The R2R3-MYB transcription factor GaPC controls petal coloration in cotton

Although a few cases of genetic epistasis in plants have been reported, the combined analysis of genetically phenotypic segregation and the related molecular mechanism remains rarely studied. Here, we have identified a gene(named GaPC) controlling petal coloration in Gossypium arboreum and following a heritable recessive epistatic genetic model. Petal coloration is controlled by a single dominant gene,GaPC. A loss-of-function mutation of GaPC leads to a recessive gene Gapc that masks the phenotype of other color genes and shows recessive epistatic interactions. Map-based cloning showed that GaPC encodes an R2R3-MYB transcription factor. A 4814-bp long terminal repeat retrotransposon insertion at the second exon led to GaPC loss of function and disabled petal coloration. GaPC controlled petal coloration by regulating the anthocyanin and flavone biosynthesis pathways. Expression of core genes in the phenylpropanoid and anthocyanin pathways was higher in colored than in white petals. Petal color was conferred by flavonoids and anthocyanins, with red and yellow petals rich in anthocyanin and flavonol glycosides, respectively. This study provides new insight on molecular mechanism of recessive epistasis,also has potential breeding value by engineering GaPC to develop colored petals or fibers for multifunctional utilization of cotton.

Role of activating transcription factor 3 （ATF3） in sublytic C5b-9-induced glomerular mesangial cell apoptosis

Sublytic complement C5b-9 complexes can cause cell apoptosis, but the mechanism of glomerular mesangial cell （GMC） apoptosis mediated by these complexes has not been well defined. The activating transcription factor 3 （ATF3） gene is an immediate early gene for the cell to cope with a variety of stress signals and can promote apoptosis of some cells. In this study, ATF3 expression and cell apoptosis in GMCs induced by sublytic C5b-9 were measured, and then the effects of ATF3 gene over-expression or knockdown on GMC apoptosis induced by sublytic C5b-9 were examined at a fixed time. The results showed that both ATF3 expression and GMC apoptosis were markedly increased and ATF3 over-expression obviously increased sublytic C5b-9-induced GMC apoptosis, whereas ATF3 gene silencing had a significant opposite effect. Collectively, these findings indicate that upregulation of ATF3 gene expression is involved in regulating GMC apoptosis induced by sublytic C5b-9 complexes.

Cardiac Fibroblast-Specific Activating Transcription Factor 3 Promotes Myocardial Repair after Myocardial Infarction

Background：Myocardial ischemia injury is one of the leading causes of death and disability worldwide.Cardiac fibroblasts （CFs） have central roles in modulating cardiac function under pathophysiological conditions.Activating transcription factor 3 （ATF3） plays a self-protective role in counteracting CF dysfunction.However,the precise function of CF-specific ATF3 during myocardial infarction （MI） injury/repair remains incompletely understood.The aim of this study was to determine whether CF-specific ATF3 affected cardiac repair after MI.Methods：Fifteen male C57BL/6 wild-type mice were performed with MI operation to observe the expression of ATF3 at 0,0.5,1.0,3.0,and 7.0 days postoperation.Model for MI was constructed in ATF3TGfl/flColla2-Cre＋ （CF-specific ATF3 overexpression group,n =5） and ATF3TGfl/flColla2-Cre-male mice （without CF-specific ATF3 overexpression group,n =5）.In addition,five mice of ATF3TGfl/flCol1a2-Cre＋ and ATF3TGfl/flCol 1 a2-Cre-were subjected to sham MI operation.Heart function was detected by ultrasound and left ventricular remodeling was observed by Masson staining （myocardial fibrosis area was detected by blue collagen deposition area） at the 28th day after MI surgery in ATF3TGfl/flColla2-Cre＋ and ATF3TGfl/flColla2-Cre-mice received sham or MI operation.Quantitative real-time polymerase chain reaction （qRT-PCR） was used to detect cell proliferation/cell cycle-related gene expression in cardiac tissue.BrdU staining was used to detect fibroblast proliferation.Results：After establishment of an MI model,we found that ATF3 proteins were increased in the heart of mice after MI surgery and dominantly expressed in CFs.Genetic overexpression of ATF3 in CFs （ATF3TGfl/flCol1a2-Cre＋ group） resulted in an improvement in the heart function as indicated by increased cardiac ejection fraction （41.0% vs.30.5%,t =8.610,P =0.001） and increased fractional shortening （26.8% vs.18.1%,t =7.173,P =0.002）,which was accompanied by a decrease in cardiac scar area （23.1% vs.11.0%,t =8.610,P =0.001）.qRT-PCR analysis of CFs isolated from ATF3TGfl/flCol1a2-Cre＋ and ATF3TGfl/flCol1a2-Cre-ischemic hearts revealed a distinct transcriptional profile in ATF3-overexpressing CFs,displaying pro-proliferation properties.BrdU-positive cells significantly increased in ATF3-overexpressing CFs than control CFs under angiotensin Ⅱ stimuli （11.5% vs.6.8%,t =31.599,P =0.001） or serum stimuli （31.6% vs.20.1%,t =31.599,P =0.001）.The 5（6）-carboxyfluorescein N-hydroxysuccinimidyl ester assay showed that the cell numbers of the P2 and P3 generations were higher in the ATF3-overexpressing CFs at 24 h （P2：91.6% vs.71.8%,t =8.465,P=0.015） and 48 h （P3：81.6% vs.51.1%,t =9.029,P =0.012） after semm stimulation.Notably,ATF3 overexpression-induced CF proliferation was clearly increased in the heart after MI injury.Conclusions：We identify that CF-specific ATF3 might contribute to be MI repair through upregulating the expression of cell cycle/proliferation-related genes and enhancing cell proliferation.

