction of molecular oxygen . CYP enzen atoms . The proen atoms . H2O2 anreaction . CYP enzreaction . CYP-relnd so on , 153. Stnd so on , 154. CYnd so on .κB, and production of cytokines and chemokines [The NADPH oxidase (NOX) family includes seven members: NOX1, NOX2, NOX3, NOX4, NOX5, dual oxidase 1 (DUOX1), and DUOX2 . These aemokines –165. Nucemokines . ROS weremokines . The impemokines . Additioemokines . It was emokines , 170. Moemokines , 172.•) and alkoxyl radicals (RO•) are intermediates of the hydroperoxide metabolism. Moreover, the arachidonic acid pathway itself could be responsible for ROS generation via NADPH oxidase activation [PTGS1 gene encodes for both COX-1 and COX-3 isoenzymes; however, COX-3 activity in humans remains unclear [PTGS2 encodes for COX-2. COX-1 predominantly regulates basal prostaglandin metabolism and normal tissue homeostasis [Cyclooxygenases (COX) and lipoxygenases (LOX) participate in polyunsaturated fatty acid (PUFA) metabolism, by producing bioactive eicosanoids. COX and LOX oxygenate arachidonic acid resulting in the formation of prostaglandin G2 and H2 (PGG2/PGH2) and fatty acid hydroperoxide, respectively, a reaction that is accompanied by ROS release . Peroxyltivation . In spittivation . COX is tivation . The PTG unclear , 178. PTeostasis . COX-2 ieostasis , 181. COeostasis , 182–184eostasis –190.LOX constitutes a large family of nonheme iron-containing dioxygenases; five LOX isoenzymes were found in humans . Dysregulated LOX expression was observed in various tumors and animal models. A dual role of LOX in tumorigenesis has been proposed, as they can be involved in both neoplastic transformation and tumor suppression .2O2 and aldehydes as by-products [Monoamine oxidases A and B (MAOA and MAOB) are mitochondrial FAD-dependent enzymes encoded by separate genes , 192. Thproducts , 194. Exproducts –197. Incproducts –203. Morproducts . Conversproducts . In thisproducts , 206.2O2, which can increase oxidative stress thereby promoting carcinogenesis. Overexpression of lysyl oxidase and its involvement in tumor progression and metastasis were shown in various cancers [Lysyl oxidase is a family of copper-dependent enzymes that play a primary role in the remodeling of the extracellular matrix (ECM). They catalyze the conversion of specific lysine residues into reactive aldehyde groups in collagen and elastin, forming protein crosslinks . The lys cancers –214.PTEN expression or mutations in the gene, as well as dysregulation of the PI3K/AKT signaling pathway, were frequently found in many tumors [The PI3K/AKT/PTEN signaling pathway is implicated in NOX-derived ROS production . PTEN  that are required for peroxiredoxins regeneration [TIGAR and ALDH4. TIGAR negatively regulates glycolysis and decreases intracellular ROS levels [ALDH4 in p53-null cells inhibits ROS generation and apoptosis [Transcription factor p53 is a widely known tumor suppressor, involved in regulating the expression of various genes encoding for both ROS-producing and antioxidant-related components . p53 indneration . Phosphaneration . Other pS levels , while aS levels . Overexppoptosis .2O2) and apoptosis in colorectal cancer cells [2O2 as signaling molecules for the induction of apoptosis [PIG3, BAX, and PUMA leading to an increase in intracellular ROS levels and induction of apoptosis. Moreover, the authors have demonstrated that p53 induction is associated with the excessive ROS release by mitochondria, which supports its prooxidant role [The function of prooxidant p53 is based on its ability to regulate the expression of genes encoding for prooxidant enzymes, such as PUMA (the p53-upregulated modulator of apoptosis), p66Shc (66 kDa Src collagen homologue (Shc) adaptor protein), and other proteins encoded by a group of p53-induced genes (PIGs). PUMA overexpression induces BCL2-associated X (BAX) protein-dependent ROS generation  cycle, while in the absence of oxygen, lactate is produced . Most tuferation . Dysreguferation –246. Incferation .The NADPH/NADP+ ratio is important for antioxidant defense; NADPH acts as a donor of reductive potential to glutathione and thioredoxin reductases. The main source of NADPH is the oxidative branch of the pentose phosphate pathway (ox-PPP) . Glucose2+, K+, Na+, H+, and other ions [Solid tumors are commonly infiltrated with different types of cells, including cancer-associated fibroblasts (CAFs), immune cells, pericytes, adipocytes, and other tissue-associated cells. This forms a distinct tumor microenvironment that comprises cell-cell and cell-extracellular matrix interactions, as well as many soluble factors , 254. Thher ions –257. Theher ions . It is aher ions , 260.2O2, which is produced at a high level by tumor cells, can diffuse into the extracellular space. H2O2 can freely cross membranes; however, the cells seem to regulate H2O2 transport by changes in membrane lipid composition, thereby maintaining cellular H2O2 concentration [2O2 [2O2 [2O2 and O2•− outside cells [2•− into the extracellular space. The superoxide radical, in turn, dismutates to H2O2 during the hypochlorous acid (HOCl) pathway. Peroxidases (POD) use the H2O2 as a substrate to generate HOCl, which further reacts with hydroxyl anions (OH−) leading to the formation of hydroxyl radicals (•OH). NOX-generated O2•− is reduced to H2O2 either by SOD3 or spontaneously; membrane CAT decomposes the produced H2O2 thereby inhibiting HOCl signaling. CAT can also decompose peroxynitrite (ONOO−) derived from the reaction between NO and H2O2 that prevents hydroxyl radical formation through the NO/peroxynitrite pathway.Relatively stable ROS, such as Hntration . Aquaporion [2O2 , 263. Mo2O2 [2O2 , while sde cells , 265. Exde cells . Briefly2O2 that induces oxidative stress in normal fibroblasts, triggering their reprogramming to CAFs and promoting field cancerization, epithelial cell transformation and growth, and cancer aggressiveness [2O2) into the tumor microenvironment [Apart from tumor cells, cancer-associated fibroblasts also release extracellular Hsiveness . Immune ironment , 268.The major mechanisms of ROS generation and detoxification are presented in ROS are generated by multiple cellular processes and can be overproduced in response to different stimuli. Normal cells can maintain oxidative homeostasis owing to the activity of various antioxidant systems which control ROS production through changes in metabolic and signaling pathways. Upon a permanent increase in ROS levels, the antioxidant defense mechanisms can promote cell death. However, oxidative stress damages many molecules, cell structures, and functions leading to the development of pathological states, such as inflammation, aging, neurodegenerative disorders, and cancer. ROS are greatly implicated in tumorigenesis, and summarizing the current data on ROS biology is important for understanding the mechanisms of tumor initiation, promotion, and progression, as well as for treatment development.


