Enterovirus A71 (EV-A71) is an important emerging virus posing a threat to children under five years old. EV-A71 infection in infants or young children can cause hand-foot-and-mouth disease, herpangina, or severe neurological complications. However, there are still no effective antivirals for treatment of these infections. In this review, we summarize the antiviral compounds developed to date based on various targets of the EV-A71 life cycle. Moreover, development of a vaccine would be the most effective approach to prevent EV-A71 infection. Therefore, we also summarize the development and clinical progress of various candidate EV-A71 vaccines, including inactivated whole virus, recombinant VP1 protein, synthetic peptides, viral-like particles, and live attenuated vaccines. Picornaviridae, represents a persistent global health problem and has caused large outbreaks in the Asia-Pacific region in recent years [Enterovirus A71 (EV-A71), a positive-strand RNA virus of the family nt years . Owing tpro, 2B, 2C, 3A, 3B, 3Cpro, and 3Dpol) that mediate viral RNA replication [The enterovirus genome encodes four structural capsid proteins  that facilitate the cellular entry and delivery of the viral genome into the cytosol of the host cell, and seven non-structural proteins  , human PAntibodies against SCARB2, PSGL-1, and DC-SIGN effectively inhibited EV-A71 infection in a dose-dependent manner , 10, 11.50 = 4 nM) and exhibited good in vivo efficiency in an EV-A71-infected mouse model [50) of EV-A71 [One compound (14) of aminopyridyl 1,2,5-thiadiazolidine 1,1-dioxides, which was capsid inhibitor, showed anti-EV-A71 infection effects  exhibited antiviral potencies against EV-A71 in HeLa cells. This peptide bound to the 2Apro active site and inhibited eIF4GI cleavage by 2Apro [2Aocessing , 22. 2Apby 2Apro .Viral protein 2B and its precursor 2 BC have been suggested to be responsible for membranous alteration and inhibition of cellular protein secretion in infected cells , 25. EV-The 2C protein plays a role in viral replication complex formation and is involved in processing nucleoside triphosphatase activity and in the synthesis of RNA-negative strands , 29. TwoThe 3A protein also plays a role in viral replication complex formation and inhibits cellular protein secretion. An enviroxime mimetic compound, AN-12-H5, was found to inhibit EV-A71 infection. Assays with resistant mutants have suggested that AN-12-H5 blocked replication by targeting 3A and also inhibited an early stage of infection by targeting VP1 and VP3 . AnotherThe 3C protein acts as a protease to cleave viral polypeptides toward their conversion to mature viral proteins during viral infection, and is thus another useful target for antiviral therapy. The compound rupintrivir  inhibited EV-A71 3C protein by mimicking the substrate of the 3C protein in vitro and protected suckling mice from EV-A71-caused limb paralysis in vivo , 34. On pro but was unstable and showed potential toxicity. Modifying the labile cyanohydrin moiety led to the discovery of the 4-iminooxazolidin-2-one-based inhibitors 4e and 4 g with potent inhibitory activity and significantly improved stability [50 value of 22.09 ± 1.07 μM by targeting 3C protease [Cyanohyfdrin (R)-1 is another potent inhibitor of EV-A71 3Ctability . One smaprotease . Luteoloprotease .pol of EV-A71 is an RNA-dependent RNA polymerase that plays a role in viral RNA synthesis. DTriP-22 is a non-nucleoside analogue that was shown to inhibit EV-A71 infection by reducing the accumulation of viral RNA [pol in vitro [pol were generated to inhibit polymerase activity and viral replication [3Diral RNA . Aurintrin vitro . As anotlication .pol and viral RNA and can be packaged into progeny virions to reduce the infectivity. APOBEC3G is also a mediator of the antiviral activity of IMB-Z, an N-phenylbenzamide derivative [Apolipoprotein B messenger RNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or A3G) can interact with viral 3Drivative .cycl and Retro-2.1 are inhibitors of several pathogens specifically targeting intracellular vesicle transport, and also participate in EV-A71 life cycle processes, including progeny virus release in vitro. Administration of Retro-2cycl at 10 mg/kg significantly protected 90% of newborn mice from lethal EV-A71 challenge [Retro-2hallenge .trans-acting factor [The 5′ untranslated region (UTR) of the EV-A71 genome is about 745 nucleotides long and highly structured, containing a cloverleaf-like structure that is critical for viral RNA synthesis and an IRES that is important for viral translation. Idarubicin (IDR) is an anthracycline compound and a USA Food and Drug Administration-approved anticancer drug. IDR inhibits EV-A71 through impaired binding between the EV-A71 IRES RNA and hnRNP A1, a known host IRES g factor .Ribavirin is a nucleotide analogue that can serve as a base analogue of either ATP or GTP, and was reported to reduce the EV-A71 titer in vitro. Ribavirin also significantly reduced the mortality, morbidity, and subsequent paralysis sequelae in EV-A71-infected mice , 46.. Short hairpin RNA (shRNA) expression plasmids or small interfering RNAs (siRNAs) that specifically targeted viral genome to inhibit viral protein expression and viral infection [RNA interference, a native and specific post-transcriptional gene silencing mechanism, has also been exploited as another antiviral tool against EV-A71 infection in vitro and in vivonfection –52.MiRNAs are approximately 19–24-nucleotide-long non-coding RNAs that post-transcriptionally repress gene expression by targeting mRNAs, and play a pivotal role in the complicated interaction networks between viruses and their hosts. MiRNAs regulate viral replication through multiple mechanisms. For example, miR-9-5p was shown to exert an anti-EV-A71 effect in cells and in a mouse model via mediating the nuclear factor-kappa B (NF-κB) activity of the RIG-I signaling pathway . In addiHS is present in the extracellular matrix, on cell surfaces, and in the intracellular granule secretions of all types of animal tissues. HS mimetics are a group of soluble synthetic or semi-synthetic compounds that are structurally related to cellular HS, and can stimulate the functions of cell-surface HS. HS is also a receptor of EV-A71. HS mimetics exhibited anti-EV-A71 activity at less than 250 mg/ml in Vero cells .GS-9620, a potent and selective agonist of Toll-like receptor 7, could inhibit EV-A71 replication mainly through the NF-κB and PI3K-AKT signaling pathways . BerberiVaccination is considered to be one of the most effective ways to protect against virus infection. Although there are many different approaches available for developing EV-A71 vaccines, including inactivating the whole virus, a live attenuated virus, virus-like particles (VLPs), recombinant subunits, and synthetic peptides, currently, only an inactivated whole virus vaccine for EV-A71 is the only candidate that has proceeded to a completed human clinical trial. To date, inactivated whole EV-A71 vaccines have been established in Taiwan, China, and Singapore. Three vaccine organizations, including Beijing Vigoo Biological Co., Ltd. (Vigoo), Sinovac Biotech Co., Ltd. (Sinovac), and the Chinese Academy of Medical Sciences (CAMS) in China completed EV-A71 vaccine phase III clinical trials in 2013 and received a license for their administration that was approved by China’s Food and Drug Administration in 2015 , 65.ClinicalTrials.gov, number NCT01508247). The efficacy of the Vigoo EV-A71 vaccine against EV-A71-associated HFMD was 90%, and that against other EV-A71-associated diseases was 80.4% during the 1-year surveillance period. In addition, the vaccine efficacy against EV-A71-associated HFMD was 100% during the second year, and no serious adverse events were reported. Thus, Vigoo claimed that their EV-A71 vaccine is safe and had good efficacy for protecting against EV-A71-associated HFMD in children. Sinovac also conducted a follow-up study for 1 and 2 years [ClinicalTrials.gov, number NCT01507857). During the 1-year surveillance period, the vaccine efficacy was 94.8% against EV-A71-associated HFMD or herpangina, and was 100% against EV-A71-associated HFMD with neurological complications. Given this success, they extended their study to follow-up the vaccine efficacy for another 12 months [ClinicalTrials.gov number, NCT01569581). The vaccine efficacy against EV-A71-associated HFMD was 97.4% [These three vaccine organizations in China used different technologies to develop an EV-A71 vaccine. CAMS used KMB-17 human diploid cells as a cell bank that were cultured using a cell factory, whereas Vigoo and Sinovac used Vero cells to amplify EV-A71 with a microcarrier bioreactor and a cell factory, respectively. All organizations selected the EV-A71 C4 subgenotype as a virus seed for vaccine development, which is the most prevalent genotype circulating in China, although they each used a different virus strain: CAMS chose the EV-A71 FY-23 strain, Vigoo chose the FY7VP5 strain, and Sinovac chose the H07 strain. The three organizations began their phase I clinical trials in 2010 to 2011, and completed their phase III clinical trials in 2013. In the Vigoo phase III clinical trial, a total of 10,245 participants aged 6–35 months randomly received a 320 U (EV-A71 antigen unit) alum-adjuvant vaccine (5120 participants) or a placebo control (5125 participants) at days 0 and 28, and were then followed-up for 1  and 2 ye 2 years , 69 in w2 months . Recentl2 months . In the as 97.4% , 71.ClinicalTrials.gov number, NCT01268787). Sixty heathy adults aged 20–60 years randomly received two intramuscular doses of either 5 μg of EV71 antigen with 150 μg of aluminum adjuvant or 10 μg of EV71 antigen with 300 μg of aluminum adjuvant, 21 days apart. The immunogenicity results indicated that the EV-A71 vaccine produced from the NHRI was safe and immunogenic in healthy adults [ClinicalTrials.gov number, NCT02777411, NCT03268083 and NCT02200237). In the clinical trial of Medigen Vaccinology Corp., a total of 365 infants or children aged 2 months to 11 years received different doses  of alum-adjuvant EV-A71 vaccine or the placebo control in a double-blind and randomized design . No vaccine-related serious adverse events were reported in this trial. In addition, the EV-A71 vaccine could elicit an immune response against not only subgenotype B4 but also B5, C4a, C4b, and C5. The EV-A71 vaccine also showed persistence for 2 years [ClinicalTrials.gov number, NCT03865238) in 2019, which is expected to be completed in 2022.In contrast to these three organizations in China, the National Health Research Institutes (NHRI) in Taiwan used the EV-A71 clinical isolate E59 strain (B4 subgenotype) as a virus seed, which was grown in Vero cells cultured with roller-bottle technology. This strain was chosen for producing the EV-A71 inactivated vaccine because of its confirmed genetic stability over several passages and its ability to grow well in Vero cells . The phay adults . Moreovey adults . Two org 2 years . Based oClinicalTrials.gov number, NCT01376479). In contrast to the organizations of China and Taiwan, Inviragen used the B3 subgenotype as the virus seed for EV-A71 vaccine production, which was named INV21. A total of 36 adults aged 21–45 years received two doses (low or high) of INV21 or placebo control 28 days apart. Inviragen claimed that INV21 induced a high immune response against HFMD caused by EV-A71. However, there has been no further clinical trial conducted in Singapore recently.In Singapore, Inviragen Inc.  completed a phase I clinical trial of an EV-A71 vaccine in April 2012 (Escherichia coli (E. coli). The purified VP1 proteins were then injected into adult female mice through an intraperitoneal route. Although the VP1 subunit vaccine could protect suckling mice against a lower challenge dose of EV-A71 (230 LD50 virus/mouse), the inactivated EV-A71 vaccine still elicited a greater immune response than the VP1 subunit vaccine and protected suckling mice against a lethal dose (2300 LD50 virus/mouse) of EV-A71. Zhou et al. [E. coli, and then vaccinated rabbits with the purified VP1 protein or heat-inactivated EV-A71 virus, which elicited comparable humoral and cellular immune responses. Moreover, maternal antibodies protect newborn mice against EV-A71 challenge. EV-A71-specific antibodies of immunized mice were elicited by purified recombinant baculovirus expressing VP1. In addition, the antisera exhibited cross-neutralization activities against different subgenotypes of EV-A71 [VP1 is not only a structural protein of EV-A71 but also exhibits strong antigenicity. Accordingly, several research groups have adopted various strategies to express EV-A71 VP1. Wu et al.  producedu et al.  also expf EV-A71 . Wang etf EV-A71  generateSalmonella enterica serovar Typhimurium expressing VP1 [Bifidobacterium longum [Saccharomyces cerevisiae [Lactococcus lactis expressing secretory VP1 [VP1 protein has also been developed as an antigen for oral vaccine development. Adult female BALB/c mice were orally immunized with transgenic tomato fruit expressing VP1 protein , attenuasing VP1 , VP1-expm longum , surfacerevisiae , or recotory VP1 . All of tory VP1  generate70–159 in VP2, P140–249 in VP2, P324–443 in VP2, and P746–876 in VP1) were combined, which induced the antisera and passively protected newborn mice against EV-A71 infection [Synthetic peptides have also been tested as an alternative strategy to develop EV-A71 vaccines, which are considered to be safe and efficacious for multivalent vaccines development. The majority of research related to antigen peptides has focused on mapping EV-A71 structural proteins . Initially, Foo et al.  found thnfection . The synnfection . Xu et anfection  generatenfection  used thenfection  sought tnfection  utilizedVLPs have been applied in the production of other viral vaccines such as hepatitis B virus and human papillomavirus, and could also be a suitable choice for EV-A71 vaccine development. The morphological characteristic and antigenicity of VLPs are similar to those of the naïve virus. Moreover, VLPs are associated with greater safety because they lack the viral genome and thus cannot replicate in the host. However, VLPs can still effectively elicit innate and adaptive immunity.neu strain) [Saccharomyces cerevisiae or Pichia pastoris, which showed protective efficacy against EV-A71 challenge in mice. In addition, maternal immunization with VLPs could also protect neonate mice against lethal EV-A71 challenge [The baculovirus expression system has been widely used for VLP production. A recombinant baculovirus co-expressing the P1 region and the viral protease 3CD of EV-A71 with different promoters was infected to insect cells for VLPs production  , 95. The strain) . EV-A71  strain) . In anot strain) . In addihallenge , 99.Chimeric VLPs, including adenovirus or varicella-zoster virus-based VLPs, have also been applied to co-express the P1 and 3CD regions of EV-A71, which could both induce an EV-A71-specific immune response and neutralization antibodies in vaccinated mice, and exhibited protective efficacy against EV-A71 infection , 101.According to the experience in developing the poliovirus Sabin vaccine, and the numerous advantages of live-attenuated vaccines, including elicitation of long-lasting immunity and cost-effective production, researchers have continued to investigate potential candidates for an EV-A71 live-attenuated vaccine. EV-A71(S1–3′) was derived from the prototype EV-A71(BrCr) strain. Five cynomolgus monkeys were inoculated with EV-A71 (S1–3′) via an intravenous route, followed by challenge with a lethal dose of EV-A71(BrCr-TR), demonstrating induction of an efficient immune response, and the sera showed neutralization activity against EV-A71(BrCr-TR) (subgenotype A) and other subgenotypes, including B1, B4, C2, and C4. However, EV71(S1–3′) caused tremor in the inoculated monkeys, and the virus was isolated from the lumbar spinal cord of inoculated monkeys on days 4 or 10 post-inoculation . TherefoBecause the detailed molecular pathogenic mechanism of EV-A71 infection remains unexplored, the virulence determinants of EV-A71 are still being investigated. The amino acid residue 145 in VP1 is considered to be an important factor for EV-A71 virulence and receptor attachment , 104. MuMoreover, the nucleotide substitutions of G64R, G64 T, and S264 L in EV-A71 3D polymerase were shown to contribute to EV-A71 replication fidelity. Enhancement of the fidelity of 3D polymerase can improve the stability and safety of live-attenuated vaccines . AnotherIn recent years, a new combination strategy of codon deoptimization and synthetic virus production has emerged for vaccine development. Tsai et al.  found thVibrio cholera, and Salmonella Typhi, and an intranasal vaccine against influenza virus. The advantages of mucosal vaccines are that they are good inducers of mucosal and systematic immunity, and the needle-free administration is more acceptable for infants and young children [The mucosal immune response, which is effectively induced by the administration of a vaccine onto the mucosal surface, is the first line of defense against pathogen invasion. Several mucosal vaccines have been licensed for use in humans, such as oral vaccines against poliovirus, rotavirus, children , 112. Alchildren –84; howechildren  found thEV-A71 is one of the most pathogenic enteroviruses infecting humans, with many outbreaks occurring worldwide causes a wide range of human diseases. However, there is still no clinically approved antiviral drug available for the prevention and treatment of the EV-A71 infection. Although the development of antiviral therapy and vaccine represents a major challenge, the progress made so far in understanding the viral replication mechanism has provided novel targets for antiviral therapy and the characterization of compounds with antiviral activity. The development of pan-enteroviruses vaccine and anti-viral drugs is an important and achievable goal in the future.


Mediator complex has been extensively shown to regulate the levels of several protein-coding genes; however, its role in the regulation of miRNAs in humans remains unstudied so far. Here we show that MED1, a Mediator subunit in the Middle module of Mediator complex, is overexpressed in breast cancer and is a negative prognostic factor. The levels of several miRNAs  were found to be regulated by MED1. MED1 induces miR-191/425 cluster in an estrogen receptor-alpha (ER-α) dependent manner. Occupancy of MED1 on estrogen response elements (EREs) upstream of miR-191/425 cluster is estrogen and ER-α-dependent and ER-α-induced expression of these miRNAs is MED1-dependent. MED1 mediates induction of cell proliferation and migration and the genes associated with it  in breast cancer, which is abrogated when used together with miR-191-inhibition. Additionally, we show that MED1 also regulates the levels of direct miR-191 target genes such as SATB1, CDK6 and BDNF. Overall, the results show that MED1/ER-α/miR-191 axis promotes breast cancer cell proliferation and migration and may serve as a novel target for therapy. Mediator was discovered in yeast as a factor required for activator-dependent transcription of genes but now it is becoming clear that it is involved in almost every step of transcription of Class II genes, including pre-initiation complex formation, initiation of transcription or promoter clearing, transcript elongation, splicing of transcripts, gene looping and termination of transcription9. In metazoans, Mediator is composed of about 30 subunits arranged in four different modules viz Head, Middle, Tail and Kinase modules. It is considered that the Head, Middle and Tail modules form the core part of the complex whereas the Kinase module reversibly associates with it as and when required. The Kinase module is mainly involved in transcriptional repression by phosphorylating CTD  heptads of RNA Pol II and some other proteins constituting the pre-initiation complex12. Being a gigantic complex, Mediator provides large and accommodating interface for several protein-protein interactions13. Generally, some specific subunits interact with specific DNA-bound transcription factors and other specific subunits interact with RNA Pol II and its accessory factors constituting transcriptional machinery, forming a communication bridge between the two. Consistent to this, Mediator is considered to be a ‘hub’ to integrate the signals from different transcription regulators and pass the ‘resultant’ signal to the transcriptional machinery3. RNA Pol II transcribes not only protein-coding genes but also the genomic regions that give rise to noncoding RNAs (ncRNAs). Role of Mediator in protein coding genes is extensively studied, but its importance in the regulation of ncRNAs is just emerging. In mouse embryonic stem cells, a stable molecular assembly formed between the Ada-Two-A-containing (ATAC) histone acetyltransferase and Mediator complex, regulates a subset of RNA pol II-transcribed unspliced ncRNA genes 14. In Arabidopsis, a set of Mediator subunits is involved in the transcription of ncRNAs that helps in recruitment of RNA pol V, a plant-specific polymerase evolved from Pol II15. In fission yeast, a triad of Med8-Med18-Med20 regulates transcription of ncRNA from centromeric region16. Thus, function of Mediator in the transcription of ncRNAs is emerging and seems to be conserved in eukaryotes.Mediator is an evolutionary conserved large multiprotein complex that plays critical role in relaying the regulatory signal from DNA-bound gene-specific transcription factors to the RNA polymerase II transcriptional machinery18. There is hardly any report but one in plants, on regulation of miRNA expression by Mediator complex. In Arabidopsis, Mediator has been shown to be involved in miRNA biogenesis by recruiting RNA Pol II to the promoters of miRNA genes15. Although miRNA genes are also transcribed by RNA Pol II in metazoans19, the direct involvement of Mediator complex in the