Activating transcription factor 3 in immune response and metabolic regulation

Activating transcription factor 3(ATF3)is a member of the ATF/cyclic adenosine monophosphate(cAMP)-response element binding protein(CREB)family of transcription factors.In response to stress stimuli,ATF3 forms dimers to activate or repress gene expression.Further,ATF3 modulates the immune response,atherogenesis,cell cycle,apoptosis,and glucose homeostasis.Recent studies have shown that ATF3 may also be involved in pathogenesis of other diseases.However,more studies are needed to determine the role of ATF3 in metabolic regulation.

Chlorophyllase is transcriptionally regulated by CsMYB308/CsDOF3 in young leaves of tea plant

Chlorophyll contributes to tea coloration, which is an important factor in tea quality. Chlorophyll metabolism is induced by light, but the transcriptional regulation responsible for light-induced chlorophyll metabolism is largely unknown in tea leaves. Here, we characterized a chlorophyllase1 gene CsCLH1 from young tea leaves and showed it is essential for chlorophyll metabolism, using transient overexpression and silencing in tea leaves and ectopic overexpression in Arabidopsis. CsCLH1 was significantly induced by high light. The DOF protein CsDOF3, an upstream direct regulator of CsCLH1, was also identified. Acting as a nuclear-localized transcriptional factor, CsDOF3 responded for light and repressed CsCLH1 transcription and increased chlorophyll content by directly binding to the AAAG cis-element in the CsCLH1 promoter. CsDOF3was able to physically interact with the R2R3-MYB transcription factor CsMYB308 and interfere with transcriptional activity of CsCLH1. In addition, CsMYB308 binds to the CsCLH1 promoter to enhance CsCLH1 expression and decrease chlorophyll content. CsMYB308 and CsDOF3 act as an antagonistic complex to regulate CsCLH1 transcription and chlorophyll in young leaves. Collectively, the study adds to the understanding of the transcriptional regulation of chlorophyll in tea leaves in response to light and provides a basis for improving the appearance of tea.

