An avian influenza A H7N9 virus emerged in March 2013 and caused a remarkable number of human fatalities. Genome variability in these viruses may provide insights into host adaptability. We scanned over 140 genomes of the H7N9 viruses isolated from humans and identified 104 positions that exhibited seven or more amino acid substitutions. Approximately half of these substitutions were identified in the influenza ribonucleoprotein (RNP) complex. Although PB2 627K of the avian virus promotes replication in humans, 45 of the 147 investigated PB2 sequences retained the E signature at this position, which is an avian characteristic. We discovered 10 PB2 substitutions that covaried with K627E. An RNP activity assay showed that Q591K, D701N, and M535L restored the polymerase activity in human cells when 627K transformed to an avian-like E. Genomic analysis of the human-isolated avian influenza virus is crucial in assessing genome variability, because relationships between position-specific variations can be observed and explored. In this study, we observed alternative positions that can potentially compensate for PB2 627K, a well-known marker for cross-species infection. An RNP assay suggested Q591K, D701N, and M535L as potential markers for an H7N9 virus capable of infecting humans. Most cases were from China, except four from the Taipei Centers for Disease Control (Taipei CDC), twelve from the Centre for Health Protection, Hong Kong SAR, one Chinese traveler reported by Malaysia, and 2 Canadian travelers returning from China. The first Taiwanese H7N9 infection was reported in April 2013 in a businessman returning from the Jiangsu province of China [Introduction of genetic variations in viral ribonucleoprotein (RNP) genes is a major virulence determinant for avian influenza viruses. Three genetic substitutions, PB2 E627K, D701N, and Q591R, have been reported to affect host cell tropism . In partThe novel influenza A H7N9 virus is an avian virus to affect humans , 19. In In this study, we determine the genome of the fourth H7N9 isolate from Chang Gung Memorial Hospital (CGMH) and describe the genome diversity of these Taiwanese isolates. We subsequently assess the genetic diversity of H7N9 genomes from cases reported between March 2013 and April 2015. In our previous studies, we performed large-scale scanning of the influenza A virus genomes and summarized a list of species-associated signatures to distinguish human and avian viruses , 28, 29.The present study aimed to characterize the genomic heterogeneity of human H7N9 viruses in the four Taiwanese patients, as well as all reported H7N9 genomes from the public databases since March 2013. Three genomes each belongs to the first three Taiwanese patients were among the database genomes we collected. These genomic sequence records are available to the general public through their web services . No patient information were ever made available through these services. The two genomes sequenced in this study were derived from an H7N9 virus isolate deposited to a virus bank maintained in the Clinical Virology Laboratory of CGMH. Such isolates were cultured from the clinical specimens collected by physicians for the purpose of medical diagnosis or public health investigation. Clinical information from any of these specimens were anonymized and de-identified prior to their inclusion to the virus bank such that the authors had no access to any identifying information at any time.The virus was isolated from sputum samples of the aforementioned patient admitted to the CGMH by using the Madin-Darby canine kidney (MDCK) cell line maintained in a Dulbecco’s modified Eagle’s medium  containing 0.1 mg/mL trypsin. For virus propagation, 200-μL of the sputum samples were injected into the allantoic cavity of 10- to 11-day-old embryonated eggs and incubated at 37°C for 3 days; subsequently, the allantoic fluid of the inoculated chicken eggs was harvested. Labels 4-CGMH1 and 4-CGMH2 represent the viruses isolated from egg passage 1 and MDCK cell passage 2, respectively, and “4” represents the fourth case of H7N9 in Taiwan.Viral RNAs were extracted from the cell culture supernatant or the allantoic fluid of the inoculated chicken eggs according to the manufacturer’s instructions using a QIAamp