CYP2D6 polymorphisms on risperidone plasma concentrations in patients with schizophrenia. Based on pharmacogenomics, we examined whether plasma concentration of risperidone is associated with clinical response and adverse side-effects.This study aimed to investigate the influence of CYP2D6 genotypes were determined using targeted sequencing. All high-frequency mutation sites of the nine exons of the gene were assayed in the present study. Plasma concentrations of risperidone and 9-hydroxyrisperidone (9-OH-RIS) were measured using high-performance liquid chromatography (HPLC). Psychiatric symptoms were monitored using The Positive and Negative Syndrome Scale (PANSS), Brief Psychiatric Rating Scale (BPRS), and Clinical Global Impression (CGI). Adverse effects were evaluated using the Barnes Akathisia Scale (BAS) and Extrapyramidal Symptom Rating Scale (ESRS). Follow-up visits were scheduled at weeks 2,4, and 8 after treatment initiation.We recruited patients with chronic schizophrenia who were then treated with risperidone. The CYP2D6*10 (81.6%), whereas CYP2D6*2 (9.2%) and CYP2D6*5 (17.1%) were relatively rare. Plasma levels of risperidone and the risperidone/9-OH risperidone ratio (R/9-OH) were significantly increased in individuals with CYP2D6*10 (P < 0.05). The change in PANSS score, weight, high-density lipoprotein (HDL) level, prolactin (PRL) level, and ESRS were significantly different from baseline, between the different genotypes (P < 0.01). Moreover, individuals with CYP2D6*10 homozygous (TT) mutations were associated with higher risperidone concentration and R/9-OH ratio than those with heterozygous mutations (CT) (P < 0.01). A change from baseline in BPRS scores was observed only during week 8 and was different between heterozygous and homozygous mutations. As for the C2851T polymorphism, the incidence of adverse metabolic effects was significantly different between the C/C and C/T genotypes (P < 0.01). Regarding the G4181C polymorphisms, the changes from baseline in GLU and TG, were different between the C/C and C/G genotypes (P < 0.01).Among the 76 patients, 100 C > T (rs1065852), 1038 C > T (rs1081003), 1662 G > C (rs1058164), 2851 C > T (rs16947), and 4181G > C (rs1135840) variants were detected. The most common allele was CYP2D6 significantly influences the plasma concentration of risperidone and may subsequently influence the adverse side-effects following risperidone treatment, while also exerting a slight influence on clinical outcomes.The genotype of The online version contains supplementary material available at 10.1186/s12888-020-03034-9. Risperidone, an atypical antipsychotic, is widely used for the treatment of psychiatric problems and is associated with better compliance and reduced incidence of adverse reactions compared with typical antipsychotics . AlthougCYP2D6 and CYP3A4 [CYP2D6 exhibits high polymorphism, and allelic variants are considered to influence the metabolic pathway of antipsychotics [CYP2D6 genotypes [CYP2D6*10/*10 variant compared with other alleles [CYP2D6*5/*10, CYP2D6*10/*10, or CYP2D6*10/*41 alleles exhibited higher levels of risperidone after administration [CYP2D6 variants influence the pharmacokinetics of risperidone. Further, an apparent association has been reported between CYP2D6 polymorphisms and clinical improvement in response to risperidone. Specifically, significant clinical improvements, assessed via the Positive and Negative Syndrome Scale – Thai (PANSS-T), have been reported in patients with the CYP2D6 “poor-metabolizer” phenotype compared with treatment outcomes in “extensive” metabolizers [CYP2D6 polymorphisms and clinical recovery [CYP2D6 genotype [CYP2D6, which may originate from the different genotypes that were analyzed in different studies. To the best of our knowledge, there have been no comprehensive studies exploring the effects of CYP2D6 variations on clinical outcomes of risperidone treatment.Risperidone is predominantly metabolized by the cytochrome P450 enzymes d CYP3A4 . The genychotics . The maiychotics . Therefoenotypes , 11. Sig alleles . Similarstration . These obolizers . Howeverrecovery , which wgenotype . These sCYP2D6*10 has been reported to be significantly associated with weight gain after risperidone treatment [CYP2D6 variants [CYP2D6 polymorphisms and serum prolactin concentrations are unclear and whether this association exists in patients with schizophrenia, remains to be investigated. Discrepancies have also been reported regarding the relationship between CYP2D6 polymorphisms and EPS, with several studies failing to detect any significant difference in the incidence of EPS in association with CYP2D6 variations, while some studies reported only a marginally significant trend [CYP2D6 polymorphism on EPS in response to risperidone are unclear and require further exploration.Adverse effects, in particular metabolic-related effects, can contribute to reduced patient compliance irrespective of symptomatic improvement . The risreatment . Other avariants , 19. Hownt trend , 21. TheCYP2D6 sequence, which was further investigated in order to obtain a more comprehensive understanding of CYP2D6 polymorphisms. The present study aimed to investigate whether CYP2D6 polymorphisms significantly effect serum/plasma concentrations of risperidone in a clinical setting. To this end, we used pharmacogenomics to investigate whether plasma concentrations of risperidone are associated with clinical responses and the incidence of adverse effects, including adverse metabolic effects and EPS.It is clear that genotypic differences contribute to the discrepancies observed in clinical outcomes. We detected all mutations in nine exons of the full-length We recruited patients that were all between 18 to 68 years of age and had been diagnosed with chronic schizophrenia , within the last 5 years, at Third People’s Hospital of Jiangyin City, Jiangsu Province between May 2018, and May 2019. All patients had to meet the following criteria: 1) had not taken any antipsychotics drugs for at least 1 year; 2) had an acute attack and were re-admitted to the hospital; and 3) were treated with risperidone monotherapy after hospitalization. Risperidone dosage was increased gradually from a low dose to a therapeutic dose within 1 week (3–6 mg/day). Exclusion criteria were as follows: 1) Had undergone electroconvulsive therapy within 3 months of study enrollment, 2) administration of other psychotropic medications, including antidepressants and mood stabilizers, 3) had obvious brain damage or serious physical illness, and 4) presented with any other condition that rendered the patient unsuitable for the trial . In total, 79 patients were recruited in the current study; however, 3 patients were dropped out of the study owing to drug adjustment or non-compliance. Ultimately, 76 patients were enrolled for subsequent data analyses.CYP2D6 genotype. The PANSS, Brief Psychiatric Rating Scale (BPRS), and Clinical Global Impression (CGI) were employed to monitor psychiatric symptoms. Treatment outcome was measured by assessing the change in clinical-symptom-scale scores over the study period. The improvement of clinical symptoms was expressed by the changes on the PANSS, BPRS, CGI scale scores from the initial assessment (week 0) to week 4 and then at week 8. The Barnes Akathisia Scale (BAS) and Extrapyramidal Symptom Rating Scale (ESRS) were used to evaluate adverse effects. Follow-up visits were made at 2,4 and 8 weeks after the initiation of treatment. During each follow-up visit, all evaluations  were repeated. Additionally, the concentrations of risperidone and 9-OH-RIS were measured at each follow-up visit.We collected data at the time of enrollment. Baseline data included demographic information, a comprehensive medical history, clinical evaluation of psychiatric symptoms, a physical examination (including weight and height), laboratory tests , and analysis of CYP2D6. The concentration/dose (C/D) ratio was calculated by dividing the total concentration of risperidone plus 9-OH-RIS (ng/mL) by risperidone dose (mg/day), to provide an index of drug elimination capacity.Fasting blood samples were collected from patients at each follow-up visit. Blood draws were consistently performed between 7 and 8 am, to ensure consistency across patients. Plasma was stored at − 20 °C until analysis. Plasma concentrations of risperidone and 9-OH-RIS were determined using high-performance liquid chromatography (HPLC) with the Agilent 1260 High Performance Liquid Chromatograph . The ratio of risperidone to 9-OH-RIS (R/9-OH ratio) was calculated from the concentration of risperidone divided by the concentration of 9-OH-RIS (ng/mL) to reflect the activity of CYP2D6 genotypes were determined in all the patients using PCR assays followed by targeted sequencing. CYP2D6 has nine exons that were all assayed in the current study. The whole experimental process consisted of primer design, polymerase chain reaction (PCR), and sequencing. Four sets of upstream and downstream primers were designed with Oligo6 software to amplify the nine exons using genomic DNA as templates. Extraction of DNA was carried out using the YC-B nucleic acid extraction reagent  according to manufacturer’s instructions. The DNA was extracted quickly then adsorbed onto a high-performance solid-phase matrix to obtain high-purity DNA by elution. The quality of genomic DNA underwent strict quality control including Nanodrop quantification and 1% agarose gel electrophoresis. The details of individual PCR programs were summarized in Supplemental Table CYP2D6*10, CYP2D6*2, and CYP2D6*65 according to the genotyping criteria for CYP2D6 .An additional 3–4 mL volume of blood was drawn once for pharmacogenetic analyses at baseline. The whole blood sample was stored at − 70 °C for subsequent extraction of genomic DNA. p-values of < 0.05 were considered to be of statistical significance. Continuous data with normal distribution are presented as mean and standard deviation, while non-normally distributed data are presented as median and interquartile range. Analysis of variance was used to compare data with normal distribution, while data with a non-normal distribution were analyzed using a 2-tailed Mann-Whitney U test to compare between two groups, or the Kruskal-Wallis test to compare multiple groups. The chi-square test was used to test differences in the distribution of categorical variables. Further comparisons between groups were carried out using the Dunn-Bonferroni post-hoc test. Multiple linear regression analysis was performed to analyze the relationship between CYP2D6 genotype and clinical response.Statistical analyses were carried out using the Statistical Package for Social Sciences . Two-tailed The mean course of disease for chronic patients was approximately 13.48 (± 7.80) years. The study sample was comprised of 76 patients. Demographic and clinical data of the study population are summarized in Table CYP2D6*10 allele. The 1038C > T (rs1081003) and 1662 G > C (rs1058164) mutations were nonsense mutations; therefore, they were excluded from subsequent analysis of single nucleotide polymorphisms. Given that the CYP2D6*10 allele is the most common allelic mutation, the heterozygous and homozygous mutations were further analyzed in our study.The majority of patients carried the P < 0.05). The Kruskal-Wallis Test revealed an apparent association between CYP2D6 genotype and plasma concentration of risperidone . Further pairwise comparisons revealed that, at all time points, plasma concentration of risperidone was significantly higher in subjects with the CYP2D6*10 genotype compared with those with the CYP2D6*2 genotype, after Bonferroni correction . Moreover, the plasma concentrations of risperidone in patients with the CYP2D6*65 and CYP2D6*2 genotypes were significantly different at week 8 , while no significant differences were observed between subjects with the CYP2D6*65 and CYP2D6*10 alleles . We did not observe any difference in 9-OH-RIS between genotypes . The R/9-OH ratio was significantly higher in subjects with the CYP2D6*10 allele than those with the CYP2D6*2 allele, at all time points, and those with the CYP2D6*65 allele at 4 and 8 weeks . The C/D ratio at 2 weeks was significantly different between subjects with the CYP2D6 genotype , with a significant difference observed between CYP2D6*65 and CYP2D6*2 genotypes ; however, the strength of this correlation decreased over time with no significant association observed at 4 or 8 weeks (both P > 0.05).Figure CYP2D6 genotypes . PANSS scores were significantly higher in individuals with the CYP2D6*2 allele than in those with the CYP2D6*65 allele after pairwise comparison using Bonferroni’s post-hoc analysis (P = 0.007). CYP2D6*10 was also higher than CYP2D6*65; however, the difference was not statistically significant (P = 0.052). Additionally, no significant difference was observed between CYP2D6*10 and CYP2D6*2 (P = 0.112). The BPRS and CGI scores were not significantly different among different genotypes from baseline to any follow-up visit .Symptom improvement in relation to genotype is presented in Table CYP2D6 polymorphisms and adverse effects are