Pueraria thomsonii Benth is an important medicinal plant. Transcriptome sequencing, unigene assembly, the annotation of transcripts and the study of gene expression profiles play vital roles in gene function research. However, the full-length transcriptome of P. thomsonii remains unknown. Here, we obtained 44,339 nonredundant transcripts of P. thomsonii by using the PacBio RS II Isoform and Illumina sequencing platforms, of which 43,195 were annotated genes. Compared with the expression levels in the plant roots, those of transcripts with a |fold change| ≥ 4 and FDR < 0.01 in the leaves or stems were assigned as differentially expressed transcripts (DETs). In total, we found 9,225 DETs, 32 of which came from structural genes that were potentially involved in isoflavone biosynthesis. The expression profiles of 8 structural genes from the RNA-Seq data were validated by qRT-PCR. We identified 437 transcription factors (TFs) that were positively or negatively correlated with at least 1 of the structural genes involved in isoflavone biosynthesis using Pearson correlation coefficients (r) (r > 0.8 or r < -0.8). We also identified a total of 32 microRNAs (miRNAs), which targeted 805 transcripts. These miRNAs caused enriched function in ‘ATP binding’, ‘defense response’, ‘ADP binding’, and ‘signal transduction’. Interestingly, MIR156a potentially promoted isoflavone biosynthesis by repressing SBP, and MIR319 promoted isoflavone biosynthesis by repressing TCP and HB-HD-ZIP. Finally, we identified 2,690 alternative splicing events, including that of the structural genes of trans-cinnamate 4-monooxygenase and pullulanase, which are potentially involved in the biosynthesis of isoflavone and starch, respectively, and of three TFs potentially involved in isoflavone biosynthesis. Together, these results provide us with comprehensive insight into the gene expression and regulation of P. thomsonii. Pueraria thomsonii Benth belongs to the perennial leguminous plants, and it is an important Chinese medicinal herb that is mainly distributed in East Asian countries, such as China and Japan. The dried roots of P. thomsonii were first described in the Shengnong Bencao Jing as a pungent diaphoretic drug, which could be used to treat influenza, body stiffness, and other illnesses [P. thomsonii are puerarin and daidzein, which accumulates in roots for Pueraria plants [Pueraria lobata (Willd.) Ohwi, another important Pueraria plant. Firstly, liquiritigenin or naringenin are hydroxylated by 2-hydroxyisoflavanone synthase (IFS) and 2-hydroxyisoflavone dehydrate (HID) to form daidzein, genistein and formononetin. Then these isoflavone scaffolds are further modified by glycosyltransferases and methyltransferases, yielding to diverse isoflavonoids [P. thomsonii are rich in starch and have a high pasting temperature, gelatinization temperature and enthalpy [llnesses . The maia plants –5. Modera plants , as wella plants . Daidzeia plants . The bioa plants –6,9,10. avonoids –5,10. Inenthalpy .P. thomsonii is limited. EST information is not available, and only 46 nucleotide sequences have been deposited in the NCBI database. Additionally, there have not been any studies on protein-coding candidate genes involved in isoflavone biosynthesis in P. thomsonii, and microRNAs (miRNAs) in P. thomsonii have not been reported. miRNAs are a class of 20–24 nt noncoding RNAs that play vital roles in plant morphogenesis, biotic and abiotic stresses, and the synthesis of secondary metabolites through either cleavage of their targets or by repressing target translation at the posttranscriptional level [However, current research on the molecular biology of al level ,13.Salvia miltiorrhiza [Dendrobium officinale [Astragalus membranaceus [Cassia obtusifolia [For species without genome information, high-throughput RNA sequencing and data analysis are powerful tools to mine for genes that have specific functions. Compared to the short-read Illumina sequencing platform, PacBio Iso-Seq can obtain long reads of up to 10 kb and does not require reconstruction of the transcripts, making it convenient and accurate to find splice variants . The fuliorrhiza  Dendrobificinale , Astragaranaceus  and Cassusifolia .P. thomsonii and carry out comprehensive analysis. In addition to the structural genes, we found regulatory genes, such as transcription factors (TFs) and miRNAs were potentially involved in isoflavone biosynthesis. We also identified alternative splicing (AS) events, some of which were related to the biosynthesis of isoflavone and starch. These results lay the foundation for breeding P. thomsonii cultivars with high isoflavone compounds through engineering.In this study, we used PacBio RS II and illumina sequencing to obtain the full-length transcripts from Two-year-old “Enge-8” plants were grown in Huazhong Medicinal Botanical Garden of Institute of Chinese Medicinal Materials, Hubei Academy of Agricultural Sciences . At that time, the temperature ranged from 19°C to 32°C, averaging approximately 25°C. Leaves, stems and roots were collected from 3 plants at an early stage on July 12, 2017, and frozen in liquid nitrogen until further use. Total RNA was extracted using RNAprep Pure Plant Kit (DP441)  according to the manufacturer's instructions. RNA degradation and contamination were assessed on 1.5% agarose gels. Then, the total RNA for the construction of the RNA sequencing libraries was quantified with a NanoDrop (Thermo Scientific), and the RNA integrity was assessed with the Agilent Technologies 2100 Bioanalyzer system  with a cutoff of an RNA integrity number (RIN) greater than 7.Equal quantities of total RNA from the nine individual samples described above were pooled for PacBio Iso-Seq library construction. Poly (A) RNA was isolated from the total RNA using oligo(dT) magnetic beads and the Poly (A) PuristTM Kit. Isolated poly (A) RNA was eluted with 20 μl of RNase-free water. One microgram of RNA was reverse transcribed using the Clontech SMARTer cDNA synthesis kit. After PCR amplification, quality control, and purification, we performed size selection using the BluePippin Size Selection System protocol, which produced two fractions containing fragments of 1–3 kb and 3–6 kb in length. These cDNA products were used to construct libraries using the SMRTBell Template Prep Kit. The concentration and quality of the cDNA library were measured using the Qubit 2.0 fluorometer and Agilent 2100 Bioanalyzer, respectively. Finally, a total of four SMRT cells were sequenced on the PacBio RS II platform .The Poly (A) mRNA from 9 samples (three organs from three plants) was enriched from the total RNA using oligo (dT) magnetic beads. Following the enrichment, the mRNA was fragmented into small pieces. Using these short fragments as templates, the first-strand cDNA was synthesized using Superscript III reverse transcriptase and random hexamer (N6) primers. Subsequently, the RNA templates were removed, and the second-strand cDNA was synthesized using dNTPs, DNA polymerase I, and RNase H. These short double cDNA fragments were purified with AMPure XP beads. After end reparation and A-tailing, the short cDNA fragments were ligated with the Illumina paired-end adaptors and purified with AMPure XP beads. Then, PCR was used to selectively enrich DNA fragments with adapter molecules on both ends and to create the final cDNA library. The concentration of the cDNA library was assessed using the Qubit2.0 fluorometer , and the quality of the cDNA library was measured using the Agilent 2100 Bioanalyzer . Finally, the libraries were sequenced from both the 5’ and 3’ ends using the Illumina sequencing system .SMRT Analysis software package (v2.3.0) was used for data processing. First, raw reads were error corrected and processed into reads of insert (ROIs) using the ToFu pipeline . Next, fhttps://github.com/Magdoll/Cogent). In general, Cogent first creates the k-mer