Drosophila model epithelium shares many characteristics with mammalian cells, indicating a high degree of similarity between the mammalian and Drosophila polyamine transport systems. In this report, we focused on the utility of the Drosophila epithelial model to identify and characterize polyamine transport inhibitors. We show that a previously identified inhibitor of transport in mammalian cells has a similar activity profile in Drosophila. The Drosophila model was also used to evaluate two additional transport inhibitors. We further demonstrate that a cocktail of polyamine transport inhibitors is more effective than individual inhibitors, suggesting the existence of multiple transport systems in Drosophila. Our findings reinforce the similarity between the Drosophila and mammalian transport systems and the value of the Drosophila model to provide inexpensive early screening of molecules targeting the transport system. Increased polyamine biosynthesis activity and an active polyamine transport system are characteristics of many cancer cell lines and polyamine depletion has been shown to be a viable anticancer strategy. Polyamine levels can be depleted by difluoromethylornithine (DFMO), an inhibitor of the key polyamine biosynthesis enzyme ornithine decarboxylase (ODC). However, malignant cells frequently circumvent DFMO therapy by up-regulating polyamine import. Therefore, there is a need to develop compounds that inhibit polyamine transport. Collectively, DFMO and a polyamine transport inhibitor (PTI) provide the basis for a combination therapy leading to effective intracellular polyamine depletion. We have previously shown that the pattern of uptake of a series of polyamine analogues in a  For both Ant444 (6) and Triamide444 (9) the EC50 values  were 10 to 15-fold lower than the concentration of Ant44  used in the assays. Maximum protection from Ant44 was observed at 10 μM 6 and 5 µM 9, respectively. These activity profiles are similar to Trimer44 (7) which is an effective transport inhibitor in mammalian Chinese Hamster Ovary (CHO) and L3.6pl cells (8) was a less effective PTI in the Drosophila model with an EC50 of 144 μM and gave maximum protection at 300 μM (Addition of Ant444 (nt44 (5) a,b. Theipl cells c 40]. I. I6) andt 300 μM d. These 5) on mammalian cells and Drosophila leg imaginal discs by competing for binding and transport via the PTS [5) were evaluated in Assay 1  and Triamide444 (9) are 3.6 μM and 2.8 μM, respectively (6 and 9 were approximately 12–15 times better than spermidine in inhibiting the toxicity of Ant44 (5).Compounds containing recognizable polyamine sequences should be able to compete for access to the polyamine receptor on the cell surface. Our previous work has shown that spermidine is able to inhibit the toxicity of Ant44  inhibition of imaginal disc development with an EC50 value of 19.7 μM and afforded complete protection at 40 μM  and Triamide444 (9) were 5-fold and 7-fold lower than spermine respectively, demonstrating that these compounds are more efficient at competing for access to the PTS than either of the native polyamines spermidine or spermine. The data for Ant444 (6) and Triamide444 (9) are similar to Trimer44 (7). In contrast, Triamide44 (8) . Spermine—a native tetraamine—was more effective than spermidine in blocking Ant44 (at 40 μM b. The EC5) in imaginal discs. Concentrations of up to 1 mM putrescine had no effect on the inhibition of imaginal disc development by Ant44 (5) (5), which is a triamine analogue. Therefore, the inability of putrescine to rescue cells from Ant44 (5) could be due to differences in relative binding affinity. An alternative interpretation is that Ant44 (5) is imported into the cell via a polyamine transporter which does not recognize putrescine. Indeed, the existence of multiple polyamine transporters with different affinities and selectivity for the native polyamines has been suggested in mammalian cells [In contrast to spermidine and spermine, the native diamine, putrescine, was unable to rescue the inhibition of Ant44 (nt44 (5) . One int6), Trimer44 (7) and Triamide444 (9) are all considerably more effective than either of the native polyamines spermidine or spermine in competing with Ant44 (5) for access to the PTS. Because putrescine could not rescue Ant44 (5) toxicity in disc development, no comparisons can be made for this native diamine.In conclusion, Ant444 (5), which accessed cells via the PTS (6), Trimer44 (7), Triamide44 (8) and Triamide444 (9) could prevent the rescue of DFMO-treated imaginal disc development by exogenous native polyamines. Essentially, we asked if these compounds could effectively compete with native polyamines for access to the PTS in DFMO-treated imaginal discs.Assay 1 tested the ability of candidate PTIs to block the toxicity of Ant44 . Data for these experiments are shown in Drosophila model assay were much higher (200–500 μM) than those observed in mammalian cells (around 1 μM). The higher doses are likely due to the fact that unlike cell culture, imaginal discs are an intact epithelial tissue surrounded by extracellular matrix, which may impede polyamine access to the PTS.Each of the three native polyamines were evaluated for their ability to rescue the development of leg discs treated with DFMO. Addition of 500 μM putrescine to the culture medium resulted in a significant increase , Trimer44 (7) and Triamide444 (9) were each found to be non-toxic to imaginal disc development at 100 μM, whereas Triamide 44 (8) was non-toxic at 300 μM.As with Assay 1, it was important to use a non-toxic dose of each PTI compound because in Assay 2 a toxic PTI would generate a false positive. To avoid introducing this bias, non-toxic concentrations of the PTI compounds were determined and used in both assays. In a series of control experiments, Ant444 (6), Trimer44 (7) and Triamide444 (9) (6), Trimer44 (7) or Triamide444 (9) is significant, our data suggest that Trimer44 (7) is less effective than Ant444 (6) or Triamide444 (9) in inhibiting the uptake of putrescine. Consistent with earlier studies, 100 μM or 300 μM Triamide44 (8) was unable to compete with putrescine for access to the Drosophila PTS  a. Imagine444 (9) a. Likewie444 (9) a. Additie444 (9) a. While hila PTS a.6), Trimer44 (7) and Triamide444 (9) were all able to significantly inhibit import of spermidine. In the presence of 10 mM DFMO and 200 μM spermidine imaginal disc development decreased from 39% to 11% in the presence of 100 μM Ant444 and to 13% in the presence of 100 μM Trimer44 (8) failed to inhibit import of spermidine even at 300 μM  did not reduce uptake of spermine, whereas 100 μM Trimer44 (7) significantly reduced imaginal disc development from 67% to 34%. Triamide444 (9) showed even greater ability to reduce spermine uptake reducing imaginal disc development from 60% to 15% (8) was unable to inhibit import of spermine even at 300 μM concentration.Finally, we tested the ability of the PTIs to inhibit import of spermine in the presence of 10 mM DFMO and 200 μM spermine. As shown in % to 15% c. Thus, 6), Trimer44 (7) and Triamide444 (9) have similar EC50 values for protection against toxicity of Ant44 (5) and a similar concentration of full protection against Ant44 (5) (6) is better at blocking uptake of putrescine, Ant444 (6) and Trimer44 (7) show similar abilities to block uptake of spermidine and Triamide444 (9) is the most potent of the PTIs at blocking spermine uptake. These findings suggest that the PTIs have different specificities for the polyamine transport systems active in the presence of DFMO. In this regard, there may be a basal and DFMO-stimulated PTS in Drosophila. The basal PTS is assessed via the Ant44 assay (Assay 1), whereas the DFMO-stimulated PTS is assessed via Assay 2 (8) in these assays is consistent with the inability of this compound to block the toxicity of Ant44 (5)  a–c, theynt44 (5) . Ant444  Assay 2 . The poont44 (5) d and sug6) and Trimer44 (7) are effective PTIs and showed different specificities towards putrescine, spermidine and spermine respectively, a combination of these inhibitors was used to block the rescue of DFMO treated leg discs by a mixture of all of the native polyamines. As shown in 6) or Trimer44 (7) alone was unable to significantly inhibit the rescue of DFMO-treated discs by the exogenous native polyamine cocktail. In contrast, a combination of 50 μM Ant444 (6) and 50 μM Trimer44 (7) significantly inhibited rescue by native polyamines even though the amount of each PTI was reduced by half compared to experiments when only one PTI was used. This result suggests that a combination of polyamine transport inhibitors will be more effective in inhibiting the import of all three native polyamines than individual inhibitors dosed alone. In the next experiments, we further examined our finding that the PTIs have different specificities for the PTS. Specifically, we asked if a cocktail of PTIs was more effective than individual PTIs in inhibiting rescue of DFMO-treated imaginal discs in the presence of all three native polyamines. In our prior experiments, we studied the effects of individual native polyamines, however, all three polyamines are present in vivo. For example in circulating red blood cells, the levels of putrescine, spermidine and spermine were found to be 3, 55 and 35 pmol/mg protein respectively . Because6), Trimer44 (7) and Triamide444 (9) towards native polyamines and the ability of a cocktail of PTIs to inhibit transport more effectively than individual PTI’s suggests the existence of multiple polyamine transport systems in Drosophila as has been observed in unicellular organisms [6) shows a greater ability to inhibit uptake of putrescine, whereas Trimer44 (7) is