Genome-wide analysis of the B3 transcription factors reveals that RcABI3/VP1 subfamily plays important roles in seed development and oil storage in castor bean(Ricinus communis)

The B3 transcription factors(TFs)in plants play vital roles in numerous biological processes.Although B3 genes have been broadly identified in many plants,little is known about their potential functions in mediating seed development and material accumulation.Castor bean(Ricinus communis)is a non-edible oilseed crop considered an ideal model system for seed biology research.Here,we identified a total of 61 B3 genes in the castor bean genome,which can be classified into five subfamilies,including ABI3/VP1,HSI,ARF,RAV and REM.The expression profiles revealed that RcABI3/VP1 subfamily genes are significantly up-regulated in the middle and later stages of seed development,indicating that these genes may be associated with the accumulation of storage oils.Furthermore,through yeast one-hybrid and tobacco transient expression assays,we detected that ABI3/VP1 subfamily member RcLEC2 directly regulates the transcription of RcOleosin2,which encodes an oil-body structural protein.This finding suggests that RcLEC2,as a seed-specific TF,may be involved in the regulation of storage materials accumulation.This study provides novel insights into the potential roles and molecular basis of B3 family proteins in seed development and material accumulation.

Ectopic expression of VvFUS3,B3-domain transcription factor,in tomato influences seed development via affecting endoreduplication and hormones

FUSCA3(FUS3)is a member of B3-domain transcription factor family and master regulator of seed development.It has potential roles in hormone biosynthesis and signaling pathways and therefore plays diverse roles in plant life cycle,especially in seed germination,dormancy,embryo formation,seed and fruit development,and maturation.However,there is limited information about its functions in seed and fruit development of grapevine.In this study,we expressed VvFUS3 in tomato for its functional characterization.Overexpression of VvFUS3 in tomato led to a reduction in seed number and seed weight without affecting the fruit size.Histological analysis found that both cell expansion and cell division in transgenic seed and fruit pericarp have been affected.However,there were no obvious differences in pollen size,shape,and viability,suggesting that VvFUS3 affects seed development but not the pollen grains.Moreover,the expression of several genes with presumed roles in seed development and hormone signaling pathways was also influenced by VvFUS3.These results suggest that VvFUS3 is involved in hormonal signaling pathways that regulate seed number and size.In conclusion,our study provides novel preliminary information about the pivotal roles of VvFUS3 in seed and fruit development and these findings can potentially serve as a reference for molecular breeding of seedless grapes.

Comparative transcriptomic analysis of Rosa sterilis inflorescence branches with different trichome types reveals an R3-MYB transcription factor that negatively regulates trichome formation

Rosa sterilis S.D.Shi is an important economic tree in China that produces fruits with high nutritional and medicinal value.Many of R.sterills’organs are covered with different types of trichomes or prickles that directly affect fruit appearance and plant management.This study used RNA sequencing technology to analyze the transcriptomes of two parts of the inflorescence branch,namely inflorescence stems with flagellated trichomes and pedicels with both flagellated and glandular trichomes.Comparative transcriptomic analysis showed that many transcription factors(TFs)are potentially involved in the formation and development of trichomes.The accumulation of RsETC1,a TF of the R3-MYB family,was significantly higher in inflorescence stems than in pedicels;quantitative reverse transcription PCR(qRTPCR)verified that its expression was significantly higher in inflorescence stems than in pedicels during the first three development stages,indicating its inhibitory action on the initiation of glandular trichomes in R.sterilis.The mRNA level of RsETC1 accumulated to significantly higher levels in trichomeless tissues than in tissues with trichromes,suggesting that this gene may inhibit the formation of trichomes in R.sterilis.Over-expression of RsETC1 in Arabidopsis resulted in glabrous phenotypes,and the expression of trichome-related endogenous genes,except for TTG1,was markedly reduced.In addition,the contents of the phytohormones jasmonic acid(JA),gibberellin A3(GA_(3)),and cytokinins(CKs)in pedicels were significantly higher than those in inflorescence stems,and the expression patterns of the genes related to hormone biosynthesis and signal transduction presented consistent responses,suggesting that the transduction of these hormones might be crucial for trichome initiation and development.These data provide a new perspective for revealing the molecular mechanism of trichome formation in R.sterilis.