Viral RNA Mini Kit . The RNA was reverse transcribed into a cDNA by using SuperScript III reverse transcriptase (RT) . The polymerase chain reaction (PCR) was performed using a proofreading DNA polymerase KOD-plus  and the specific primers listed in http://www.ncbi.nlm.nih.gov/genomes/FLU/) [™ Database from the Global Initiative on Sharing Avian Influenza Data  [The full genome sequences of A/Taiwan/4-CGMH1/2014 and A/Taiwan/4-CGMH2/2014 are available in GenBank (KM244508–KM244523). Other H7N9 sequences included in this study were downloaded from the Influenza Virus Resource of National Biotechnology Information Center   and the id.org/) .http://mistic.leloir.org.ar/) to detect coupled mutations [Protein translation and multiple sequence alignment were performed using BioEdit (Version 7.2.5) . The posutations .A homology model of the PB2 protein for A/Taiwan/4-CGMH2/2014(H7N9) was constructed using SWISS-MODEL , which wThe RNP complex coding sequences derived from A/Anhui/1/2013(H7N9) were synthesized using DNA synthesis (IDT or GeneArt), cloned into the pcDNA3.1/myc-HisA and pFLAG-CMV5.1 vectors, and labeled H7N9-PB1, -PB2, -PA, and -NP. Site-directed mutagenesis was performed using pFLAG-CMV5.1-H7N9-PB2 to generate -K627E, -V139I&K627E, -K191E&K627E, -V511I&K627E, -M535L&K627E, -N559T&K627E, -M570I&K627E, -Q591K&K627E, -I647V&K627E, -M676V&K627E, and -D701N&K627E plasmids by mutagenic primer sets . PlasmidRNP activity was measured using a chloramphenicol acetyl transferase enzyme-linked immunosorbent assay . First, 293T cells were cotransfected with 1 μg of the H7N9 RNP components cloned into pcDNA3.1. Concurrently, a plasmid containing a reporter gene (CAT) flanked by the viral promoters (pPOLI-CAT-RT) was transfected into the cells. The total cell lysate was extracted 48 h after transfection using the 1× lysis buffer provided in the CAT ELISA kit. After quantifying the protein content, each protein sample was diluted to 5 μg/μL and serially diluted using the lysis buffer. Subsequently, the sample was assayed according to the manufacturer’s instructions to determine the CAT levels.With 10 amino acid substitutions, PB2 had the highest mutation , followeAmong the 46 positions listed in rd and 4th patients in 2014, suggesting that the H7N9 viruses have drifted away from the vaccine candidate. Many avian H7N9 viruses exhibited diverse genetic makeups at the listed amino acid positions in nd through the 4th Taiwanese patients. Even in avian H7N9 viruses, this mutation affecting receptor-binding was already seen in 396 of 460 HA sequences we examined. It was mentioned that two PB2 mutations Q591K and D701N could enhance polymerase activity of avian viruses in human 293T cells [Amino acid residues for the H7N9 vaccine candidate A/Anhui/1/2013 were included in 3T cells . PB2 591Over 140 H7N9 genomes recorded between late March 2013 and late April 2015 were retrieved from the NCBI and GISAID databases to comprehensively explore the H7N9 amino acid transitions. In a previous study, we reported 47 species-associated signatures that potentially marked a genetic boundary at which an avian influenza A virus can efficiently transmit to or replicate in humans . Table 2Only four human-avian signature positions developed human-like residues that dominated the H7N9 population . No humaOnly four . M535L is another mutation exhibiting the compensatory effect with 627E . However, the other seven amino acid substitutions that covaried with K627E exhibited no such effect.A CAT reporter RNP activity assay was performed to evaluate the effects of the PB2 K627E covariations. Only 10 mutations . These affirmed the finding by Mok et al. [P < 0.05). Although the two positions 591 and 627 were structurally close with apparent involvement in the same host factors, the residues 535 and 701 were spatially distant from 627, suggesting the involvement of a different mechanism. Of the 147 PB2 sequences we analyzed, only five had Q591K, and four of them occurred at an extremely early stage of H7N9 endemic in March and April 2013. In all, 15 D701N substitutions were observed, and none occurred after January 2014. Conversely, 23 M535L substitutions were observed in all three seasons, signifying its regular presence and potential role in the increasing H7N9 