profile of nonredundant transcripts, calculates pairwise distances, and then clusters transcripts into families based on their k-mer similarity. Each transcript family is further reconstructed into one or several unique transcript model(s) (UniTransModels) using a De Bruijn graph method. Then, nonredundant transcripts were mapped to UniTransModels using GMAP (v2014-08-04) [The AS events were obtained as previously described . First, 4-08-04) . Splicin4-08-04) .https://blast.ncbi.nlm.nih.gov/Blast.cgi), Swissprot [Nonredundant transcripts were annotated based on the following databases: Nr (NCBI nonredundant protein sequences)  . Genes thttp://www.mirbase.org) [P. thomsonii microRNAs manually [Glycine max or Medicago truncatula miRNA were chosen as a reference. The minimal folding free energy index (MFEI) was calculated as described previously [Known microRNA sequences were downloaded from miRBase  . Due to ase.org) . The crimanually ,35. Wheneviously . Targetseviously .http://bioinfo.ut.ee/primer3/). The primers used in this study are listed in -ΔΔCt method [For each tissue type, the RNA samples extracted from three individuals were used as biological replicates for qRT-PCR analyses. Each biological replicate had three technical replicates. Then, RNA was digested with RNase-free DNase I (Promega) to remove genomic DNA contamination. Reverse transcription was performed using 1 μg total RNA for each example with 200 U M-MLV Transcriptase (Takara) in a 20 μl volume. The reaction was carried out at 70°C for 10 min, 37°C for 60 min and 70°C for 15 min. The resulting cDNA was diluted to 800 μl with sterile water. qPCR was carried out in triplicate reactions using the BIO-RAD CFX system (BIO-RAD). Gene-specific primers were designed by Primer3  RNA samples were constructed and subjected to SMRT sequencing by the PacBio RS II platform. To avoid the preference of different transcripts based on sequence length, 1–3 kb and 3–6 kb libraries were constructed and sequenced by 2 SMRT cells. In total, 300,584 polymerase reads were generated for these two libraries. After discarding the adapter sequence and sequences shorter than 50 bp, 207,253 and 237,356 subreads representing 5.13 Gb and 5.88 Gb were obtained, respectively  of P. thomsonii were subjected to sequencing by Illumina HiSeq. As a result, we obtained 23,147,850, 23,885,655, 22,604,617, 40,145,343, 41,575,513, 34,787,110, 25,765,267, 44,547,852 and 27,659,075 clean reads, which is representative of 848.9 Gb . The IllTo categorize the nonredundant transcripts, sequences were compared against Nr, Swissprot, KEGG, GO, COG, eggNOG, KOG and Pfam using BLASTX; as a result, 43,083, 32,079, 17,391, 24,720, 40,870, 25,626 and 35,525 transcripts were found to be annotated in the abovementioned databases, respectively. These results are summarized in P. thomsonii. However, isoflavone-related genes in P. thomsonii have not been reported to date. To fill this gap, we first annotated the 44,339 nonredundant transcripts of P. thomsonii with KEGG database. In total, 17,391 transcripts were annotated, which could be divided into 128 categories . Amylopectin synthesis involves more than 17 genes encoding different types of starch synthase (SS), starch branching enzyme (SBE), starch debranching enzyme (DBE) and phosphate transporter PHO1 (PHO1) [GBSS Ⅰa, uncharacterized GBSS, GBSS Ⅱ, SSⅢ, SSⅣ, SSSⅠ, SSSⅢ, pullulanase-DBE (PULⅠ), PHO1 and PHO1-like.The roots of n starch . Therefo1 (PHO1) . In thisPueraria plants. To analyze TFs in P. thomsonii, sequences of the nonredundant transcripts were compared to the sequences in the iTAK database using the default parameters [It has been reported that TFs play important roles in plant growth and development and in the biosynthesis of secondary metabolites . Until rrameters . As a reThen, the expression levels of the nonredundant transcripts were determined by FPKM. Compared with the expression levels in the plant roots, those of transcripts with a |fold change| ≥ 4 and FDR < 0.01 in the leaves or stems found by DESeq were assigned as DETs, because the roots accumulate more isoflavone compounds  and someAmong the 9,225 DETs, the comparison of the roots and leaves resulted in 8,138 DETs. Of these, 4,610 were upregulated, and 3,528 were downregulated. KEGG enrichment was carried out, and the top 20 enriched pathways, including the phenylpropanoid- and isoflavone-biosynthesis pathways, are shown in DRF) (PB10444), HID (PB16218), IFS (PB13397), PlUGT2 (PB16388), IF7MAT (PB14269), granule-bound starch synthase (PB19045) and PHO1 , which are potentially involved in the biosynthesis of flavone, isoflavone or starch, respectively, were validated by qRT-PCR , CHI (PB36892), HID (PB16218), IFS (PB13397), PlUGT2 (PB16388), IF7MAT (PB14269) and PlUGT43 (PB16388). As a result, we found a total of 437 TFs that were coexpressed with at least one of the 7 structural genes involved in isoflavone biosynthesis using the cutoff r > 0.8 or r < -0.8  of the expression profiles of the 508 TFs with those of r < -0.8 . Among tr < -0.8 . The 437P. thomsonii, we identified the miRNAs and analyzed their target genes. Because P. thomsonii small RNAs were not sequenced, based on the conservation of miRNAs, we used psRobot software to predict the P. thomsonii miRNAs by comparing the known miRNAs in miRBase (version 22) to the nonredundant transcripts to find matches with no more than three mismatches and less than two bulges in the hairpin structures. In total, we identified 32 miRNAs that belong to 12 families , LOB (2 transcripts) and GRAS (2 transcripts) TFs, respectively. Some miRNAs targeted 2 families of TFs. For example, MIR156 targeted SBP (16 transcripts) and MYB (1 transcript). MIR1507 targeted C3H (3 transcripts) and FAR1 (1 transcript). Some miRNAs targeted TFs from more than 2 families. For example, MIR319 targeted TCP (5 transcripts), C2C2-Dof (2 transcripts) and HB-HD-ZIP (2 transcripts), while MIR396 targeted GRF (3 transcripts), HB-WOX (3 transcripts), RWP-RK (1 transcript), WRKY (1 transcript) and Trihelix (1 transcript). Interestingly, we found that SBP  was targeted by MIR156a. The expression profile of PB22265 was negatively correlated with that of IFS, and PB8085 was negatively correlated with CHS and IFS . We also found that TCP (PB17199) and HB-HD-ZIP (PB19367) were targeted by MIR319. The expression profile of PB17199 was negatively correlated with that of CHS, CHI, HID, IFS, PlUGT2, IF7MAT and PlUGT43, and the expression profile of PB19367 was negatively correlated with that of CHS, CHI, and IFS . Then, AS events were detected by aligning the nonredundant transcripts back to the UniTransModels with SUPPA using the default setting. In this study, we found a total of 1,482 UniTransModels occurrences of AS, involving 2,690 AS events  was negatively correlated with that of CHS, CHI, IFS, IF7MAT and plUGT43 in P. thomsonii. In addition to MYBs, we also found TFs of WRKY, C2C2, HB, GRAS, bHLH, GARP and SBP that were potentially involved in isoflavone biosynthesis by activating or repressing multiple structural genes Click here for additional data file.S2 FigThe X-axis represents the enrichment factor and log10 . The Y-axis indicates the different KEGG pathways.(PNG)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOC)Click here for additional data file.S6 Table(DOC)Click here for additional data file.S7 Table(DOC)Click here for additional data file.S8 Table(XLS)Click here for additional data file.S9 Table(XLS)Click here for additional data file.S10 Table(XLS)Click here for additional data file.S11 Table(XLS)Click here for additional data file.S12 Table(XLS)Click here for additional data file.S13 Table(XLS)Click here for additional data file.S14 Table(XLS)Click here for additional data file.S15 Table(XLS)Click here for additional data file.S16 Table(XLS)Click here for additional data file.S17 Table(XLS)Click here for additional data file.