A NAC transcription factor MNAC3-centered regulatory network negatively modulates rice immunity against blast disease

NAC transcription factors(TFs)are pivotal in plant immunity against diverse pathogens.Here,we report the functional and regulatory network of MNAC3,a novel NAC TF,in rice immunity.MNAC3,a transcriptional activator,negatively modulates rice immunity against blast and bacterial leaf blight diseases and pathogen-associated molecular pattern(PAMP)-triggered immune responses.MNAC3 binds to a CACG cis-element and activates the transcription of immune-negative target genes OsINO80,OsJAZ10,and OsJAZ11.The negative function of MNAC3 in rice immunity depends on its transcription of downstream genes such as OsINO80 and OsJAZ10.MNAC3 interacts with immunity-related OsPP2C41(a protein phosphatase),ONAC066(a NAC TF),and OsDjA6(a DnaJ chaperone).ONAC066 and OsPP2C41 attenuate MNAC3 transcriptional activity,while OsDjA6 promotes it.Phosphorylation of MNAC3 at S163 is critical for its negative functions in rice immunity.OsPP2C41,which plays positive roles in rice blast resistance and chitin-triggered immune responses,dephosphorylates MNAC3,suppressing its transcriptional activity on the target genes OsINO80,OsJAZ10,and OsJAZ11 and promoting the translocation of MNAC3 from nucleus to cytoplasm.These results establish a MNAC3-centered regulatory network in which OsPP2C41 dephosphorylates MNAC3,attenuating its transcriptional activity on downstream immune-negative target genes in rice.Together,these findings deepen our understanding of molecular mechanisms in rice immunity and offer a novel strategy for genetic improvement of rice disease resistance.

Silencing of signal transducer and activator of transcription 3 expression by RNA interference suppresses growth of human hepatocellular carcinoma in tumor-bearing nude mice

AIM： To explore the effect of silencing of signal transducer and activator of transcription 3 （STAT3） expression by RNA interference （RNAi） on growth of human hepatocellular carcinoma （HCC） in tumorbearing nude mice in vivo.METHODS： To construct the recombinant plasmid of pSilencer 3.0-H1-STAT3-siRNA-GFP （pSHI-siRNA- STAT3） and establish the tumor-bearing nude mouse model of the HCC cell line SMMC7721, we used intratumoral injection together with electroblotting to transfect the recombinant plasmid pSHI-siRNA- STAT3 into the transplanted tumor. The weight of the nude mice and tumor volumes were recorded. STAT3 gene transcription was detected by semi-quantitative reverse transcription polymerase chain reaction （RT- PCR）. Level of protein expression and location of STAT3 were determined by Western blotting and immunohistochemical staining. STAT3-related genes such as survivin, c-myc, VEGF, p53 and caspase3 mRNA and protein expression were detected in tumor tissues at the same time. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） assay was used to detect apoptosis of tumor cells.RESULTS： The weight of the treated nude mice increased, and the tumor volume decreased markedly compared with those of the mock-treated and negative control groups （P 〈 0.01）. The results of RT-PCR and Western blotting showed that mRNA and protein levels of STAT3 declined markedly in the treated group. The change in STAT3-related gene expression in tumor tissues at the mRNA and protein level also varied, the expression of survivin, VEGF and c-myc were obviously reduced, and expression of p53 and caspase3 increased （P 〈 0.01）. Most of the tumor tissue ceils in the treated group developed apoptosis that was detected by TUNEL assay.CONCLUSION： Silencing of STAT3 expression by RNAi significantly inhibits expression of STAT3 mRNA and protein, and suppresses growth of human HCC in tumor-bearing nude mice. The mechanism may be related to down-regulation of survivin, VEGF and c-myc and up-regulation of p53 and caspase3 expression. Accordingly, the STAT3 gene may act as an important and effective target in gene therapy of HCC.