shown in Table F = 7.514, P = 0.001). Post-hoc tests for pairwise comparisons between the various genotypes identified significant differences between subjects with the CYP2D6*10 and CYP2D6*65 alleles (P < 0.001), and between those with the CYP2D6*65 and CYP2D6*2 (P = 0.025) alleles. The differences between HDL from baseline to week 8 were also significantly different between genotypes , and persisted between individuals with the CYP2D6*10 and CYP2D6*65 alleles, when between-group comparisons were conducted (P = 0.012). Prolactin levels were significantly associated with genotype . Post-hoc pairwise comparisons indicated that the change in prolactin levels in subjects with the CYP2D6*65 allele was significantly higher than other genotypes from baseline to week 8, especially the CYP2D6*10 allele (both P < 0.05). The occurrence of EPS was significantly different between different groups , which is mainly attributed to the higher occurrence of EPS in individuals with the CYP2D6*10 allele compared with the other two genotypes.The relationships between CYP2D6*10 allele, 26 exhibited homozygous (TT) mutations, while the remaining 21 carried heterozygous mutations (CT). The plasma concentration of risperidone was significantly higher in subjects with homozygous mutations of CYP2D6*10 compared with heterozygous mutations, at each time point . The difference in R/9-OH ratio was also significant at each time point , while 9-OH-RIS was only significantly different at week 2 . The C/D ratio was significantly different between CYP2D6*10TT/CT carriers at weeks 4 and 8  but not at week 2. The change in the BPRS score from baseline to week 8 was significantly different between subjects with heterozygous or homozygous mutations  (Table P > 0.05) (Table Of the 47 subjects who carried the 5) Table .Fig. 2BC100T, C2851T, or G4181C) and presented in Table G4181C polymorphism in the C/C genotype . No other significant differences were identified in age or gender between the polymorphisms (both P > 0.05). Additionally, there were no significant differences in plasma concentration of risperidone, PANSS, BPRS, or CGI at baseline between subjects with different polymorphisms .The demographics and disease-specific characteristics of subjects are stratified by single nucleotide polymorphism . Further pairwise comparisons identified no other significant difference between the C/C and C/T genotypes in subjects with C100T (P > 0.05). In terms of G4181C, a significantly higher plasma concentration of risperidone was identified in the C/C genotype compared with the G/C genotype at each time point . However, the 9-OH-RIS was not significantly different between genotypes in both C100T and G4181C at any time point . The C/D ratio was significantly different between subjects with C100T and G4181C polymorphisms  and between subjects with CC/TT and CT/TT genotypes in further pairwise comparisons at any time point . For the C2851T polymorphism, plasma drug concentration, R/9-OH, or C/D ratio were not significantly different for any genotype .Figure C100T and C2851T polymorphisms . However, for the G4181C polymorphism, improvement in CGI-S and CGI-I scores at week 4 were significantly different between the C/C and C/G genotypes . Analysis of the predictive values of the alleles  and SNPs  with regard to clinical response (defined as 25% improvement in PANSS from baseline), identified 30 and 40 subjects as treatment responders at weeks 4 and 8, respectively. After controlling for confounding factors, regression analysis showed that plasma concentrations of risperidone at week 2 were a statistically significant predictor of clinical response . The relationship between adverse effects and SNPs are shown in Table C100T polymorphism . In patients with the C2851T polymorphism, adverse metabolic effects, including weight change from baseline to week 4 , changes in HDL from baseline to week 4  and week 8 , and change in PRL from baseline to week 8 , were significantly higher among patients with the C/T genotype compared with the C/C genotype. In contrast, the incidence of EPS, as assessed by ESRS or BAS, did not differ significantly in relation to genotype. With regards to the G4181C polymorphism, the change in GLU from baseline to week 4 (Z = -2.401 P = 0.016) and the TG level from baseline to week 4 , were significantly higher in subjects with the C/C genotype compared with C/G, however, by week 8, this difference was not statistically significant . There were no significant differences in any other adverse metabolic effects or the incidence of EPS, between genotypes of the G4181C polymorphism .The associations between SNPs and clinical responses to risperidone treatment are presented in Table CYP2D6 genotype significantly influenced the plasma concentration of risperidone. This finding also suggests that among patients treated with risperidone, genotype may influence adverse drug reactions. However, the CYP2D6 genotype exerts a slight effect on improvement of clinical symptoms.The present study demonstrates that among patients with schizophrenia, the CYP2D6 alleles or SNPs. Similarly, significant differences were also observed between heterozygotes and homozygotes among the most common CYP2D6*10 alleles. Different CYP2D6 alleles or SNPs were significantly associated with adverse drug effects, including adverse metabolic reactions and EPS. No obvious differences were observed between heterozygotes and homozygotes among the CYP2D6*10 allele. Only a slight difference was reported in the improvement of clinical symptoms among individuals with different CYP2D6 genotypes.The main finding of the current study is that the plasma drug concentrations of risperidone were significantly different among different CYP2D6 genotype influenced the metabolism of risperidone, evidenced by increased plasma concentration and metabolic ratio (RIS/9-OH-RIS) [CYP2D6*10, while plasma concentration of 9-OH-RIS was not found to be significantly correlated [CYP2D6*10 to be associated with plasma concentrations of both risperidone and 9-OH-RIS, conflicting with our results [Our study revealed that the -OH-RIS) , 16, 22.-OH-RIS) , 23, as rrelated . HoweverCYP2D6*10 mutations, confirming previous studies that reported subjects who had homozygous mutations CYP2D6*10 to have higher plasma concentrations of risperidone compared with single-allele carriers [CYP2D6 gene product is significantly lower in CYP2D6*10-homozygous subjects compared with wild-type homozygotes, while the CYP2D6*10-heterozygous subjects exhibit moderate enzyme activity [CYP2D6*10 is associated with lower enzymatic activity. Our study revealed that subjects with CYP2D6*2 have lower plasma concentrations of risperidone, indicating increased enzymatic activity after treatment, compared with the other two genotypes. Thus, there appears to be a significant relationship between CYP2D6 genotype and CYP2D6 activity; however, to the best of our knowledge, no study has directly compared CYP2D6 activity between these three genotypes. Indeed, research regarding CYP2D6*65 and CYP2D6*2 is limited. Considering the small sample size of the present study, the differences that we have identified should be considered preliminary and interpreted with caution; further long-term large-scale research is warranted.We found that after treatment, the plasma concentration of risperidone, was significantly different depending on the homo- or heterozygosity of carriers , 25. Thiactivity .. TherefC100T, C2851T, and G4181C) and plasma concentrations of risperidone, RIS/9-OH-RIS ratio, and C/D ratio. To the best of our knowledge, this is the first exploratory research to investigate the relationships between CYP2D6 SNPs, plasma concentration of antipsychotics, and clinical response relative to antipsychotic treatment, in patients with schizophrenia. Similar research involving psychiatric patients has shown the CYP2D6 1584C > G polymorphism as having a significant influence on thioridazine:mesoridazine plasma ratio, although the correlation between polymorphisms and clinical response was not investigated in this study [C100T and G4181C polymorphisms were associated with differences in plasma concentration of risperidone and RIS/9-OH-RIS ratio. These findings indicate that even single-nucleotide mutations are sufficient to affect the activity of metabolic enzymes. Furthermore, the C/D ratio was obviously different between SNPs, suggesting that plasma clearance varies according to allelic variants. These factors may contribute to the clinical treatment response, and these findings provide new insight for individualized drug therapy.Significant associations were observed between SNPs  compared with those with a *1/*1 genotype [CYP2D6*10 and CYP2D6*2 were found to be associated with increased weight gain compared with CYP2D6*65, which contradicts some previous reports but agrees with others. A previous study suggested that individuals with the poor-metabolizer phenotype have increased plasma drug levels after treatment, resulting in serious antipsychotic-induced toxicity with consequent dose-dependent complications [CYP2D6*65 should be related to increased weight again while CYP2D6*2 should not be. Lane et al. reported a significant correlation between CYP2D6*10 and weight gain in patients receiving risperidone [CYP2D6*65 was associated with lower HDL levels following risperidone treatment compared with the other genotypes. The relationship between HDL and CYP2D6 genotype has not been reported previously. In contrast, we found that in response to risperidone treatment, CYP2D6*65 was associated with higher levels of prolactin compared with the other alleles, particularly CYP2D6*10. Previous reports on the association between CYP2D6 polymorphisms in relation to prolactin concentrations, are controversial. One study explored the impact of CYP2D6 polymorphisms on the prevalence of risperidone-induced adverse effects, including hyperprolactinemia, revealed that the poor-metabolizer phenotype influenced the frequency of adverse effects and poor treatment compliance [CYP2D6 ultra-rapid metabolism may contribute to increased prolactin levels in children, consist with our observations [CYP2D6*10 genotype. However, the predictive value of genotype for extrapyramidal side-effects is controversial [CYP2D6*10 or CYP2D6*65 are more likely to suffer EPS. With respect to SNPs, our study revealed that weight change and HDL or PRL alterations from baseline in response to treatment, differed significantly between subjects with C2851T wild-type compared with mutant, while GLU and TG differed significantly between subjects with G4181C homozygous mutations compared with heterozygous mutations. Previous studies on SNPs and their association with adverse reactions are scarce, with only one published report describing the association between the 1846G > A polymorphism of CYP2D6 and extrapyramidal side effects [Consistent with previous research, we found clear associations between genotype and adverse effects, including adverse metabolic reactions and EPS. A study involving children and adolescents receiving antipsychotics identified a significant difference in weight gain between patients with the allele*4 . Anothergenotype . In the ications . Anotherications . Theoretperidone . These fmpliance . Althougrvations . Also corvations , we founoversial , 35. Somoversial , 34, whi effects . Our resCYP2D6 gene had a significant effect on drug concentration and adverse drug effects. Previous studies have shown that polymorphisms of the CYP2D6 gene display different enzyme activity including decreased, increased, or nonfunctional enzymatic activity [CYP2D6*10, were associated with more severe adverse effects, including weight gain and increased HDL and EPS, supporting the assumption that CYP2D6 gene polymorphisms may be associated with different adverse reactions via alterations in enzyme activity. However, the slight improvement in clinical symptoms did not seem to be associated with CYP2D6 genetic variations.Collectively, the current study showed that polymorphisms of the activity . Differeactivity . Our stuCYP2D6 polymorphisms on the long-term clinical responses and side-effects, which may affect our conclusions. Additionally, patients who were non-compliant with treatment were excluded from the analysis and the study could be missing some key patient characteristics that influence drug treatment response. Finally, we only identified three significant single-nucleotide gene-effective mutation sites. Therefore, comparisons between more mutation sites could not be performed. Further verification through studies involving large sample sizes and comparing more genotypes are warranted.The present study has some limitations which should be acknowledged. Firstly, the sample size was small. Secondly, subjects were followed-up for only 8 weeks; therefore, we cannot comment on the effects of CYP2D6 polymorphisms and the plasma concentration of risperidone, and that CYP2D6 genotypes may serve as a predictor of adverse reactions, but not improvement of clinical symptoms, after risperidone treatment.In summary, our study reveals the association between Additional file 1.