Vernicia fordii), an economically important woody oil plant, is a monoecious and diclinous species with male and female flowers on the same inflorescence. The extremely low proportion of female flowers leads to low fruit yield in tung orchards. The female flower normally develops along with stamen abortion; otherwise sterile ovules will be produced. However, little knowledge is known about the molecular basis of the female flower development in tung tree. In this study, integrated analyses of morphological and cytological observations, endogenous phytohormone assay and RNA-seq were conducted to understand the molecular mechanism of the female flower development in tung tree. Cytological observation suggested that the abortion of stamens in female flowers (SFFs) belongs to the type of programmed cell death (PCD), which was caused by tapetum degeneration at microspore mother cell stage. A total of 1,366 differentially expressed genes (DEGs) were identified in female flowers by RNA-seq analysis, of which 279 (20.42%) DEGs were significantly enriched in phenylpropanoid biosynthesis, phenylalanine metabolism, flavonoid biosynthesis, starch and sucrose metabolism, and plant hormone signal transduction. Stage-specific transcript identification detected dynamically expressed genes of important transcription regulators in female flowers that may be involved in PCD and floral organ development. Gene expression patterns revealed that 17 anther and pollen development genes and 37 PCD-related genes might be involved in the abortion of SFF. Further analyses of phytohormone levels and co-expression networks suggested that salicylic acid (SA) accumulation could trigger PCD and inhibit the development of SFF in tung tree. This study provides new insights into the role of SA in regulating the abortion of SFF to develop normal female flowers.Tung tree ( Zea mays  , and SA controls the timing and extent of PCD in the hypersensitive response (HR) . As an eitis nil . In Arab3 (NPR3) . Moreove3 (NPR3) . SA can 3 (NPR3) .Vernicia fordii), native to China, is widely planted in China and other countries for its ornamental purpose and tung oil production  under natural conditions. The flower was randomly collected every 5 days from March to April, 2017. Male and female flowers at stage 2 in 30 days before flowering . The samples were prefixed and observed with the paraffin sectioning method described by Total RNAs were isolated using RNAprep pure plant kit DP441 , according to the manufacturer’s protocol. RNA sequencing was performed by Illumina Hiseq 2000 . Our reference genome sequences and annotations of tung tree were appliced for the analysis of RNA-seq data. Raw reads of the RNA-seq data are found in NCBI Sequence Read Archive database with the Accession Nos. SRX3843588; SRS3089151; SRS3089154; SRS3089148; SRS3089147; and SRS3089150.de novo assembly and functional annotation were performed as described by FoldChange absolute value of ≥ 1 were marked as significantly different between the two samples. Stage-specific genes were selected as described by The detailed processes of p-value of ≤ 0.05. In order to identify genes which were correlated in expression with those genes involved in anther and pollen development, PCD, and SA, the top 50 genes in each significant module were used for network construction according to correlation degree. The co-expression networks were visualized using the Cytoscape v.3.5.1 program1  of ≥ 1  in at least one of the six samples were utilized to construct a co-expression network using the weighted correlation network analysis (WGCNA) R software package . Modulesprogram1 .Elongation Factor 1-α (EF1α) was used as the internal control  was used for cDNA synthesis. QPCR was performed on a Bio-Rad CFX96 Real Time PCR system using SYBR Premix ExTaq II . The primers in this step were listed in  control . The relT method . PhytohoAll experiments were performed with three biological repeats. The data were analyzed by one-way ANOVA procedure in SPSS 22.0 . All figures showed the average value of three repeats. All data were expressed as means plus or minus standard deviations (mean ± SD).Tung tree is a diclinous species and the functionally normal female flower develops along with stamen abortion in tung tree . HoweverThe SFF showed delayed development in comparison with stamens in male flowers (SMFs). The SFF developed into sporogenous cell stage at 35 DBF (stage 1) , while tTaken together, the functionally normal female flower developed along with stamen abortion in tung tree. The SFF developed normally at stages 1 and 2, but degenerated from stages 3 to 6. Our morphological and cytological observation further confirmed that the abortion of SFF in tung tree belongs to the type of PCD. The tapetum cell degeneration should contribute to the abortion of SFF, finally leading to PCD in MMC.In order to elucidate the molecular mechanism responsible for the abortion of SFF in tung tree, the male and female flowers at stage 2 , stage 4 , and stage 6  were RNA-sequenced. Based on our tung tree genome sequence, a total of 22,344 genes were identified, of which 18,334 genes were expressed  across six flower samples.Further, we identified 3,147 DEGs in male flowers  and 1,366 DEGs in female flowers  . The PCAKEGG annotation showed that 496 (15.76%) DEGs in male flowers were enriched in 110 KEGG pathways , of whicEarly flower 4 (ELF4) gene and ELF4-LIKE 4 (EFL4) gene involved in the flowering time and embryoid’s morphogenesis of Arabidopsis, and male fertility genes, like G-type lectin S-receptor-like serine/threonine-protein kinase RLK1, Non-specific lipid-transfer protein 8, and Myb family transcription factor At1g14600 , Argonaute 2 (AGO2). These genes are well-known to function in anther or pollen development , a total of 115 genes were specifically expressed in C1 that were enriched for ribosome biogenesis in eukaryotes (ko03008) and synthesis and degradation of ketone bodies (ko00072) by KEGG enrichment analysis , includit1g14600 . A totalelopment  , Arabinogalactan peptide 16 (AGP16), Calcium-binding protein CML 38 (CML38), HVA22-like protein A (HVA22A), and E3 ubiquitin-protein ligase PUB 23 (PUB23). These genes have been reported to function in PCD , bHLHs , MYB (MYB108), etc. , SFF began to degenerate in C2, while SMF developed at microspore stage. KEGG enrichment analysis showed that a total of 168 stage-specific genes were mainly enriched in sesquiterpenoid and triterpenoid biosynthesis (ko00909), plant hormone signal transduction (ko04075), alpha-Linolenic acid metabolism (ko00592), Amino sugar and nucleotide sugar metabolism (ko00520), including n in PCD  , etc. , indicat8), etc. .HVA22-like C (HVA22C) and Protein ASPARTIC PROTEASE IN GUARD CELL (ASPG), ovule development controlling gene, Agamous-like 11 (AGL11), and petal development controlling gene, AGL32 were also included in C3 , a total of 201 specific genes were identified in C3, while 1189 specific genes were in X3 . KEGG ened in C3 . In X3, ed in C3 .MYB-related protein Zm38 (MYB35/TDF1), MYB24, ODORANT 1 (MYB103/ODO1), BTB/POZ and TAZ domain-containing protein 3 (TAZ1), BARELY ANY MERISTEM 1 (BAM1), Cytochrome P450 703A2 (CYP703A2), MALE STERILITY (MS1), and Fatty acyl-CoA reductase 2 (MS2) (LRR receptor-like serine/threonine-protein kinase RPK 2 (RPK2), SPOROCYTELESS/NOZZLE (SPL/NZZ), ABORTED MICROSPORES (AMS) Somatic embryogenesis receptor-like kinase 1 (SERK1), TAZ1, EXCESS