STAT3 activation of tumor-associated macrophages is associated with cytokines of tumor microenvironment and prognostic factors in breast cancer

Objective:The aim of this study was to investigate the correlation of STAT3 activation of tumor-associated macrophages(TAMs) and local cytokines(IL-1β,TNF-α,TGF-β and IL-12) and prognostic factors in breast cancer.Methods:TAMs in 50 primary breast cancers and macrophages in 15 normal breasts were examined by immunohistochemistry.And STAT3 DNA-binding activity of TAMs in 33/50 primary breast cancers was measured by transcription factor DNA-binding ELISA.In addition,the concentrations of IL-1β,TNF-α,TGF-β and IL-12 were measured in the 33 primary breast cancers extracts by ELISA.The correlation between STAT3 activity of TAMs and concentrations of IL-1β,TNF-α,TGF-β and IL-12 were analyzed.The correlation between STAT3 activity of TAMs and conventional clinicopathologic parameters were also evaluated.Results:The macrophages density showed a significant increase in primary breast cancers compared to normal breasts.STAT3 DNA-binding activity of TAMs in breast cancer was significantly higher than that of monocytes/macrophages from peripheral blood of the patients.Furthermore,STAT3 activity of TAMs was correlated significantly with the levels of IL-1β,TNF-α and TGF-β in breast cancer tissues.But an inverse association was observed between STAT3 activity of TAMs and IL-12.In addition,STAT3 activity of TAMs was higher in high histological type than in low histological type,and STAT3 activity of TAMs was higher in CerBb-2 positive than CerBb-2 negative.Conclusion:STAT3 activation of TAMs may be associate with increasing of IL-1β,TNF-α and TGF-β and decreasing of IL-12 in breast cancer.STAT3 activation of TAMs may also be correlated with histological grade and CerBb-2 status of breast cancer.

Runx3 might participate in regulating dendriti cell function in patients with irritable bowel syndrome

Objective:To evaluate the expression levels and correlations among the expressions of transforming growth factor-β1（TGF-β1）,Kunx3 and CD83 in colonic mucosal specimens from IBS patients.Methods:A total of 40 patients were selected,who were confirmed as IBS by Rome III standard and 40 healthy volunteers served as control.Colonic mucosal specimens of each subject were collected from colon sigmoideum with biopsy forceps.Runx3,TGF-β1?and CD83(the marker for immunecompetent mature dendritic cells（DCs）mRNA in the sigmoid colon tissue were measured by real-time fluorescence quantitative PCR.Results:Compared with the control group,CD83 mRNA expressions were higher in patients with IBS than in healthy controls（P【0.05）and were associated with runt-related transcription factor 3（Runx3）mRNA levels（r=-0.361,P【0.05）.Meanwhile,Runx3 mRNA levels were associated with TGF-β1,mRNA expressions in irritable bowel syndrome（IBS）patients（r=0.402,P【0.05）.However,there was no correlation between the mRNA expressions of TGF-β1 and CD83（P】0.05）.Conclusions:The increase of abnormal dendritic cells might influence the occurrence and development of IBS.TGF-β1 signal pathway might not be involved in Runx 3-regulated maturation of dendritic cells in IBS.

Tyrosine 23 Phosphorylation of Annexin A2 Promotes Proliferation,Invasion,and Stat3 Phosphorylation in the Nucleus of Human Breast Cancer SK-BR-3 Cells

Objective To investigate the role of tyrosine 23 （Tyr23） phosphorylation of Annexin A2 （Anxa2） in regulating the proliferation and invasion of human breast cancer SK-BR-3 cells. Methods A panel of lentivirus plasmids expressing Anxa2-wide type （Ana2-WT）, Anxa2-Y23A, and Anxa2-Y23D was generated and infected with SK-BR-3 cells. The monoclonal strains were screened. The expression of Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D was determined by Western blot analysis. The ability of the cells to proliferate was detected through an MTT [3-（4,5-Dimethylthiazol- 2-yl）-2,5-diphenyltetrazolium bromide] test. Boyden chamber assays were employed to examine migration and invasion abilities. The interaction between Anxa2 and Stat3 was analyzed by immunoprecipitation analyses. Nucleoprotein and cytosolic protein were extracted from SK-BR-3, Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D cells to analyze the expression and localization of Stat3 phosphorylation. Results The monoclonal strains constitutively expressing Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D were screened. Both Anxa2-WT and Anxa2-Y23D enhanced the proliferation, migration and invasion abilities of SK-BR-3 cells （P〈0.05）. Immunoprecipitation analysis revealed that Anxa2 and Stat3 interacted with each other, and the expression of Stat3 phosphorylation in the nucleus was enhanced by Anxa2-Y23D. Conclusions Tyr23 phosphorylation of Anxa2 promotes the proliferation and invasion of human breast cancer SK-BR-3 cells and the phosphorylation of Stat3 in the nucleus.