adaptation to humans.In addition to mutations at PB2 627, mutations at PB2 591 and 701 were strongly involved in mammalian host restriction . In thisSeveral other amino acids exhibited covarying patterns with K627E, which were consistent with those exhibited by Q591K, M535L, and D701N. However, the RNP activity assays suggested no correlation between the amino acid changes at these positions with PB2 K627E. Some of these substitutions, however, formed their own coevolving patterns. For example, we found {V139I, S286G, T569A, M676V}, occurring only in season II (November 2013–January 2014), in 12 strains from Shenzhen and Hong Kong, regardless of whether they were accompanied by 627E or K. Such amino acid coevolutions suggest their possible involvement in polymerase activities. We did not rule out any other coevolving residues that emerged in other H7N9 genes or across different genes. Further investigation is warranted to determine whether these changes occurred spontaneously or were correlated with host adaptability or other functions.RNP activity is measured by a constitutional assay. Influenza viral genes PA, PB1, PB2 and NP were cotransfected into cells to express RNP complex. Meanwhile, a reporter gene, such as luciferase, CAT, or GFP flanked by viral promoters was also transfected into cells to be driven by RNP complex . RNP actChen et al. proposed species-specific signatures that have been widely used as markers to assess the possibility of human infection from avian influenza viruses. These signatures were determined according to an analysis of 306 human and 95 avian virus genomes in 2006 ; these sNumerous human-isolated avian influenza A viruses exhibit PB2 627K, which is a human-specific signature, and strong biological evidence indicates that an E627K mutation promotes avian viral replication in mammals. However, 45 of the 147 human H7N9 isolates investigated in this study exhibited an avian signature E at this position. Additional sequence analysis suggested that 10 PB2 substitutions could potentially increase the efficiency of an avian virus in infecting humans in the absence of PB2 627K. We used a reporter assay to test all of these substitutions and showed that either Q591K, M535L, or D701N mutation increases the viral RNP activity in human cells with PB2 627E, suggesting that E627K, Q591K, M535L, and D701N are crucial markers for assessing the potential of an avian virus to infect humans, as well as for potentially increasing adaptation for the virus to gain human-to-human transmission capability in leading to a pandemic in the future.S1 Fighttp://mistic.leloir.org.ar/). MI scores are labeled on the arcs connecting these amino acids. Arcs are of variable thickness to approximate the MI scores. An MI threshold of 6.5 was used according to the server default setting. K627E was found coupled with D701N, N559T, K191E, K526R and Q591K. Two nearly remote clusters were also identified, including  and .Based on the alignment in (TIF)Click here for additional data file.S2 FigPB2 sequences of H7N9 viruses listed in (TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.


Human ovarian cancer cells (A2780), the corresponding cisplatin resistant cells (A2780cisR), and human embryonic kidney cells (HEK-293) were each incubated for 24 h and 72 h with [1]6+ and compared to untreated cells. Different responses were obtained depending on the cell type and incubation time. Most pronounced changes were found for lipids, choline containing compounds, glutamate and glutathione, nucleotide sugars, lactate, and some amino acids. Possible contributions of these metabolites to physiologic processes are discussed. The time-dependent metabolic response patterns suggest that A2780 cells on one hand and HEK-293 cells and A2780cisR cells on the other hand may follow different cell death pathways and exist in different temporal stages thereof. Following the success of platinum-based anticancer drugs, with cisplatin  being th50 values [p-cymene)6Ru6(tpt)2(dhnq)3](CF3SO3)6, [1]6+, shown in In our group, a series of water-soluble hexacationic arene ruthenium prisms have been prepared and probed for their cytotoxic activity and interactions with biological ligands –15. This0 values , (iii) a0 values  can lead0 values ,19 makinAlthough