MICROSPOROCYTES1/EXTRA SPOROGENOUS CELLS (EXS1/EMS1), MYB33 and MYB65   which we 2 (MS2) . Patternnd MYB65 , which wnd MYB65 . QRT-PCRnd MYB65 –G. TogetLeucine-rich repeat receptor-like serine/threonine/tyrosine-protein kinase (SOBIR1), AGP16, and BAG family molecular chaperone regulator 7 (BAG7) etc. (Lesion Simulating Disease 1 (LSD1), Calcium/calmodulin-regulated receptor-like kinase 2 (CRKL2), Homologies of Respiratory Burst oxidase homolog protein A (RBOHA) and so on  etc. . These gG7) etc. . Likewisnd so on  , gibberellin (GA), jasmonic acid (JA), cytokinin (CK), and SA in tung flowers at different stages were measured . The malThe levels of JAs and CKs showed different patterns in comparison with IAA, ABA, and GA . JA was According to the analysis of phytohormone levels, SA is likely to play an important role in the abortion of SFF instead of other phytohormones.Arabidopsis, there are four homologs encoding PAL (PAL1-4), three homologs encoding CM (CM1-3), and two homologs encoding ICS (ICS1/2) (CM members (CM1-3), one PAL and one ICS (ICS2). The three CMs displayed different expression patterns. Generally, FPKM of CM1 was higher than CM2 and CM3 across all samples. CM1 displayed different expression profiles in male and female flowers, and was up-regulated with the development of female flowers  . In tung flowers . CM2 was flowers . CM3 was flowers . PAL was flowers .Arabidopsis SA signaling pathway, three receptors are reported, including NON-EXPRESSOR OF PR1 (NPR), transcription factor TGA (TGA), PATHOGENESIS-RELATED (PR) (NPR (NPR1/3/5), four TGAs , and two PRs  were found in tung tree genome  were highly expressed in C2 of female flowers (In TED (PR) . Three he genome . The exp flowers . Among t flowers . Interes flowers .ICS2, CM2, NPR5, TGA3, PR-1, and PR-4B, while only two genes (CM3 and NPR3) were significantly correlated with MESA , C2 with MEpink module , and C3 with MEbrown module  . Consequently, 21 co-expression modules were identified for each sample . An effe = 0.03) .TAZ1, MS1, MS2), PCD-related genes , and SA-related genes  (TAZ1 (2324 edges), MS1 (2583), MS2 (2297), RING1 (2926), POB1 (3159), CM3 (2512), NPR3 (2534), RGA1 (2990), and RGA4 (3132) had significantly high edge numbers, suggesting their prominent roles in anther and pollen development of female flowers , WRKY32 (3178), WRKY39 (2992), suggesting their important biological functions in regulating anther and pollen development of female flowers in tung tree  . The TAZ flowers . Furtherung tree .VPS25 (354 edges), CAX3 (326), HVA22A (373), and HVA22F (375); (2) the SA-related genes of PAL (408), PR-4B (435), and SABP2 (375), (3) the ethylene-related genes of ethylene-responsive transcription factors (ERF2 (378), ERF5 (379), ERF17 (373), and RAP2-3 (409))  was identified as having high connectivity in MEpink module ) . Interesk module . Based oAGP16, RAN1, BAG7, FLAVIN-DEPENDENT MONOXYGENASE1 (FMO1), ASGP1, HVA22C, CM1, TGA7, and PAL, etc. (AGP16 (1872 edges), RAN1 (1509), BAG7 (1925), and FMO1 (1010), (2) the SA-related genes, including CM1 (2114), and PAL (1660)  . This reTaken together, the analyses of gene co-expression networks suggested that SA might regulate the anther and pollen development and the PCD, resulting in the abortion of SFF in tung tree.A. deliciosa, Quercus sube, and Diospyros kaki (The production of unisexual flowers in flowering plants has evolved more than 1000 times . Unisexuros kaki , 2018, tAsparagus officinalis (In tung tree, we found that the SFF was arrested in C2 before the formation of tetrads in microspore meiosis , which wicinalis . More imicinalis . In tungSPL/NZZ, and EXS1/EMS1 have important roles in early anther development and differentiation, while AMS, MS1, MYB33, MYB65, and MYB103 are involved in tapetum and pollen wall development (The use of classical genetic screens has uncovered a large number of genes involved in anthers development and in the production of viable pollen. In our study, 17 anther and pollen development genes were highly expressed in C1, but lowly expressed or silenced in C2 and C3 . It is welopment . Thus, lThymelaea and Meliaceae, the gynoecium in male flowers is aborted by PCD at meiosis stage (Programmed cell death is a controlled cellular suicide which plays an important role in plant development. The nuclear DNA degradation, vacuolization, or the activation of specific proteases belongs to specific morphological and biochemical features of PCD . In tungis stage . Hence, is stage .SOBIR1, ASPG16, and BAG7, which are well-known to positively regulate PCD (RBOHs, which are the source of reactive oxygen species in the oxidative burst and negatively regulate cell death (Programmed cell death is controlled by both positive regulators and negative regulators. In this study, positive regulators were highly expressed in C2 or C3 of female flowers, like late PCD . Negativll death . These rCucumis sativus and Cucumis melo (Populus tomentosa, Jatropha curcas, and Castanea henryi (A variety of plant hormones are likely to be involved in the regulation of reproductive organ abortion and development pathways in unisexual flowers . For examis melo . CK proma henryi . In tunga henryi . Only SAa henryi . TherefoNPR5, TAG3, PR-1, and PR-4B in SA pathway showed high expression in C2 of female flowers. Interestingly, ICS2 also exhibited the highest expression level in C2 among all samples. In Arabidopsis, isochorismate pathway is the main source of SA accumulation (mulation . Furthermulation . TogetheWRKYs were the important connectors between SA-related genes and anther and pollen development genes, and SA- and PCD-related genes (Arabidopsis, members of WRKYs have interaction with SA-related genes. For example, WRKY58 interacts with NPR1 to regulate SA accumulation in plant defense (NPR1-NPR3 interaction, enabling NPR1 to restrict the spread of PCD (lsd1 mutant. In addition, FMO1 is not only necessary for systemic accumulation of SA and downstream signaling after pathogen infection but also regulates PCD in Arabidopsis (Salicylic acid mediates PCD in plants under biotic stress . In our ed genes . In Arab defense . The lowd of PCD . SA-deped of PCD , which abidopsis . In ricebidopsis . Herein,bidopsis .Additionally, we constructed a hypothetical model for the development of normal female flowers in tung tree. At the early stage, SFF develops normally in female flowers with low level of SA . With SAWe confirmed that the abortion of SFF in tung tree belongs to the PCD type and demonstrated that tapetum degeneration at MMC stage is the major reason causing the abortion of SFF. Furthermore, we constructed a model for the abortion of SFF in tung tree based on integrated analyses of morphological and cytological observations, endogenous phytohormone assay and RNA-seq. During the middle stage in female flowers, SA accumulation triggers PCD activation and arrests anther and pollen development, which ultimately results in the abortion of SFF in tung tree. This study provides valuable information for better understanding the development of female flowers in tung tree.LZ, XT, and ML conceived and designed the experiments. ML, WL, XF, and GZ performed the experiments. ML, XF, HL, MS, GZ, YF, and LZ analyzed the data. ML, WL, GZ, XT, HL, YF, and LZ contributed reagents, materials, and analysis tools. ML and LZ wrote the manuscript. All authors discussed results and commented on the manuscript.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.