Alternate day fasting aggravates atherosclerosis through the suppression of hepatic ATF3 in Apoe^(-/-)mice

Atherosclerosis is the major contributor to cardiovascular mortality worldwide.Alternate day fasting(ADF)has gained growing attention due to its metabolic benefits.However,the effects of ADF on atherosclerotic plaque formation remain inconsistent and controversial in atherosclerotic animal models.The present study was designed to investigate the effects of ADF on atherosclerosis in apolipoprotein E-deficient(Apoe^(-/-))mice.Eleven-week-old male Apoe^(-/-)mice fed with Western diet(WD)were randomly grouped into ad libitum(AL)group and ADF group,and ADF aggravated both the early and advanced atherosclerotic lesion formation,which might be due to the disturbed cholesterol profiles caused by ADF intervention.ADF significantly altered cholesterol metabolism pathways and down-regulated integrated stress response(ISR)in the liver.The hepatic expression of activating transcription factor 3(ATF3)was suppressed in mice treated with ADF and hepatocyte-specific overexpression of Aft3 attenuated the effects of ADF on atherosclerotic plaque formation in Apoe^(-/-)mice.Moreover,the expression of ATF3 could be regulated by Krüppel-like factor 6(KLF6)and both the expressions of ATF3 and KLF6 were regulated by hepatic cellular ISR pathway.In conclusion,ADF aggravates atherosclerosis progression in Apoe^(-/-)mice fed on WD.ADF inhibits the hepatic ISR signaling pathway and decreases the expression of KLF6,subsequently inhibiting ATF3 expression.The suppressed ATF3 expression in the liver mediates the deteriorated effects of ADF on atherosclerosis in Apoe^(-/-)mice.The findings suggest the potentially harmful effects when ADF intervention is applied to the population at high risk of atherosclerosis.

Bexarotene improves motor function after spinal cord injury in mice

Spinal cord injury is a challenge in orthopedics because it causes irreversible damage to the central nervous system.Therefore,early treatment to prevent lesion expansion is crucial for the management of patients with spinal cord injury.Bexarotene,a type of retinoid,exerts therapeutic effects on patients with cutaneous T-cell lymphoma and Parkinson's disease.Bexarotene has been proven to promote autophagy,but it has not been used in the treatment of spinal cord injury.To investigate the effects of bexarotene on spinal cord injury,we established a mouse model of T11–T12 spinal cord contusion and performed daily intraperitoneal injection of bexarotene for 5 consecutive days.We found that bexarotene effectively reduced the deposition of collagen and the number of pathological neurons in the injured spinal cord,increased the number of synapses of nerve cells,reduced oxidative stress,inhibited pyroptosis,promoted the recovery of motor function,and reduced death.Inhibition of autophagy with 3-methyladenine reversed the effects of bexarotene on spinal cord injury.Bexarotene enhanced the nuclear translocation of transcription factor E3,which further activated AMP-activated protein kinase-S-phase kinase-associated protein 2-coactivator-associated arginine methyltransferase 1 and AMP-activated protein kinase-mammalian target of rapamycin signaling pathways.Intravenous injection of transcription factor E3 sh RNA or intraperitoneal injection of compound C,an AMP-activated protein kinase blocker,inhibited the effects of bexarotene.These findings suggest that bexarotene regulates nuclear translocation of transcription factor E3 through the AMP-activated protein kinase-Sphase kinase-associated protein 2-coactivator-associated arginine methyltransferase 1 and AMP-activated protein kinase-mammalian target of rapamycin signal pathways,promotes autophagy,decreases reactive oxygen species level,inhibits pyroptosis,and improves motor function after spinal cord injury.