more effective in inhibiting uptake of spermine (5)  , consistDrosophila imaginal disc assay as an early and inexpensive system in which to evaluate compounds targeting the mammalian PTS. There are several advantages to our approach. First, mammalian cell culture is not a natural cellular environment because cells lack cell-cell contacts and extracellular matrix, both of which are factors influencing drug accessibility to cells in vivo. In contrast, the imaginal disc assay tests the effects of medicinal compounds on cells in a more natural environment. Second, inexpensive early animal model testing of promising compounds can reduce the time it takes successful compounds to reach the clinic by up to fifty percent. Mice are more expensive to use in the early stages of drug development where most compounds will fail, therefore a cheaper system such as our Drosophila assay is useful. Third, experiments in mice can only be performed on a small scale, whereas we can assay relatively large numbers of imaginal discs, typically more than 100 per assay. Our work reinforces the value of the Drosophila and mammals. Our work suggests that this is the case. In previous work, we compared the uptake of nine polyamine analogs in mammalian CHO and L1210 cells and Drosophila imaginal discs [5) and N1-(3-aminopropyl)-N4-(anthracen-9-ylmethyl)butane-1,4-diamine (Ant43) gain entry to mammalian cells via the polyamine transport system as evidenced by spermidine competition experiments and greatly reduced uptake in CHO-MG cells, which lack a functional transport system [Drosophila imaginal discs, suggesting that they do not utilize the transport system to gain access to cells in either system. In addition, Trimer44 (7) has previously been shown to be an effective inhibitor in mammalian cells, whereas Triamide44 (8) was not [Drosophila assay. Of course, the imaginal disc assay is only useful to understand mammalian transport if the polyamine transport system is similar in al discs . Two of t system . In imag was not  and thesDrosophila imaginal disc assay has added to our knowledge of polyamine transport inhibitors. We show that two compounds that exhibit toxicity in mammalian cell culture, Ant444 (6) and Triamide444 (9), are non-toxic in the Drosophila assay and are effective PTIs with activity profiles similar to that of Trimer44 (7). The reduced toxicity of Ant444 and Triamide444 in Drosophila may due to a lower effective concentration of these compounds reaching the cell surface due to the presence of intact cell-cell adhesions and extracellular matrix. We also provide activity data for the PTIs against all three native polyamines  whereas most mammalian cell culture studies focus on spermidine uptake. This approach revealed differences in the ability of each PTI to inhibit uptake of individual polyamines suggesting the existence of multiple transport systems. This view is further reinforced by our finding that a mixture of two PTIs is more effective than either PTI alone at inhibiting uptake of a cocktail of all three native polyamines.Use of the i values for several of these compounds in terms of competing with 3H-radiolabeled spermidine for the putative cell surface receptors in L1210 murine leukemia cells. The L1210 Ki values for putrescine, spermidine and spermine are 208.2, 2.46 and 1.34 μM, respectively [i values for Ant44 (5), Ant444 (6) and Trimer44 (7) are 1.8 μM, 0.05 μM and 0.49 μM, respectively [i value of Triamide44 (8) was not determined in L1210 cells, a comparative study of the Trimer44 and Triamide44 compounds in human L3.6pl pancreatic cancer cells revealed Ki values of 36 nM and 398 nM, respectively [In this study, we assayed the ability of PTIs to inhibit the rescue of DFMO treated imaginal discs in the presence of exogenous polyamines. This approach is clinically relevant in that many tumors circumvent DFMO treatment via upregulation of their polyamine transport systems. Our previous work indicates that the PTIs inhibit polyamine uptake. Our data are consistent with the reported Kectively . The L12ectively ,43. Althectively , suggesti values of Ant44, Ant444 and Trimer44 suggest that these compounds compete with the native polyamines for uptake. For example, Ant44 (a triamine) has a L1210 Ki value of 1.8 μM and provides a fluorescent molecule with similar affinity for the polyamine transport system as the native polyamines spermidine (L1210 Ki = 2.46 μM) and spermine (L1210 Ki = 1.34 μM). We speculate that in order to be successfully imported, compounds must bind and release from the cell surface receptors. The Ki values of the native polyamines (spermidine and spermine) suggest that Ki values in the low μM range are optimal for these binding and releasing properties. The related Ant444 compound 6 (a tetraamine) has a significantly lower L1210 Ki value (51 nM) indicating high affinity for the cell surface receptors. Using confocal microscopy, we have demonstrated that this higher affinity of Ant444 was observed as a compound which could not be washed off the surface of L1210 cells by phosphate buffered saline (PBS). In contrast, the triamine Ant44 could be readily washed off the surface of L1210 cells by PBS and appeared to have improved uptake past the cell membrane [5:48 h L1210 IC50 = 0.3 μM) compared to Ant444 (6:48 h L1210 IC50 = 7.5 μM) [The low Kmembrane . This da 7.5 μM) . In summ6) and Triamide444 (9) are effective PTIs expands our understanding of the chemical rules governing an effective PTI design. Inhibitors presenting diamine arms, like Triamide44 (8), are ineffective transport inhibitors. In contrast, compounds containing a higher number of charges in their polyamine arms such as Trimer44 (7) and Triamide444 (9) are effective PTIs. In this regard, N1-substituted triamine and tetraamine analogues can be used to design efficient ligands and inhibitors of polyamine transport. Our work and previous studies suggest that presentation of at least three or more positive charges is necessary for efficient competitive binding to the PTS. Our finding that Ant444 , was recently shown to behave in a similar manner as AMXT-1501 (11) both in its ability to shrink tumors in vivo as well as to beneficially modulate the immune response [Drosophila model can be used to pre-screen PTIs prior to more expensive testing in mouse models. Having a cheap model system for early animal testing will reduce the time from conceptual PTI design to future validation in clinical trials. A combination therapy using DFMO and a PTI has shown promise in cancer growth inhibition ,49. Whil11, 12 , which iresponse . Thus, t


Toxoplasma gondii is an opportunistic protozoan apicomplexan and obligate intracellular parasite that infects a wide range of animals and humans. Rhoptry proteins 5 (ROP5), ROP16, ROP18 and dense granules 15 (GRA15) are the important effectors secreted by T. gondii which link to the strain virulence for mice and modulate the host’s response to the parasite. Little has been known about these molecules as well as GRA3 in type Chinese 1 strains that show polymorphism among strains of archetypical genotypes. This study examined the genetic diversity of these effectors and its correlated virulence in mice among T. gondii isolates from China.I/III), and the other 4 as type II (ROP16II). The strains investigated may be divided into four groups based on GRA3 amino acid alignment, and all isolates of type Chinese 1 belong to the μ-1 allele except Wh6 which is identical to type II strain.Twenty-one isolates from stray cats were detected, of which 15 belong to Chinese 1, and 6 to ToxoDB #205. Wh6 isolate, a Chinese 1 strain, has an avirulent phenotype. PCR-RFLP results of ROP5 and ROP18 presented few variations among the strains. Genotyping of GRA15 and ROP16 revealed that all the strains belong to type II allele except Xz7 which carries type I allele. ROP16 amino acid alignment at 503 locus demonstrated that 17 isolates are featured as type I or type III  contains supplementary material, which is available to authorized users. Toxoplasma gondii is an obligate intracellular protozoon that can infect a broad spectrum of vertebrate hosts including humans. The infection with T. gondii in domestic animals causes abortion and leads to great economic losses in livestock production. In humans, Toxoplasma infection usually does not lead to obvious clinical symptoms and signs [Toxoplasma infection, however, may become activated in immunocompromised individuals, causing severe or life-threatening disseminated toxoplasmosis such as encephalitis or lethargy [Toxoplasma infection [Toxoplasma infection [Toxoplasma infection [nd signs . Latent lethargy . Recent nfection  and morenfection . Long-tenfection . Additionfection .Toxoplasma contains only one species, Toxoplasma gondii. However, genotyping results of the strains collected from human and animals around the world show a rich genetic diversity [T. gondii genotypes varies greatly with geographical locations [T. gondii database (http://toxodb.org/toxo). In North America and Europe, T. gondii has three archetypal clonal lineages known as types I, II and III which exhibit remarkable phenotypic differences [ToxoDB#9) dominates in the ten types identified [In the biological taxonomy, the genus iversity  which poocations . So far ferences . Comparaentified –19, whicToxoplasma gondii has evolved a number of strategies to subvert its host’s immune responses. Previous studies indicated that ROP16I/III carried in types I and III strains induces alternatively activated macrophage (M2) in host innate immunity to Toxoplasma infection, modulating host signaling pathways and producing virulence in mice [I, while all strains (100%) isolated from meat were avirulent for