BRAF mutations were almost exclusively V600E  and primarily found in junctional and compound naevi. NRAS mutations were either Q61K or Q61R and frequently identified in dermal naevi. Recurrent non-V600E BRAF, KIT, NF1, and TERT promoter mutations, present in acral melanoma, were not identified. Our study identifies BRAF and NRAS mutations as the primary pathogenic event in acral naevi, however, distributed differently to those in non-acral naevi. The mutational profile of acral naevi is distinct from acral melanoma, which may be of diagnostic value in distinguishing these entities.Acral naevi are benign melanocytic tumors occurring at acral sites. Occasionally they can progress to become malignant tumors (melanomas). The genetics of acral naevi have not been assessed in larger studies. In our study, a large cohort of 130 acral naevi was screened for gene mutations known to be important in other naevi and melanoma subtypes by targeted next-generation sequencing. Mutation status was correlated with clinicopathological parameters. Frequent mutations in genes activating the MAP kinase pathway were identified, including  Naevi are benign proliferations of melanocytes that usually appear in the first decades of life, but may also be present at birth . The most frequent naevi arising on the skin are designated common acquired naevi. Naevi arising in acral sites can be located on the dorsal or volar aspect of the hands and feet. Acral naevi are generally acquired naevi. Whereas conventional naevi are more common in fair-skinned individuals and associated with UV exposure , acral nHistologically, naevi can be categorized into subtypes, depending on whether they are confined to the epidermis , the dermis , or show both epidermal and dermal components (compound naevi). Acral naevi differ somewhat histologically from their more common non-acral cutaneous counterparts, and may demonstrate factors such as asymmetry, irregular pigmentation, or nuclear hyperchromasia ,8. In paBRAF V600E mutations (80–90%) [NRAS mutations  . In contof cases ). Other and BRAF ,18,19,20er gains . Activatue naevi ,23,24. Butations , which hutations . Deep peutations . In summBRAF-mutant (~50%), RAS-mutant (20–30%), NF1-mutant (10–15%), or triple wild-type (10–15%). BRAF V600 mutations are relevant therapeutically in melanoma, enabling treatment with BRAF and MEK inhibitors [The genetics of melanoma have been well studied ,29,30,31hibitors .BRAF and NRAS are still the most frequent mutations present , followed by NF1 and KIT mutations. Two studies analyzing cohorts of 21 [BRAF mutations.More recent genetic studies have focused on less frequent melanoma subtypes, such as acral melanoma ,33,34. TUnderstanding the genetics of naevi enables a better understanding of the pathogenesis of these tumors and the genetic underpinnings of their progression to malignant tumors. This knowledge may help design ancillary assays or algorithms predicting the clinical behavior of tumors difficult to classify solely based on histomorphological evaluation.The goal of our study was to investigate the frequency of activating mutations in a large cohort of acral naevi (130 samples). A custom targeted next-generation sequencing approach was used to assess various genes with high sensitivity, and associations of identified mutations with clinical pathologic parameters were explored.The study cohort consisted of 130 acral naevus samples from 123 patients  with an average age of 41 years (range 10 to 79). The tumors included 20 (15.4%) junctional, 76 (58.4%) compound, 17 (13.1%) primarily dermal, and 17 (13.1%) dermal naevi. All samples were primary tumors. Available clinical data are listed in BRAF alterations were the most frequent mutations , 86 (99%) being c.1799A>T, V600E, and 1 (1%) being a 1799_1801delTGA, V600E/K alteration. NRAS Q61 mutations were identified in 24 cases (18%), comprising 13 (54%) c.182A>G Q61R and 11 (46%) c.181C>A Q61K alterations. One MAP2K1 305_307delAGA, E102_I103V, c.309_311delCAA, K104del mutation was detected. Recurrent KIT, HRAS, KRAS, or TERT promoter mutations were not observed. All identified mutations were mutually exclusive of one another (Targeted amplicon sequencing showed that  another .BRAF-mutant (mean 40 years) and NRAS-mutant (mean 48 years) tumors occurring at older ages than wild-type tumors . Wild-type naevi were more often junctional than BRAF-/NRAS-mutant tumors, while BRAF-mutant tumors were often compound and NRAS-mutant tumors were often dermal (p < 0.0001). Complete details are presented in An analysis with available clinico-pathological data was performed. Statistically significant associations were found between oncogene mutation status and patient age, with BRAF and NRAS mutations.Applying a targeted next generation sequencing approach, we analyzed the largest cohort of acral naevi reported to date, identifying highly recurrent BRAF mutation frequency of 67% is lower than that described in non-acral cutaneous naevi (78–82%) [BRAF mutations, with only one exception, a single tumor with a 1799_1801delTGA, V600E/K alteration. This differs from melanoma, in which around 20% of BRAF V600 mutations are non-V600E  [BRAF mutations in melanoma were found to be more common in older patients, these mutations consistently represented between 10 and 20% of BRAF mutations in the 20–29, 30–39, 40–49, and 50–59 age groups [The detected (78–82%) ,35. V600 others) ,31,36. WNRAS mutation frequency we identified in acral naevi is considerably higher than ~6% NRAS mutations reported in cutaneous common acquired naevi, of which larger studies identified NRAS mutations in around 6% of samples [NRAS mutations (<5 per study) were identified [NRAS mutations and distributed relatively equally.The 18%  samples ,37. The entified ,37. Howeentified ,31,38, wNRAS- and BRAF-wild-type (15%) , with a larger panel covering 29 genes recurrently mutated in cutaneous or uveal melanoma [MAP2K1 mutation , highly reminiscent of E102_I103del mutations reported to be activating with similar consequences to BRAF mutations [BRAF and NRAS mutations the most common activating mutations detected in acral naevi.Sequencing the 19 tumors initially found to be melanoma , we onlyutations . In the CDKN2A and TP53, are not frequently altered in common naevi [Tumor suppressor genes, including on naevi . In our p < 0.0001) of mutation status with naevi location . NRAS mutations were more frequent in deeper naevi . In addition, the estimated naevus volume also varied, depending on mutation status  [NRAS mutation frequencies were relatively comparable between melanoma and naevi  ,33,43. Hund 20%) ,22,31). tely 20% ,22,31). erations ,31,33, rAll of the identified mutations are assumed to have arisen somatically. UV-exposure is expected to play a carcinogenic role, most likely more so in tumors arising on sun-exposed dorsal areas than palmar or plantar areas. However, other mechanisms, such as sporadic non-carcinogen-induced mutations, probably play a greater pathogenic role in these tumors than in non-acral cutaneous naevi.BRAF, KIT, or NF1 mutations in naevi, this may imply that acral melanomas harboring these mutations arise primarily de novo, whereas BRAF V600E or NRAS Q61 mutant acral melanomas may more frequently arise from pre-existing naevi. Larger studies of acral melanoma with strong epidemiological data would be required to see if such an association does exist.Melanomas can arise with or without transformation from pre-existing naevi. Most naevi do not transform into melanoma, and many melanomas arise without knowledge of a preexisting naevus ,44,45. TOur study has some caveats. We analyzed a maximum of 29 genes, and rarer mutations may have been missed. This also applies to translocations, which are rare in acral melanoma ,43, but BRAF V600E mutations were most frequent (66%), identified primarily in superficial  naevi. However, NRAS Q61 mutations were also detected (18%), particularly in naevi with a more dermal location. The mutation profile identified in acral naevi differs considerably from acral melanoma, which could prove to be of diagnostic value.In summary, our study presents the most comprehensive genetic analysis of a large group of acral naevi to date. n = 101), Germany. All cases were screened by at least one experienced board-certified dermatopathologist (KGG or EH). The study was done with the approval of the Ethics Committee of the University of Duisburg-Essen, under the IRB-number 18-8426-BO. The study was performed with patient informed consent and conducted in accordance with the Declaration of Helsinki.Samples of acral naevi were obtained from the databases of the Department of Dermatology University Hospital Essen and Dermatopathologie bei Mainz , according to the manufacturer’s instructions.