regulation of miRNA expression in metazoans has not been shown.MicroRNAs (miRNAs) are a family of well-studied small ncRNAs that regulate gene expression at post-transcriptional level by sequence-specific targeting of mRNAs causing translational repression or mRNA degradation21. We show that estrogen (E2)-activated ER-α targets MED1 to recruit Mediator complex to the promoter of miR-191/425 cluster inducing their transcription. Both miR-191 and MED1 promote breast cancer cell proliferation and migration. MED1-mediated increase in cell proliferation and migration is inhibited upon miR-191 downregulation, suggesting the presence of a MED1/ER-α/miR-191 oncogenic axis that could serve as an effective target for breast cancer therapy. MED1 is also shown here to be involved in the regulation of other breast cancer related miRNAs suggesting the crucial and yet unexplored role of Mediator complex in breast cancer pathogenesis through regulation of miRNAs.In this report, we show that MED1, a subunit in the Middle module of Mediator complex, is altered in upto 20% of breast cancer patients and is correlated to poor survival. MED1 is known to play an essential role in ER-α-mediated gene transcription23. Analyzing 1105 invasive breast cancer cases for genetic alterations, we observed that MED 1 is amplified in 10% and mutated in 0.6% of BC patients , a hormone implicated in breast cancer, through transcriptional regulation by ER25.Next we wanted to know if MED1 is directly involved in the regulation of these miRNAs. Interestingly, we found a significant positive correlation between some of the highly induced miRNAs- and MED1 levels in breast cancer TCGA dataset  present in the regulatory region of the target genes. Our previous study demonstrated the recruitment of ERs on the EREs present in region A (27bp upstream of miR-191) and region B (~4.2 kb upstream of miR-191) in response to estrogen treatment24 Fig. . Thus, w24 Fig. . Importa24 Fig. . Similar24 Fig. . Thus, b24 Fig. . In orde24 Fig. . To know24 Fig. . Altoget32. Since recruitment of MED1 to the EREs present in the promoter region of miR-191/425 cluster is ER-α dependent and ER-α-regulated transcription of this cluster is MED1 dependent, we sought to study the interaction between ER-α and MED1. To address this, we performed co-immunoprecipitation experiment in the presence of increasing dose of estrogen. Interestingly, we found that estrogen enhances the physical interaction between ER-α and MED1  along with its control were transfected with/without MED1 overexpression and effect on cell survival was observed Fig. . MED1-meIn line with these observations, increase in the expression of genes involved in breast cancer cell proliferation or migration  was observed in response to both miR-191overexpression and MED1 overexpression Fig. . We foun24 . Notably, both MED1 and miR-191 showed similar effects on these genes further suggesting the existence of MED1-miR-191 axis  and breast cancer cell lines (n = 6)36. Here we have shown that MED1 promotes breast cancer cell proliferation and migration. The levels of several proliferation and migration associated genes were induced upon MED1 overexpression. Based on analyses of various datasets using TCGA clinical data, high MED1 levels were found to correlate with poor prognosis of breast cancer patients23. Additionally, recent studies demonstrated that MED1 knockdown makes breast cancer cells more sensitive to anti-estrogen fulvestrant or tamoxifen treatment38. Overall, MED1 is involved in human breast carcinogenesis and treatment response and thus, understanding its functioning is imperative. Our results here identified MED1 as a regulator of several miRNAs known to be involved in breast cancer such as miR-10b-5p, -100-5p, -17-5p, 18a-5p, -191-5p, 193b-3p, 205-5p, -326, -422a and -425 suggesting its importance in breast cancer pathogenesis. MED1 was also shown to have significant positive correlation with the levels of miR-191-5p, miR-425-5p, miR-422a and miR-100-5p in breast cancer patients. There are several reports that suggest that Mediator complex plays an important role in both activation and repression of RNA pol II-mediated transcription of genes. For instance, MED14, which like MED1 is also targeted by several nuclear factors has been found to repress transcription of several genes in yeast and plants40. Similarly, MED15 and MED16 are known to be involved in both positive and negative regulation of transcription41. It is suggested that transcriptional regulation by Mediator subunits depends on the structure of the promoter, combination of cis elements and overall chromatin organization in the context specific manner43. In accordance with this, human Mediator has been found to act as both an activator and repressor, depending on the conditions used in the cell free transcription assay46. Interestingly, we noticed that some of the miRNAs that have been reported to show positive correlation with ER levels such as miR-100, -17, -18a,-191, -422a and miR-425 were MED1 induced while those known to show negative correlation with ER levels such as miR-10b and miR-155 showed downregulation by MED1 with exceptions such as miR-326, known to be induced by ER but downregulated by MED128. An interesting negative feedback loop was also identified here since a miRNA known to target MED147, miR-205, was found to be downregulated by MED1. It is also likely that MED1 mediated regulation of all miRNAs is not limited to estrogen receptor context. MED1 is known to interact with several transcription factors such as Thyroid hormone receptor, Androgen receptor, Glucocorticoid receptor, BRCA1, Hepatocyte nuclear factor 4 alpha, Peroxisome proliferator-activated receptor gamma, p53 etc49 and thus, MED1 mediated regulation of specific miRNAs may be guided by its interactions with various transcription factors. Besides, whether all the miRNAs are direct MED1 targets or not has not been investigated yet for any miRNA except miR-191 and miR-425. It may be that some miRNAs are getting regulated due to indirect regulation of its activators or repressors by MED1.Based on TCGA data analyses of a dataset with >1000 breast cancer patients, MED1 was found to be altered (majorly amplifications) in upto 20% of these patients and its transcript levels correlated well with the copy number variations30. Our results here show that estrogen induced transcription of miR-191/425 is also MED1-dependent suggesting commonality in the two in terms of their target promoters. Indeed, ER-α and MED1 occupy same EREs in the regulatory region of miR-191/425 cluster. Furthermore, occupancy of MED1 on these EREs is ER-α-dependent indicating interaction between the two is imperative for miR-191/425 cluster regulation. This study reveals that interaction of ER-α and MED1 is ligand-dependent since binding of estrogen to ER increases occupancy of both ER-α and MED1 on the EREs present in the upstream region of miR-191/425 cluster. It is well established that Mediator stabilizes and facilitates pre-initiation complex (PIC) formation51. On the basis of these results and earlier reports, we propose a working model of estrogen induced expression of miR-191 and miR-425  of miR-191 in breast cancer47. MED13, a subunit in the Kinase module is targeted by miR-378 and miR-208a to regulate mitochondrial metabolism, insulin sensitivity and glucose tolerance in mice53. Thus, in animals it seems that miRNAs play important role in regulating Mediator subunit composition and so its activity. However, role of Mediator or its subunits in the regulation of miRNAs in animals is not yet discovered. Our study reveals the involvement of a Mediator subunit, MED1, in the regulation of expression of miRNA genes, miR-191 and miR-425, in humans and provides the mechanism for it. Recently, in Arabidopsis, a dicot plant, three Mediator subunits were implicated in the transcription of few miRNA genes14. This is interesting because unlike metazoans, plant genomes do not code for nuclear receptors and MED1, which we found to be the main regulator of miRNA gene transcription in this study. However, plants do have their own set of plant specific TFs. More than 40 percent of TFs found in Arabidopsis and rice are specific for plants. Thus, though different sets of TFs and Mediator subunits are involved in transcription of miRNA genes in animals and plants, involvement of Mediator in miRNAs regulation seems to be conserved in two major eukaryotic kingdoms suggesting that this mechanism of gene regulation evolved before the diversification of plantae and animalia.In a miRNA screen with human placental trophoblasts under hypoxic conditions, MED1 was identified as a target of miR-2052 in incubator (Shell labs).The human breast cancer cell line, MCF7, was a kind gift from Dr. Mircea Ivan . MDA-MB-231 and ZR-75 cells were purchased from NCCS cell line repository, Pune. Cells were maintained in RPMI 1640 (GIBCO) medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin and 10% fetal bovine serum and incubated at 37 °C in 5% CO5 cells/well) in 6-well plates. In case of plasmids , 2.5 µg of plasmid was transfected into 6-well plates using Lipofectamine 2000 (Invitrogen). In case of siRNAs , 100 nM while in case of esiRNAs , 2 µg/µl  were transfected using Lipofectamine 2000. After 6 hrs, media was changed and the samples were assayed after 48 hrs for miRNA levels (through stem-loop RT-PCR) or other cellular assays. Each experiment was repeated three times.Cells were seeded , according to manufacturer’s instructions. Stem-loop RT-PCR was done to determine the level of all the miRNAs and RNU6B  in all the samples. 