G12/G13 mutation were measured by ddPCR, yielding high consistency (ICC = 0.956). Receiver operating characteristic (ROC) curves were constructed to determine KRASG12/G13 mutational status based on mutant allele frequency generated by ddPCR. Using the best threshold cutoff , ddPCR had superior diagnostic sensitivity (100%) and specificity (100%) relative to the two other techniques. Thus, ddPCR is effective for detecting the KRASG12/G13 mutation in colorectal adenocarcinoma tissue samples. By allowing definition of the optimal cutoff, ddPCR represents a potentially useful diagnostic tool that could improve diagnostic sensitivity and specificity.KRAS  is a major predictive marker for anti-epidermal growth factor receptor treatment, and determination of KRAS mutational status is crucial for successful management of colorectal adenocarcinoma. More standardized and accurate methods for testing KRAS mutation, which is vital for therapeutic decision-making, are required. Digital droplet polymerase chain reaction (ddPCR) is an advanced digital PCR technology developed to provide absolute quantitation of target DNA. In this study, we validated the clinical performance of ddPCR in determination of KRAS mutational status, and compared ddPCR results with those obtained by Sanger sequencing and peptide nucleic acid-clamping. Of 81 colorectal adenocarcinoma tissue samples, three repeated sets of KRAS Colorectal cancer (CRC) is one of the most common cancers in the United States . ApproxiKRAS is a major predictive marker for anti-EGFR treatment, and determination of KRAS mutational status is crucial for successful management of CRC patients. Mutation of KRAS is detected in 35–45% of CRC patients, and most of these mutations affect codons 12 and 13 ,7,8. To Digital droplet PCR (ddPCR) is a recently developed sequencing method capable of sensitively detecting target DNA in varying backgrounds of wild-type DNA in a small amount of material . By partG12/G13 mutation (KRAS codon 12 and codon 13) using CRC patient tissue samples. KRASG12/G13 mutant allele frequency (MAF) generated by ddPCR was measured repeatedly to assess the consistency of ddPCR. Receiver operating characteristic (ROC) curves were constructed based on the MAF to determine KRASG12/G13 mutational status with the optimal cutoff value. The diagnostic performance of ddPCR was compared with gold standard methods for KRASG12/G13 mutational analysis: Sanger sequencing and PNA-clamping assay (PCR with PNA-mediated clamping).In this study, we sought to validate a ddPCR platform for detection of KRASThis study was performed using 81 formalin-fixed paraffin-embedded (FFPE) surgically resected colorectal adenocarcinoma (CRAC) tissue samples collected at a single institutional center  from January 2014 to December 2017. Tissue samples included primary and metastatic  tissues from CRAC patients. Hematoxylin/eosin-stained slides of selected cases were pathologically reviewed by two pathologists (M-KY and GEB), and the most representative areas were selected. Twenty non-neoplastic colon FFPE tissue samples  were included for negative control. Sixteen serums from healthy persons were also included negative control.G12/G13 mutation using ddPCR, Sanger sequencing, and PNA-clamping PCR. All bio-specimens and data used for this study were provided by the Biobank of Chungnam National University Hospital, a member of the Korea Biobank Network. The study was approved by Chungnam National University Hospital institutional review board (IRB file no. 2018-10-012-001). The study was retrospective, and a waiver of consent was approved by the Institutional Review Board.Twenty-micron thick sections of FFPE samples were prepared and deparaffinized in xylene. DNA was isolated using the QIAamp DNA FFPE Tissue Kit and QIAamp Circulating Nucleic Acid kit . All extracted DNA was diluted to 10 ng/μL. Extracted DNAs were evaluated for KRASG12/G13 multiplex kit (#1863506) for screening of codons 12/13  using the ddPCR Bio-Rad KRASns 12/13 A. ReactiThe QuantaSoft software  classifies droplets by first determining a fluorescence threshold B. Some dExtracted DNA (20 ng) from CRAC tissue samples were sent for Sanger sequencing .For mutation analyses in codons 12 and 13 of the KRAS gene, primer sequences for exon 2, 5′-GTAAAACGACGGCCAGTGTGTGACATGTTCTAATATAGTCA-3′(forward) and 5′-GCGGATAA CAATTTCACACAGGGAATGGTCCTGCACCAGTAA-3′ (reverse) and for exon 3, 5′-TAATA CGACTCACTATAGGGGTGCTTAGTGGCCATTTGTC-3′ (forward) and 5′-GCTAGTTATTGC TCAGCGGTATGCATGGCATTAGCAAAG -3′ (reverse) were utilized for the PCR reaction.PCR amplification conditions were as follows: 95 °C 5 min; 95 °C 30 s, 60 °C 30 s, 72 °C 1 min for 35 cycles; 72 °C 7 min. PCR products were purified using Millipore plate MSNU030 . The purified PCR products were then Sanger-sequenced with the BigDye terminator v3.1 sequencing kit and a 3730xl automated sequencer . Nucleotide sequence data were analyzed with Variant reporter computer software version 1.1 .Extracted DNA (7 μL) from CRAC tissue samples was tested by PNA-clamping assay using the PNA clamp KRAS mutation detection kit  for screening of codons 12/13/59/61/117/146. Reaction mixtures contained 7 µL DNA template, 3 μL of each PNA mix, and 10 μL of 2× premix, and amplification was performed in a CFX96 real-time PCR instrument (Bio-Rad) with the following thermal program: pre-incubation at 94 °C for 5 min, followed by 40 cycles of amplification at 94 °C for 30 s (s), 70 °C for 30 s, 63 °C for 30 s, and 72 °C for 30 s.The efficiency of PNA-mediated PCR clamping was determined by measuring the threshold cycle (Ct) value. The Ct values for the control and mutation assays were obtained by observing the SYBR Green amplification plots. The delta Ct (ΔCt) value was calculated ([Control Ct] − [Sample Ct] = ΔCt) and the cutoff ΔCt was defined as 2 for the all mutations.G12/G13 detection by the ddPCR platform, cases in which mutations were detected by Sanger sequencing were considered as positive references. KRASG12/G13 mutation detection by ddPCR was performed three times , and the mean MAF of KRASG12/G13 was used to develop ROC curve. Internal consistency of the scales was assessed by Cronbach’s alpha via the intraclass correlation coefficient and kappa coefficient.To develop ROC curves for KRASG12/G13 mutation by ddPCR, Sanger sequencing, and PNA-clamping assay. All statistical analyses were performed using SPSS version 26.0 for Windows  and MedCalc version 19.2.0 for Windows .Diagnostic value  was calculated for the detection of KRASG12/G13 mutant) 3480 for channel 2 (KRASG12/G13 wild) . Samples with fewer than 10,000 generated droplets were excluded from analysis. Threshold horizontal lines were set at 9474 for channel 1 (KRAS13 wild) B. MAF waG12/G13 mutant droplets. Twenty non-neoplastic colon tissue samples showed 0 to 6 KRASG12/G13 mutant droplets and the MAF were 0 to 0.55% (mean: 0.06%). A total of 81 CRAC tissue samples showed KRASG12/G13 mutant droplets from 0 to 1121 droplets and the MAF were 0 to 81.17% (mean: 11.13%).Sixteen serums from healthy persons showed no KRASG12/G13 wild type by Sanger sequencing and PNA clamping assay. A total of 81 CRAC, 51 (63%) CRAC were KRASG12/G13 wild type and 30 (37%) CRAC were KRASG12/G13 mutant (23 cases of codon12 and 7 cases of codon 13) by Sanger sequencing. A total of 81 CRAC, 48 (59%) CRAC were KRASG12/G13 wild type and 33 (41%) CRAC were KRASG12/G13 mutant (27 cases of codon12 and 6 cases of codon 13) by PNA clamping assay.Sixteen serums from healthy persons and 20 non-neoplastic colon tissue samples were all KRASG12/G13 by ddPCR as “mutant” or “wild-type,” a cutoff value for MAF was required. To this end, we generated ROC curves to determine KRASG12/G13 mutational status from MAFs generated by ddPCR  non-neoplastic colon; (B) non-neoplastic colon and KRASG12/G13 wild-type CRAC by Sanger sequencing (p < 0.001) (G12/G13 wild CRAC by Sanger sequencing) was 0.943 and optimal cutoff was 7.9% (p < 0.001)  A. The AU< 0.001) B.G12/G13 mutation using the calculated cutoffs  coefficient for the three sets of MAFs and the mean was 0.956 (p < 0.001), which is an excellent concordance rate.To assess the reproducibility of the ddPCR platform, we measured three sets of KRASG12/G13 mutation status to the CRAC tissue samples . In 53 KRASG12/G13 wild-type cases, MAF ranged from 0% to 7.53% (the mean of wild-type MAF = 0.67%).We validated KRASng assay . Twenty-G12/G13 mutation, the concordant rate of the ddPCR and Sanger sequencing was 93% (75/83); the ddPCR and PNA-clamping assay was 89% (72/81); Sanger sequencing and PNA-clamping assay was 81% (66/81). Six discordant cases  were identified between ddPCR and Sanger sequencing. Except 1 case (T54), Sanger sequencing detected KRASG12/G13 mutation, otherwise, ddPCR  and PNA clamping assay did not detect KRASG12/G13 mutation. The MAF of discordant cases were 0.17%, 0.26%, 0.39%, 0.54%, 1.78%, and 7.9%. Nine discordant cases  were identified between ddPCR and PNA clamping assay. Except 2 cases , PNA clamping assay detected KRASG12/G13 mutation, otherwise, ddPCR and Sanger sequencing did not detect KRASG12/G13 mutation. The MAF of discordant cases were 0.11%, 1.04%, 2.14%, 2.42%, 4%, 5.48%, 7.53%, 18.2%, and 19.36%. T52  and T53  cases showed discordant results in 3 repetitive ddPCR results.Comparing two methods to detect KRASG12/G13 mutation was detected by two or more methods were defined as positive references. Twenty-eight of 81 CRAC tissue samples (35%) harbored the KRASG12/G13 mutation from the results of positive references. Fifteen of 81 cases (18.5%) yielded discrepant results (p < 0.001); the κ value was 0.842 (p < 0.001) for the Sanger sequencing; 0.764 (p < 0.001) for PNA-clamping assay. The κ value of ddPCR with the Sanger sequencing was 0.842 (p < 0.001) and 0.764 (p < 0.001) with PNA-clamping assay.For comparative analysis, cases in which KRAS results , *. ddPCG12/G13  and low specificity (30.61%). With 7.9% cutoff generated from ROC curve validated with KRASG12/G13 wild-type samples, diagnostic value of ddPCR yielded a high sensitivity (84.38%) and high specificity (97.96%). We decided that 7.9% MAF cutoff might decrease the possibility of FP results and be more appropriate for clinical application using FFPE patient samples.The sensitive PCR method has the possibility to lead to false positive (FP) results. An FFPE sample is very commonly used for clinical sequencing because it is easy to match tumor and normal tissue in the slides and it can be stored at room temperature. However, sequencing from DNA-extracted FFPE samples can yield errors due to fragmentation of genomic DNA and chemical processing damage to the samples . The limG12/G13 mutation have the possibility of KRASG12/G13 mutant type. The sanger sequencing detected KRASG12/G13 mutation in cases with low MAF (0.17%~1.78%) that 7.9% MAF cutoff can be rather