ruthenium complexes share some structural elements with platinum drugs, the mechanisms of action for most of the ruthenium complexes seem to follow different pathways which are not yet fully understood . For ins1]6+ (1H high resolution magic angle spinning (HR-MAS) NMR spectroscopy. In recent years, HR-MAS NMR has emerged as a powerful non-invasive tool for recording the profile of small metabolites in biological material such as cells or tissues 6+  was used tissues ,25. It h tissues ,27. In p tissues –31, doxo tissues , tamoxif tissues , or doce tissues  under vap-cymene)6Ru6(tpt)2(dhnq)3](CF3SO3)6 (= [1](CF3SO3)6) with tpt = 2,4,6-tri(pyridin-4-yl)-1,3,5-triazine and dhnq = 5,8-dihydroxy-1,4-naphthoquinonato was prepared as previously described (CF3SO3)6 in DMSO (c = 1 mM) to reach a final drug concentration of 0.9 μM for A2780 cells, 0.84 μM for A2780cisR cells, and 0.44 μM for HEK-293 cells corresponding to c = 2*IC50 for each cell line. Cells cultured in medium containing the same amount of DMSO only were used as controls. As IC50 values were collected at 96 h drug incubation, exposure times to [1]6+ were chosen well below this value (24 h) and at a longer period of 72 h. At these two time points (t = 24 h and 72 h) the cells were harvested by trypsination and centrifuged at 2000 rcf for 5 min. The supernatant was discarded and the cells suspended in 1 mL freezing medium . The freezing medium acts as cryoprotectant allowing for maintenance of cell integrity 6+ . Spectral regions between 0 and 6.4 ppm from the 1D noesy spectra were taken as basis for the analysis. The corresponding PCA scores plot for the first 3 principal components, explaining 56.8% of the variance, is shown in Principal component analysis (PCA) was applied to the Interestingly, a clear distinction could not just be observed for the different cell types but also for cells of the same cell line obtained from two different growth periods of 24 h and 72 h –S13 Figs1]6+ was tested against the three cell lines and compared to the toxicity of cisplatin assessed in the same experimental set-up. The corresponding IC50 values are given in 1]6+ was clearly higher against A2780 cancer cells with an IC50 value below 1 μM. Moreover, [1]6+ has an IC50 value in a similar range against the cisplatin resistant cell line (A2780cisR) indicating that the cytotoxic action seems to bypass the resistance mechanism, which is effective against cisplatin. However, the low IC50 value of [1]6+ against HEK-293 cells indicates that the complex despite its potency to overcome resistance does not exhibit selectivity. Our previous studies have suggested that compared to analog large ruthenium complexes the high cytotoxicity of the present compound may be related to its relatively high stability towards biological ligands [The cytotoxicity of [ ligands –15.1]6+ onto the metabolic profile of the three different cell lines, the spectral regions between 0 ppm and 6.4 ppm of the noesy1d HR-MAS spectra were divided into 97 individually sized buckets and evaluated by multivariate analysis. For this, the data were combined to 6 pairs of control and drug-treated sample sets according to the 3 cell lines and 2 incubation times used in this study. PCA applied to the 6 data sets resulted in a complete separation of control and drug-treated samples for each group  are shown in 1H HR-MAS spectra derived from control and drug-treated cell samples from both incubation times are shown for the spectral regions in which the main lipid signals  was observed after 24 h drug incubation, while the remaining lipid resonances did not contribute to separation from control cells (2)n and lactate—CH3 signals overlap n resonance, which was split into 3 buckets (2-CH = and—CH = CH-), respectively, while the unsaturated lipid resonances in A2780cisR cells were only increased by about 20% (p<0.01 for—CH = CH- after 24 h). The observed changes in choline containing compounds were approximately on the same order of magnitude for the three cell lines with a significant increase (after 24 h) and subsequent decrease (after 72 h) for PC and GPC in A2780 cells, while in A2780cisR and HEK-293 cells, only PC exhibited a significant increase after 24 h and 72 h, respectively  and alanine (Ala) with increased levels