500 ng of total RNA was used for cDNA formation using RevertAid first strand cDNA synthesis kit (Fermentas). Specific RT primers are used for each miRNAs:miR-191RT- GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCAGCTG, RNU6BRT-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAATATGGAAC, miR-425RT- GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAACG, miR- 100RT- GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCACAAG, miR-205RT- GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCAGACTC, miR-326RT- GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTGGAG, miR-422aRT- GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCCTTC, miR-557RT- GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTTCAGT, miR-17RT- GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTACCT, miR-18aRT- GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTACCT, miR-206RT- GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCACAC, miR-193bRT- GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGCGGGA, miR-145RT- GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGGGAT, miR-10bRT- GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCACAAA. Taqman miRNA probe, Assay ID-Hs03910067_s1 (Ambion) was used for miR-155.qPCR was performed using miRNA specific forward and a common Stem-loop reverse universal primer (Reverse Primer- GTGCAGGGTCCGAGGT). The sequence of forward primers used are: miR-191F- CGCGCAACGGAATCCCA, miR-425F- GCCGAATGACACGATCACTCC, RNU6BF- GCCCCTGCGCAAGGATGAC, miR-100F- GCAACCCGTAGATCCG, miR-205F- GCTCCTTCATTCCACCG, miR-326F- GATACCTCTGGGCCCTTC, miR-422aF- GCGCGCACTGGACTTAGGGTC, miR-557F- GCGCGCTACAGTACTGTG, miR-17F- GCGCGCAAAGTGCTTACAGTG, miR-18aF- GCGCGTAAGGTGCATCTAGTGC,miR-206F- GGAGTAGTGGAATGTAAGGAAGT, miR-193bF- GTCAACTGGCCCTCAAAG, miR-145F- AGACGGCAGGTCAGGTCCAC, miR-10bF- GCGTACCCTGTAGAACCG, GAPDHF- GTCCATGCCATCACTGCCAC.For gene transcript level quantification, the primers used are: GAPDHR- AGACGGCAGGTCAGGTCCAC, MED1F- CGAGTTAGGATCTGGATGAAGG, MED1R- GAAGTTGACTTCATGTCCTTGC, JUNF- GTGTGCACGAGTGGGAAGG, JUNR- GATCGAATGTTAGGTCCATGGAG, FOSF- AATGACCCTGAGCCCAAGCC, FOSR- AGCTCTGTGGCCATGGCC, VEGFF- GCTACTGCCATCCAATCGAGAC, VEGFR- CTATGTGCTGGCCTTGGTGAG, SATB1F- TCAGCAACAGCAGCAGCAAC, SATB1R- GATTCCCAAGGCTTCCACTG, CDK6F- GTGTGGAAATTCACTGCCTGG, CDK6R- TCAGAGAGCTGTGCTGCACC, BDNFF- AACTCAGGCCGAATGATCAAG, BDNFR- AAATGGCAGAGGTGAGGCAG, EGFRF- TGAGCAAGGAGCACAAGCCAC, EGFRR- AGAATTCCATCCCCTCCGTTTC, MMP1F- AGAGCAGATGTGGACCATGCC, MMP1R- TGAGCATCCCCTCCAATACCTG, ERBB4F- TCTCCCTGGCTGGTGTGTCTC, ERBB4R- TCCCTCTCTCACCCAAACTGATG.et al.24. The estrogen response element-A (ERE-A), the binding site for ER-α and MED1 in the upstream region of miR-191/425 cluster was mutated through site-directed mutagenesis with primers spanning the ER-α/MED1 binding site with indicated mutations  that encompass estrogen responsive elements present upstream of miR-191 and miR-425 promoter fragments. 2 EREs upstream of miR-191, with 100% match with ER-α consensus were cloned downstream of a luciferase promoter vector PGL3-tk-luciferase, (Promega) as previously described in Nagpal ERE-A-cloning-F- ATTCTCGAGCAGCTGCTTTTGGGATTCCGERE-A-cloning-R- ATAACGCGTCACCAGGGAAGCTCAACGGERE-A-mutF- AGGGCGAGTGTGGTGGGGGCAGGAGCTCCERE-A-mutR- GGAGCTCCTGCCCCCACCACACTCGCCCTERE-B-cloning-F- ATACTCGAGGCATGAGGTATGGCAGAGGERE-B-cloning-R- ATAACGCGTACCACTGCCCTTATCTTGCCTGFor luciferase assays, MCF7 cells (10000 cells/well) were cotransfected with 500 ng/well of each construct ERE-A and ERE-B along with ER-α/MED1/parent vector (PC) using Lipofectamine 2000 (Invitrogen). To confirm role of MED1 in binding of ER-α at these promoter fragments, 500 ng/well of each construct was cotransfected along with 500 ng each of ER-α/parent vector (PC) and 2 µg/µl of inhibitor for MED1; esiMED1/esiCtrl using Lipofectamine 2000 (Invitrogen). pRL-TK (10 ng/well) was transfected in all the wells for normalization of transfection efficiency. The activities of Firefly  and Renilla (Renilla reniformis) luciferase were quantified with the Dual Luciferase Reporter Assay (Promega) 48 hrs post-transfection.Cells were transfected with esiMED1/esiCtrl and 48 hrs post-transfection, cells were lysed. 150–200 µl (per well of a 6-well) of RIPA buffer was used for cell lysis and harvested lysate was stored at −80 °C till further use. The protein concentration was determined by using Bradford Reagent (Sigma Aldrich). Equal amount of protein lysates were separated with 8–10% SDS-PAGE and transferred to nitrocellulose membrane (Amersham Hybond ECL). The membrane was then probed with a specific primary antibody at a dilution of 1:2000  followed by washing and incubation with secondary antibody . The specific protein band was visualized by autoradiography using an ECL kit (Amersham ECL Prime). The quantification of band density was then done by using GelQuantNET software (Biochem lab solutions).−8 M) was given for 2 hrs and cells were then crosslinked, washed and resuspended in RIPA buffer for CHIP. To confirm the interaction of MED1 and ER, MCF7 cells were starved for estrogen for 3–4 days (as described above) and estrogen treatment (0–10 nM) was given for 45 min. followed by cell crosslinking and lysis using RIPA buffer. Immunoprecipitation was performed as previously described in Nagpal et al.24. Briefly, cells were crosslinked, washed and lysed followed by sonication. Specific antibodies against ER-α/MED1 (Santa Cruz Biotechnology), salmon sperm DNA (Sigma) and pre-equilibrated protein A-sepharose beads (30 µl) were added to the chromatin extract and incubated overnight, washed and eluted with 0.5%w/v SDS solution. For protein-protein interaction (ER-MED1), the immunoprecipitated proteins were eluted in SDS loading buffer and are separated by using SDS-PAGE gel (8%) followed by western detection using MED1-specific was used as control antibody while the chromatin extract without any antibody/beads treatment was used as positive control. For DNA sequence specific quantification qPCR was done. For chromatin immunoprecipitation, the precipitated chromatin extract was decrosslinked at 65 °C for 4–5 hrs and DNA was then purified with PCR purification kit (HiMedia). Rabbit IgG of chromatin extract using sequence specific primers . Each experiment was repeated twice.To confirm recruitment of MED1 to the promoter of miR-191/425 cluster, 70–80% confluent MCF7 cells were crosslinked, washed, and resuspended in RIPA buffer as described above. For ER-α mediated recruitment of MED1, cells were transfected with siRNA for ER-α or siCtrl and 48 hrs post transfection cells were crosslinked and used for CHIP analysis. For ligand-dependent effect of MED1, MCF7 cells were starved for estrogen (cultured in phenol red free medium with dextran-stripped serum for 3 days) and tranfected with esiMED1/esiCtrl. 48 hrs post transfection, estrogen treatment (1 × 10https://cancergenome.nih.gov). Gene and miRNA expression data and the corresponding clinical information for the BRCA  dataset were obtained through Firebrowse (http://firebrowse.org/) data portal. The normalized reads count for mRNA  and mature miRNAs (RPM) were downloaded and log2 transformed. From the BRCA dataset, we selected samples with miRNA, mRNA and overall survival (OS) data. The correlations between the expression levels of miRNA and mRNA were calculated using Pearson correlation (GraphPad software). Survival analysis was performed using Kaplan-Meier curve log-rank testing, using Cutoff finder54 for best MED1 high- and low-expression selection. Comparison of MED1 and miRNA expression in BC and normal breast was performed using two-tailed Mann Whitney test.The datasets analyzed during the current study are available in The Cancer Genome Atlas (TCGA) data portal  were overexpressed transiently using lipofectamine2000 and 48 hrs post transfection effect on cell survival and cell migration was observed using MTT -2,5-Diphenyltetrazolium Bromide) and wound healing assay.The cellular assays were performed as already described in ref.To confirm that miR-191 is responsible for the MED1 mediated effects, MED1 overexpression was coupled with miR-191 inhibition using anti-miR-191/Ctrl oligos and effect on cell survival and migration was looked for using the similar assays as described above. For wound healing assay, the area of migration was estimated by choosing an arbitrary unit scale.Supplementary Information


These aggregates could be disassembled into dimer and/or monomer by (TGnT) tetramolecular G-quadruplexes , which were affected by the linker length of dimeric cyanine dyes and layers of G-quartets. The 1H-NMR titration results suggest that the binding mode of dimeric cyanine dye with TGnT might be on both ends—stacking like a clip. This binding mode could clearly explain that matching structures between dimeric cyanine dyes and TGnT quadruplexes could regulate the disassembly properties of aggregates. These results could provide clues for the development of highly specific G-quadruplex probes.Cyanine dyes have been widely applied in various biological systems owing to their specific photochemical properties. Assembly and disassembly process of cyanine dyes were constructed and regulated by special biomolecules. In this paper, dimeric cyanine dyes with different repeat units (oligo-oxyethylene) in linker (TC-Pn) ( Clearly, the resonance signals for G5-NH and T6-H6 gradually disappear. T6-H6 and T1-H6 show a remarkable downfield shift. The changes of the chemical shifts of T1 and T6 protons (>0.1 ppm) are much larger than those of others, suggesting that the binding site for TC-P4 with TG4T is probably located on T1 and T6. T6 is 3′-terminal G-quartet and T1 is 5′-terminal G-quartet. It is reasonable that TC-P4 could stack on T1 and T6 through both end-stacking modes. Based on this binding mode, TC-P5 and TC-P4 both have a better matching length to interact with four and five G-tetrads of TGnT tetramolecular G-quadruplex. TC-P3 with a short linker length could not bind to TG8T in the form of monomer like a clip due to its short linker length, while TC-P6 aggregates could be induced into dimer and monomer due to a matching linker length. In order to clarify binding sites of dimeric cyanine dye with tetramolecular G-quadruplex, ormation ,22. In FThe synthesis of TC-P3-P6 has been described in our previous study . All the4 (TG3T), [d(TGGGGT)]4 (TG4T), [d(TGGGGGT)]4 (TG5T), [d(TGGGGGGT)]4 (TG6T) and[d(TGGGGGGGGT)]4 (TG8T) were purchased from Sangon Biotech Co., Ltd.  and purified by HPLC. Ultrapure water was prepared by a Milli-Q gradient ultrapure water system . All these oligonucleotides of [d(TGGGT)]2PO4-K2HPO4, 70 mM KCL, 1 mM EDTA, pH 7.4) was used to dissolve the oligonucleotides. The absorbance at 260 nm was used to determine the concentrations of oligonucleotides. All oligonucleotides were heated to 95 °C for 5 min, rapidly cooled to 4 °C and kept overnight at 4 °C. Their folding topologies were identified by circular dichroism (CD) before usage. The stock solution of 100 μM dimeric cyanine dyes was prepared by dissolving into DMSO and storing in the dark at room temperature. PBS buffer  equipped with a Peltier effect heated cuvette holder in a 10 mm quartz cell. All the CD spectra were recorded on a JASCO J-815 spectrophotometer  in a 10 mm quartz cell at room temperature. All CD spectra were collected with a scan speed of 500 nm/min and a response time of 0.5 s between 200 nm and 350 nm with three scans averaged. 1H-NMR titration spectra were carried out by a Bruker Avance 600 spectrometer  equipped with a BBI probe. The stocking solution of TG4T was prepared by dissolving in PBS . The spectra was recorded immediately by addition of TC-P4 dissolved in DMSO-d6. Bruker pulse program p3919 gp was used to suppress water peak. The 1H-NMR titration results provide binding sites of dimeric cyanine dyes location at both ends of tetramolecular G-quadruplex. This work enriches our understanding on designing specific ligands of recognition of G-quadruplex and highlights the application potential of these G-quadruplex structures in regulation of supramolecular assembly of cyanine dye. In this study, the spectra characteristics of dimeric cyanine dye with tetramolecular G-quadruplex were investigated. In PBS, dimeric cyanine dyes could form H-aggregates or mixture aggregates. These aggregates could be transformed into dimer and/or monomer by tetramolecular G-quadruplex with proper layers of G-quartets.