Alternative Splicing in the Anthocyanin Fruit Gene Encoding an R2R3 MYB Transcription Factor Affects Anthocyanin Biosynthesis in Tomato Fruits

Tomato(Solanum lycopersicum)fruits are typically red at ripening,with high levels of carotenoids and a low content in flavonoids.Considerable work has been done to enrich the spectrum of their healthbeneficial phytochemicals,and interspecific crosses with wild species have successfully led to purple anthocyanin-colored fruits.The Aft(Anthocyanin fruit)tomato accession inherited from Solanum chilense the ability to accumulate anthocyanins in fruit peel through the introgression of loci controlling anthocyanin pigmentation,including four R2R3 MYB transcription factor-encoding genes.Here,we carried out a comparative functional analysis of these transcription factors in wild-type and Aft plants,and tested their ability to take part in the transcriptional complexes that regulate the biosynthetic pathway and their effi-ciency in inducing anthocyanin pigmentation.Significant differences emerged for SlAN2like,both in the expression level and protein functionality,with splicing mutations determining a complete loss of function of the wild-type protein.This transcription factor thus appears to play a key role in the anthocyanin fruit pigmentation.Our data provide new clues to the long-awaited genetic basis of the Aft phenotype and contribute to understand why domesticated tomato fruits display a homogeneous red coloration without the typical purple streaks observed in wild tomato species.

Qingnaoyizhi decoction suppresses the formation of glial fibrillary acidic protein-positive cells in cultured neural stem cells by inhibiting the Janus kinase 2/signal transducer and activator of transcription 3 signaling pathway

OBJECTIVE: Inactivation of the Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3) signaling axis plays a crucial role in determining the fate of neural stem cells(NSCs).Qingnaoyizhi decoction(QNYZD) has been used for the treatment of vascular dementia and has shown to improve synaptic remodeling. The aim of this study was to evaluate the effect of cerebrospinal fluid(CSF) containing QNYZD(CSF-QNYZD) on the differentiation of cultured NSCs and the involvement of the JAK2/STAT3 pathway.METHODS: The protein expression levels of glial fibrillary acidic protein(GFAP), tubulin, drosophila mothers against decapentaplegic protein(SMAD-1), STAT3, and phosphorylated-STAT3 were detected by western immunoblot analysis in the groups: control, CSF, JAK/STAT inhibitor(AG490),CSF-QNYZD, and CSF-XDZ(CSF-Xidezhen). The differentiation of NSCs was determined by immunofluorescence staining. The proliferation of NSCs was measured using the Cell Counting Kit-8 proliferation assay.RESULTS: Compared with the control group,CSF-QNYZD and AG490 significantly increased the number and expression of tubulin-positive cells, reduced the number and expression of GFAP-positive cells, and down-regulated the expression of p-STAT3. However, CSF-QNYZD also decreased the expression of SMAD-1 and STAT3.CONCLUSION: Enhanced neuronal differentiation may be associated with the down-regulation of glial differentiation instead of promoting proliferationin treated NSCs. Furthermore, QNYZD may play a direct role in suppressing the formation of GFAP-positive cells and enhancing neuronal differentiation by inhibiting JAK2/STAT3 activation. Overall, these results provide insights into the possible mechanism underlying QNYZD-mediated neurogenesis.

Edaravone protects against oxygen-glucose-serum deprivation/restoration-induced apoptosis in spinal cord astrocytes by inhibiting integrated stress response

We previously found that oxygen-glucose-serum deprivation/restoration（OGSD/R） induces apoptosis of spinal cord astrocytes, possibly via caspase-12 and the integrated stress response, which involves protein kinase R-like endoplasmic reticulum kinase（PERK）, eukaryotic initiation factor 2-alpha（eIF2α） and activating transcription factor 4（ATF4）. We hypothesized that edaravone, a low molecular weight, lipophilic free radical scavenger, would reduce OGSD/R-induced apoptosis of spinal cord astrocytes. To test this, we established primary cultures of rat astrocytes, and exposed them to 8 hours/6 hours of OGSD/R with or without edaravone（0.1, 1, 10, 100 μM） treatment. We found that 100 μM of edaravone significantly suppressed astrocyte apoptosis and inhibited the release of reactive oxygen species. It also inhibited the activation of caspase-12 and caspase-3, and reduced the expression of homologous CCAAT/enhancer binding protein, phosphorylated（p）-PERK, p-eIF2α, and ATF4. These results point to a new use of an established drug in the prevention of OGSD/R-mediated spinal cord astrocyte apoptosis via the integrated stress response.