in A2780 cells after 24 h  and decrcpmg spectra. In this region there is less overlap and the number of metabolite resonances is much smaller as compared to the aliphatic region  and phenylalanine (Phe), which were increased in A2780 cells and HEK-293 cells (only Tyr) at 72 h drug incubation .1]6+ was not found to be uniform for the three different cell lines but seemed rather specific for each cell line. Moreover, the time course of the metabolite changes has to be considered since observed changes did not simply correlate with incubation time but were in part even opposite after prolonged periods of 72 h. In The metabolic response to treatment with 6+. This indicates that the lipid biosynthesis may not be enhanced but rather that existing lipids undergo transformation. Interestingly, a similar selective increase of poly-unsaturated fatty acids (PUFAs) with no concomitant increase of total lipids has been recently reported for osteosarcoma cells responding to doxorubicin and methotrexate treatment [In A2780cisR cells the overall lipid content remained more or less unchanged while there was an increase in the unsaturated lipid signals (significant for—CH = CH-) upon treatment with [reatment . Moreovereatment  or to otreatment  and was reatment  also leareatment .The results obtained from the downfield spectral region  support 1]6+ and GSH, however, [1]6+ catalyzed the oxidation of GSH [1]6+ most likely reflect enhanced GSH production to induce the detoxification. However, this cellular defense mechanism seems not to be effective against [1]6+. A possible reason for GSH accumulation may be that the GST catalyzed oxidation is inhibited by the metallaprism. Dose-dependent GST inhibition by polyphenolic compounds leading to increased intracellular GSH levels has been previously reported [1]6+ (An interesting observation was the increase in glutamate (Glu) and glutathione (GSH) found throughout in all cell lines both at short and long incubation times . In mostn of GSH . The increported . The dhned [1]6+  may play1]6+ depending on the cell line and the incubation time. The cell-type specific responses suggest different possible pathways of cell death induced by [1]6+. Pronounced effects induced on A2780 cancer cells may be caused by early necrotic stages while the cisplatin resistant A2780cisR and HEK-293 cells more likely seem to follow apoptotic cell death pathways. Metabolic profiles of untreated cells underwent time-dependent changes representing different stages of cellular growth in culture. Thus, HR-MAS NMR presents a snapshot of the metabolic state of the cells. Against this background metabolic transitions from short to long incubation times with [1]6+ observed for HEK-293 and cisplatin resistant A2780cisR cells on one side, and for A2780 cells on the other side, may reflect different stages towards their suggested cell death pathways.In conclusion, the presented HR-MAS NMR based metabonomic data revealed different metabolic responses towards treatment under same conditions with the hexacationic ruthenium metallaprism [1]6+. In A2780 and HEK-293 cells mainly the lipid metabolism was affected, however, with changes in opposite directions. For cisplatin resistant A2780 cells increased nucleotide sugars and unsaturated lipid components suggest that lipid biogenesis and glycosylation pathways are involved in the treatment response. Increased GSH levels found in all responding cell lines suggested the potential of [1]6+ to bypass cellular detoxification processes, which may be part of the cytotoxic active principle of [1]6+.Analysis of the most pronounced chemical alterations provides indications of possible mechanistic processes involved in the cytotoxic effects of [1]6+ may serve as biomarkers for treatment response and may be helpful for monitoring future studies aimed at the design of more selective Ru complexes as potential new alternative anti-cancer drugs.Nevertheless, the suggested cell death pathways and metabolic processes involved remain hypothetical and additional investigations including cell morphological studies will be required to further support the conclusions drawn from the present study. Resonance signals which proved to be sensitive towards [S1 Fig1H-spectra comparing freshly harvested and thawed