high and can miss mutant cases with low MAF. We set arbitrary cutoff to increase the specificity to use ddPCR as a diagnostic assay using FFPE tissue samples. Considering the biggest merit of the ddPCR is the sensitivity, we need to set appropriate MAF cut offs depending on the sample types (blood or urine) and detection probes (TaqMan probe or multiplex kit). In a previous study, CRAC patients with below 1% MAF KRAS mutation showed more therapeutic response with anti-EGFR therapy than above 1% MAF KRAS mutation [Cases with wild type by ddPCR KRASmutation . ClinicaG12/G13. Three sets of measurements yielded seven discrepant results that 5 discordant cases raised at the first measurement and 2 cases were small-sized specimens (less than 1 cm). We guessed that an unaccustomed technique in pipette handling during droplet generation and small-sized specimens could be the reason. Low fractions of mutant DNA raised the possibility of false-positive or false-negative results. When using FFPE tissue samples, sensitivity could be limited by the amount of mutant DNA; however, in such a case, repetitive measurement could guarantee the results and increase diagnostic sensitivity. Statistically, ddPCR yielded an excellent intraclass correlation, allowing us to conclude that the ddPCR platform has the potential to be used as a sensitive and reliable method to detect KRASG12/G13 mutation.ddPCR is a fluorescent probe-based PCR assay to partition sample DNA into ~20,000 droplets that fluorescence emitted from each droplet is measured to quantitate the number of target DNA molecules . The numG12/G13 DNA in samples. However, the Sanger sequencing has been employed in diagnostic laboratories and is considered a gold standard for evaluating KRASG12/G13 with high specificity. In addition, PNA-clamping is also commonly used in diagnostic laboratories for evaluating KRASG12/G13 with high specificity [G12/G13 mutation, the concordant rate of the ddPCR and Sanger sequencing was 93% and the ddPCR and PNA-clamping assay was 89%. Sanger sequencing detected KRASG12/G13 mutation in cases with low MAF by ddPCR (mean MAF = 0.4%). PNA clamping assay detected KRASG12/G13 mutation in cases with MAF by ddPCR (mean MAF = 3.7%), however, showed wild type with high MAF by ddPCR (18.2% and 19.36%). The ddPCR showed more comparable results to Sanger sequencing. PNA clamping assay had the possibility of lower specificity compare to ddPCR and Sanger sequencing.This study has some limitations. First, when developing ROC curves to calculate ddPCR cutoffs, we defined positive references based on results from Sanger sequencing. However, the mutational status assessed by Sanger sequencing did not represent the true properties of KRAScificity . CompariG12/G13 status, but showed a low sensitivity and positive predictive value. ddPCR, Sanger sequencing, and the PNA-clamping assay showed comparable results for detecting KRASG12/G13 mutation, however, the required amount of DNA (1 μL) for ddPCR is much less than for Sanger sequencing (20 ng) and PNA-clamping assay (7 μL). This technical advantage is useful detecting KRASG12/G13 mutation in small biopsied tissues or even liquid biopsy samples. Furthermore, the KRASG12/G13 multiplex kit could not discriminate the mutation codon site and did not cover the full spectrum of mutation sites in KRAS. The ddPCR platform includes two fluorescence filters and supports at least duplex reactions. The development and optimization of higher-order multiplexing techniques for ddPCR are still required.In a comparison of Sanger sequencing and PNA-clamping assay, ddPCR with applied clinical cutoff eliminated false-positive results and preserved high sensitivity (100%) and specificity (100%) relative to the Sanger sequencing and PNA-clamping assay. In comparison with NGS panel sequencing , ddPCR aG12/G13 mutation tend to have more advanced tumors and shorter survival, implying that KRASG12/G13 could be used as a prognostic factor [G12/G13 is a highly informative and useful marker for the management of CRAC. By allowing the optimal cutoff value to be defined, ddPCR has potential for use as a diagnostic tool that could improve diagnostic sensitivity and specificity. Because ddPCR has high sensitivity and reproducibility, it would be suitable for daily application in laboratories seeking to detect KRASG12/G13 mutations in CRAC tissue samples.Determining KRAS mutational status has become crucial for successfully managing CRC patients, as well as in applications of anti-EGFR therapy. Furthermore, patients with the KRASc factor . Thus, K


Vitamin D is a steroid hormone crucial for bone mineral metabolism. In addition, vitamin D has pleiotropic actions in the body, including anti-cancer actions. These anti-cancer properties observed within in vitro studies frequently report the reduction of cell proliferation by interruption of the cell cycle by the direct alteration of cell cycle regulators which induce cell cycle arrest. The most recurrent reported mode of cell cycle arrest by vitamin D is at the G1/G0 phase of the cell cycle. This arrest is mediated by p21 and p27 upregulation, which results in suppression of cyclin D and E activity which leads to G1/G0 arrest. In addition, vitamin D treatments within in vitro cell lines have observed a reduced C-MYC expression and increased retinoblastoma protein levels that also result in G1/G0 arrest. In contrast, G2/M arrest is reported rarely within in vitro studies, and the mechanisms of this arrest are poorly described. Although the relationship of epigenetics on vitamin D metabolism is acknowledged, studies exploring a direct relationship to cell cycle perturbation is limited. In this review, we examine in vitro evidence of vitamin D and vitamin D metabolites directly influencing cell cycle regulators and inducing cell cycle arrest in cancer cell lines. Cancer can be described as a heterogeneous disease characterized by uncontrolled cell growth, invasion, and spread from the original site of the disease to several other sites in the body (metastasis) . Cancer 2D3. Since then, numerous pre-clinical studies on the anti-cancer actions of vitamin D metabolites have shown promising results . Fi. Fi19]. Cyclin-dependent kinases (CDKs) are a family of heterodimeric serine/threonine protein kinases that enable the cell to progress sequentially in the cell cycle . CDKs arIn mammalian cells, cyclin/CDK complexes require further activation by post-translational modification before they mediate cell cycle progression. Firstly, cyclin activating kinases (CAKs)—a trimolecular complex of Cdk7, cyclin H, and Mat1—phosphorylate Thr160 and secondly, Cdc25 phosphatase dephosphorylates Thr14 and Tyr15 to form an active complex . In contCDK inhibitors (CDKIs) finely modulate cell cycling by temporarily blocking the activity of CDK-cyclin complexes . CDKI exINhibitors of CDK4 and consist of members: p16INK4A, p15INK4B, p18INK4C, and p19INK4D. INK4 inhibitors specifically prevent the binding of CDK4 and CDK6 to cyclin D but do not bind other CDKs [CIP1 (Cdk Interacting Protein 1), p27KIP1 (Kinase Inhibitory Protein 1), and p57KIP2 (Kinase Inhibitory Protein 2), which bind CDK2 activity. In this way, CDKIs provide fine regulation of control between the phases of the cell cycle.The two families of CDKIs are the INK4 and the CYP/Kip inhibitors, and respectively prevent the binding of CDKs to their cognate cyclins, or disrupt activated cyclin-CDK complexes, thereby causing transcriptional inhibition ,21. Firsher CDKs ,21. SecoMitogenic signals converge on cyclin D-CDK4 and result in the progression of the cell to the restriction point . Cyclin The pRB pocket protein family includes three members: pRB, p107, and p130, and they are named after a conserved binding pocket region through which pRB, p107, and p130 bind cellular factors such as the E2F family of transcription factors and viral oncoproteins . pRB is C-MYC, MYCN, and MYCL, which code for the oncoproteins C-MYC, N-MYC, and L-MYC, respectively [The MYC oncogene family consists of ectively . These oectively  involvedectively ,29. MYC ectively . The MYCectively .MYC expression is tightly controlled at transcriptional and post-transcriptional levels in healthy cells; however, MYC expression is frequently dysregulated in human cancers ,27. C-MYHowever, MYC is considered a double face transcription factor: MYC overexpression promotes cell proliferation in malignancy but also concomitantly sensitizes healthy cells to apoptosis . HealthyOnce the cell in the S phase has faithfully replicated its genome, it enters the G2 phase . During Vitamin D derivatives inhibit the progression of the cell cycle in various cell and tumor types. To date, the anti-proliferative action of vitamin D derivatives has been well established in malignant keratinocytes, and this action has also been well studied in breast and prostate cancer cells. The best-described direct action of vitamin D on cell cycle regulators is the role of vitamin D in the G1/G0 cell cycle arrest. The G1/G0 cell cycle arrest is predominantly mediated by the upregulation of CDKIs p21 and p27. The mechanisms of p21 and p27 upregulation by calcitriol are varied. This section will explore the role of vitamin D and its metabolites on p21 and p27 expression. Furthermore, other mechanisms of G1/G0 cell cycle arrest, such as inducing pRB expression and inhibition of C-MYC expression, will also be explored. Lastly, the role of calcitriol in arresting the cell at the G2/M checkpoint will also be discussed.The upregulation of p21 and p27 CDKIs in numerous types of cells treated with vitamin D is frequently documented within in vitro studies . Conside2D3 increases the expression of p21 n of p21 . A functabolites . Functioabolites ,57, breaabolites ,38,58, aabolites . Therefo2D3 inhibited cell growth but also decreased p21 expression [2D3, and this resulted in a G1 arrest [Within in vitro cell differentiation models, such as myeloid leukemia HL60 and SCC cell lines, p21 showed variable response to calcitriol treatment. In addition to cell cycle arrest, p21 is also associated with cellular differentiation. Despite the presence of VDRE at the p21 gene promoter, studies on calcitriol treatment reveal cell-specific results. In HL60 cells, calcitriol increased p21 expression and induced G1/G0 cell cycle arrest [pression . In the 1 arrest . However1 arrest . The ini2D3 has been shown to regulate the p53-p21 axis. Cross-talk between VDR and p53 family members is important in tumor suppression [p21 and p53 co-operates with VDR to regulate the transactivation of p21 mRNA [MDM2) gene independent of p53 [In addition, p21 is also transcriptionally regulated by p53 protein . This p5pression . For exap21 mRNA . Mechanip21 mRNA ; the regp21 mRNA  and the p21 mRNA . Additiot of p53 , which et of p53  which ca2D3 treatment in HL60 cells [1/G0 cell cycle arrest