In this work, atmospheric-pressure (AP) cold plasma was employed to activate the Au/P25-As catalyst prepared by a modified impregnation method. The influence of cold plasma working gas  on the structure and performance of the obtained Au/P25 catalysts was investigated. X-ray diffraction (XRD), UV-Vis diffuse reflectance spectroscopy (DRS), transmission electron microscopy (TEM), and X-ray spectroscopy (XPS) were adopted to characterize the Au/P25 catalysts. CO oxidation was used as model reaction probe to test the Au/P25 catalyst. XRD results reveal that supporting gold and AP cold plasma activation have little effect on the P25 support. CO oxidation activity over the Au/P25 catalysts follows the order: Au/P25-O2P > Au/P25-As > Au/P25-ArP ≈ Au/P25-H2P > Au/P25-AirP. Au/P25-AirP presents the poorest CO oxidation catalytic activity among the Au/P25 catalysts, which may be ascribed to the larger size of gold nanoparticles, low concentration of active [O]s, as well as the poisoning [NOx]s. The poor catalytic performance of Au/P25-ArP and Au/P25-H2P is ascribed to the lower concentration of [O]s species. 100% CO conversion temperatures for Au/P25-O2P is 40 °C, which is 30 °C lower than that over the as-prepared Au/P25-As catalyst. The excellent CO oxidation activity over Au/P25-O2P is mainly attributed to the efficient decomposition of gold precursor species, small size of gold nanoparticles, and the high concentration of [O]s species.Commercial TiO The highest [O]s is observed for Au/P25-As , low concentration of active [O]s, as well as the poisoning [NOx]s s ,25.2P > Au/P25-As > Au/P25-ArP ≈ Au/P25-H2P > Au/P25-AirP. The poor catalytic performance of Au/P25-ArP and Au/P25-H2P is ascribed to the lower concentration of [O]s species. Au/P25-AirP presents the poorest CO oxidation catalytic activity among the Au/P25 catalysts, which may be ascribed to the larger size of gold nanoparticles, low concentration of active [O]s, as well as the poisoning [NOx]s. 100% CO conversion temperatures for Au/P25-O2P is 40 °C, which is 30 °C lower than that over the as-prepared Au/P25-As catalyst. The excellent CO oxidation activity over Au/P25-O2P is mainly attributed to the efficient decomposition of gold precursor species, small size of gold nanoparticles, and the high concentration of [O]s species. AP oxygen cold plasma activation is found to be an efficient method for synthesizing high performance Au/P25 catalysts.AP cold plasma was adopted to activate the Au/P25-As catalyst prepared by a modified impregnation method, and the influence of working gas on the structure and performance of the obtained Au/P25catalysts was investigated. XRD analyses confirm that supporting gold and AP cold plasma activation have little effect on the P25 support. All of the Au/P25 catalysts exhibit high CO catalytic oxidation activity. Catalytic performance of the Au/P25 catalysts is closely related with the working atmosphere of AP cold plasma. CO oxidation activity over the Au/P25 catalysts follows the order: Au/P25-O


Understanding the spatio-temporal patterns of cone degeneration is important for evaluating potential treatments. In the present study we performed a systematic characterization of the spatio-temporal patterns of S- and M/L-opsin+ outer segments was fundamentally complete by P21 in the central retina, and 90% complete by P45 in the peripheral retina. In comparison, degeneration of S- and M/L-opsin+ cell bodies proceeded at a slower rate. There was a marked hemispheric asymmetry in the rate of S-opsin+ and M/L-opsin+ cell body degeneration. M/L-opsin+ cones were more resilient to degeneration in the superior retina, whilst S-opsin+ cones were relatively preserved in the inferior retina. In addition, cone outer segment and cell body degeneration occurred far more rapidly in the central than the peripheral retina. At P14, the superior retina comprised a minority of genuine S-cones with a much greater complement of genuine M/L-opsin cones and dual cones, whilst the other three retinal quadrants had broadly similar numbers of genuine S-cones, genuine M/L-cones and dual cones. At P60, approximately 50% of surviving cones in the superior, nasal and temporal quadrants were dual cones. In contrast, the inferior peripheral retina at P60 contained almost exclusively genuine S-cones with a tiny minority of dual cones. Microglial number and activity were stimulated during rod breakdown, remained relatively high during cone outer segment degeneration and loss of cone somas in the central retina, and decreased thereafter in the period coincident with slow degeneration of cone cell bodies in the peripheral retina.Outer segments of S- and M/L-cones degenerated far more rapidly than their somas. Loss of both S- and M/L-opsinThe results of the present study provide valuable insights into cone degeneration in the Rd1 mouse, substantiating and extending conclusions drawn from earlier studies. Retinitis pigmentosa (RP) comprises a diverse group of hereditary retinal dystrophies. Although the genetic mutations in RP are usually expressed in rods, in most phenotypes, secondary cone degeneration eventually ensues with loss of the remaining central vision . NeuroprIn recent decades, a variety of animal models of RP have been developed. The Pde6brd1  mouse is a very widely used model of RP , which iTo date, a number of studies—employing a variety of experimental methodologies—have shed light on certain aspects of cone degeneration in the Rd1 mouse –14. NotaMice possess two types of cones that are distinguished by the portion of visible light spectrum to which they are sensitive. Short wavelength light stimulates S-cones, whilst medium to long wavelengths stimulates M/L-cones. These cones can be distinguished using well-characterised antibodies that specifically bind to the light-sensitive opsin protein in question. In wild-type (WT) mouse retinas, S- and M/L-opsins are expressed in the outer segments of S- and L/M-cones, respectively , while i+ cone outer segment and cell body degeneration in Rd1 mice, to describe the distribution and proportion of dual cones in Rd1 retinas, and to examine the kinetics of microglial activation during the period of cone degeneration in the Rd1 retina. The combined data will provide a valuable baseline for future studies investigating cone neuroprotection in the Rd1 mouse.The aims of the present study were to perform, for the first time, a systematic characterization of the spatio-temporal patterns of S- and M/L-opsinThis study was approved by the Animal Ethics Committees of SA Pathology/Central Adelaide Local Health Network and the University of Adelaide  and conformed with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, 2013, and with the ARVO Statement for the use of animals in vision and ophthalmic research. C3H/HeJArc  mice and C57BL/6J mice were obtained from Animal Resources Centre . The Pde6brd1 mouse is an inbred strain homozygous for the PDE6b mutation. Rd1 mice were inbred to produce offspring. All animals were housed in a temperature and humidity controlled room with a 12-h light and 12-h dark cycle, and provided with food and water and environmental enrichment ad libitum. Rd1 mice were humanely killed at various postnatal day (P) time points for detection of cone opsin proteins by immunohistochemistry and for detection of mRNA expression of cone opsins. For the spatio-temporal characterisation of cone degeneration by immunohistochemistry on wholemounts, the number of samples analysed was as follows: P14 (n = 7), P21 (n = 5), P30 (n = 7), P45 (n = 7), P60 (n = 7), P75 (n = 4), P90 (n = 6), P150 (n = 5) and P300 (n = 5). Six WT (C57BL/6) mice were humanely killed at P60. For determination of the proportion of genuine S-cones, genuine M/L-cones and dual cones, the number of samples analysed was as follows: P14 (n = 5), P60 (n = 24). For real-time RT-PCR, the number of samples analysed was as follows: WT (n = 6), P14 (n = 6), P30 (n = 6), P45 (n = 9), P60 (n = 15), P90 (n = 6). For the characterisation of microglia by immunohistochemistry on wholemounts and transverse sections, the number of samples analysed was as follows: transverse sections: P7, P14, P21, P30, P45, P60, P75 ; wholemounts P21 (n = 4), P45 (n = 6), P60 (n = 6), P300 (n = 4).All mice were euthanized by transcardial perfusion with physiological saline under terminal anaesthesia (100 mg/kg body weight ketamine and 10 mg/kg body weight xylazine) followed by decapitation. The superior aspect of each cornea was marked before globes were enucleated. For wholemount immunohistochemistry, eyes were fixed in 4% (w/v) neutral buffered formalin for 24 h and dissected into posterior eye-cups. The corneal mark was used to orient the eye and a small radial cut was made in the superior retina while it was still attached to the retinal pigment epithelium (RPE) in the eye-cup. Retinas were removed and prepared as flattened wholemounts by making another four radial cuts. Retinal wholemounts were then stored in phosphate buffered saline (PBS). For double labeling immunohistochemistry, retinas were incubated in PBS containing 1% (v/v) Triton X-100 detergent (T) for 1 h at room temperature. Next, retinas were incubated in PBS-T containing 3% (v/v) normal horse serum (NHS-T) for 1 h at room temperature to block non-specific antibody binding. Retinas were then incubated overnight at 4 °C with a combination of primary antibodies diluted in NHS-T. OPN1SW antibody was used to detect short-wave sensitive cones, whilst anti-R/G opsin antibody was used to detect medium/long wave sensitive opsin cones , 3 parts 100% ethanol, 1 part glacial acetic acid and 3 parts water, is the preferred fixative for whole eyes as it provides optimal tissue morphology while avoiding retinal detachment. Globes were embedded sagittally and 4 μm serial sections were cut. Colorimetric immunohistochemistry was performed as previously described , 21. In For double-labeling immunofluorescence of transverse retinal sections, samples were treated exactly as described above except that a combination of primary antibodies was used. Detection of signal was achieved by incubation with the appropriate combination of AlexaFluor-488 and -594 conjugated secondary antibodies  instead of streptavidin–peroxidase conjugate. Sections were mounted using anti-fade mounting medium.Confirmation of the specificity of antibody labeling was judged by the morphology and distribution of the labeled cells, by the absence of signal when the primary antibody was replaced by isotype/serum controls, and, by comparison with the expected staining pattern based on our own, and other, previously published results. Retinal wholemounts and transverse sections were examined under a fluorescence microscope  equipped with a scientific grade, cooled CCD camera. Wholemount images were taken using a Hamamatsu NanoZoomer 2.0-HT fluorescence module and viewed using the Hamamatsu NanoZoomer Digital Pathology system. Photomicrographs were captured using an excitation wavelength of 488 nm to detect Alexafluor 488-labeled antibodies and an excitation wavelength of 594 nm to detect Alexafluor 594-labeled antibodies. Transverse retinal sections were photographed using a light microscope.For quantification of the spatio-temporal characterisation of cone degeneration by immunohistochemistry on wholemounts, four photomicrographs of the central retina (measuring 720 μm × 540 μm) were taken directly superior, temporal, inferior, and nasal to the centre of the optic disc, and, four photomicrographs of the peripheral retina (measuring 720 μm × 540 μm) were taken 1.5 mm superior, temporal, inferior, and nasal from the centre of the optic disc, yielding a total of 8 images per fluorescent channel per retina. For determination of the proportion of genuine S-cones, genuine M/L-cones and dual cones, four photomicrographs of the peripheral retina (measuring 526.5 × 422.5 µm), one in each of the retinal quadrants, were photographed 1.5 mm from the optic disc. Quantification of cone survival was performed using Image-J software . Initially, however, images were processed in Photoshop CS3  for uneven lighting (using a flatten filter), sharpened, levels enhanced, and finally converted to 8-bit mode. Cone cell bodies were identified by boththeir wide ovoid morphology, segments by their narrow, bundle-like appearance. Outer