cell suspensions for A2780, A2780cisR, and HEK-293 cell suspension in PBS, spectral region 0.5–4.6 ppm.HR-MAS (TIF)Click here for additional data file.S2 Fig1H spectra for a A2780cisR cell suspension in PBS recorded directly (0h), after 3h, 6h, 9h, and 13h under MAS conditions (MAS 3kHz) and T = 37°C. Spectral region 0.5–4.7 ppm and expansions.HR-MAS (TIF)Click here for additional data file.S3 Fig1H1H-TOCSY spectrum for a A2780 cell suspension in PBS, spectral region 0.5–5.5 ppm.HR-MAS (TIF)Click here for additional data file.S4 Fig1H1H-TOCSY spectrum for a A2780 cell suspension in PBS, spectral region 5.0–8.8 ppm.HR-MAS (TIF)Click here for additional data file.S5 Fig1H-J-resolved spectrum for a A2780 cell suspension in PBS, spectral region 0.5–4.5 ppm.HR-MAS (TIF)Click here for additional data file.S6 Fig1H-J-resolved spectrum for a A2780 cell suspension in PBS, spectral region 5.0–8.8 ppm.HR-MAS (TIF)Click here for additional data file.S7 Fig(A): 0.5–2.5 ppm, (B): 2.5–4.5 ppm, (C): 4.9–6.5 ppm.Individually sized bucket selection shown on an overlay of spectra obtained for A2780cisR control & drug treated cells (72h). The filled regions were excluded due to contaminations from ethanol, DMSO, and proximity to the residual water signal. (TIF)Click here for additional data file.S8 FigA, B), A2780cisR  and HEK-293 cells  at incubation times of 24h and 72h. Red: 95% confidence interval.PCA scores plots based on 1D noesy HR-MAS spectral regions between 0.5 and 6.5 ppm (97 buckets) of control (blue circles) and drug treated (green crosses) cells for A2780 ((TIF)Click here for additional data file.S9 FigPLS scores plot based on 1D noesy HR-MAS spectral regions between 0.5 and 6.5 ppm (97 buckets) of control cells A2780, A2780cisR, and HEK-293 cells grown for 24h. Blue line: 95% confidence level.(TIF)Click here for additional data file.S10 FigA) Loadings on the first PLS component LV1 and (B) for the second PLS component LV2. For bucket assignments, see Loading plots for PLS scores shown in (TIF)Click here for additional data file.S11 Fig(A) PLS scores plot based on 1D noesy HR-MAS spectral regions between 0.5 and 6.5 ppm (97 buckets) of A2780 control cells grown for 24h (red) and 72h (blue). Blue line: 95% confidence level (B) Corresponding PLS loadings for LV-1. For bucket assignments, see (TIF)Click here for additional data file.S12 Fig(A) PLS scores plot based on 1D noesy HR-MAS spectral regions between 0.5 and 6.5 ppm (97 buckets) of A2780cisR control cells grown for 24h (red) and 72h (blue). Blue line: 95% confidence level. (B) Corresponding PLS loadings for LV-1. For bucket assignments, see (TIF)Click here for additional data file.S13 Fig(A) PLS scores plot based on 1D noesy HR-MAS spectral regions between 0.5 and 6.5 ppm (97 buckets) of HEK-293 control cells grown for 24h (red) and 72h (blue). Blue line: 95% confidence level. (B) Corresponding PLS loadings for LV-1. For bucket assignments, see (TIF)Click here for additional data file.S14 Fig(A) 24h and (B) 72h. Red line: arbitrary threshold set to a load value of +/- 0.1.Loading plots for the first PLS component LV1 and 97 variables (= buckets) for PLS comparing A2780 control cells versus drug treated at (TIF)Click here for additional data file.S15 Fig(A) 24h and (B) 72h. Red line: arbitrary threshold set to a load value of +/- 0.1.Loading plots for the first PLS component LV1 and 97 variables (= buckets) for PLS comparing A2780cisR control cells versus drug treated at (TIF)Click here for additional data file.S16 Fig(A) 24h and (B) 72h. Red line: arbitrary threshold set to a load value of +/- 0.1.Loading plots for the first PLS component LV1 and 97 variables (= buckets) for PLS comparing HEK-293 control cells versus drug treated at (TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Vitamin D is required not only for bone health but also has been reported to play a role in a range of ailments such as autoimmune disease, cardiovascular disease, type 2 diabetes, hypertension, depression, and certain types of cancer. Several studies have reported that poor vitamin D status during childhood and adolescence is related to obesity and metabolic syndrome. Vitamin D deficiency has been associated with hypertension and increased risk of cardiovascular disease while 25(OH)D