® , according to the manufacturer’s instructions. Tumors not demonstrating a mutation in the 16 genes were further sequenced by a 29-gene panel . The rep-value of p ≤ 0.05 was considered statistically significant.We investigated associations of mutation status with available clinical and pathological parameters, using chi-squared tests and Fisher exact tests, as appropriate. All statistical analyses were performed using IBM SPSS Statistics software . A BRAF V600E mutations (66%), primarily in superficial  locations, and frequent NRAS Q61 mutations (18%), particularly in more dermal localized tumors. Differences in mutation profile between acral naevi and melanoma may prove to be of diagnostic value.Acral naevi demonstrate frequent


OPHN1 ranging from exon 11–15. This is the first report of an OPHN1 deletion in a Japanese patient with DWM.Dandy-Walker malformation (DWM) is a rare congenital malformation defined by hypoplasia of the cerebellar vermis and cystic dilatation of the fourth ventricle. Oligophrenin-1 is mutated in X-linked intellectual disability with or without cerebellar hypoplasia. Here, we report a Japanese DWM patient carrying a novel intragenic 13.5-kb deletion in  The frequency of DWM in the U.S. is ~1 in 25,000–35,000 liveborn infants . DWM becomes apparent in early infancy, is complicated by macrocephaly, and occurs along with increased intracranial pressure, spastic paraparesis, and hypotonia2. In addition, motor deficits, such as delayed motor development, hypotonia, and ataxia, as well as intellectual disability (ID), are often seen2. To date, various chromosomal abnormalities, such as trisomy 9, −13, −18 and partial duplications/deletions of chromosomes, in DWM patients have been reviewed1. Additionally, heterozygous deletions of cerebellum-specific Zinc-finger genes, ZIC1 and ZIC4, on chromosome 3q24 are associated with DWM3. X-linked DWM with ID is also caused by an AP1S2 mutation4.Dandy-Walker malformation (DWM) is a midbrain–hindbrain malformation characterized by cerebellar vermis hypoplasia and dysplasia, cystic dilatation of the fourth ventricle and an elevated torcula, often accompanied by hydrocephalusOPHN1 encodes oligophrenin 1, which is a Rho-GTPase activating protein involved in synaptic morphogenesis and functions through the regulation of the G protein cycle5. OPHN1 (NM_002547) consists of 25 exons and spans ~391 kb on chromosome Xq12 (UCSC Genome Browser: https://genome.ucsc.edu). Oligophrein 1 is an 802 amino-acid protein harboring multiple domains, such as a BAR domain, PH domain, Rho-GAP domain, and three proline-rich sequences6. OPHN1 was originally identified as a disrupted gene by a translocation t in a female patient with mild mental retardation7. To date, 10 point mutations, four splicing mutations, six small insertion/deletion or duplication mutations, and 17 chromosomal rearrangements in OPHN1 have been identified in patients with neurodevelopmental disorders, including cerebellar hypoplasia, intellectual disability (ID), epilepsy, seizure, ataxia and schizophrenia . In addition, Schwartz et al.6 recently expanded the clinical spectrum of OPHN1-associated phenotypes in comparison to the phenotypes described in previous reports. Moortgat et al.8 also described four families with intellectual disability without cerebellar hypoplasia.OPHN1.Here, we report a Japanese DWM boy carrying an intragenic deletion in https://ncbiobank.org/index-e.html). We collected DNA samples, clinical information, and cell lines, with informed consent, from 583 families with neurodevelopmental diseases that were diagnosed between 2004 and 2016. The study was approved by the ethical committee of NCNP. The cell lines were developed by the immortalization of peripheral lymphocytes with Epstein-Barr virus.The biobank at the National Center of Neurology and Psychiatry (NCNP) is a unique biorepository for neuropsychiatric, muscular, and developmental diseases in Japan (FMR1 and FMR2) and Sanger sequencing . We performed direct sequencing of PCR amplicons using an ABI3730 capillary sequencer  according to the standard protocol. We determined the breakpoint by comparing the sequence of the patient with sequences from an unaffected person.We conducted the candidate gene approach on chromosome X to identify the causative gene in this patient. The following 19 known causative genes for XLID were analyzed by repeat expansion analyses (The patient (III-1) was a 2-year-old boy, and he was referred for developmental delay at the age of 11 months. His maternal uncle was affected with hydrocephalus Fig. . The boyL1CAM mutation by sequencing and a gross copy number variation in chromosome X by a BAC-based array-CGH9, which were both negative, we performed mutation screening of 19 known causative genes for XLID in the patient. Consequently, we found an intragenic deletion in OPHN1 involving exon 11–15, which include the PH and GAP domains . In addition, we did not find the deletion in a 1254 Japanese general population data set created by high-depth whole genome sequencing10.After initial screening for an ins Fig. . To moreins Fig. . The delOPHN1 mutations have been reported in cases with XLID with cerebellar hypoplasia, strabismus, epilepsy, hypotonia, ventriculomegaly, and distinctive facial features8. Additionally, OPHN1 mutations have also been reported in individuals with autism or childhood onset schizophrenia11, so OPHN1-associated clinical phenotypes are variable11. DWM and hydrocephalus in the present patient are likely the most severe imaging findings observed in patients with OPHN1 mutations. The novel intragenic deletion in OPHN1 eliminated exon 11–15, which encode PH and GAP domains. This deletion leads to a premature truncation, c.934_1276del (p.Gly312Ilefs*24), of OPHN1; the transcript might presumably be degraded by nonsense-mediated mRNA decay. Altogether, we concluded that the deletion in OPHN1 is the pathogenic genetic abnormality in this patient who showed profound ID and DWM.In past studies, Supplementary Figure 1