in HL60 cells. To date, several different mechanisms of 1,25(OH)2D3 regulation of p27 have been reported 60 cells . In late60 cells  which in60 cells . Taken treported .2D3 treatment did not change p27 mRNA levels, but reduced mRNA expression of p45/Skp2 and Cks1 which are responsible for p27 protein ubiquitinylation and degradation [2D3 may increase p27 expression and sustain the G1 cell cycle block as p27 regulates cyclin E-CDK2 activity [The dominant mechanism of p27 regulation by vitamin D is by proteosome-dependent protein degradation. For example, in both ovarian  and prosradation . The netradation ,78. Thisradation  and humaradation . By protactivity .2D3 also mediates p27 by two transcriptional mechanisms. Firstly, VDR enhanced the expression of transcription factors responsible for the increase in p27 gene expression, Sp1, and NFY, in SW620 colon cancer cell line and LNCaP prostate cancer cell line [p27 gene expression [In addition to the aforementioned post-translational mechanism of p27, 1,25(OH)ell line . Secondlpression . Akt propression ,85. Ther2D3 regulates p27 expression by decreasing microRNA 181a expression, and it has been reported in myeloid cells [2D3 in a dose- and time-dependent manner [p27 mRNA whereas cells transfected with pre-miR181a constructs abrogated the 1,25(OH)2D3-induced upregulation of p27 [Furthermore, 1,25(OH)id cells . MicroRNid cells . Wang ett manner  which den of p27 .Hence, several mechanisms of p27 regulation have been reported, and data suggests a cell-specific response to calcitriol treatment. Additional studies on p27 regulation by vitamin D metabolites in other cell lines and cancer cell types are needed to fully elucidate the mechanism of p27 upregulation by vitamin D metabolites in cancer.2D3 directly decreases the action of downstream targets, cyclin D-CDK4/6, and cyclin E-CDK2 complexes, respectively. The result of these perturbations of cell cycle regulatory proteins is that pRB remains in a hypophosphorylated state complexed to E2F [2D3 prevents the transcriptional activity of E2F transcription factors and denies entry into the S phase of the cell cycle. In addition to this indirect role of 1,25(OH)2D3 on E2F expression, 1,25(OH)2D3 can increase the expression of pRB in myeloid leukemia cells approximately 10 h after vitamin D treatment [By increasing CDKIs p21 and p27, 1,25(OH)reatment , althougIn addition to its regulation of pRB, 1,25(OH)2D3 has demonstrated downregulation of the prominent pRB target gene, C-MYC. Several studies have demonstrated a significant reduction of C-MYC expression after 1,25(OH)2D3 treatment in colon ,90 and p2D3 treatment reduced C-MYC mRNA by 50% and resulted in a significant reduction in C-MYC protein [2D3 promoted VDR/β-catenin interaction and prevented the β-catenin translocation into the nucleus [2D3 may indirectly inhibit C-MYC gene transcription via the APC/β-catenin pathway. C-MYC therefore appears to be an important target of calcitriol in policing cancer cells to cause G1/G0 arrest.The mechanism for C-MYC repression by calcitriol seems to be cell-specific. In prostate cancer C4-2 cells, 1,25(OH) protein . In SW48 nucleus . β-caten2D3, with the general consensus that G1/G0 cell cycle arrest is the primary target of vitamin D metabolites. However, there are a selected number of studies that have shown G2/M cell cycle arrest in cells treated with 1,25(OH)2D3. In HL60 myeloid leukemia cells treated with 1,25(OH)2D3, a G2/M arrest was evident in cell cycle analyses [2D3-induced G2/M cell cycle arrest. In ovarian cancer cell lines, 1,25(OH)2D3 increased GADD45α  and subsequently induced cell cycle arrest at the G2/M phase [GADD45α gene [Currently, there is limited evidence of the induction of G2/M arrest by 1,25(OH)analyses ,94. Only/M phase . Interes45α gene ; howeverThe VDMS, at an autocrine level, controls genes that regulate cell proliferation and cell death. CpG islands span the promoters of CYP2R1, CYP24A1, and VDR, while a CpG island is located within the CYP27B1 gene locus .Liganded VDR signaling has been shown to be attenuated in cancer . EpigeneIn addition, expression of CYP27B1 is often downregulated in cancer which may be accounted for by CpG islands also present in the gene locus. In breast cancer cell lines, the CYP27B1 gene showed reversible DNA hypermethylation which caused CYP27B1 silencing. Similarly, Novakovic et al.  demonstrThe regulation of CYP24A1 by DNA methylation demonstrates cell-specificity. Hypermethylation of the CYP24A1 promoter region, with associated epigenetic silencing of CYP24A1 expression, has been identified in prostate cancer cell line PC3  and tumoCollectively, these studies demonstrate that the epigenetic regulation of gene expression of the VDMS is altered in cancer mainly by DNA hypermethylation. This altered state leads to abnormal protein expression levels, which may favor carcinogenesis in a cell-specific manner. Therefore, autocrine regulation of the VDMS may impact G1/G0 and G2/M cell cycle arrest in cell lines and in clinical studies. Epigenetic alteration of the VDMS may also provide insight into the discordant alignment between in vitro cancer studies and clinical studies. Exploration of epigenetic marks of the VDMS on cell cycle regulators may thus provide a possible explanation of the inhibition of cell arrest in cancer patients. Future studies can also target altered autocrine activation of vitamin D precursors, autocrine catabolism of activated vitamin D hormone, and abrogated signaling of liganded VDR, and their collective association with perturbations of vitamin D-induced cell cycle arrest.Vitamin D metabolites target numerous cell cycle regulators in order to arrest the cell cycle. G1/G0 arrest is the primary mode of cell cycle arrest described within in vitro cell lines treated with vitamin D compounds, although a few studies have described G2/M cell cycle block with vitamin D treatments. Within the G1/G0 arrest, several studies have demonstrated the upregulation of CDKIs, p21, and p27, while few studies have also shown decreased C-MYC expression and increased pRB expression. In addition, the mechanisms of cell cycle arrest by vitamin D demonstrate cell specificity. More data are needed to fully elucidate the mechanistic action of vitamin D on cell cycle proteins, especially to resolve the role of vitamin D metabolites’ anti-cancer actions in pre-clinical and clinical studies. The altered vitamin D metabolizing system by epigenetic marks may partly account for dysregulated vitamin D activation and signaling, and studies exploring this topic may provide insight into the perturbations of cell cycle arrest in cancer. The exploitation of cell arrest mechanisms by vitamin D and its analogs is a promising though currently unresolved therapeutic target.


Albatrellus flettii had potent anti-cell viability activity. Using bioassay-guided fractionation, mass spectrometry and nuclear magnetic resonance, we successfully isolated three known compounds . These compounds represent the major anti-cell viability components from the ethanol extracts of A. flettii. We also identified a novel biological activity for these compounds, specifically in down-regulating KRAS expression in two human colon cancer cell lines. Relatively little is known about the anti-cell viability activity and mechanism of action of confluentin. For the first time, we show the ability of confluentin to induce apoptosis and arrest the cell cycle at the G2/M phase in SW480 human colon cancer cells. The oncogenic insulin-like growth factor 2 mRNA-binding protein 1 (IMP1) has been previously shown to regulate KRAS mRNA expression in colon cancer cells, possibly through its ability to bind to the KRAS transcript. Using a fluorescence polarization assay, we show that confluentin dose-dependently inhibits the physical interaction between KRAS RNA and full-length IMP1. The inhibition also occurs with truncated IMP1 containing the KH1 to KH4 domain (KH1to4 IMP1), but not with the di-domain KH3 and KH4 (KH3&4 IMP1). In addition, unlike the control antibiotic neomycin, grifolin, neogrifolin and confluentin do not bind to KRAS RNA. These results suggest that confluentin inhibits IMP1-KRAS RNA interaction by binding to the KH1&2 di-domains of IMP1. Since the molecular interaction between IMP1 and its target RNAs is a pre-requisite for the oncogenic function of IMP1, confluentin should be further explored as a potential inhibitor of IMP1 in vivo.In our search for bioactive mushrooms native to British Columbia, we determined that the ethanol extracts from fruiting bodies of the terrestrial polypore  Albatrellus flettii and, specifically, its ethanol extracts.There have been relatively few studies exploring the medicinal potential of compounds from wild mushrooms native to British Columbia (BC), Canada. Therefore, we have begun to study medicinal properties of selected native fungi, focusing on three bio-assays relevant to cancer: anti-cell viability, immuno-stimulatory and anti-inflammatory activities ,2. ScreeAlbatrellus flettii (family Albatrellaceae) is an attractive terricolous polypore, often with a distinct blue-gray colouring on the cap; it is commonly found in western North America ranging from Alaska to California and Mexico, including throughout BC, Alberta, Washington, Oregon, Idaho and Wyoming (TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 Fig+ peak at m/z = 329.3.The ESIMS spectrum exhibited a [M + H](TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S9 Fig(TIF)Click here for additional data file.S10 Fig(TIF)Click here for additional data file.S11 Fig(TIF)Click here for additional data file.S12 Fig+ peak at m/z = 327.2.The ESIMS spectrum exhibited a [M + H](TIF)Click here for additional data file.S13 Fig(TIF)Click here for additional data file.S14 Fig(TIF)Click here for additional data file.S15 Fig(TIF)Click here for additional data file.S16 Fig(TIF)Click here for additional data file.S17 Fig(TIF)Click here for additional data file.S18 Fig(TIF)Click here for additional data file.S19 FigHT29 cells were treated with 20 μM (A) or 50 μM (A and B) of grifolin, neogrifolin and confluentin for 48 hours. Isolated cell lysates were then subjected to Western blot analysis.(TIF)Click here for additional data file.S20 Fig(A) A summary of 3’ and 5’ end truncated KRAS RNA fragments corresponding to nts 1–185, and their relative binding affinity for IMP1. (B) Electrophoretic mobility shift assay of 3’ and 5’ end truncated KRAS RNA fragments corresponding to nts 1–185. (C) A summary of 3’ and 5’ end truncated KRAS RNA fragments corresponding to nts 93–185, and their relative binding affinity for IMP1. (D) Electrophoretic mobility shift assay of 3’ and 5’ end truncated KRAS RNA fragments corresponding to nts 93–185.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Raw Images(PDF)Click here for additional data file.