mice. Interestingly, we found that all the Chinese 1 isolates, including virulent Wh3 and avirulent Wh6 strains, shared the genes of GRA15II with type II strains and ROP16I/III with types I or III strains [rop16I/III deficient Wh3 strain (data unpublished) did not result in a remarkable attenuation of virulence in mice, suggesting that ROP16I/III in the parasite with GRA15II background is not closely associated in the virulence of Chinese 1 strains. in mice . Alvarez in mice  analyzed strains . We obseT. gondii identified the rhoptry kinase ROP18 and rhoptry pseudokinase ROP5 as virulence factors of the three archetypal clonal lineages. They function together to block innate immune mechanisms activated by IFN-γ in murine hosts [Previous genetic mapping of crosses between clonal type I, II, and III strains of ne hosts –26. Howene hosts  and playne hosts . Our prene hosts , suggestT. gondii in mice.Consequently, we analyzed the polymorphism of rhoptry proteins of ROP16, ROP18, ROP5 and dense granule proteins of GRA15 and GRA3 of type Chinese 1 strains to (i) explore the characteristics of the effectors that are associated with host immunity subversion and mouse virulence among the strains and (ii) to reveal the polymorphism-related pathogenesis of Female Swiss Webster (SW) mice (specific pathogen free) aged 6 to 8 weeks were obtained from the Biomedical Research Institute of Nanjing University, China. The mice were treated in compliance with the Care and Use of Laboratory Animals of the National Institutes of Health. Ethical permission was obtained from the Institutional Review Board of the Institute of Biomedicine at Anhui Medical University (Permit No. AMU26–081108).T. gondii strains were extracted from the ascitic fluid of mice infected with each isolate using a QIAamp® DNA Mini kit  following the manufacturer’s protocols. Genomic DNA stocks were stored at -80 °C until analyzed.Genomic DNA of the GRA15, ROP16 and ROP18 PCR-RFLP protocols were followed in accordance with a previous study . ROP5 alROP16 nucleotides 1424–1614  and the T. gondii strains was tested based on the previous mouse bioassay data. The virulence difference between Wh3 and Wh6 strains has been noted in long-term serial mouse passages. Here mice were infected with 1000 tachyzoites by intraperitoneal injection and observed for 30 days post-infection. Toxoplasma gondii strains that caused 0–29%, 30–79% and 80–100% mortality of mice were considered non-virulent, intermediate and virulent, respectively [Virulence of We assessed the mouse mortality of type Chinese 1 and ToxoDB #205 strains in combination with the data of previous studies. Mice were intraperitoneally infected with 1000 tachyzoites and mouse survival was observed for 30 days post-infection. The results showed that all of these strains presented a virulent phenotype in mice except for the Wh6 isolate which has the only avirulent phenotype [rop18 allele 2 except for Xz7 strain which carries rop18 allele 1 .rop5 allele 7 was found to be a unique genotype which could not be digested by FspBI.ROP5 is the major determinant of acute virulence in mice and was found to have allele 5 in all Chinese 1 strains but allele 7 in ToxoDB#205 and allele 5 in Xz8 strains  of ROP16 and found that all strains of Chinese 1 and strains of Xz7 and Xz8 of ToxoDB #205 are characterized by carrying leucine at position 503 while Xz9, Xz37, Xz39 and Xz40 strains of ToxoDB#205 presented serine. Additionally, amino acid of ROP16 at position 486 in Chinese 1 and ToxoDB#205 strains are homologous to that of archetypal type II strains except for Xz7 and Xz8 strains that are identical to archetypal type I strain, as shown in Table gra3 full length of nucleotides was amplified in the strains investigated. The coding sequences were aligned, followed by construction of the phylogenetic tree. We found that the sequences could be categorized into 5 types, the archetypal types I, II and III and μ-1 and μ-2 . Amino acid alignments revealed 96% or 99% positivities of ROP18 of the Chinese 1 Wh3 strain in comparison with that of GT1 or ME49, respectively, suggesting that sequence variations of ROP18 indeed exists but was not able to be identified by the PCR-RFLP analysis in the present study.T. gondii strains. The results revealed that the ROP18 and ROP5 gene allele types could be used to predict strain virulence in mice. ROP18 has 4 alleles; alleles 1 and 4 show association with a virulent phenotype, while allele 2 and 3 strains show a low virulence in mice [2 and ROP55 show a virulent phenotype in mice except the Wh6 strain. This result is inconsistent with the previous report using RFLP [2 with ROP182 and ROP17 [Shwab et al.  analyzed in mice . However in mice . Strainsing RFLP , suggestnd ROP17 , and/or II in type II strains mediates NF-kB nuclear translocation, drives macrophages towards a classically activated phenotype (M1) and promotes host innate immunity against Toxoplasma infection [I/III of types I and III has the ability to phosphorylate both STAT3 and STAT6 which results in the alternative activation of macrophages (M2), facilitating replication of parasites within the host cells [GRA15nfection . Meanwhist cells , 34, 35.II at position 503 in type II strains  is serine (ROP16 S503) which has no phosphorylation activity of STAT3/6 kinase. The types I and III strains , however, are featured with ROP16 L503 (ROP16I/III) instead of S503 (ROP16II). ROP16I/III is able to phosphorylate STAT3/6, drive macrophage to M2 polarization and help the parasite with its multiplication in macrophages [Toxoplasma infection. Interestingly, in the present study, we found that the amino acid residue of ROP16 at position 503 is leucine  in all of the isolates of type Chinese 1 and some isolates  of ToxoDB#205 identified in China mainland. Additionally, we noted that all of the strains of type Chinese 1 and some of ToxoDB#205, regardless of their mouse virulence, carry GRA15II, which is identical to GRA15II in type II strains [T. gondii that have both key effectors of ROP16I/III and GRA15II implicates the unique mechanism of the host immune response and pathogenesis in Chinese 1 T. gondii infection.Yamamoto et al.  have shorophages . This ma strains . The chaDense granule protein 3 (GRA3) is known to be secreted by the parasites after invasion and appears at the parasitophorous vacuole membrane (PVM) . It playToxoplasma [Furthermore, a more comprehensive analysis of type Chinese 1 strains is needed at the genomic DNA and transcriptome level. The development and application of CRISPR-Cas9 technology will enable us to precisely manipulate target genes to extend the power of reverse genetics in xoplasma .T. gondii from China. We demonstrated that the majority of those isolates are featured with the phenotypic ROP16I/III of type I and type III strains and GRA15II of type II strains. This strongly suggests that the characterized polymorphism of the crucial effectors of ROP16 and GRA15 in Chinese 1 strains may result in a significantly different outcome of Toxoplasma infection through the subversion of the host’s innate immunity. Furthermore, the Wh6 strain contains type II GRA3 which is unique in Chinese 1 strains and may lead to phenotype differences. Additionally, the present results indicate that genotyping of the loci including ROP5 and ROP18 with a PCR-RFLP strategy that is globally used may be inadequate to predict the virulence of Chinese 1 isolates of T. gondii.The present study examined sequence variations of ROP5, ROP16, ROP18, GRA3 and GRA15 genes in 21 Chinese 1 and ToxoDB#205 isolates of Additional file 1: Figure S1.. Analyzing the full length of GRA15, the result shows that all Chinese 1 and ToxoDB #205 strains (but not Xz7 strain) were identical to the type II strain, which has an 84 aa deletion from position 519 to 602. (TIFF 3957 kb)GRA15 translations alignmentAdditional file 2:GRA3 translations alignment. Aligning the full length of GRA3 translations of those strains, shows that Xz7 is the only one which has a longer length compared with the others. GRA3 of Wh6 and Xz8 strains are homologous to that of type II. The amino acid sequence from 131 to 180 is the mutation cluster region constituting a major part of all mutations. (TIFF 4457 kb)


Ubiquitin-like with PHD and RING Finger domains 1 (UHRF1) is a potent oncogene overexpressed in various solid and haematological tumors and its high expression levels are associated with decreased expression of several TSGs including p16INK4A, BRCA1, PPARG and KiSS1. Using its several functional domains, UHRF1 creates a strong coordinated dialogue between DNA methylation and histone post-translation modification changes causing the epigenetic silencing of TSGs which allows cancer cells to escape apoptosis. To ensure the silencing of TSGs during cell division, UHRF1 recruits several enzymes including histone deacetylase 1 (HDAC1), DNA methyltransferase 1 (DNMT1) and histone lysine methyltransferases G9a and Suv39H1 to the right place at the right moment. Several in vitro and in vivo works have reported the direct implication of the epigenetic player UHRF1 in tumorigenesis through the repression of TSGs expression and suggested UHRF1 as a promising target for cancer treatment. This review describes the molecular mechanisms underlying UHRF1 regulation in cancer and discusses its importance as a therapeutic target to induce the reactivation of TSGs and subsequent apoptosis.Epigenetic silencing of tumor suppressor genes (TSGs) through DNA methylation and histone changes is a main hallmark of cancer.  N. N105]. promoter . We havepromoter . In accopromoter . EGCG  that targets the SRA domain with subsequent impact on DNMT1/UHRF1 interactions and decrease in global DNA methylation .