concentration is known to be independently associated with both insulin sensitivity and beta cell function. A recent study reported that low serum 25(OH)D to be associated with obesity and metabolic syndrome in Korean children. We investigated the association between serum 25(OH)D concentrations and the presence of metabolic syndrome components in Korean children and adolescents. We also compared the components of metabolic syndrome according to vitamin D status in Korean children and adolescents.2) waist circumference, and blood pressure were performed. Serum lipid, fasting plasma glucose (FPG), insulin and 25(OH)D levels were measured. HOMA-IR (homeostasis model assessment for insulin resistance) = [glucose (mmol/L) x insulin]/22.5 values were calculated. Metabolic syndrome defined by modified National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATP III).The study included 141 Korean children and adolescents who were aged 6 to 18 in Kangdong Sacred Heart Hospital. Anthropometric measurements, including height, weight, body mass index  children and adolescents had metabolic syndrome. Children and adolescents who have metabolic syndrome have significantly lower serum 25(OH)D concentration than those who do not have (12.35 ± 4.65 vs. 14.81 ± 4.63 ng/mL) (p=0.015). Waist circumference, SBP, fasting plasma glucose, insulin, HOMA-IR are significantly correlated with serum 25(OH)D concentration (p<0.05). Only LDL-cholesterol is significantly different according to tertile groups of serum 25(OH)D concentration. The odds ratio of group I (25(OH)D <11.50 ng/mL) is 1.826 compared to group III (25(OH)D >16.30 ng/mL).Children and adolescents who have metabolic syndrome have lower serum 25(OH)D concentration. The prevalence of metabolic syndrome may be higher in children and adolescents with severe 25(OH)D deficiency. Further investigation will be needed to identify the influence of 25(OH)D on metabolic syndrome.


Arabidopsis NAC transcription factor NAC072 has been reported as a transcriptional activator in Abscisic acid (ABA)-responsive gene expression. However, the exact function of NAC072 in ABA signaling is still elusive. In this study, we present evidence for the interrelation between NAC072 and ABA-responsive element binding factor 3 (ABF3) that act as a positive regulator of ABA-responsive gene expression in Arabidopsis. The transcript of NAC072 is up-regulated by ABF3 in ABA response, and NAC072 protein interacts with ABF3. Enhanced ABA sensitivity occurs in nac072 mutant plants that overexpressed ABF3. However, overexpression of NAC072 weakened the ABA sensitivity in the abf3 mutant plants, but instead of recovering the ABA sensitivity of abf3. NAC072 and ABF3 cooperate to regulate RD29A expression, but are antagonistic when regulating RD29B expression. Therefore, NAC072 displays a dual function in ABF3-mediated ABA-responsive gene regulation.The NAM, ATAF1/2, and CUC2 (NAC) domain proteins play various roles in plant growth and stress responses.  Arabidopsis, the ABA-responsive element binding factor (ABF/AREB) members  can bind the ABA-responsive element (ABRE) and function in ABA-dependent signaling pathway  is an important signaling molecule that enables plants to tolerate unfavorable environmental stresses such as drought, salt, cold, heat, and oxidation . Core co pathway . Principelopment .Arabidopsis. For example, ORESARA1 (ORE1/AtNAC2) positively regulates the salt stress response  TFs contain a plant-specific highly conserved N-terminal domain and play various roles in plant development, senescence and the response to environmental stress . The molresponse ; JUNGBRUresponse ; NAC016 response ; and NACresponse . These rnac019nac055nac072 was only slightly more sensitive to ABA or dehydration than the single mutants of nac019, nac055, and nac072 or the double mutants of nac019nac055, nac055nac072, and nac019nac072 , nac072 (SALK_083756), nac055 (SALK_014331), and nac019 (SALK_096295) were obtained from the Arabidopsis Biological Resource Center (ABRC1). The seeds for nac072abf3, nac055/72, nac019/72, and nac055/072/019 (3P) were obtained by hybridization. All seeds were surface sterilized in 70% ethanol containing 0.5% Tween-100. The supernatant was discarded and the seeds were suspended in ethyl alcohol