segments label for cone opsins with high fluorescent intensity. In comparison, cell bodies label with lower fluorescent intensity. These different cellular structures can therefore be analysed and quantified separately. The area in pixels was calculated with the “analyse particles” function. Essentially, the same method was used to quantify Iba1 and CD68 labeled microglia. To quantify the number of Iba1-positive cells in transverse sections of the retina, four images were captured across each immunolabeled retina, a process which was independently repeated for a second labeled section.For characterisation of the proportion of genuine M/L-cones, genuine S-cones, and dual cones in P14 and P60 Rd1 mice, initial image processing was performed using Photoshop CS3. Each pair of S-opsin and M/L-opsin images were corrected for uneven lighting using a flatten filter, and where necessary the linear gradient tool, and subsequently adjusted to be of approximately the same labeling intensity. The images were then merged , sharpened, levels enhanced, filtered for “noise/dust” and finally converted to 8-bit mode. In Image-J software, colour thresholding was performed on each merged image to determine the proportion of “red”, “yellow” and “green” cones, which correspond to “genuine S-cones”, “dual cones”, and “genuine M/L-cones”, respectively. To facilitate this goal, the total area of “all cones” within the image was first determined by manually thresholding using the “brightness” scale, with “saturation” and “Hue” set at the maximum range of 0–255. Next, to delineate the proportion of individual cone types, the “Hue” scale was altered to individually detect red, yellow, and green colours. For each “Hue” setting, the area in pixels was calculated using the “analyze particles” function, and a mask of the labeled area was produced.http://bioinfo.ut.ee/primer3-0.4.0/primer3/) and were selected wherever possible to amplify sequences that spanned at least one intron. Primer sequences were analyzed for Tm (melting temperature), secondary structure and primer-dimer formation with NetPrimer analysis software (http://www.premierbiosoft.com/netprimer). PCR assays were performed using the CFX cycler (Bio-Rad) and all samples were run in duplicate. After the final cycle of the PCR, primer specificity was checked by the dissociation (melting) curve method and by electrophoresis of PCR products on 3% agarose gels. All mRNAs were amplified with high efficiency and linearity during real-time PCR. Mean amplification efficiencies, as determined by plotting cycle threshold as a function of initial cDNA quantity, were in the range of 1.90–2.00. Results obtained from the real-time PCR experiments were, therefore, quantified using the comparative threshold cycle (CT) method (ΔΔCT) for relative quantitation of gene expression, with a minor correction for amplification efficiency. All values were normalised to a pool of two different endogenous reference genes, GAPDH and cyclophilin, and expressed relative to WT retinas and relative to the earliest time point analysed, P14. Statistical analysis was carried out by one-way ANOVA followed by post hoc Dunnett’s Multiple Comparison Test.All mice were killed by transcardial perfusion with physiological saline under deep anaesthesia. Retinas were carefully dissected, total RNA was isolated, and first strand cDNA was synthesized from DNase-treated RNA samples. Real-time PCR reactions were then carried out in 96-well optical reaction plates using the cDNA equivalent of 10 ng total RNA for each sample in a total volume of 20 μl containing 1× SYBR Green PCR master mix (BioRad) forward and reverse primers. Thermal cycling conditions were 95 °C for 3 min and 40 cycles of amplification comprising 95 °C for 12 s, 63 °C for 30 s and 72 °C for 30 s. Primer pairs . Relative to WT retinas, the levels of both S-opsin and M/L-opsin mRNAs were lower by P14, albeit the differences did not reach significance at this time  compared with the superior , nasal , and temporal  quadrants. This results exemplifies the remarkable survival of S-opsin+ cones in the peripheral regions of the inferior retina  Representative high magnification images of M/L-opsin+ and S-opsin+ cones in retinal wholemounts of Rd1 mice from postnatal day 60 (B). Outer segments are identified by high fluorescent intensity (white arrows). In comparison, cell bodies label with lower fluorescent intensity (white arrowheads). These different cellular structures can, therefore, be analysed and quantified separately. (B) Image thresholding of M/L-opsin+ cone segments. The left image shows M/L-opsin+ cones in the superior peripheral quadrant of a retinal wholemount. Insets 1 and 2 are magnified views of two regions from the photomicrograph. The right panel shows the ML-opsin+ image overlaid with the mask derived from image thresholding to isolate outer segments (white represents areas to be quantified). It can be seen that the mask recapitulates the distribution of immunolabeled segments. Scale bar A = 50 μm; B= 100 μm.Additional file 3. Representative images of rhodopsin+-rods in transverse sections of the Rd1 mouse central retina from postnatal day (P) 14 to P21. At P14, the outer nuclear layer is reduced to 3–4 cells in thickness. By P21, rod degeneration is almost complete. Scale bar 50 μm.Additional file 4. Representative images of M/L-opsin+-cones in transverse sections of the Rd1 mouse mid-retina from postnatal day (P) 14 to P60. At P14, outer segments are typically swollen and misshapen, while ectopic redistribution of M/L-opsin to the cell body is frequently evident. By P21, outer nuclear layer thinning is very advanced, and M/L-opsin+ outer segment degeneration is considerable. M/L-opsin cell body degeneration progresses gradually from P21 to P60. Scale bar 50 μm.Additional file 5. Representative images of S-opsin+-cones in transverse sections of the Rd1 mouse mid-retina from postnatal day (P) 14 to P60. At P14, outer segments are typically swollen and misshapen, while ectopic redistribution of S-opsin to the cell body is uniformly evident. By P21, outer nuclear layer thinning is very advanced, and S-opsin+ outer segment degeneration is considerable. S-opsin cell body degeneration progresses gradually from P21 to P60. Scale bar 50 μm.Additional file 6. Representative, high magnification, images of S-opsin+ cones, M/L-opsin+ cones and their merged image in retinal wholemounts of C57BL/6 wild-type mice. Images from the superior (A-C), inferior  and temporal (J-L) quadrants are shown. Double labeling immunofluorescence was performed using antibodies directed against S-opsin (red) and M/L-opsin (green). Scale bar: 100 μm.Additional file 7. Representative images of genuine S-cones, genuine M/L-cones and dual cones in the inferior peripheral retina of C57/BL/6 wild-type mice. Double labeling immunofluorescence of retinal wholemounts was performed using antibodies directed against S-opsin (red) and M/L-opsin (green). (A) S-opsin+ cones; (B) M/L-opsin+ cones; (C) merged image ; (D) mask of genuine S-cones, (E) mask of genuine M/L-cones (F) mask of dual cones; (G) merged image  overlaid with mask of genuine S-cones; (H) merged image  overlaid with mask of genuine M/L-cones; (I) merged image  overlaid with mask of dual cones. Scale bar: 100 μm.Additional file 8. Representative images of genuine S-cones, genuine M/L-cones and dual cones in the superior peripheral retina of Rd1 mice at postnatal day 14. Double labeling immunofluorescence of retinal wholemounts was performed using antibodies directed against S-opsin (red) and M/L-opsin (green). (A) S-opsin+ cones; (B) M/L-opsin+ cones; (C) merged image ; (D) mask of genuine S-cones, (E) mask of genuine M/L-cones (F) mask of dual cones; (G) merged image  overlaid with mask of genuine S-cones; (H) merged image  overlaid with mask of genuine M/L-cones; (I) merged image  overlaid with mask of dual cones. Scale bar: 100 μm.Additional file 9. Representative images of genuine S-cones, genuine M/L-cones and dual cones in the inferior peripheral retina of Rd1 mice at postnatal day 14. Double labeling immunofluorescence of retinal wholemounts was performed using antibodies directed against S-opsin (red) and M/L-opsin (green). (A) S-opsin+ cones; (B) M/L-opsin+ cones; (C) merged image ; (D) mask of genuine S-cones, (E) mask of genuine M/L-cones (F) mask of dual cones; (G) merged image  overlaid with mask of genuine S-cones; (H) merged image  overlaid with mask of genuine M/L-cones; (I) merged image  overlaid with mask of dual cones. Scale bar: 100 μm.Additional file 10. Representative images of genuine S-cones, genuine M/L-cones and dual cones in the nasal peripheral retina of Rd1 mice at postnatal day 14. Double labeling immunofluorescence of retinal wholemounts was performed using antibodies directed against S-opsin (red) and M/L-opsin (green). (A) S-opsin+ cones; (B) M/L-opsin+ cones; (C) merged image ; (D) mask of genuine S-cones, (E) mask of genuine M/L-cones (F) mask of dual cones; (G) merged image  overlaid with mask of genuine S-cones; (H) merged image  overlaid with mask of genuine M/L-cones; (I) merged image  overlaid with mask of dual cones. Scale bar: 100 μm.Additional file 11. Representative images of genuine S-cones, genuine M/L-cones and dual cones in the temporal peripheral retina of Rd1 mice at postnatal day 14. Double labeling immunofluorescence of retinal wholemounts was performed using antibodies directed against S-opsin (red) and M/L-opsin (green). (A) S-opsin+ cones; (B) M/L-opsin+ cones; (C) merged image ; (D) mask of genuine S-cones, (E) mask of genuine M/L-cones (F) mask of dual cones; (G) merged image  overlaid with mask of genuine S-cones; (H) merged image  overlaid with mask of genuine M/L-cones; (I) merged image  overlaid with mask of dual cones. Scale bar: 100 μm.Additional file 12. Representative images of genuine S-cones, genuine M/L-cones and dual cones in the superior peripheral retina of Rd1 mice at postnatal day 60. Double labeling immunofluorescence of retinal wholemounts was performed using antibodies directed against S-opsin (red) and M/L-opsin (green). (A) S-opsin+ cones; (B) M/L-opsin+ cones; (C) merged image ; (D) mask of genuine S-cones, (E) mask of genuine M/L-cones (F) mask of dual cones; (G) merged image  overlaid with mask of genuine S-cones; (H) merged image  overlaid with mask of genuine M/L-cones; (I) merged image  overlaid with mask of dual cones. Scale bar: 100 μm.Additional file 13. Representative images of genuine S-cones, genuine M/L-cones and dual cones in the inferior peripheral retina of Rd1 mice at postnatal day 60. Double labeling immunofluorescence of retinal wholemounts was performed using antibodies directed against S-opsin (red) and M/L-opsin (green). (A) S-opsin+ cones; (B) M/L-opsin+ cones; (C) merged image ; (D) mask of genuine S-cones, (E) mask of genuine M/L-cones (F) mask of dual cones; (G) merged image  overlaid with mask of genuine S-cones; (H) merged image  overlaid with mask of genuine M/L-cones; (I) merged image  overlaid with mask of dual cones. Scale bar: 100 μm.Additional file 14. Representative images of genuine S-cones, genuine M/L-cones and dual cones in the nasal peripheral retina of Rd1 mice at postnatal day 60. Double labeling immunofluorescence of retinal wholemounts was performed using antibodies directed against S-opsin (red) and M/L-opsin (green). (A) S-opsin+ cones; (B) M/L-opsin+ cones; (C) merged image ; (D) mask of genuine S-cones, (E) mask of genuine M/L-cones (F) mask of dual cones; (G) merged image  overlaid with mask of genuine S-cones; (H) merged image  overlaid with mask of genuine M/L-cones; (I) merged image  overlaid with mask of dual cones. Scale bar: 100 μm.Additional file 15. Representative images of genuine S-cones, genuine M/L-cones and dual cones in the temporal peripheral retina of Rd1 mice at postnatal day 60. Double labeling immunofluorescence of retinal wholemounts was performed using antibodies directed against S-opsin (red) and M/L-opsin (green). (A) S-opsin+ cones; (B) M/L-opsin+ cones; (C) merged image ; (D) mask of genuine S-cones, (E) mask of genuine M/L-cones (F) mask of dual cones; (G) merged image  overlaid with mask of genuine S-cones; (H) merged image  overlaid with mask of genuine M/L-cones; (I) merged image  overlaid with mask of dual cones. Scale bar: 100 μm.