Kcnj10 mutations identified in both mouse and human cohorts. However, genetic analyses of epilepsy phenotypes in mice to date have been carried out as acute studies in seizure-naive animals or in Mendelian models of epilepsy, while humans with epilepsy have a history of recurrent seizures that also modify brain physiology. We have applied a repeated seizure model to a genetic reference population, following seizure susceptibility over a 36-d period. Initial differences in generalized seizure threshold among the Hybrid Mouse Diversity Panel (HMDP) were associated with a well-characterized seizure susceptibility locus found in mice: Seizure susceptibility 1. Remarkably, Szs1 influence diminished as subsequent induced seizures had diminishing latencies in certain HMDP strains. Administration of eight seizures, followed by an incubation period and an induced retest seizure, revealed novel associations within the calmodulin-binding transcription activator 1, Camta1. Using systems genetics, we have identified four candidate genes that are differentially expressed between seizure-sensitive and -resistant strains close to our novel Epileptogenesis susceptibility factor 1 (Esf1) locus that may act individually or as a coordinated response to the neuronal stress of seizures.Epilepsy has many causes and comorbidities affecting as many as 4% of people in their lifetime. Both idiopathic and symptomatic epilepsies are highly heritable, but genetic factors are difficult to characterize among humans due to complex disease etiologies. Rodent genetic studies have been critical to the discovery of seizure susceptibility loci, including  Epilepsies in humans are highly heritable syndromes that are modified by complex interactions between genes and the environment. In the US, ∼2.2 million people will develop some form of epilepsy during their lifetime .Identification of gene by environment interactions for individuals with epilepsy is limited by complex disease etiologies and the inability to identify at risk populations prior to disease onset. Consequently, the majority of epilepsy genome-wide association studies (GWAS) carried out to date compare affected populations to seizure-naive controls rather than following epilepsy progression in a population from a seizure-naive to an epileptic state . ExperimPreclinical epilepsy models have implicated multiple gene mutations as contributors to seizure disorders. This success is due in part to strong genetic influences that segregate among rodent models of seizure susceptibility . UnfortuSzs1 locus  that may mitigate the effects of Szs1. This hypothesis is supported by interactions between Szs1 and our novel epileptogenesis susceptibility locus on chromosome 4; Epileptogenesis susceptibility factor 1 (Esf1). Systems genetics analysis of the Esf1 region has uncovered a coregulated set of expression QTL (eQTL) that have been implicated in neurological stress responses that are now also implicated in epileptogenesis.We have utilized a repeated-flurothyl-induced seizure model delivered to the Hybrid Mouse Diversity Panel (HMDP) to identify genetic susceptibility factors that modify epileptogenesis (n = 8); BTBR +Ltpr3tfT/J ; BXD#/TyJ ; BXD##/RwwJ ; C57BL/6J ; C57BL/10SNJ ; C57BL/10J ; C57BL/6NJ ; C57BLKS/J ; CAST/EiJ ; CBA/J ; CE/J ; DBA/2J ; FVB/NJ ; LG/J ; MRL/MpJ ; NOD/ShiLtJ ; NZW/LacJ ; PWD/PhJ , and Sm/J . Male and female mice were obtained from the Jackson Laboratories  and (1) were acclimated to the animal facility for 1 wk before seizure testing commenced, or (2) were further bred at the Wadsworth Center and exposed to the repeated-flurothyl seizure model. Mice were housed on a 12 hr light–dark cycle with ad libitum access to food and water.The rationale for using the HMDP and its advantages with respect to statistical power and genetic resolution have been previously described (n = 150) from this cohort were genotyped with the Mouse Universal Genotyping Array  and mapping  was performed using R/qtl  ether diluted in 95% ethanol] infused via a syringe pump onto a gauze pad suspended at the top of the chamber at a rate of 100 μl/min . Once a P < 4.4 × 10−6  were isolated from 11- to 12-wk-old B6 and D2 seizure-naive or seizure-exposed mice 90 min after completion of the repeated-flurothyl model as in Brains .Actb ; Per3 ; Park7 ; Camta1 ; and Vamp3 . Primer sequences are available upon request or from the manufacturer (www.idtdna.com/site/order/qpcr/predesignedassay). Real-time 5× HOT FirePol qPCR master mix purchased from Mango Biotechnology was used to perform PCR reactions according to the manufacturer’s recommendations (Solis BioDyne) using a 7500 Fast Real-Time PCR system (Applied Biosystems). All qPCR reactions were quantified using the relative standard curve approach based on the manufacturer’s recommendations. Candidate genes Camta1, Park7, Per3, and Vamp3 were normalized to β actin prior to determining B6:D2 expression ratios that exceeded a P value <0.05 assuming unequal variance between the populations.Relative RNA expression levels were quantified using the following Primetime premade 5′ nuclease assays from Integrated DNA Technologies: t-tests using JMP version 10.0 (SAS Institute). Groups were defined for interaction studies between Szs1 and Esf1 based on their genotype at single nucleotide polymorphisms (SNPs) rs8259388 for Szs1 and rs13478053 for Esf1. Correlational and eQTL mapping tools using BXD strains are freely available using GeneNetwork (http://www.genenetwork.org).Statistical analyses evaluating genetic interactions between GSTs by trial were carried out by either one-way ANOVA or Student’s Esf1 were selected using the tools available on GeneNetwork. We initially used expression data from the UTHSC BXD Aged Hippocampus data generated from Affymetrix mouse gene 1.0 ST exon level arrays  for correlational analyses with retest GST values. Additional datasets used to confirm these analyses included GN72 provided by Genome Explorations Inc. for the NIAAA as part of an SBIR grant to Dr. David Patel; GN46 that was generated by the UTHSC-SJCRH cerebellum transcriptome profiling consortium; and published datasets GN206 and GN281  or the UTHSC Genome Browser (http://ucscbrowserbeta.genenetwork.org).Cis-eQTLs adjacent to http://www.genenetwork.org/).All seizure threshold data are available from the GeneNetwork website under accession numbers 18963–18981 , based on their average daily GST scores of 275 sec for the most sensitive groups (D2 and FVB mice) compared to 350 sec for the intermediate groups . We calculated the slope of induction phase GSTs  as a quantitative surrogate for the kindling effect seen previously (P < 0.0001) between strains . As shown in Calmodulin Binding Transcription Activator 1 (Camta1) gene  gene .Bis1, congenic analyses performed by others in conjunction with our mapping results exclude the retest association from overlap with this locus (Epileptogenesis susceptibility factor 1 (Esf1); a novel seizure susceptibility locus that is dependent on prior seizure exposure to manifest its effects.Chromosome 4 GWAS associations were detected at two distinct time points and locations during the repeated-flurothyl seizure model. We first detected a locus with maximal effect (−log p 2.29 e−07) at rs32914632 near 138 Mb on chromosome 4 in the 6-d GST data , while rSzs1 and Esf1 effects in our model may indicate an interaction between these loci driven by repeated seizures. We tested for interdependency between the two genetic states by segregating BXD strains based on their genotype at Szs1  to compare respective GSTs at either end of the seizure protocol (GST1 and retest GST). As expected, the average response between B6 and D2 Szs1 groupings was significantly different for GST1 (P = 0.001), but not for retest GST , but not GST1 response. Notably, when the B6 and D2 Szs1 subsets were further divided based on their genotypes at Esf1, we only observed significant differences between the D2-Szs1/B6-Esf1 compared to the D2-Szs1/D2-Esf1 subset   were detected within four genes: Camta1, Period 3 (Per 3), Parkinson protein 7 (Park 7), and Vesicle Associated Membrane Protein 3 (Vamp3)  . These cfor Esf1 .cis-eQTL and Esf1, we correlated the expression values for each probe set with retest GST values. Most probe sets with LRS over 25 were significantly correlated (P < 0.05) with retest GST  were observed in two Camta1 probes present in exons 7 and 10 of Camta1 (To better define the relationship between these test GST . Exons felations . The mosf Camta1 .cis-eQTLs were found in other expression datasets using distinct arrays and brain tissues (cis-eQTL (selected based on a LRS score of 15 or greater within 2 Mb of a given locus) trended toward tissue-specific patterns. For example, while Camta1 and Park7 had cis-eQTL in hypothalamus  were observed between B6 and D2 hippocampus in the seizure-naive set for Camta1, Park7, and Per3, but not in cerebellar mRNA. In mice undergoing repeated-flurothyl seizures, there was an induction of Park7 and Vamp3 in B6 cerebellum that did not occur in D2 cerebellum. However, only Camta1 expression in hippocampus was significantly and differentially expressed between B6 and D2 mice after seizures.We confirmed the presence of differential expression between B6 and D2 inbred strains using qPCR. We compared control cerebellum and hippocampus from age-matched B6 and D2 animals as well as similar sets that had undergone the full flurothyl induction protocol, collecting tissue 90 min after the last seizure. As shown in This work demonstrates a novel preclinical approach to discover gene by environment interactions that mediate epileptogenesis. While rodent genetic studies have uncovered important seizure mechanisms among mammals  could influence Szs1 effects, either by suppression of an existing Szs1 mutation, like the reported Kcnj10 mutation, or via alterations in other genes in this region. Multiple QTL affecting a variety of neurological traits in mice are present on distal chromosome 1 (Szs1 effects on seizure susceptibility.While association of the Kcnj10) have been observed in both human temporal lobe epilepsy and experimental epilepsy models (r2 = −0.71) could be due to suppression of Kcnj10 activity in subsequent seizure trials. Reductions in Kcnj10 activity may be a common feature of repeated seizures that are mediated by uncharacterized mechanisms of epileptogenesis. In our model, suppressing both normal and mutant alleles of Kcnj10 as a means of seizure adaptation would consequently remove the Szs1 effects from associations after the initial seizure. The mechanisms responsible for these effects may be within the Szs1 interval or could be trans-acting factors that modify the effects of Szs1. While environmental confounds like animal age could contribute to seizure progression, all animals in our study were 6–7 wk old at the onset of the analysis. The presence of significantly different rates of kindling among HMDP strains suggests genetic factors mediate this activity within the HMDP. However, poor correlation between HMDP strain haplotypes within Szs1 and kindling in our data indicates that the uncharacterized kindling modifier and Szs1 are likely unlinked.Reductions in astroglial Kir4.1 channels , whose inhibition is reported to result in epileptiform activity (Significant SNP associations reappear on day 6 in the