Chlamydia pneumoniae. This structure defines the interface between these essential T3S proteins and reveals that CdsO engages the periphery of the export gate that may allow the ATPase to catalyze an opening between export gate subunits to allow cargo to enter the export apparatus. We also demonstrate through structure-based mutagenesis of the homologous export gate in Pseudomonas aeruginosa that mutation of this interface disrupts effector secretion. These results provide novel insights into the molecular mechanisms governing active substrate recognition and translocation through a T3SS.Type III protein secretion systems (T3SS) deliver effector proteins from the Gram-negative bacterial cytoplasm into a eukaryotic host cell through a syringe-like, multi-protein nanomachine. Cytosolic components of T3SS include a portion of the export apparatus, which traverses the inner membrane and features the opening of the secretion channel, and the sorting complex for substrate recognition and for providing the energetics required for protein secretion. Two components critical for efficient effector export are the export gate protein and the ATPase, which are proposed to be linked by the central stalk protein of the ATPase. We present the structure of the soluble export gate homo-nonamer, CdsV, in complex with the central stalk protein, CdsO, of its cognate ATPase, both derived from  Many pathogenic Gram-negative bacteria utilize T3SS to export virulence factors in a well-regulated manner. Most component proteins of the T3SS are highly structurally conserved, capable of recognizing and secreting diverse effectors, which are recruited to the cytoplasmic sorting complex by chaperones. This work describes the molecular architecture of two essential components of a T3SS, identifies the interface between the components, and establishes the necessity of this interaction for effector secretion. Bordetella, Chlamydia, EHEC/EPEC, Pseudomonas, Salmonella, Shigella, and Yersinia. Although T3S effectors are generally not conserved across species, the secretion apparatus itself (the T3S injectisome), is well-conserved. In addition, the injectisome shares structural similarities with the flagellar T3SS, which bacteria utilize for motility .,28.18,28rganisms . These sChlamydia trachomatis . The presence of a small residue with its sidechain pointed along the coiled-coil results in a backbone- mediated interaction between CdsO25-110 and CdsV  was useled-coil . The Cdsand CdsV . This insequence  and in psequence . This inC: CdsO25-110 complex in the context of the full injectisome, the CdsO25-110 structure was manually extended to contain residues 1–162 of the 168 residues of full-length CdsO from C. pneumoniae, using the C. trachomatis CdsO as a template  and bacteria were evaluated for secretion competency. Presence of the effector proteins ExoT and ExoS, and translocator proteins PopB and PopD, in Pseudomonas aeruginosa PA01 ΔexsE culture medium was compared with the presence of secreted proteins in the L635A/D636A double mutant and in wild type PcrD  suggests that CdsV likely interacts in vivo with ATPase-bound CdsO. While no direct measurement for CdsO concentration in Chlamydia is known, it is not among the ~470 relatively abundant proteins assessed by quantitative mass spectrometry, and is likely less abundant than CdsV, which was observed [25-110 exits the CdsVC ring at an ~60° angle  is mediated by conserved residues Phe72 and Leu76 of FliJ . Mutatioled-coil . Conversecretion . Howeverecretion . These rIn summary, we present the atomic features and interaction interface of two critical components of the T3SS cytoplasmic sorting complex- the export gate CdsV with the ATPase’s central stalk protein CdsO. Suprisingly, CdsO does not engage the central pore of CdsV, but instead docks at a peripheral intersubunit interface and is positioned to create an opening between CdsV subunits allowing a route for bound cargo to enter the secretion apparatus. Further biophysical studies will be essential to describe how the energetics of ATP hydrolysis and the proton motive force are coupled to drive contraction and dilation of the export gate to promote virulence.Chlamydia pneumoniae and cloned into a pET28 expression vector, to utilize the vector’s N-terminal hexa-His-tag and thrombin cleavage site. Protein expression was performed at low temperature (18°C for 16 hours) in BL21 Star (DE3), after addition of 1 mM isopropyl β-d-thiogalactopyranoside for induction. Bacteria were collected by centrifugation and flash frozen in liquid nitrogen for later use. Bacteria were lysed with an Emulsiflex homogenizer (Avestin) in 25 mM sodium phosphate pH 8.0, 150 mM NaCl, with 10 μg/mL leupeptin, 1 μg/mL hen egg white lysozyme, 1 mM PMSF, 1 μg/mL DNase I and 0.7 μg/mL pepstatin. The lysate was clarified by centrifugation, and CdsVC was purified with Talon metal affinity resin followed by gel-filtration in 10 mM HEPES pH 7.5, 150 mM NaCl . Nonameric peak fractions (CdsVC is a mixture of monomer/dimer and nonomer during gel-filtration) were pooled and concentrated with an Amicon ultrafiltration cell to 2mg/ml for crystallization. The CdsVC L638/D639A mutant was purified following the same protocol as for the WT CdsVC.CdsV residues 345–710 was amplified from Chlamydia pneumoniae and cloned into pET28. Protein expression and purification were performed as described above for CdsV, with the exception that the final gel-filtration buffer contained 300 mM NaCl.CdsO residues 25–110 were also amplified from C was crystallized by hanging drop vapor diffusion from a reservoir containing 100 mM HEPES pH 6.75 and 5% polyethylene glycol-6000 (PEG-6000), at 21°C. Crystals were obtained after ~ 2 weeks, cryoprotected using crystallization buffer supplemented with 20% glycerol, and cryo-cooled in liquid nitrogen. For heavy atom derivates, crystals were soaked in 1 mM heavy atoms in mother liquor for 2 days and harvested as for native crystals. X-ray data were collected at 100 K at LS-CAT Sector 21 at the Advance Photon Source . The data-collection statistics are given in 2-soaked crystal, with no evidence of bound gold, was used as a native. The data obtained from the crystals soaked in three heavy atoms- AuCl3, PtCl4 and UO2(CH3CO2)2, as well as an AuCl3 soaked “native” used as the input to SHARP [CdsVto SHARP  to solveto SHARP ,39. PhasC and residues 25–110 of CdsO (CdsO25-110). CdsVC and CdsO25-110 were mixed with ~10% molar excess of CdsO25-110 and crystals were grown from 100 mM Bis-Tris pH 6.0, 4% PEG 3350, and 200 mM ammonium acetate. The CdsVC -CdsO25-110 structure was determined by molecular replacement using CdsVC in Phenix.Crystallization CdsV-CdsO was performed using multiple CdsO constructs, with the final structure including CdsVfree sets.A partial model of CdsV was built in COOT  and usedC:CdsO complex. Refinement statistics are listed in https://consurf.tau.ac.il/), and the PISA server (http://www.ebi.ac.uk/pdbe/prot_int/pistart.html) [Refinement was performed in Phenix, and included individual B-factors, TLS refinement, non-crystallographic symmetry restraints, and secondary structure restraints. Grouped B-factors were used in refinement of the CdsVrt.html) . Structurt.html) .25-110. Calculations were performed using the TA instrument ITC Analyze software.Isothermal titration calorimetry measurements were performed on a TA Instruments Nano ITC. Measurements were performed at 20°C with a 300 μL cell volume and 24 x 2 μL injections with a stirring rate of 150 rpm. All proteins were in 10 mM HEPES pH 7.5, 500 mM NaCl. The respective protein concentrations were 15.0 uM, 12.1 uM, and 2.2 mM for nonameric CdsVc, nonameric CdsVc L638A/D639A, and monomeric CdsOC and the CdsVC L638/D639A double mutant were assessed by thermal unfolding based on intrinsic tryptophan fluorescence. The WT and mutant CdsVC proteins were diluted to 50 μM with gel filtration buffer and loaded into Tycho NT.6 capillaries . Experiments were performed using a NanoTemper Tycho NT.6 instrument. The temperature gradient monitored was from 35 to 95°C, increasing by 0.5°C sec-1. Protein unfolding was recorded by measuring changes in tryptophan fluorescence at emission wavelengths of 330 and 350 nm as a function of temperature. Inflection temperatures were determined by automatic fitting of fluorescence ratios (350/330 nm) with a polynomial function, where the maximum slope corresponds to the peak of its first derivative.The stability of WT CdsVpcrD open reading frame was constructed by pairing primers pcrDVG2-3-1 (5-ATTAGGAAAAGTGTACACGGACATCGAGATGAACAGGTTGGGCAAAAACGACCTGAGCGGGCTTCTCG-3') and pcrDVG2-5-2 (5'-ATGTCCGTGTACACTTTTCCTAATCTATTCATTTCAATATCTGTATAGTTCATTCCCGCGCCTCCAGCTCCAGC-3') with flanking primers pcrDC5X (5'-AAAAAtctagagACCTTCCTGGCTCTCGCGCTGCT-3') and pcrD3H . Flanking PCR products were combined by splicing by overlap extension PCR. The PCR products specifying mutations were then cloned as XbaI/HindIII fragments into the allelic exchange vector pEXG2 [P. aeruginosa PAO1 ΔexsE [Primers specifying the alanine mutations at codons 635 and 636  were paired with flanking primers  to generate flanks, which were subsequently joined by spicing by overlap extension PCR . The conor pEXG2 . The plaO1 ΔexsE  by matinexsE, PAO1 ΔexsE pcrD-VG2 and PAO1 ΔexsE pcrD(L635A+D636A)-VG2 were grown in LB supplemented with 2.5 g/L NaCl to late log phase. Cultures were harvested and resuspended in 2 mL LB with or without 5 mM EGTA. After 30 min, 1 mL of culture was pelleted, and protein was precipitated from 500 uL of supernatant. The pellets were resuspended and normalized to a final OD600 of 2.5. Samples were separated by SDS-PAGE on a 10% gel (BioRad) and transferred to a PVDF membrane. With the exception of RpoA and VSV-G-tagged PcrD, the indicated proteins were detected by Western Blot using affinity purified rabbit antisera. RpoA was detected using a commercial mouse monoclonal antibody (BioLegend), and VSV-G using a commercial rabbit antibody (Thermo).PAO1F ΔS1 Table(DOCX)Click here for additional data file.S1 FigFor CdsO, residues corresponding to α-helix 1 and 2 are indicated, as are the transmembrane and cytoplasmic domains for CdsV. The residue numbers of the protein regions used in the experiments described in this paper are highlighted in red.(TIF)Click here for additional data file.S2 FigC structure  to purple (conserved)), as determined by the ConSurf server, and based on the alignment of CdsVC orthologs from Pseudomonas, Shigella, Yersinia, Bordetella, Salmonella, and Vibrio (C include (clockwise from upper left) bottom, top, lateral, and slab views.Sequence conservation displayed on the CdsVd Vibrio . Surface(TIF)Click here for additional data file.S3 FigResidues corresponding to chain A are underlined. Chain A is colored as in (TIF)Click here for additional data file.S4 FigC from Chlamydia (Uniprot ID Q9Z8L5), Pseudomonas (Q9I327), Shigella (P0A1I5), Yersinia (P0C2V3), Bordetella (Q84CT3), Salmonella (A0A0F7J9S2), and Vibrio (A0A2A2ND56). Residues involved in the CdsVC oligomeric interface are highlighted in green. Conserved lysines and arginines that line the pore are indicated with red stars. Prolines 656 and 658 are highlighted in blue.Primary sequence alignment of CdsV(TIF)Click here for additional data file.S5 FigConserved lysines and arginines that line the pore can be observed in the slab view (lower left).(TIF)Click here for additional data file.S6 FigC structure (middle) and the CdsVC:CdsO structure (right). Apo-CdsVC is displayed to the left and colored by subdomain, for reference. (B) Helix 3 of subdomain 2 shifts toward subdomain 4 by ~5 Å when CdsVC is bound to CdsO (cyan), as compared to the apo structure (magenta). (C) β-strands 8 and 11 adjust by ~2.3 and ~2.5 Å in the presence of CdsO.(A) Superposition of protomers from the apo-CdsV(TIF)Click here for additional data file.S7 FigShigella flexneri (4A5P [Helicobacter pylori (3MYD [Despite the loops of subdomains 2 and 4 extending into the SD2-4 cleft (A-B), the unbound (A) and CdsO-bound CdsV (B) structures more closely align with the “open” conformation of FlhA, observed in both chaperone-bound forms (C-D). In the “closed” state of MxiA and FlhA (E-F), subdomains 2 and 4 dramatically shift to close the SD2-4 cleft. (A) Apo-CdsV, colored by subdomains as for ri (4A5P ); (F) Flri (3MYD ).(TIF)Click here for additional data file.S8 FigChlamydia (Q9Z7J9) with orthologs from Pseudomonas (A0A0C6F691), Shigella (P0A1K3), Yersinia (A0A0E1NFR4), Bordetella (A0A0E8FIJ9), Salmonella (P0A1K2), Vibrio (A0A0H6WY40), and Escherichia (B7UMA5). Thr 67, the residue at the center of the loop connecting the two helices of the CdsO coiled-coil, is indicated with a black star. (B) Representative cartoon of CdsO determined in this work, colored according to sequence conservation (using ConSurf), with an extended model of CdsO, shown as both cartoon and surface representation. More conserved residues are located near the N- and C-termini of the coiled-coil. (C) An extended model of CdsO, colored by electrostatic potential . The two views are obtained by 180° rotation about the y-axis.(A) Sequence alignment of CdsO from (TIF)Click here for additional data file.S9 FigChlamydia trachomatis CdsO structure (3K29) as a guide, residues of CdsO not visible in our crystal structure were modeled in COOT (colored in grey). (B) Comparison of the CdsVC-bound CdsO (left) with CdsO from C. trachomatis . (C) The structure of FliJ manually docked into the CdsO binding site of CdsV, with FliJ residues proposed to influence export gate binding and secretion [C and EscO from EscN from the crystal structure and cryo-EM structures (6NJP), respectively, are noted.(A) Using the DB 3K29; ); FliJ fe; 3AJW; ); and Ysecretion  shown asecretion . The cor(TIF)Click here for additional data file.S10 Fig25-110. (A) CdsVc binds CdsO25-110 with a Kd of 28 ± 3 μM and displays an exothermic isotherm. The L638A/D639A mutant does not show detectible binding toward CdsO25-110 an has an endothermic isotherm. (C) Thermal unfolding curves for CdsVc and the L638A/D639A mutant, revealing that both are quite stable with melting temperatures of 58°C and 55 C°, respectively.(A) and (B) show representative isothermal titration calorimetry traces for CdsVc and the L638A/D639A mutant titrated with CdsO(TIF)Click here for additional data file.S11 FigC (A) and CdsVC:CdsO (B).Top and side views, with the unit cell, of the crystal packing of CdsV(TIF)Click here for additional data file.