Despite the apparent clinical benefits of high-dose cytarabine (Ara-C) over lower dose Ara-C in acute myeloid leukemia (AML) therapy, the mechanism behind high-dose Ara-C therapy remains uncertain. In this study, a LC-MS-based method was carried out to investigate the metabolic alteration of ribonucleotide and deoxyribonucleotide in human promyelocytic leukemia cells (HL-60) after treatment with Ara-C to reveal its antitumor mechanism. The metabolic results revealed that four nucleotides  could be used as potential biomarkers indicating the benefit of high-dose Ara-C over lower dose Ara-C treatment. Combining metabolic perturbation and cell cycle analysis, we conjectured that, apart from the acknowledged mechanism of Ara-C on tumor inhibition, high-dose Ara-C could present a specific action pathway. It was suggested that the pronounced rise in AMP/ATP ratio induced by high-dose Ara-C can trigger AMP-activated protein kinase (AMPK) and subsequently Forkhead Box, class O (FoxO), to promote cell cycle arrest. Moreover, the significant decrease in CDP pool induced by high-dose Ara-C might further accelerate the reduction of dCTP, which then aggravates DNA synthesis disturbance. As a result, all of these alterations led to heightened tumor inhibition. This study provides new insight in the investigation of potential mechanisms in the clinical benefits of high-dose Ara-C in therapy for AML. Cytarabine  is a nucleotide-analog chemotherapeutic drug used alone or in combination with other antineoplastic drugs to treat different forms of leukemia. Multiple clinical trials have demonstrated complete remission (CR) rates of 50%–60% and overall survival rates of 30%–40% among acute myeloid leukemia (AML) patients receiving Ara-C-based therapy ,2,3. MorThe anti-leukemia effect of Ara-C is the result of active uptake into target cells and the subsequent metabolism of Ara-C into its active metabolite. As shown in Ara-CTP constitutes the main cytotoxic component, while dCK activity comprises the rate-limiting step for Ara-CTP formation. However, the concentration achieved by high-dose Ara-C treatment exceeds by far the concentration for saturation of dCK. Thus, the ability to synthesize intracellular Ara-CTP is saturable ,17. DespIt is well known that the action of nucleoside analogues against cancer can disturb endogenous ribonucleotide (RN) and deoxyribonucleotide (dRN) pool sizes, which play essential roles in a broad range of key cellular functions. In contrast, the action can also be affected by RN and dRN pool sizes ,19. An uConsidering the importance of RNs and dRNs as the most affected metabolites during Ara-C treatment, a LC-MS method was used to investigate the metabolic alteration of nucleotides in human promyelocytic leukemia cells (HL-60) after treatment with different doses of Ara-C to reveal its antitumor mechanism. The obtained LC-MS data were analyzed to determine potential biomarkers and related metabolic pathways. Furthermore, cell cycle analysis was carried out to assist metabolic illustration.50 of Ara-C was close to 2.5 μM. It is noteworthy that, when HL-60 cells were treated with Ara-C at the range between 2.5 and 20 μM for 24 h, its viability was approximately 50%, which presented small variation.The antiproliferative effect of Ara-C on HL-60 cells was investigated with MTT assay to assess the selected drug concentrations for the following studies. As shown in p < 0.01. There was a progressive increase in G0/G1 phase arrest from 4 to 24 h. Finally, treatment with Ara-C at 2.5 and 15 µM for 24 h induced cell cycle arrest in G0/G1 phase from 33.5 ± 3.6% to 63.1% ± 9.5% and 75.5% ± 4.2%, respectively, both p < 0.01. These results indicated that Ara-C could block HL-60 cells in G0/G1 phase to inhibit cell growth or cancer progression. Furthermore, high-dose Ara-C (15 µM) induces more severe arrest in G0/G1 phase than does low-dose Ara-C (2.5 µM).The active metabolite of Ara-C can incorporate into DNA to inhibit DNA synthesis. In order to investigate the effect of difference dosage Ara-C on the cell cycle phase distribution in HL-60 cells, the cell cycle analysis was carried out. After drug treatment, the cell cycle distribution significantly changed . SpecifiFor dRN pool sizes, after 4 and 24 h incubation, dATP and dTMP levels demonstrated significant increases in routine-dose Ara-C groups, while dCTP, dCDP, and dGDP levels demonstrated significant decreases in high-dose Ara-C groups. As Ara-C exerts efficacy by competing with dCTP for incorporation into DNA and inhibiting RRs through its active metabolite Ara-CTP, the perturbation of dCDP and dCTP levels would be the most pronounced. The depletions of dCDP and dCTP in high-dose Ara-C groups were more evident than that in routine-dose Ara-C groups, which suggested that high-dose Ara-C therapy could more severely and persistently perturb cellular metabolism than routine-dose Ara-C therapy. The severely perturbed cellular metabolism, especially dCDP and dCTP metabolism, could be one potential mechanism of the clinical benefits gained from high-dose Ara-C therapy.t-test (p < 0.01) combined with a VIP value generated in PLS-DA (VIP > 1) and only four metabolites  were eventually selected as potential biomarkers.To summarize the potential biomarkers and pathways disturbed by high-dose Ara-C to reveal possible action mechanisms, a metabolic pathway scheme was given in AMP-activated protein kinase (AMPK) is one of the key regulators of cellular metabolism, which can be regulated by the cellular AMP/ATP ratio . The conThus, we conjectured that the significant decrease in the ATP pool induced by high-dose Ara-C resulted in a pronounced AMP/ATP ratio rise, which triggered AMPK and subsequently FoxO to promote cell cycle arrest. This conjecture was consistent with the cell cycle observation of more severe cell cycle arrest in G0/G1 phase in the high-dose Ara-C group compared with the routine-dose Ara-C group. Moreover, the significant decrease in the CDP pool induced by high-dose Ara-C further accelerated the reduction of dCTP pool, which then aggravated DNA synthesis disturbance. As a result, all these alterations in metabolism led to heightened tumor inhibition. The hypothesis could partly explain the mechanism behind the clinical benefits from high-dose Ara-C in therapy for AML.13C1015N5-triphosphate (ATP13C15N) were purchased from Sigma-Aldrich Chemical Co. . LC-MS grade methanol, acetonitrile, and acetic acid were purchased from Anaqua Chemical Supply Co. . Ultra-pure water was obtained from the Milli-Q Gradient Water System .Cytarabine for injection used in this study was purchased from Actavis Italy S.p.A. Human acute promyelocytic leukemia cell line HL-60 was supplied by American Type Culture Collection (ATCC) . Phosphate buffer saline (PBS), RPMI medium 1640, 0.25% Trypsin-EDTA solution, penicillin-streptomycin solution, and fetal bovine serum (FBS) were obtained from GIBCO Invitrogen Co. . A Cell Cycle Analysis Kit was purchased from Signal-way Antibody Co., Ltd. . Trichloroacetic acid (TCA), hexylamine (HA), diethylamine (DEA), trioctylamine, 1,1,2-trichlorotrifluoroethane and stable isotope labeled adenosine-2. After overnight culture, cells were divided into two groups: a control group and an experimental group. Cells of the control group were incubated in a medium only. Cells of the routine-dose group were incubated for 4 and 24 h with 2.5 µM Ara-C, which is almost equivalent to the routine-dose regimens with 0.5–1 g/m2 Ara-C plasma concentrations. Cells of the high-dose group were incubated with 15 µM Ara-C, which is almost equivalent to the plasma concentrations (2–3 g/m2 Ara-C) achieved during high-dose Ara-C regimens [The HL-60 cells were seeded in 100 mm × 20 mm dishes  and cultured in RPMI Medium 1640 supplemented with 10% FBS, 100 UI/mL penicillin, and 100 μg/mL streptomycin in humidified air at 37 °C with 5% CO4 per well. After incubation, they were treated with Ara-C at different concentrations for 24 and 48 h. MTT solution  was added to each well and incubated further for 4 h. The medium was removed and 100 μL of DMSO was added to each well to dissolve the purple crystals of formazan. Absorbance was measured at 570 nm with a microplate UV-Vis spectrophotometer ; reference wavelength was 650 nm. IC50 values of Ara-C were calculated by GraphPad Prism Software Inc. .The cytotoxicity of cell growth by Ara-C was investigated via the MTT assay. HL-60 cells were seeded in 96-well plate  at a density of 2 × 105 cells/well in 6-well culture plates in duplicate, and incubated with Ara-C at 2.5 (routine-dose) and 15 µM (high-dose) for 4 and 24 h. They were then harvested and fixed in 70% (v/v) cold ethanol overnight at 4 °C. The fixed cells were collected by centrifugation and re-suspended in PBS. Subsequently, the re-suspended cells were incubated with 5 mg/mL propidium iodide (Sigma-Aldrich) and 10 mg/mL RNase A (Sigma-Aldrich) at room temperature for 30 min in the dark. The cells were then analyzed on a flow cytometer . Finally, the percentages of cells in different phases  were calculated using Modfit software .Cells were seeded at 4 × 10g; 4 °C), and the pellets were frozen in liquid nitrogen and stored at −80 °C for further analysis. Extraction was performed by addition of 150 µL of 15% TCA containing 7.5 µL of 20.0 µM ATP13C15N as an internal standard and placed on ice for 10 min. After centrifugation at 13,500 rpm for 15 min in a cold room, the acidic supernatant was separated and neutralized twice with 100 µL of a mixture of trioctylamine and 1,1,2-trichlorotrifluoroethane . Samples were stored at −80 °C until analysis within two days.After removal of the culture medium, cells were washed once with precooled PBS and then treated with trypsin. Each digested sample was re-suspended in 10 mL of PBS, followed by centrifugation  consisting of an Accela Autosampler, an Accela pump, and a Quantum Access triple quadrupole mass spectrometer. Chromatography was performed on an XTerra-MS C18 column  . The column was maintained at 35 °C and the flow rate was 0.3 mL/min. The mobile phase was composed of Solvent A  and Solvent B (50% acetonitrile in water). The column was eluted with a linear gradient system: 0–15 min, 100%–88% A; 15–35 min, 88%–72% A; 35–45 min, 72%–45% A; 45–50 min, 45%–100% A; 50–60 min, 100%–100% A. For all RN and dRN, the following optimized parameters were obtained. The sheath gas pressure reached 40 psi. The ionspray voltage was set at 3000 V for negative mode and 4000 V for positive mode at a temperature of 350 °C and an auxiliary gas pressure of 15 psi. Quantification was performed using multiple reactions monitoring (MRM) as previously published . Data act test. p < 0.05 was considered to be significant and p < 0.01 to be very significant.Results are mean ± SD and evaluated using the independent-samples Student’s SIMCA-P software  was used to conduct partial least squares-discriminant analysis (PLS-DA) model for pattern recognition. The PLS-DA model was used to separate samples into two blocks and obtain clear discrimination between the control- and Ara-C-treated (2.5 and 15 µM) groups. Variables that made significant contributions to discriminating between groups were considered to be potential biomarkers. The variable importance in the projection (VIP) values was used to select biomarkers. Variables with a VIP exceeding 1 showed a higher than average influence on the classification.t-test and a VIP value generated by SIMCA-P in the PLS-DA model of more than 1.0. These metabolites were eventually selected as potential biomarkers.Ultimately, the differences among samples were evaluated for individual metabolites by using an independent-samples In this study, a LC-MS-based method was used to investigate the metabolic alteration of ribonucleotide and deoxyribonucleotide in HL-60 cells after treatment with different doses of Ara-C to reveal its antitumor mechanism. The statistic results revealed that four nucleotides  could be used as potential metabolic biomarkers. Combining metabolic alterations and biological results, we conjectured that, apart from the acknowledged mechanism of Ara-C on tumor inhibition, high-dose Ara-C could present a specific action pathway. It was suggested that the pronounced rise in AMP/ATP ratio due to the significant reduction of ATP induced by high-dose Ara-C triggered AMPK and subsequently FoxO to promote cell cycle arrest. Moreover, the significant decrease in CDP pool induced by high-dose Ara-C further accelerated the reduction of dCTP pool, which then aggravated DNA synthesis disturbance. As a result, all these alterations led to heightened tumor inhibition. This study provides new insight in the investigation of the potential mechanism in the clinical benefits of high-dose Ara-C in therapy for AML, which can assist in its improvement and applications.