induction phases that correspond with the timing of the GST plateau effect seen in B6 mice (Esf1 is the predominant effect controlling seizure susceptibility with the top five associations all occurring in Camta1.Seizure-driven epileptogenesis is predicted to continue into the incubation phase of the repeated-flurothyl model due to a period of spontaneous seizures where B6 mice have as many as six seizures a day remitting to less than one seizure a day by the end of our model . When reKcnj10 segregating in the HMDP are associated with initial seizure effects that diminish over time, which may also be true in humans with epilepsy, raising the question of the nature of the association of Kcnj10 with epilepsy. A possibility is that the effect of Kcnj10 deficiency on seizure susceptibility is twofold. Its primary effect on hyper-excitability is mitigated by repeated seizures, but its effects on baseline K+ buffering in the interictal period remains abnormal. Such imbalances in K+ would be amplified in the context of repeated seizures leading to imbalances in downstream signaling cascades, in particular, Ca2+ signaling that has been shown to be altered by the presence of functional Kir4.1 channels (Complex networks of neuronal adaptation are activated by seizures (Szs1, which is likely due in part to deficits in Kcnj10, has a significant impact on retest GST when these mice are further segregated based on their Esf1 genotype, while those carrying a B6 Szs1 haplotype have reduced effect on Esf1 outcome. Thus, differences in Szs1 could prime the ultimate outcomes that are observed in Esf1, possibly through an imbalance in steady-state K+ levels that exacerbate alternative mechanisms of epileptogenesis either within astrocytes or through effects on neuronal networks.We show that strains carrying D2 alleles of cis-eQTL around Esf1 was previously associated with other neuronal complex traits, including stress response (Per3. We also found differential baseline expression in Per3 in B6 and D2 mice (Camta1 expression differences.Utilization of BXD strains was important to our project in two ways. First, it added increased power in our association analyses that would not have been possible with inbred strains alone . The pre D2 mice , dependicis-eQTL adjacent to Esf1 was also observed in the hippocampus of B6 and D2 parental strains by qPCR. While three of the four candidate loci showed significant differences in mRNA prior to seizures, only Camta1 maintained significant differences after completion of the repeated-flurothyl model. The differential expression of these loci was consistently higher in B6 mice indicating that a global regulatory control may impact this effect. Thus, our observation that the two SNPs rs369709 and rs3697097 that account for all of the cis-eQTL with LRS over 20 map to a region proximal to Esf1 could provide a good place to compare these genomes for transcriptional elements that may facilitate this kind of coordinated differential transcription.Confirmation of the differential expression of Esf1 have plausible ways they could contribute to epileptogenesis. As mentioned above, Per3 has previously been implicated in neuronal stress response and other gene family members have been implicated in induced seizures in mice (Park 7 protects against oxidative stress and may protect neurons from death (Vamp3 is a downstream target of MeCP2 that mediates SNARE-dependent exocytosis of glutamate (Each of these potential candidates for Camta1 is an excellent candidate for epileptogenesis susceptibility. In addition to having the five most significant SNPs associated with retest GST, and the best correlation with eQTLs in our system genetics data, it has several known functions that make it a good candidate from a biological perspective. First, calmodulin is a well-known mediator of postictal brain activity and has been implicated in brain adaptation to chemical (+ buffering capacity that is an aspect of our model. Finally, mutations in CAMTA1 are associated with multiple neurological phenotypes, including seizure susceptibility in humans (chemical  and elec


TET1 as one of the differentially methylated regions with gene-body hypermethylation. Herein, we characterize mechanisms that control TET1 gene activity at the transcriptional level. We show that treatment of CLL cell lines with 5-aza 2´-deoxycytidine (DAC) results in the activation of miR26A1, which causes decrease in both mRNA and protein levels of EZH2, which in turn results in the decreased occupancy of EZH2 over the TET1 promoter and consequently the loss of TET1 expression. In addition, DAC treatment also leads to the activation of antisense transcription overlapping the TET1 gene from a cryptic promoter, located in the hypermethylated intronic region. Increased expression of intronic transcripts correlates with decreased TET1 promoter activity through the loss of RNA Pol II occupancy. Thus, our data demonstrate that TET1 gene activation in CLL depends on miR26A1 regulated EZH2 binding at the TET1 promoter and silencing of novel cryptic promoter by gene-body hypermethylation.The Ten-eleven-translocation 1 (TET1) protein is a member of dioxygenase protein family that catalyzes the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine. TET1 is differentially expressed in many cancers, including leukemia. However, very little is known about mechanism behind TET1 deregulation. Previously, by characterizing global methylation patterns in CLL patients using MBD-seq, we found  TET2, while less is known about TET1 and 3 [TET1 was identified as a fusion partner of mixed lineage leukemia (MLL) gene in AML patients [TET1 coding sequences in hematologic cancers [Aberrant DNA methylation profiles are well documented epigenetic alterations that are implicated in several disorders including hematopoietic malignancies. The DNA methylation pattern across the genome is a culmination of a balance between DNA methylation and demethylation, which is brought by dynamically regulated functional interplay between DNA methyl transferases (DNMTs) and Ten-Eleven Translocation protein family (TETs) . Active T1 and 3 , 6. TET1patients . TET1 hapatients –11. TET1patients . Since c cancers , its exa cancers .Chronic lymphocytic leukemia (CLL) is a common incident in the west and characterized by diverse clinical behavior and heterogeneous disease course . Recent Recently, our comprehensive methylation analysis by Methyl-CpG-binding domain protein enriched genome-wide sequencing (MBD-Seq), identified several differentially methylated regions, including promoter elements and gene-body regions . It is vTET1 gene-body (Intronic region) but not its promoter region showed hypermethylation in CLL patient samples compared to normal healthy sorted B cell samples. Both IGHV mutated and unmutated CLL prognostic groups showed significantly hypermethylated specific peak in the TET1 intronic region compared to normal sorted B cell sample   compared to normal B cell samples   Figure . Consist) Figure . No sign) Figure .TET1 gene showed significant differential expression in CLL samples (n= 96) compared to normal sorted B cells (n =9)  Figure . We furts Figure . As showTET1 gene expression, we wanted to explore whether it has any functional link to TET1 expression. For this purpose, TET1 expression levels were analyzed in four leukemic cell lines: two CLL cell lines (HG3 and MEC1) and two MCL cell lines (GRANTA 519 and Z138) and all four cell lines express TET1 gene   , indicats Figure  and 3C. 2 Figure . We nexte Figure . Taken tFrom the published evidence it is known that promoter methylation correlates with gene repression  while thTET1 promoter. As shown in Figure Based on the promoter activity data of the HMR, we proceeded further to check for the presence of any intronic transcripts in the HMR region. To detect intronic transcripts encoded by the HMR region, the total cDNA was synthesized using DNase 1 treated total RNA and RT-qPCR analysis was performed using primers spanning, downstream and upstream of the HMR region. To check if amplification is from genomic DNA contamination, mock cDNA samples without RT enzyme were used as controls for all the respective cDNA samples. As shown in Figure TET1, which encode antisense transcripts spanning across the HMR of its sense counterpart TET1. We next wanted to investigate whether induction of cryptic promoter activity has any functional connection to the TET1 promoter activity upon DAC treatment. To this end, ChIP assay was performed on TET1 promoter TSS site, HMR region and downstream to HMR region using antibodies against RNA Pol II on DAC treated and untreated MEC1 cell line samples. Interestingly, following DAC treatment, the occupancy of RNA Pol II at the TET1 promoter decreases while at the HMR downstream region the RNA Pol II occupancy increases  was amplified containing the predicted cryptic promoter region and assayed for the promoter activity using luciferase assays. As shown in Figure Genes are regulated at the levels of transcriptional initiation, transcription elongation, pre-mRNA 3’ processing and mRNA degradation. These gene regulatory steps further controlled by complex and coordinated multi-level cross talks between transcription factors, chromatin remodelers and long and small noncoding RNAs , 30. In TET1 gene-body [TET1 gene-body hypermethylation correlates with TET1 gene activation, we wanted to understand the functional role of gene-body hypermethylation in TET1 gene expression. DAC treatment to induce demethylation across the genome has been widely used to explore the functional role of DNA methylation in gene expression. Thus by treating four different CLL cell lines with DAC, we have shown that demethylation of hypermethylated TET1 gene-body leads to the activation of methylation dependent cryptic promoter. Both strand-specific RT-PCR and promoter luciferase assays clearly demonstrated that cryptic promoter encodes antisense transcripts, which span across the TET1 promoter. Activation of cryptic transcription across the TET1 promoter correlates with the loss of RNA Pol II occupancy and TET1 gene silencing, indicating that antisense cryptic transcription may be regulating TET1 gene expression in part by occluding the transcription initiation machinery from the TET1 promoter. Thus our data is consistent with the functional role of gene-body methylation in repressing aberrant cryptic promoter activity. Interestingly, we observed presence of many SINE and LINE Alu repeats located in the HMR region where the intronic transcripts are expressed. It would be interesting to know the role of these transcribed repetitive elements in TET1 expression.Gene-body hypermethylation has lately been recognized as a transcriptional regulatory step in regulating alternative splicing  and crypene-body . Since TmiR26A1, EZH2, in the TET1 gene expression. Previously, miR26A1 has been implicated in the regulation of TET1 [TET1 promoter in the UCSC genome browser utilizing the ENCODE/Broad Institute ChIP-seq datasets, revealed presence of EZH2 peaks at the TET1 promoter in several cell types. Thus we wanted to investigate the functional interplay between EZH2, TET1 and miR26A1. Previously, we documented that miR26A1 is hypermethylated in CLL samples compared to normal B cells [miR26A1 can regulate TET1 at the transcriptional level via modulating the levels of EZH2 at the TET1 promoter. Interestingly, in this context EZH2 acts as an activator in the TET1 gene regulation, which is in contrast to its widely accepted function as transcriptional repressor via catalyzing H3K27me3 modification. Previously, in a few instances EZH2 has been implicated in the gene activation function which is independent of histone methyltransferase activity [miR26A1 directly regulates TET1 at the post-transcriptional level or via regulating EZH2 occupancy at the TET1 promoter there by interfering with TET1 activity indirectly at the transcriptional level. According to our data  treatment as shown in the Figure miR26A1, decrease in EZH2 levels and induced expression of intronic transcripts at HMR region resulting in reduced RNA Pol II occupancy at the TET1 promoter, leading to reduced TET1 expression levels. Thus our data demonstrate a novel regulatory loop between miR26A1-EZH2 and TET1 and thus providing an explanation for consistent upregulation of TET1 and EZH2 in CLL patients while miR26A1 is hypermethylated. Hence, our study opens up a new avenue in the further exploration of regulatory connection of EZH2 and TET1.Finally, a schematic representation of overall data of this study is shown in Figure Total 40 CLL patient samples were included in the present study. All the peripheral blood mononuclear cells (PBMCs) CLL samples were collected from different hematology departments in the western part of Sweden after written consent had been obtained. 