We reported here our development of a P24-VP8* nanoparticle-based trivalent vaccine. First, we established a method to produce tag-free P24-VP8* nanoparticles presenting the VP8*s of P[8], P[4], and P[6] rotaviruses, respectively, which are the three predominantly circulating rotavirus P types globally. This approach consists of a chemical-based protein precipitation and an ion exchange purification, which may be scaled up for large vaccine production. All three P24-VP8* nanoparticle types self-assembled efficiently with authentic VP8*-glycan receptor binding function. After they were mixed as a trivalent vaccine, we showed that intramuscular immunization of the vaccine elicited high IgG titers specific to the three homologous VP8* types in mice. The resulted mouse sera strongly neutralized replication of all three rotavirus P types in cell culture. Thus, the trivalent P24-VP8* nanoparticles are a promising vaccine candidate for parenteral use against multiple P types of predominant rotaviruses.Rotavirus causes severe gastroenteritis in children. Although vaccines are implemented, rotavirus-related diarrhea still claims ~200,000 lives annually worldwide, mainly in low-income settings, pointing to a need for improved vaccine tactics. To meet such a public health need, a P Rotaviruses are non-enveloped, double-stranded RNA viruses belonging to the family Reoviridae. The virus is capsulated by three-layered capsids that are made by multiple viral proteins organizing in an icosahedral symmetry. Rotavirus is an important pathogen causing infectious acute gastroenteritis in young children, commonly resulting in dehydration and even death. Several commercial rotavirus vaccines are currently available in the international market and have helped to substantially diminish rotavirus disease burden globally ,2. It isWhile the mechanisms behind the observed impaired effectiveness of the live rotavirus vaccines in the developing world remain elusive , increas24-VP8* nanoparticle has been generated as a rotavirus vaccine candidate /VP8* and VP7, respectively. Worldwide surveillance indicates that P[8] and P[4] are the two predominant P types in the developed world , account24-VP8* nanoparticles of rotavirus P[8] type were produced previously by expressing the P-VP8* fusion proteins with glutathione S-transferase (GST) tags for purification purposes , P[4], and P[6] rotaviruses, respectively. This includes a selective protein precipitation by ammonium sulfate, followed by a single step of ion exchange chromatography, which may be able to scale up to large production of the P24-VP8* nanoparticle vaccine. The P24-VP8* nanoparticles were characterized biochemically, morphologically, and functionally and then mixed as a trivalent vaccine. The vaccine was studied immunologically, proving it as a promising vaccine candidate against multiple rotavirus P types circulating around the globe.The Ppurposes ,41. Sinc24 nanoparticles , of a P[8] (strain 13851), a P[4] (strain BM5256), and a P[6] strain 11597) rotavirus, were PCR amplified from our lab stock plasmids  and clon97 rotaviarticles ,18. The Escherichia coli (E. coli)  induced by 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at room temperature (~25 °C) overnight [4)2SO4] at 1.0 M end-concentrations for 30 min to precipitate the P-VP8* proteins. After centrifugation at 5000 rpm for 20 min with an Avanti J26XP centrifuge  using a JA-17 rotor, the protein pellets were washed twice using 1 M (NH4)2SO4 solution in PBS. The P-VP8* protein precipitations were then solved in 20 mM Tris buffer (pH 7.5). The proteins were further purified by an anion exchange chromatography and then analyzed by gel-filtration chromatography (see below). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze protein quality using 10% separating gels. It was also used to quantitate proteins using serially diluted bovine serum albumin  with known concentrations as standards on the same gels , P[4], and P[6] rotaviruses, in equal molar ratio as a trivalent vaccine at 30 µg/mouse/dose; (2) the P24-VP8* nanoparticles of P[8] type at 10 µg/mouse/dose; (3) the P24-VP8* nanoparticles of P[4] type at 10 µg/mouse/dose; (4) P24-VP8* nanoparticles of P[6] type at 10 µg/mouse/dose; (5) the P24 nanoparticles without the VP8* antigens  proteins were eluted mainly in two peaks , respectively, were expressed in the 24-VP8* nanoparticles efficiently, as shown by a major peak . Each of the three individual nanoparticles displaying the VP8* antigens of P[8], P[4], and P[6] rotaviruses, as well as the P24 nanoparticle without the VP8* antigen, were also immunized to mice for comparisons. The outcomes . These results support the notion that the P24-VP8* nanoparticle trivalent vaccine is a potent one against multiple major rotavirus types circulating in both developed and developing worlds.The 50% neutralization titers of the mouse sera after administration with the trivalent vaccine were determined by fluorescence-based plaque reduction assays . Consist24-VP8* (this study) and S60-VP8* [Rotavirus P types are determined by the surface spike protein VP[4] with its distal head VP8* interacting with host glycan receptors to initiate viral infection, making the VP8* antigens an ideal vaccine target. The P[8] rotaviruses are the most detected rotavirus P type worldwide, followed by P[4] and P[6] types. Importantly, two recent studies ,51 indicS60-VP8* ,35 nanop24-VP8* nanoparticle vaccine against the three predominant P-type rotaviruses. We first designed three types of tag-free P24-VP8* nanoparticles and produced them using a scalable approach that consists of simple selective precipitation by (NH4)2SO4, followed by a further purification through an anion exchange chromatography. After proving their nanoparticle formation with correct size, morphology, and ligand binding function, these three P24-VP8* nanoparticles displaying the VP8* antigens of the P[8], P[4] and P[6] rotaviruses were mixed as a trivalent vaccine. Mouse immunization study showed that the trivalent vaccine elicited high antibody responses in mice to VP8*s of all three P types and the resulted mouse sera neutralized rotavirus replication of all three rotavirus P types in cell cultures. These data strongly suggested that our trivalent P24-VP8* nanoparticles are a promising parenteral vaccine candidate against rotavirus-associated diseases.In this study, we generated and evaluated a trivalent P24-VP8* nanoparticles. This GST removal step will impose a negative impact on future large production of the P24-VP8* nanoparticle vaccine. Thus, a simpler, scalable production approach is needed. We presented evidence here that tag-free P-VP8* protein can be selectively precipitated by (NH4)2SO4. After a further purification step by anion exchange chromatography, the P-VP8* protein reaches a high purity. Importantly, this method also increases the production yields of the P-VP8* proteins compared with those using the GST-tag method based on our lab-scale evaluation (data not shown). The tag-free P-VP8* proteins were shown to self-assemble into P24-VP8* nanoparticles efficiently with the expected glycan-binding function, validating the production and purification methodology for vaccine purpose.The P-VP8* proteins were made previously as GST-tagged fusion proteins for purification purposes , and the24-VP8* nanoparticles induced significantly lower IgG titers to the two heterologous VP8* antigens. These results indicate a necessity of a cocktail or trivalent P24-VP8* nanoparticle vaccine for a broader efficacy. Sequence alignments of the three VP8*s show that P[8] and P[4] VP8*s share higher sequence identity (84.3%) than those between P[8]/P[4] and P[6] (59.7%/60.4%), which explains the observed discrepancies in IgG cross reactivity. Similarly, consistent with their IgG titers, the mouse sera after immunization with the trivalent vaccine showed high neutralizing titers against all three P-type rotaviruses in cell culture. Although it has not yet been done, we anticipate that the mouse sera after immunization with each single-valent P24-VP8* nanoparticle would show lower neutralizing titers against the two heterologous P-type rotaviruses based on their cross-reactive IgG titers. These data support the trivalent P24-VP8* nanoparticles being a potent vaccine with broad effectiveness. Ideally, we should also examine the cross-antigenic reactivity and cross-neutralization titers against more heterologous P types of VP8*s and rotaviruses for a comprehensive understanding of the further broad efficacy of our trivalent vaccine. However, this could not be completed at this time due to the lack of necessary resources. To reach this goal in the near future, we plan to prepare more recombinant VP8* proteins representing further rotavirus P types to evaluate the breadth of the cross antigenic reactivity of the trivalent vaccine-immunized mouse sera. We also plan to reach out to other labs for additional cell-culture-adapted rotavirus strains of other heterologous P types for future neutralization study.The trivalent vaccine elicited similar high IgG titers to all P[8], P[4], and P[6] VP8*s, and the mouse sera revealed equally high neutralization titers to all the three rotavirus P types, while the single valent P24-VP8* nanoparticles representing the three VP8* types, respectively, in the current trivalent vaccine is based on the hypothesis that each monovalent antigen elicits similar immune responses by the same amount of antigens. This hypothesis has now been proved by comparing the similar IgG responses of the trivalent vaccine towards each of the three VP8* types (The equal molar ratio of the three individual P8* types . In fact24-VP8* nanoparticle, which has 24 VP8* antigens per nanoparticle and a large molecular mass of 1.25 mDa. Another VP8*-based vaccine candidate is the S60-VP8* pseudovirus nanoparticles that are composed of a T = 1 icosahedral norovirus inner shell with 60 surface-displayed VP8* antigens, having a large molecular weight of 1.47 mDa , P[4], and P[6] types are a promising parenteral vaccine candidate for broad protection against rotavirus infection and diarrhea in both developing and developed worlds.The outcomes of this study collectively demonstrated that the trivalent P