B-lymphocyte differentiation is one of the best understood developmental pathways in the hematopoietic system. Our understanding of the developmental trajectories linking the multipotent hematopoietic stem cell to the mature functional B-lymphocyte is extensive as a result of efforts to identify and prospectively isolate progenitors at defined maturation stages. The identification of defined progenitor compartments has been instrumental for the resolution of the molecular features that defines given developmental stages as well as for our understanding of the mechanisms that drive the progressive maturation process. Over the last years it has become increasingly clear that the regulatory networks that control normal B-cell differentiation are targeted by mutations in human B-lineage malignancies. This generates a most interesting link between development and disease that can be explored to improve diagnosis and treatment protocols in lymphoid malignancies. The aim of this review is to provide an overview of our current understanding of molecular regulation in normal and malignant B-cell development. Immunoglobulin (Ig) gene recombination and both positive and negative selection events to ensure proper functionality (reviewed in [The generation of B-lymphocytes in the bone marrow (BM) is a highly complex process guiding multipotent hematopoietic stem cells to become immunoglobulin-expressing B-cells. The differentiation process depends on the orchestrated activities of transcription factor (TF) networks and extracellular signals acting in conjunction to drive the expansion and maturation of progenitor populations. The process is complicated by the fact that cells must undergo iewed in ). Even tiewed in ,3,4. Furiewed in ). The ai+ Fraction A compartment [+ cells.In order to understand the process controlling the generation of highly specified blood cells, it is of critical importance to identify and prospectively isolate cells at defined maturation stages. B-lymphocyte development has been suggested to proceed from the hematopoietic stem cell, through the lymphoid primed multipotent progenitor (LMPP)  stage, tpartment , proceed+ population with preserved combined B and T-lineage potential within the classical CLP compartment [While the progenitor cells within the classical CLP compartment retain lymphoid linage potentials and display a reduced capacity to generate myeloid cells , the incpartment ,12. Henc− B-cell progenitors express B220, functional analysis fails to link B220 expression exclusively to B-lineage-committed progenitors. Rather, a fraction of the B220+ cells retain T-cell [One of the earliest markers associated with B-cell progenitors is B220, a heavily glycosylated splice form of the CD45 protein (CD45R) (reviewed in ). Expresn T-cell ,12,15, Nn T-cell , and even T-cell . − B-lineage committed progenitors could be a consequence of non-linear developmental paths not subject to the restrictions predicted from a hematopoietic tree  promoter [Igll1 gene, encoding one of the surrogate light chains [+ “CLP” population was largely restricted to the production of B-lineage cells; subsequently, these progenitors were denoted B-lymphocyte progenitors BLPs [While conventional surface marker expression did not allow for the prospective isolation of committed CD19promoter  allowed promoter . Despitet chains , is not t chains ,29, the t chains ,31. Contt chains . Transplors BLPs . While tors BLPs . This inors BLPs  , CD49E, CD11A, CD54, and CD43 ,18,35,36Rag1 and Dntt [FoxO1 gene [+ “CLP” compartment being intact or even increased in the absence of EBF1 [+ compartment is dramatically decreased [With our increasing understanding of the developmental trajectories in early B-cell development, it has been possible to explore molecular interplay at defined developmental stages. While lineage-specific gene expression is often associated with functional lineage commitment, it has been reported that the expression of lineage-associated genes can be detected already in multipotent progenitors ,39. Thesand Dntt . The actand Dntt ,43,44, Iand Dntt , SPI1  gene [Notch1 [Ebf1 gene [Ebf1 and Pax5 genes display lineage plasticity [Even though EBF1 has the ability to repress genes associated with alternative cell fates ,62 and tin vitro ,69,70,71ms) gene  and Notc [Notch1 . Even th [Notch1 , the fin [Notch1  links thbf1 gene ,76, creaasticity ,78.Ikzf3 gene [Rag gene expression critical for recombination of the Ig light chain genes [In addition to their function in stable lineage commitment, EBF1 and PAX5 are critical regulators of genes encoding proteins forming the pre-B cell receptor (pre-BCR) ,80,81,82zf3 gene , encodinzf3 gene . This rezf3 gene , to drivin genes ,92,93. Fin genes . This hain genes . Hence, PAX5, EBF1, or IKZF1 genes are observed in a majority of B-ALL patients [Myb [Spi1 [Bcl11a [Ikzf1 [Ebf1 [Pax5 gene in mice does not appear to result in any dramatic developmental block [PAX5 deletions are often found in combination with complex karyotypes and other genetic aberrations, including recurrent translocations like t (ETV6-RUNX1) or t (TCF3-PBX1) [PAX5 mutations are also found in combination with genetic alterations in other TFs such as IKZF1 and EBF1 [Ebf1/Tcf3 [Ebf1/Runx1 [Ebf1/Pax5 [While these TF networks clearly play crucial roles in normal B-cell development, it is becoming increasingly clear that they are closely connected to malignant transformation (reviewed in ). This ipatients ,95,96. Epatients ,98 or fopatients ,102,103,patients ,95,96. Epatients , the redpatients ,95,96. Tnts [Myb , Spi1 [1yb [Spi1 , Bcl11a  [Bcl11a , Ikzf1 [a [Ikzf1 , or Ebf1f1 [Ebf1  gene resal block ,110, it al block . FurtherF3-PBX1) ,96. PAX5and EBF1 ,96, likebf1/Tcf3 , Ebf1/Ruf1/Runx1 , or Ebf1bf1/Pax5  results Pax5 can be converted into other lineages with preserved malignant features [Even though leukemia is generally considered to be confined to one defined hematopoietic lineage, about 7% of patients display a more complex disease ,115. Thefeatures ,118. Furfeatures . Additiofeatures , furtherfeatures ,115, novfeatures ,122. ThiEven though intrinsic cell events such as regulatory loops created by TF networks are of critical importance for B-cell differentiation, the normal development and expansion of progenitors depend on extracellular signals in the microenvironment. While some of these signals are shared with other hematopoietic progenitors, others are restricted to lymphoid progenitor compartments. Among the former are Kit ligand , acting via the receptor tyrosine kinase cKIT . This reFlt3 gene being repressed by PAX5 [+ B-cell compartments [− compartments in the mouse BM [B-cell development is also influenced by cytokines with more restricted activity. These include FLT3 ligand (FL) acting through the FLT3 receptor expressed in the earliest lineage-restricted progenitor cells. Both the LMPP  and the  by PAX5 . Ectopic by PAX5  and disr by PAX5 . Howeverartments . The pheartments . In lineartments , lineageartments . The IL7artments  or the lartments  results mouse BM ,142. Themouse BM . IL7 andmouse BM .Ebf1 transcription, potentially driving the progenitor towards B-cell fate [Many cytokines would appear to be permissive, stimulating proliferation and reducing apoptosis in the B-cell progenitors rather than driving development to a specific lineage in an instructive manner. However, while T-cell development can be largely rescued by overexpression of BCL2 in mice deficient in IL7 signaling, this does not fully rescue B-cell development ,146. Thiell fate . Furtherell fate  or the Bell fate . While tell fate . Furtherell fate , can be ell fate . In all,ell fate .Pax5 or Ebf1, in combination with transgenic expression of a constitutive active STAT5, causes a synergistic increase in the formation of B-lineage leukemia [Despite the function of Il7 in human B-cell development being somewhat disputed , inactivleukemia . Hence, While the detailed understanding of maturation pathways is often considered a subject mainly relevant to developmental biology, our increased understanding of molecular events involved in malignant transformation highlights the relevance of cell differentiation in malignant transformation. Understanding developmental trajectories can be important for diagnosis since, even though leukemia is caused by expansion of progenitor B-cells, the heterogeneous expression of surface IG suggests that leukemia can reside in both the pro- and pre-B cell compartments ,115. The