5 DNA samples of CD+19 sorted normal B cells from healthy age matched controls were purchased from 3H biomedical . All samples were diagnosed according to recently revised iWCLL criteria , showingDNA was extracted from CLL PBMC samples using DNA extraction kit  according to the manufacturer's protocol. RNA extractions were done using miRNeasy Mini Kit  along with on column DNase1treatment  to remove any residual genomic DNA. The total cDNA synthesis was performed using Superscript III FS synthesis supermix kit  according to the manufacturer's protocol.TET1 promoter TSS  . All the primer sequences used for Pyrosequencing assay are listed in The hypermethylated region (HMR) peak from obtained from MBD seq data  is 687 bThe expression levels of TET1 and EZH2 genes were analyzed with Taqman gene expression assays  . For the expression of intronic transcripts, 7 sets of custom primers were designed using Primer 3 software, spanning the intronic region between Promoter TSS and HMR on TET1 gene and the RT-qPCR analysis was done using Power SYBR Green PCR master mix . Differences in expression were calculated using the ΔΔCt method. All the primer sequences and the product size of the amplified region were listed in the Strand specific PCR is performed using specifically designed 5’GSP (Gene specific Primer) and 3’GSP for synthesizing gene specific cDNA from DNase1 treated RNA samples. The 5’GSP specifically binds to antisense strand and the 3’GSP binds to the sense strand. After cDNA synthesis, to quantify the strand specific cDNA synthesis we used two sets of primers which are located downstream to the 5’GSP and 3’GSP. The PCR product size and the primer sequences used were listed in In order to identify the promoter activity of HMR and downstream cryptic promoter regions, we constructed several PGL3 dual-luciferase reporter vectors  containing the target sequence for checking the promoter activity. Luciferase activity was determined 48 h after transfection as described earlier , by use ChIP was performed with the shearing module kit  and one-day ChIP kit , according to the manufacturer's instructions. Western blot analysis was performed using total cell lysates lysed from transfected CLL cell line samples. The details of ChIP assay, western blot analysis along with the Antibodies used are described in Supplementary Materials and Primer sequences are provided in


Saccharomyces cerevisiae and was later found to be involved in many aspects of cell functions and many biological and pathological processes. The role of MAP4K4 in immunity, inflammation, metabolic and cardiovascular disease has been recognized. Information regarding MAP4K4 in cancers is extremely limited, but increasing evidence suggests that MAP4K4 also plays an important role in cancer and MAP4K4 may represent a novel actionable cancer therapeutic target. This review summarizes our current understanding of MAP4K4 regulation and MAP4K4 in cancer. MAP4K4-specific inhibitors have been recently developed. We hope that this review article would advocate more basic and preclinical research on MAP4K4 in cancer, which could ultimately provide biological and mechanistic justifications for preclinical and clinical test of MAP4K4 inhibitor in cancer patients.The serine/threonine kinase MAP4K4 is a member of the Ste20p (sterile 20 protein) family. MAP4K4 was initially discovered in 1995 as a key kinase in the mating pathway in  MAP4K4, also known as HGK  or NIK  is a serine/threonine (S/T) kinase that belongs to the mammalian family of Ste20 protein kinases due to their shared homology to the budding yeast kinase Ste20p . Ste20 fMAP4K4 contains ~1200 amino acids with a molecular mass of ~140 KDa , 8. The As summarized in Table Despite different localizations of their catalytic domains (N-terminus vs. C-terminus), mammalian Ste20 kinases share similar features in the kinase domain . In geneTaking the global phosphoproteomics approach and by comparing the corresponding SILAC (Stable Isotope Labeling by Amino acids in Cell) ratios of EGF  stimulation and erlotinib (EGFR inhibitor) treatment in lung adenocarcinoma cells, a prior study identified two serine sites S648 and S708 in the middle domain of MAP4K4 as EGFR  signaling-dependent phosphorylation sites . Serine The C-terminal domain of MAP4K4 contains a citron-homology domain (CNH) that appears to determine MAP4K4 association with other factors . For insTaken together, current evidence, as summarized above, strongly supports that the MAP4K4 kinase activity can be positively or negatively regulated by upstream kinases. The identities of the kinases remain largely unexplored. If multiple kinases are involved, it is highly likely that the selection within a repertoire of candidate kinases is context-dependent, depending on the cell type, the nature of the external stimuli, and the cell state. The biochemical and biological consequences of phosphorylation could also be context-dependent. In addition to negatively or positively regulating MAP4K4 kinase activity, phosphorylation may also determine MAP4K4 subcellular localization and substrate-selection.MAP4K4 gene contains a nearby p53 binding sites cluster downstream of the promoter and six potential p53 binding sites in the first intron, four of which are confirmed by chromatin immunoprecipitation (ChIP) experiments [A recent study points to a possibility that in T cells of type 2 diabetes patients, mRNA level of MAP4K4 might be affected by enhanced methylation of CpG islands in its promoter region , suggesteriments , 29. IndDespite the fact that evidence from genetic studies using mouse model is still lacking, emerging evidence from preclinical and patient association studies strongly suggests that MAP4K4 may play an important role in many types of cancer and could serve as a novel actionable target for cancer treatment. The first evidence suggesting a role of MAP4K4 in cancer came from observations that MAP4K4 is highly expressed in 40 of the NCI-60 human tumor cell lines and can modulate cellular transformation, adhesion and invasion . In thisLittle information exists regarding how MAP4K4 is involved in cancer. As shown in Fig. In addition to above mentioned candidate downstream mediators of MAP4K4, MAP4K4 also participated in the regulation of other cancer-related signaling pathways or factors including insulin pathway, hippo signaling (LATS1/2 and YAP/TAZ) and mTOR/AMPK , 47–52. How MAP4K4 regulates its downstream effectors or signaling mediators remains largely unexplored. Since MAP4K4 is a kinase, one would expect that the primary job of MAP4K4 is to phosphorylate its substrate. Indeed, MAP4K4 can directly phosphorylate TRAF2 at serine 35 to promote its degradation in T cells . HoweverAs summarized in Table Current evidence supports but not yet provides sufficient biological and mechanistic justification for MAP4K4 as a novel cancer therapeutic target. Evidence definitely linking MAP4K4 to the development and progression of any types of cancer is still lacking. To this end, it is essential to examine the impact of genetic manipulation of MAP4K4 in mouse models of cancer. When interpreting results from experiments using MAP4K4 knockout mice (whole-body or tissue-specific knockout), potential redundancy and functional compensation among MAP4Ks should be taken into consideration. Since small-molecule inhibitors of MAP4K4 are available, in order to eventually use these inhibitors in clinic, future studies should attempt to test their tumor prevention and antitumor activity in mouse models of cancer.Currently there is no sufficient information suggesting in which type of cancer MAP4K4 inhibitor can be used as a novel promising therapy. Target overexpression is an overrated predictor of efficacy since it may also represent a cellular attempt to limit unbridled growth, unless functional results from genetic manipulation of MAP4K4 in that particular tumor model are available, it is difficult to predict cancer types responsive to MAP4K4 inhibitor treatment. It is possible that MAP4K4 could promote tumor development and progression in certain types of cancer and functions as a tumor repressor in other types of cancer, or plays different roles at different stages during tumor development and progression. Therefore it is crucial to gain a thorough understanding of how MAP4K4 is involved in a particular type of cancer functionally and mechanistically before conducting clinical testing of MAP4K4 inhibitor in cancer patients.No information is available about whether and how MAP4K4 is involved in resistance to standard cancer therapy. If MAP4K4 contributes to cancer development and progression, it is highly likely that MAP4K4 can also be involved in treatment resistance. Therefore in addition to examine the potential anti-tumor activity of MAP4K4 inhibitors as a standalone therapy, it is also important to test if MAP4K4 inhibitors can be used in combination to overcome resistance to chemotherapy, radiation therapy, targeted therapy and immunotherapy.Detailed molecular understanding of how MAP4K4 is involved in cancer biology is essential for firmly establishing MAP4K4 as a target for that particular type of cancer. Crucial to this effort is to identify key upstream regulators and downstream effectors including substrates of MAP4K4. To develop efficient methods to block MAP4K4, it is also crucial to understand how MAP4K4 functionally interacts with Ste20 family members.Cancer remains a largely incurable disease, indicating an urgent and unmet need for novel effective therapeutic approaches. Identifying a novel cancer therapeutic target that could be amenable to pharmacologic intervention is challenging. To this end, we believe that a better understanding of biological functions and underlying mechanisms of MAP4K4 in cancer could have far-reaching implications for new directions in cancer therapy.