As a neurotropic virus, enterovirus A71 (EV-A71) emerge and remerge in the Asia-Pacific region since the 1990s, and has continuously been a threat to global public health, especially in children. Annually, EV-A71 results in hand-foot-and-mouth disease (HFMD) and occasionally causes severe neurological disease. Here we reviewed the global epidemiology and genotypic evolution of EV-A71 since 1997. The natural selection, mutation and recombination events observed in the genetic evolution were described. In addition, we have updated the antigenicity and virulence determinants that are known to date. Understanding EV-A71 epidemiology, genetic evolution, antigenicity, and virulence determinants can expand our insights of EV-A71 pathogenesis, which may benefit us in the future. Enterovirus A71 (EV-A71) has caused various symptoms and diseases ranging from hand-foot-and-mouth disease (HFMD), herpangina, rashes, and diarrhea, to aseptic meningitis, pulmonary edema, acute flaccid paralysis (AFP), brainstem encephalitis and Guillain–Barré syndrome . AlthougEnterovirus and the family Picornaviridae, enterovirus A71 (EV-A71) is a non-enveloped positive single strand RNA virus, containing 7.4-kb long RNA with a large open reading frame (ORF) flanked by the 5′ and 3′ untranslated region (UTR) [pro) of poliovirus can cleave the P1 capsid protein from the polyprotein. In contrast, the 3CD protease cleaved the P1, P2, and P3 precursors [pro can inhibit retinoic acid-inducible gene I (RIG-I) -mediated interferon response [pro assists in the interaction of 5′ UTR with RNA-dependent RNA polymerase (RdRp) (3Dpol) [As a member of the genus on (UTR) . The 5′ on (UTR) , 17. Theecursors . The matecursors ). The P1ecursors –22. VP4,ecursors , 24. Tyrecursors . PSGL-1 ecursors , while Secursors . In addiecursors , are antresponse . Also, 3 (3Dpol) . The RdR (3Dpol) . Since tThe first EV-A71 isolate was identified in 1969 in the United States . During − 4 to 10− 6 mutations per nucleotide [Due to the error-prone RdRp, RNA viruses generate 10cleotide , which lcleotide . Geneticcleotide . EV-A71 Analyzing the genotypic changes according to the recent evolutionary studies reports, inter-genotype shifts appeared in Taiwan and Japan. The predominant strain of the 1998 outbreak in Taiwan was EV-A71 genotype C2 with 90% having recombination with coxsackievirus A8 (CV-A8) and the remaining 10% were genotype B4 isolates , 61. Theet. al., recombination was found to be a replicative process that is RdRp-mediated [et. al. showed that the sequence resulted in a recombination of genotypes B3 and B2 [et. al. (2010) had observed recombination events at the 2A-2B junction in EV-A71 genotypes B with genotype C, and EV-A71 genotype B with CV-A16 strain G-10 in the 3C region of EV-A71 viruses, while CV-A16 strains were found to possess recombination at the 2A-2B junction between CV-A16 strain G-10 and EV-A71 genotype A [et. al. (2019) had developed a cell-based assay in order to observe recombination events of EV-A71 and found that recombination events were highest in C2 genotype followed by C4 then B5 [et. al. (2010) had previously reported on recombination breakpoints and recombination frequencies of EV-A71. Recombination breakpoint locations may vary depending on the strain and country, however, the 3Dpol coding region shown to have the highest frequency at which recombination occurs as a unit [Recombination of EV-A71 viruses is another common phenomenon. In poliovirus, high nucleotide sequence identities within a region, mainly seen in P2 and P3, of parental strains are in favor of homologous recombination via a ‘copy-choice’ mechanism, resulting in a possible combination that may favor survival during the natural selection process , 68. Accmediated . Both inmediated . Recombimediated , 71–75. mediated , and EV-mediated , 71. Durmediated , 76. Fro3 and B2 . For the3 and B2 . Yip et.notype A . Recombinotype A . Woodman then B5 . Chen etet. al. (2012) had also found that VP1Q145E enhances binding of EV-A71 to mouse neuroblastoma  observed that, in cooperation with VP1Q145E, an amino acid change in VP2 at position 149 from lysine (K) to methionine (M) (VP2K149M) is associated with the increase of RNA accumulation, viral cytotoxicity and uncoating in mice neuronal cells, and an increase in mouse lethality in vivo [et. al. (2012) had observed that an amino acid change from glutamic acid (E) to glutamine (Q) in VP1 position 145 (VP1E145Q) was found in more severe cases of EV-A71 infections [145G/Q mutation had been found to be associated with the virus’ ability to bind to the receptor PSGL-1 while VP1145E was associated with the inability to bind to PSGL-1 [et. al., (2018) they had found infectivity of the human airway organoids were EV-A71 strain-dependent. Coincidentally, the well-known position VP1–145 was also found to be a key determinant of infectivity of human airways. Van der Sanden et. al. had identified VP1145Q as a key determinant of increased infectivity in human airway organoids. In addition, in the absence of VP1145Q, viruses with relatively high replication rates were found to have both VP198K and VP1104D mutations in genotype C5 strains. Therefore, VP198K and VP1104D may be potential infectivity markers in specific viral strains [et. al. (2017) had demonstrated the evolution of EV-A71 virus within a single autopsy case from the 1998 EV-A71 outbreak in Taiwan. EV-A71 viruses were isolated from various tissues and analyzed, thus showing the evolution of the virus within the host, as well as tissue tropism. They had identified a dominant haplotype switch from VP1-31D to VP1-31G, with VP1-31G being dominant in the central nervous system (CNS), indicating possible contribution to CNS invasion of the virus. The VP1D31G mutation was also found to enhance EV-A71 entry into neuroblastoma, increase virus growth rate and fitness in human neuronal cells, and had a higher proportion in the virus population in fatal patients than in HFMD patients [et. al. (2012) had analyzed the EV-A71 genome from various tissues of an immunocompromised patient. They had found that the mutation VP1L79R, located in the BC loop region, plays a critical role in cell tropism and affects the viral binding ability and fitness in neuronal cells in vitro [107A was found to regulate the maturation of EV-A71. Zhang et. al. had discovered that VP1107A allowed greater flexibility of the VP1 BC loop and regulated the efficient cleavage of VP0, influencing maturation and viral uncoating, thus increasing viral replication [et. al. (2012) found that a VP1K244E mutation is critical in mouse adaptation and virulence [et. al. (2013) had also found that mutations at VP1–244 can abolish virus binding to PSGL-1, while mutations at VP1–242 can influence virus binding. They had also proposed that depending on the protein at VP1–145, by its influence to control the exposure of the side-chain VP1244K, VP1–145 can act as a switch which controls PSGL-1 binding [For the last two decades, increasingly more effort has been placed in understanding EV-A71. Several virus virulence determinants have been identified for EV-A71. Since the generation of mouse-adapted EV-A71 , an aminet. al. 012 had aogenesis . Huang e in vivo . In contfections . In addio PSGL-1 . In a st strains . More repatients . Similarin vitro . In anotlication . Zaini eirulence . Nishimu binding .Table 2U158C was found to be associated with translation and virulence in mice [et. al. had also reported on other mutations that were associated with higher prevalence in severe cases of EV-A71: VP1145G/Q/R, VP1164E, and 2A68K [et. al. (2005) had also reported that temperature-sensitive mutants that are located in the 5′ UTR, 3Dpol, and 3′ UTR can cause an attenuation in neurovirulence [pro has also been found to influence replication and virulence of EV-A71. A 3CN69D mutation had shown to attenuate virulence by impacting the substrate-binding site and catalytic active site. This mutation had also diminished 3Cpro activity and its ability to shutoff host cell metabolism, inhibition of host cellular transcription and host immune system [The 5′ UTR in mice . Other 5 in mice . Li et. nd 2A68K . Apart fnd 2A68K . The 3C nd 2A68K , 94. Ariirulence . The 69te system . With bee system .et. al. (2000) had demonstrated that adult mice were resistant to multiple EV-A71 challenges, thus producing neutralizing antibodies post-infection that play a role in limiting severity of EV-A71 infection. These neutralizing antibodies showed a protective role against EV-A71 by administering hyperimmune serum (1:128) 1-day post-infection in a mouse model. The anti EV-A71 NAbs was found to be able to effectively protect neonatal mice when passively immunized and when delivered and fed by an immunized dam [et. al. observed that guinea pig antisera against genotypes B2 and C1 had higher neutralization titers against genotypes B2, B4, and B5 but lower titers against genotypes A, C1, C2, and C4. Similarly, van der Sanden et. al. had similar results with rabbit antisera against genotypes B2 with higher neutralizing titers against genotypes B1 and B2 but lower titers against genotypes C1, C2, and A. However, rabbit sera against genotype C1 showed higher neutralization activity with EV-A71 genotypes A, B, and C [et. al. had previously investigated the cross reactivity and antigenic property of human antiserum from EV-A71 infected patients from 1998 to 2008 against human pathogenic EV-A71. Using sero-microneutralization data, an antigenic map was constructed which showed the antigenic diversity of the different genotypes. Based on this map, they had found that genotype B1 and B4 viruses were clustered closely together, genotype C2 and C4 formed a separate cluster from genotype B that was more spread out. Genotype B5, however, had formed its own cluster within the map  had confirmed that the amino acid residues VP1–98, − 145, and − 164 worked in a cooperative manner as antigenic determinants for B4 and B5 strains (Fig. With the increase in mutations, a change in antigenicity can also occur in addition to viral virulence. The capsid proteins have long been a target in producing antibodies against EV-A71 for immune system recognition. VP1 capsid region has been found to possess many antigenic determinants and is considered to play an important role in characterizing antigenicity . Neutralized dam . Cross-nized dam , 100. Miserum 1:1 1-day poGenotype shifts, changes in antigenic properties, and recombination events have shown to contribute to the evolution of EV-A71. Although there only genotype C4 available EV-A71 vaccine from China to date, increasing knowledge of the virus will better aid in the development of a vaccine that is able to protect against the different genotypes. Therefore, continuous surveillance of EV-A71 is required in order to better understand its epidemiology and viral evolution.