Epithelial to mesenchymal transition (EMT) is an essential program happened in the initial phase of cancer metastasis. We found that in HCC cells, TDO2 promoted the EMT process evidenced by altered levels of biomarkers for EMT. Mechanically, TDO2 regulated the Kyn production in HCC cell via activated aryl hydrocarbon receptor (AhR). Together, these results indicate that TDO2 promotes the EMT of hepatocellular carcinoma through activating Kyn-AhR pathway, thereby participating in the metastasis and invasion of HCC.Tryptophan 2,3-dioxygenase (TDO2), an enzyme involved in tryptophan (Trp) metabolism has been linked with some malignant traits of various cancers. Kyn, the main product of Trp metabolism pathway catalyzed by TDO2 and indoleamine 2,3-dioxygenase (IDO) in tumor cells, was also demonstrated to activate aryl hydrocarbon receptor (AhR), which may regulate cancer growth and invasion in some malignancies. However, whether TDO2 participates in the metastasis and invasion of HCC has not been explored before. The underlying mechanism played by TDO2 in this process still requires further investigation. Here, we demonstrated that overexpression of TDO2 correlates with advanced stage or malignant traits in HCC patients. Knockdown or inhibition of TDO2 suppressed the migration and invasion of HCC cells  Liver cancer, the fifth most common cancer, ranks the second leading cause of cancer-related death worldwide , 2. HepaTryptophan 2,3-dioxygenase (TDO2), encoded by gene Tdo2, is expressed normally at high levels in the liver. It acts as the first and rate-limiting step of tryptophan (Trp) metabolism along kynurenine (Kyn) pathway and maintains systemic tryptophan levels . Kyn, thvia activation of AhR leading to increased EMT.Epithelial to mesenchymal transition (EMT), a process that epithelial cells lose their polarized organization and acquire migratory and invasive capabilities, is considered to contribute to cancer metastasis , 12. Thein vitro and vivo experiments. Mechanistically, we demonstrated that TDO2 was responsible for the metabolism of Trp along Kyn pathway in HCC cells, and regulated the EMT process at least partly through Kyn-AhR pathway. Together, our results indicate that the overexpression of TDO2 promotes HCC metastasis capability through Kyn-AhR mediated induction of EMT.Here, we report our studies of the role of TDO2 in the metastasis and invasion of HCC, we searched TCGA database and mined data of the expression of TDO2 in different cancers. We found that the expression level varied in HCC and stomach adenocarcinoma according to different stages. The TDO2 expression was relatively high in HCC with vascular invasion and so was it in stomach adenocarcinoma with advanced stages in clinical samples. The effect of knockdown or inhibition of TDO2 on the EMT associated metastasis ability of HCC cell lines was investigated by Huh7 and LM3 HCC cell lines were transfected with two puro plasmid expressing sh-Tdo2 and scrambled control using transfection reagent . Two shRNAs were designed to knockdown of TDO2, shown as following.TDO2 inhibitor 680C91, Tryptophan and Kynurenine were purchased from Sigma-Aldrich, AhR inhibitor CH-223191 were purchased from Selleck Chemicals .pLKO.1puro-shhTDO2-AFCCGGGGAAAGAACTCCAGGTTTACTCGAGTAAACCTGGAGTTCTTTCCTTTTTpLKO.1puro-shhTDO2-ARAATTAAAAAGGAAAGAACTCCAGGTTTACTCGAGTAAACCTGGAGTTCTTTCCpLKO.1puro-shhTDO2-BFCCGGTCATAAGGATTCAGGCTAACTCGAGTTAGCCTGAATCCTTATGATTTTTpLKO.1puro-shhTDO2-BRAATTAAAAATCATAAGGATTCAGGCTAACTCGAGTTAGCCTGAATCCTTATGAAll clinical specimens were obtained with informed consent of patients in Shanghai general hospital and confirmed by pathologists. Twenty-three cases of paired specimens, HCC tissue and the adjacent normal tissue, and 16 cases of gastric cancer tissue were collected and stored in liquid nitrogen. Another 28 cases of HCC specimens were collected and stored in formalin. Human liver cancer cell lines  and normal human liver cell (LO2) were obtained from Type Culture Collection of the Chinese Academy of Science . Cell lines except for HepG2 were cultured in DMEM medium and HepG2 in MEM medium, with 10% fetal bovine serum (FBS)  and 1% penicillin-streptomycin under a humidified atmosphere containing 5% CO2 at 37°C.5 cells were firstly seeded per well in 6-well plates and cultured for 24 h, after which scratching with a 200 μl micropipette tip in the center of the well were performed. Then, the cells were cultured with serum-free medium and corresponding treatment. Images were captured at 0 and 48 h after scratch. The width of wound healing was measured, and migration rate was calculated.Scratch wound assays and Transwell chamber assays were applied to test the migration and invasion capabilities of HCC cells. For scratch wound healing assay, 5×10Transwell chamber assays with and without Matrigel-coated were performed to show cell migration and invasiveness. Cells were seeded at 20,000 cells or 40,000 per well in DMEM medium in the upper chamber without or with Matrigel coated, respectively. Six hundred μl Medium containing 10% FBS was added to the bottom chamber. Forty-eight hours later, cells in the upper surface of transwell chamber were erased with swab and cells transferred to the lower surface of the chamber were fixed in 4% paraformaldehyde and then stained with aniline violet for visualization and photography.Three independent experiments were carried out. Mean ± standard error of mean (SEM) was calculated for each of the experiments.Specimens and Cells were lysed in RIPA with 1% PMSF. Nuclear and cytoplasmic separation were guided according to the manual of Nucleo-cytoplasmic protein extraction kit . Cell protein extracts (50 μg) were denatured by boiling, separated on SDS-PAGE gels, and electrotransferred to polyvinylidene fluoride membranes . After the membranes were blocked with 5% skim milk dissolved in Tris-buffered saline containing 0.1% Tween20, they were incubated with primary antibodies overnight at 4°C, including anti-TDO2 , anti-AhR , anti-Cyp1b1, anti-E Cadherin, anti-N Cadherin, anti-MMP9 , or anti-Vimentin antibodies. The antibodies above were applied at the concentration of 1:500. Horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG was used as secondary antibodies, at the concentration of 1:2,000. Immunocomplexes were visualized with an ECL luminescence reagent . Glyceraldehyde 3-phosphate dehydrogenase  was used as an internal control. Densitometry quantification was performed using Image J.Total RNA was isolated using the traditional Trizol methods. cDNA was synthesized with ProFlex™ PCR system using PrimeScript™ RT Master Mix reagent kit . Quantitative reverse transcription PCR (qRT-PCR) was performed in Roche LightCycler 96 using SYBR Premix Ex Taq™ . All fold-change data were normalized to GAPDH. The 2-ΔΔCt method was used to calculate relative expression levels.2O2 was applied to incubate the tissue arrays for 10 min to quench endogenous peroxidase activity. After blocked with 1% bovine serum albumin for 20 min, tissue arrays were incubated with primary antibody, including TDO2 antibody, anti-N cadherin or anti-E cadherin, with 1:100 dilution overnight at 4°C, prior to incubation with biotinylated secondary antibody for 30 min at 37°C. Coloration lasted for 1 min in DAB . Images were recorded using Lax software under the unified parameters.The HCC tissue paraffin sections were subjected to de-paraffinization in xylene, rehydration through graded ethanol  and distilled water, prior to boiling in 10 mM citrate buffer solution (pH 6.0) for 15 min for antigen retrieval. Three percent HCells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Cells were permeabilized with 0.3% TritonX-100 in PBS for 20 min and then blocked with 1% BSA in PBS containing 0.3M glycine. Subsequently, the primary anti-AhR antibody (1:100 dilution) was added to the cells and incubated overnight at 4°C. The secondary FITC combined antibody  was diluted in 1:100 and added to cells for 30 min at room temperature. 40,6-diamidino-2-phenylindole (DAPI) was last added for 5 min to visualize the nuclear of cells. Immunostaining was observed at 400 magnification using the Leica TCS SP8 confocal microscope and images were captured using the Leica LAS-AF software .High performance liquid chromatography (HPLC) was used to analyze the concentration of Trp and Kyn. Cell culture supernatant was collected, centrifuged, and transferred to fresh tubes and frozen until subjected to analysis. Two hundred μL sample was precisely pipetted, and 200 μM perchloric acid was added for purification. The sample was mixed with vortex for 30 s and placed at room temperature for 5–10 min, followed by centrifugation for 5 min at 10,000 r/min. The supernatant was collected for test. VWD C18 column  was used as detector. Twenty μl sample was injected with the speed of 1.0 ml/min and measured at 225 nm wavelength. Fifteen mmol/L acetic acid: sodium acetate  was applied as mobile phase. The concentrations were calculated based on standard solutions.6 cells in 25μl PBS containing 25% Matrigel were injected into the subcostal region of the left lobe. Mice were sacrificed 6 weeks later, and the livers were removed, imaged, and embedded in paraffin. Hematoxylin and eosin staining were performed to confirm tumor metastasis. All animal experiments were approved by the Institutional Animal Care and Use Committee of Shanghai General Hospital.A total of 20 BALB/c nude mice were used for the orthotopic mouse model of HCC according to previous study . Mice weP value <0.05 is considered statistically significant.Statistical analyses were carried out and graphics were generated using GraphPad Prism 7.00. Results are shown as representative images or as mean ± SEM of at least three independent experiments. Data according with Gaussian distribution were analyzed using the unpaired t-test. The basic information parameters of patients were analyzed using Fisher’s exact test. Data shown in graphical format represented as means (± SEM) or medians with interquartile range. To investigate whether the expression of TDO2 correlates with HCC progress and other digestive maliganancies, we utilized the public data available in TCGA (The Cancer Genome Atlas) database. We found that stomach adenocarcinoma and esophageal carcinoma in advanced stages had higher TDO2 expression than that in early stages  and rectum adenocarcinoma , thymoma , testicular Germ Cell Tumors , and Uveal melanoma (UM)  .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.