There are many single-gene causes of steroid-resistant nephrotic syndrome (SRNS) and the list continues to grow rapidly. Prompt comprehensive diagnostic testing is key to realising the clinical benefits of a genetic diagnosis. This report describes a bespoke-designed, targeted next-generation sequencing (NGS) diagnostic gene panel assay to detect variants in 37 genes including the ability to identify copy number variants (CNVs).This study reports results of 302 patients referred for SRNS diagnostic gene panel analysis. Phenotype and clinical impact data were collected using a standard proforma. Candidate variants detected by NGS were confirmed by Sanger sequencing/Multiplex Ligation-dependent Probe Amplification with subsequent family segregation analysis where possible.NPHS1 or NPHS2.Clinical presentation was nephrotic syndrome in 267 patients and suspected Alport syndrome (AS) in 35. NGS panel testing determined a likely genetic cause of disease in 44/220 (20.0%) paediatric and 10/47 (21.3%) adult nephrotic cases, and 17/35 (48.6%) of haematuria/AS patients. Of 71 patients with genetic disease, 32 had novel pathogenic variants without a previous disease association including two with deletions of one or more exons of Gene panel testing provides a genetic diagnosis in a significant number of patients presenting with SRNS or suspected AS. It should be undertaken at an early stage of the care pathway and include the ability to detect CNVs as an emerging mechanism for genes associated with this condition. Use of clinical genetic testing after diagnosis of SRNS has the potential to stratify patients and assist decision-making regarding management. Patients with nephrotic syndrome (NS) suffer a breakdown of the glomerular filtration barrier at the level of the podocyte leading to massive proteinuria, hypoalbuminaemia and oedema.COL4A3, COL4A4 or COL4A5 and may present with proteinuria (some with FSGS on biopsy), and more commonly haematuria.COL4A3 and COL4A4 have been identified in association with FSGS, together with thin basement membrane nephropathy but without the extrarenal features of AS.Pathogenic variants in single genes affecting podocyte function are a common cause of SRNS, reported in up to 29.5% of a predominantly childhood cohort.COL4A3, COL4A4, COL4A5).The proportion of single-gene cases identified inversely correlates with the age of onset with 69.4%–100% of congenital-onset disease reported as having a genetic aetiology.Timely genetic testing can considerably alter patient management and facilitate a greater understanding of the genetic complexity of the condition.We have developed a clinically approved gene panel test for 37 SRNS and collagen-related genes  using a In total, 302 patients were referred with informed consent for diagnostic gene panel analysis over a 26-month period. The diagnostic test has been formally assessed for validity, and socio-legal/ethical implications by the UK Genetic Testing Network and UK National Health Service (NHS) commissioners through the gene dossier process, and was undertaken in an accredited UK NHS Laboratory. Data presented pertain only to anonymised auditing of routine diagnostic testing; therefore, this study was not subject to ethical approval.The sequencing and bioinformatics protocol is described in brief with further details as 10.1136/jmedgenet-2017-104811.supp1Supplementary file 110.1136/jmedgenet-2017-104811.supp2Supplementary file 2cis or trans) and segregation. CNVs in patients with single heterozygous variants in recessive genes were identified by CONTRAWhere possible, relative testing was undertaken using Sanger sequencing to determine phase  being issued in 22 calendar days.An average gene coverage of 99.26% coding sequence at a minimum read depth of 30× was achieved on a typical 12-patient run. The per-gene coverage is shown in Targeted gene panel testing of all 302 patients identified 71 (23.5%) with a likely genetic cause for disease. The genetic diagnostic rate among the group with SRNS was 21.2%, including 44/209 (21.1%) paediatric and 10/46 (21.7%) adult nephrotic cases . In patiThe spectrum of pathogenic variants is summarised in NPHS1, WT1 and NPHS2 in 12 (4.7%), 11 (4.3%) and 7 (2.7%) patients, respectively. In the Alport group, the variants were all in collagen genes: COL4A3, COL4A4 and COL4A5 in 5 (14.3%), 2 (5.7%) and 10 (28.6%) patients, respectively. Of note, five SRNS/FSGS patients had LP variants in COL4A3, COL4A4 and COL4A5  including a single novel LP COL4A3 variant, c.698G>A, p.(Gly233Glu), in a patient with a dominant family history of FSGS (patient 4) which tracked with disease in an affected brother. Among patients with SRNS/FSGS who were found to have genetic disease (excluding those with collagen variants), there was an autosomal-dominant mode of inheritance in 16/41 (36.6%) of those with disease onset ≤18 years compared with 6/7 (85.7%) of those >18 years.The most frequently detected LP variants in the SRNS group (n=255) were in NPHS1 and NPHS2; and 5.8% each in INF2, MYH9, PLCE1 and PTPRO. Of the 71 patients with likely genetic disease, 11 cases had 12 additional VUS in genes other than the main causative one for that patient, most frequently collagen genes in 41.7% and WT1 in 16.7%. Overall, of the 64 recorded VUS, 31.3% were in collagen genes, 6.3% in NPHS2, 6.3% in NPHS1 and 6.3% in MYH9.In addition to the patients with LP variants, a further 40 patients had one or more VUS .NPHS2 exon 2 in combination with a paternally inherited c.1032delT variant. The c.1032delT variant has been previously reported as the most frequent pathogenic variant in NPHS2 in Poland (Kashubian region).ACTN4 (1 patient), COL4A3 (2), COL4A4 (1), COL4A5 (10), INF2 (2), LAMB2 (2), NPHS1 (5), NPHS2 (2), SMARCAL1 (1), TRPC6 (1) and WT1 (3).Among the 71 patients with a genetic cause for disease, 32 had variants without a previous disease association including 26 with one or more novel variants absent from population databases. Two patients had gene deletions of one or more exons detected by CNV analysis . PatientINF2 LP variants were detected in two adult-onset NS patients: p.(Tyr50Asp) (patient 25) and p.(Leu165Arg) (patient 26). Both were missense variants affecting highly conserved residues within the diaphanous inhibitory domain of the INF2 protein consistent with the previously reported spectrum of disease-causing variants. Segregation supports pathogenicity, p.(Tyr50Asp) co-segregating with disease in five affected family members and p.(Leu165Arg) present in one affected family member and absent in two unaffected family members. An additional sensory neuropathy phenotype previously reported in 12.5% of INF2 casesNovel WT1 variants were identified in the known hotspot region (exons 6–9)WT1 exon 8 (patient 65) was associated with age of onset of 30 years and was inherited from an affected father who was diagnosed in his 30s. Although WT1 is normally associated with childhood-onset disease, these findings are consistent with a previously described biphasic childhood and adulthood presentation of variants in WT1.Two novel missense patient 6 was assoNPHS1 or NPHS2 that has been previously published as disease causing. No CNVs were identified. The finding of these variants may be incidental. However, given the low incidence of SRNS, the young age of onset  and the low frequency/absence of the variants in databases of subjects without known renal disease it is possible that there are additional NPHS1/NPHS2 variants in unsequenced intronic or promoter regions which may act in combination to cause the phenotype in these patients. These findings were reported as compatible with the phenotype but insufficient to make a diagnosis.In six clinically affected SRNS cases (patients 112–117 in online sNPHS1. Patient 112 developed SRNS under the age of 4 years with a heterozygous p.(Asp105Asn) variant previously reported in a Japanese patient with CNS where a second variant was not detected.NPHS1 or other genes in the panel. Patient 117 had a single heterozygous missense variant in NPHS2: c.872G>A, p.(Arg291Gln) previously reported as pathogenic in the homozygous/compound heterozygous state. Patient 117 also had a novel single heterozygous variant in NPHS1: c.2512C>A, p.(Pro838Thr) which is not reported in population databases and prediction tools suggest is deleterious.Three of these patients  with early-onset SRNS were heterozygous for previously reported rare pathogenic variants in NPHS2 p.(Arg138Gln) (exon 3) and p.(Leu156Phefs*11) (exon 4) in a compound heterozygous state with the NPHS2 non-neutral polymorphism p.(Arg229Gln). Tory et alNPHS2 is contributing to these patients’ phenotypes.Two further patients 115 and 116) presenting with classical NS had single previously reported pathogenic variants in  and 116 NPHS1 (12 patients), LAMB2 (3 patients), NPHS2 (2 patients) and WT1 (1 patient). For cases of SRNS with known age of onset >18 years, the rate was 28.6% (6/21) with LP variants in INF2 (two patients), WT1 (two patients), SCARB2 (one patient) and TRPC6 (one patient).The age of disease onset was known with certainty for 164 (64.3%) of 255 patients referred with a clinical presentation of SRNS and online s10.1136/jmedgenet-2017-104811.supp4Supplementary file 4LAMB2 and NPHS2 pathogenic variants. At least nine family tests are known to have helped inform suitability for donor transplant treatment. Other familial testing has provided diagnoses for relatives and aided in clarifying variant pathogenicity. Other responses confirmed that genetic testing impacts on diagnosis and prognosis after transplant.Physicians were asked whether the results of genetic testing would alter immunosuppressive management in patients with SRNS. Responses were obtained in relation to 71 (27.8%) of these patients. The response rate was not high enough to make definitive conclusions, but broadly clinicians reported that results would influence decisions to reduce or stop immunosuppression and one physician indicated that treatment decisions would be made after the gene panel results were known. The diagnostic test result from 67 patients resulted in the subsequent testing of 148 family members including two prenatal tests for Gene panel testing is becoming more and more relevant for screening rare diseases, with greatly improved cost/benefit. The knowledge of the genetic basis of SRNS has expanded considerably over the past five years with several national and international cohorts recently publishedThe frequency of likely pathogenic variants among patients with SRNS was 21.2%. This is marginally lower than 24%–34% reported in other studies which included predominantly subjects with childhood-onset disease.EMP2, KANK1 and KANK2 have been described in family studies but evaluation in larger cohorts did not identify additional patients with pathogenic variants.KANK2 was included in the gene panel reported here and no likely pathogenic variants were identified. The updated version of the panel includes EMP2, KANK1 and KANK4. Our current recommendation for clinicians is not to use the NGS gene panel test for patients with persistently SSNS unless there are specific reasons to suspect a genetic aetiology.Twelve patients with SSNS were referred for genetic testing and none had a potentially pathogenic variant. Thus far, no purely monogenic causes for SSNS have been identified.In this study, 100% of LP variants identified by NGS meeting diagnostic variant-calling quality parameters have subsequently been confirmed as being present on Sanger sequencing. Sanger confirmation of NGS LP variants is currently in line with best practice ACGS guidance. It also provides a confirmation of sample identity following pooling of samples during the NGS process and establishes a familial test for relatives. As further evidence is collected, confirmatory testing may become redundant.WT1 variant without any manifesting extrarenal features. In addition, variants in secondary genes which may potentially contribute to the phenotype of the patient can be identified by a panel approach.The use of gene panel testing supersedes stepwise screening protocolsGene panel screening identified 32 likely pathogenic variants without a previous disease association. Absence or rarity in population data is used as evidence to support pathogenicity; however, it is acknowledged that some ethnicities in this global cohort were either not known or may currently be insufficiently represented in population databases. Where possible, segregation analysis was performed to provide additional evidence to support pathogenicity; however, family members were not always available for testing, reflecting the use of this panel in a clinical setting.The interpretation of variants of unknown significance in a global cohort is also a challenge of panel testing. As well as segregation analysis, future expansion of population databases will allow improved filtering of population-specific variants and functional work may also aid the interpretation of variants.NPHS1 or NPHS2 pathogenic variant detected, suggesting deep intronic or regulatory variants if these genes are truly recessive in mechanism. Future whole-genome sequencing in these patients may help to elucidate a genetic pathogenesis.This study has demonstrated two cases of CNVs present in NS genes; therefore, CNV analysis of NGS data should be routinely undertaken as part of the variant analysis pipeline, together with confirmation using a second method such as MLPA. It is also apparent that there are a number of clinically typical cases with only a single known COQ2, COQ6 or PDSS2, coding for proteins the coenzyme Q10 pathway, may indicate the potential for benefit from treatment with this enzyme.PLCE1 variants who responded to treatment with steroids and ciclosporin.PLCE1 variants as their affected relatives suggest a more complex genotype–phenotype interplay and raise the possibility of spontaneous improvement rather than a true response to medication.WT1 variants have responded to steroids and immunosuppression.WT1, should prompt search for other features of an associated syndrome, such as Frasier syndrome and risk of gonadoblastoma.The timing of testing in relation to disease onset and the speed of genetic reporting are important for clinical utility. It is potentially possible to generate results within 1–2 weeks, thereby avoiding diagnostic biopsy in some cases. In certain contexts, earlier testing and more rapid turnaround are important because results may have direct consequences for prenatal testing and patient treatment. Pathogenic variants in The presence of a causative variant in SRNS is associated with a lower risk of post-transplantation recurrence of disease, occurring in 25.8% of patients testing negative for genetic disease compared with 4.5% of those with an identified variant in a European cohortWe report that NGS gene panel testing with bioinformatics analysis for SNVs and CNVs at an early stage after diagnosis of SRNS or suspected AS with results in a clinically relevant time scale has the potential to improve patient stratification and the care pathway.