Ziyin Huatan Recipe, a Chinese herbal compound, inhibits migration and invasion of gastric cancer by upregulating RUNX3 expression

Objectives: Ziyin Huatan Recipe(ZYHT), a traditional Chinese medicine comprised of Lilii Bulbus, Pinelliae Rhizoma, and Hedyotis Diffusa, has shown promise in treating gastric cancer(GC). However, its potential mechanism has not yet been clearly addressed. This study aimed to predict targets and molecular mechanisms of ZYHT in treating GC by network pharmacology analysis and to explore the role of ZYHT in GC both in vitro and in vivo.Methods: Targets and molecular mechanisms of ZYHT were predicted via network pharmacology analysis. The effects of ZYHT on the expression of metastasis-associated targets were further validated by Western blot and quantitative real-time polymerase chain reaction. To explore the specific molecular mechanisms of the effects of ZYHT on migration and invasion, the runt-related transcription factor 3(RUNX3) gene was knocked out by clustered regularly interspaced short palindromic repeats/Cas9, and lentiviral vectors were transfected into SGC-7901 cells. Then lung metastasis model of GC in nude mice was established to explore the anti-metastasis effect of ZYHT. Western blot and immunohistochemistry were used to explore the impact of ZYHT on the expression of metastasis-related proteins with or without RUNX3 gene.Results: The network pharmacology analysis showed that ZYHT might inhibit focal adhesion, migration,invasion and metastasis of GC. ZYHT inhibited the proliferation, migration and invasion of GC cells in vitro via regulating the expression of metastasis-associated targets. Knocking out RUNX3 almost completely reversed the cell phenotypes(migration and invasion) and protein expression levels elicited by ZYHT.In vivo studies showed that ZYHT inhibited the metastasis of GC cells to the lung and prolonged the survival time of the nude mice. Knocking out RUNX3 partly reversed the metastasis of GC cells to the lung and the protein expression levels elicited by ZYHT.Conclusion: ZYHT can effectively inhibit the invasion and migration of GC in vitro and in vivo, and its molecular mechanism may relate to the upregulation of RUNX3 expression.

TGF-β1-regulated miR-3691-3p targets E2F3 and PRDM1 to inhibit prostate cancer progression

Transforming growth factor-β1(TGF-β1)acts as a tumor promoter in advanced prostate cancer(PCa).We speculated that microRNAs(miRNAs)that are inhibited by TGF-β1 might exert anti-tumor effects.To assess this,we identified several miRNAs downregulated by TGF-β1 in PCa cell lines and selected miR-3691-3p for detailed analysis as a candidate anti-oncogene miRNA.miR-3691-3p was expressed at significantly lower levels in human PCa tissue compared with paired benign prostatic hyperplasia tissue,and its expression level correlated inversely with aggressive clinical pathological features.Overexpression of miR-3691-3p in PCa cell lines inhibited proliferation,migration,and invasion,and promoted apoptosis.The miR-3691-3p target genes E2F transcription factor 3(E2F3)and PR domain containing 1,with ZNF domain(PRDM1)were upregulated in miR-3691-3p-overexpressing PCa cells,and silencing of E2F3 or PRDM1 suppressed PCa cell proliferation,migration,and invasion.Treatment of mice bearing PCa xenografts with a miR-3691-3p agomir inhibited tumor growth and promoted tumor cell apoptosis.Consistent with the negative regulation of E2F3 and PRDM1 by miR-3691-3p,both proteins were overexpressed in clinical PCa specimens compared with noncancerous prostate tissue.Our results indicate that TGF-β1-regulated miR-3691-3p acts as an anti-oncogene in PCa by downregulating E2F3 and PRDM1.These results provide novel insights into the mechanisms by which TGF-β1 contributes to the progression of PCa.