Wide local excision followed by radiotherapy was offered as an alternative to mastectomy to 93 women on 96 occasions. Complications attributable to the surgery occurred in 4 per cent whilst 37.5 per cent developed complications secondary to the radiotherapy. The complications were generally of a mild nature. Nine patients (9.3%) have developed regional recurrence and seventeen (17.7%) distant metastases with a mean follow up of 53.4 months.


We document the descriptive statistics and detailed regression outputs for educational attainment and measured 2D:4D ratios, based on the RLMS data . Regression analysis is conducted using STATA 13, gologit2 which is a special code for the generalized ordered logit regression in STATA environment. We provide graphs of differences in means of 2D:4D ratios by educational attainment. Information about the distribution of self-identified nationalities and fields of university degrees of respondents is presented. Specifications TableValue of the data.•Descriptive statistics of national self-identities, major fields, educational attainment and fingers’ 2D:4D measurements are helpful in the case of analyzing possible associations between education and 2D:4D.•Detailed generalized ordered logit regressions’ outputs are helpful in understanding of the general patterns of 2D:4D associations at different levels of education obtained. Inclusion of classical predictors (education of parents) is helpful in foreseeing their correlations with different levels of educational attainment in the studied samples.•Graphs of means are helpful in comparison of differences between averaged 2D:4D of individuals with university degree and averaged 2D:4D of individuals who completed lower levels of education.1Measurements were taken by a special team of 2 trained assistants, while other information was taken from the RLMS survey which contains questions regarding an individual׳s socioeconomic characteristics and family background 2See


RUNX1 is a member of the RUNX family of transcription factors that also includes RUNX2 and RUNX3 [CDH1 promoter to positively regulate E-Cadherin expression.The functional consequences of RUNX1 loss or gain of expression that contribute to breast cancer development and progression have not been fully elucidated. A role for RUNX1 in suppressing ER oncogenic signaling is suggested by the fact that combined loss of Runx1 with either TP53 or Rb1 leads to hyperproliferation of ER+ luminal cells in the mouse mammary gland . InactivIn apparent conflict with the role of RUNX1 as a suppressor of EMT in ER+ luminal tumors, downregulation of RUNX1 expression in TN tumors inhibits tumor invasion and metastasis . How do In summary, increasing evidence supports a role for RUNX1 in breast cancer progression. Understanding how RUNX1 functions in a cell context-dependent manner will be essential for determining the mechanism(s) by which this transcription factor impacts tumor biology.


Bifidobacterium lactis HN019 reduces transit time, but its mechanisms of action and effects on motility patterns are poorly understood. The aim of this study was to investigate changes in GI motility induced by an extract of HN019 on distinct patterns of colonic motility in isolated rat large intestine, compared with a known promotility modulator, prucalopride. The large intestines from male Sprague Dawley rats (3–6 months) were perfused with Kreb's buffer at 37°C in an oxygenated tissue bath. Isometric force transducers recorded changes in circular muscle activity at four independent locations assessing contractile propagation between the proximal colon and the rectum. HN019 extract was perfused through the tissue bath and differences in tension and frequency quantified relative to pre-treatment controls. Prucalopride (1 μM) increased the frequency of propagating contractions (by 75 ± 26%) in the majority of preparations studied (10/12), concurrently decreasing the frequency of non-propagating contractions (by 50 ± 11%). HN019 extract had no effect on contractile activity during exposure (n = 8). However, following wash out, contraction amplitude of propagating contractions increased (by 55 ± 18%) in the distal colon, while the frequency of non-propagating proximal contractions decreased by 57 ± 7%. The prokinetic action of prucalopride increased the frequency of synchronous contractions along the length of colon, likely explaining increased colonic rate of transit in vivo. HN019 extract modified motility patterns in a different manner by promoting propagating contractile amplitude and inhibiting non-propagations, also demonstrating prokinetic activity consistent with the reduction of constipation by B. lactis HN019 in humans.Attention is increasingly being focussed on probiotics as potential agents to restore or improve gastrointestinal (GI) transit. Determining mechanism of action would support robust health claims. The probiotic bacterium  Constipation is a common functional gastrointestinal (GI) disorder affecting 20% of the general population worldwide   and incubated at 37°C in an anaerobic workstation  containing 10% CO2, 10% H2 and 80% N2 for 48 h. A 50 mL secondary culture was inoculated with 0.5 mL of primary broth (adjusted to an OD600 of 1.5) and incubated anaerobically for 16 h. Two 150 mL secondary cultures were inoculated with 1.5 mL of secondary broth and incubated anaerobically for 16 h to the stationary phase. The bacterial cell culture was harvested and processed into extract under anaerobic conditions, then used in the motility assays freshly each day. Bacterial cells were collected by centrifugation  and resuspended in 5 mL of anaerobic Krebs solution . The bacterial cell pellet was washed twice in Krebs solution, resuspended in 5 mL and incubated on ice for 10 min. The mixture was then sonicated on ice using 20 s pulses with 30 s intervals, power level 2 at 40% duty for 10 min . The sonicated mixture was then centrifuged  to remove the cell debris and the resulting supernatant was ultra-centrifuged to remove any remaining cell membranes  and to prevent excessive frothing of the solution in the motility experiment phase. The final supernatant was a cell-free extract (HN019 extract) that was adjusted to pH 7.4 (with 5 M sodium hydroxide) and diluted 1/10 dilution in Kreb's solution for use in the motility assay.A bacterial extract was used in this study as opposed to live bacteria because ) Espey, . Therefoad libitum.This study was carried out in strict accordance with the recommendations of the New Zealand Animal Welfare Act 1999. The protocol was approved by the AgResearch Limited (Grasslands) Animal Ethics Committee . Male adult Sprague Dawley rats, 3–6 months of age, weighing 250–400 g were obtained from AgResearch Ruakura . The rats were housed under a 12 h light/dark cycle, and fed Sharpes Diet 86 . Food and water were available 2, 5% CO2) whilst the colon was gently flushed with Krebs solution to expel fecal pellets and the entire tissue was then mounted in an organ bath (approximately 350 mL capacity), a stainless steel rod (35 cm in length and 2 mm in diameter) was inserted through the lumen of the colon, which was then perfused at 20 mL/min with Krebs buffer at 35 ± 1°C. The lumen was also perfused with Krebs buffer at 1.5 mL/min which was pumped aborally using a constant flow pump because this provided the pressure required to record consistent propagating contractions. Changes in circular muscle tension were recorded from four sites simultaneously along the length of large intestine, using four custom-made metal hooks anchored 3 cm from both oral and anal ends of the preparation and evenly spaced apart at approximately 4 cm intervals. These hooks were connected via silk thread to force transducers and contractions measured after applying 2 g of tension. Muscle contraction data were recorded using isometric force transducers (MLT0201) connected to an eight-channel bridge amplifier, integrated using PowerLab 8/35 hardware and acquired and analyzed using LabChart 8 software. All recording equipment hardware and software were from ADInstruments Pty Ltd., Bella Vista, NSW, Australia.The protocol for recording motility in isolated intact whole large intestine has recently been described  with one experimental factor (the three treatments) and one repeated factor (four time points) was used to analyze differences in the frequency and amplitude of synchronous contractions within experiments, and also compared with the Krebs treatment control. All analyses were carried out using the R software version 3.2.3. ANOVA assumptions were met through log or square root transformation where necessary. Linear mixed effects models were used with appropriate variance function for modeling heterodasticity.Data were excluded from statistical analysis where they were contrary to the main effects observed: 2/12 preparations for prucalopride and 2/10 preparations for HN019, and are described in the results section.Contractile patterns were recorded from the circular muscle layer at four locations simultaneously along the length of the large intestine , before and after addition of treatment conditions. Motility patterns were quantified with respect to changes in frequency and amplitude of synchronous and non-synchronous phasic contractions, and contractions that occurred only in the proximal colon Figure .n = 8) synchronous contractions occurred between proximal colon and rectum. Although no changes in synchronous contractions were detected over the course of the experiment  over the initial 30 min of exposure and by 75 ± 26% over 30–60 min exposure. This occurred without any change in amplitude  to the large intestine resulted in no detectable change in contractile activity over 60 min, yet it was found that within the first 30 min following washout of HN019, the contractile amplitude increased by 55 ± 18% in the proximal colon Figures , 2A,B. PHN019 extract decreased the amplitude of non-synchronous contractions in the proximal colon to 30% lower than that for the corresponding Krebs treatment control at 60 min Figure . The inhThe ratio of non-synchronous to synchronous contractions averaged 5.2:1 across the pre-treatment controls  and was reduced to 1.0:1 after 60 min of prucalopride, and 1.6:1 for HN019 extract post-treatment, yet remained high at 5.0:1 for the Krebs control  . AL was supported by a Palmerston North Medical Research Foundation Summer Scholarship. Experiments in this project were in part conducted in the laboratory of NS and funded by the NH&MRC of Australia (#1067335).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.