and then air dried on filter paper. The seeds were sown on 0.5x MS medium with 0.8% agar containing 2% sucrose. After 2 days stratification at 4°C, the seeds were germinated and grown in a climate chamber under a daily cycle of 16 h light and 8 h dark at 20 ± 2°C. To examine the effects of ABA on germination and the development of green cotyledons, surface sterilized seeds were sown on 0.5x MS medium with 0.8% agar containing various concentrations of ABA . The root growth experiment was conducted at 3 days after germination, for which the seedlings were transferred to new 0.5x MS media with 0.8% agar containing 10, 30, or 50 μM ABA.The ABF3 or NAC072 were amplified and cloned into the modified pCAMBIA1301 plasmids (p35S: mCherry-6Myc or p35S:eGFP-3Flag) to obtain p35S:ABF3-mCherry-6Myc or p35S:NAC072-eGFP-3Flag, respectively. The primers used to construct these plasmids are listed in Supplementary Table Arabidopsis by the floral dip method using Agrobacterium tumefaciens strain GV3101 as previously reported  and NAC072-C (168–297aa) were amplified and cloned into pGBKT7 or pGADT7 (Clontech), respectively. The sequence encoding full-length ABF3 was amplified and cloned into pGADT7 or pGBKT7. The primers used to construct these plasmids are listed in Supplementary Table TM RT reagent Kit  was used for reverse transcription. Real-time PCR was performed in an ABI 7500 system with the SYBR® Premix DimerEraserTM , each reaction consisted of 20 ng of cDNA, 0.1 μM primers and 10 μL 2x SYBR Premix ExTaq in a final volume of 20 μL. The reactive cycle used 95°C for 30 s, then 40 cycles at 95°C for 5 s and 60°C for 34 s. The gene expression quantity was calculated using the relative 2-ΔΔCT method as previously reported , and a PrimeScriptreported . The relescribed . The prepRD29A:GUS or pRD29B:GUS reporter constructs, a ∼1200 bp region of the RD29A promoter or a ∼600 bp region of the RD29B promoter was cloned into pGreen vectors that was kindly provided by Hou XL Lab. The effector constructs (p35S:ABF3-mCherry-6Myc or p35S:NAC072-eGFP-3Flag) were generated by amplifying the corresponding cDNAs and then cloning into pGreen-35S. The p35S:luciferase (LUC) plasmid was used as an internal control to evaluate protoplast transfection efficiency. Arabidopsis mesophyll protoplasts were prepared and subsequently transfected as previously described , respectively. The protein expression level of NAC072 and ABF3 was confirmed using WB with Flag or c-Myc antibody. The presented data are the average of three technical replicates, and experiments were biologically repeated at least three times with similar results.To create the escribed , after wpGreen binary vector containing C- or N-terminal fusions of EYFP, generated Cp35S:ABF3-EYFP and N.p35S:NAC072-EYFP Then both of them cotransformed into Arabidopsis mesophyll protoplasts as previously described , ABF3 (AT4G34000), ACT2 (AT3G18780), RD29A (AT5G52310), RD29B (AT5G52300), NAC055 (AT3G15500), and NAC019 (AT1G52890).Sequence data from this article can be found in the NAC072 improves ABA sensitivity in Arabidopsis, whereas loss of NAC072 function in plants reduces ABA sensitivity . The NAC072 expression was dramatically reduced in abf3 after ABA treatment, whereas the transcript level of ABF3 was not affected in the NAC072 deficient mutant nac072 . It suggests that the expression of NAC072 is mediated by ABF3 under ABA treatment. Meanwhile, RD29A and RD29B are rapidly induced by drought stress or ABA, and are thus considered to be stress-response marker genes . These suggest that NAC072 may cooperate with ABF3 in the regulation of RD29A gene expression, while acting antagonistically with ABF3 in the regulation of RD29B gene expression. Thus, NAC072 seems to play a dual role in ABA-inducible gene expression in combination with ABF3.Previous researches  and our er genes . Both RDabf3, nac072, and abf3nac072 mutants by the physiological experiments on germination, the development of green cotyledons and axial root growth. During the germination process, no significant difference was observed between WT, abf3, nac072, and abf3nac072 under 0, 0.5, or 2 μM of ABA treatments . However, remarkably, these mutants showed ABA insensitivity during the development of green cotyledons and axial root growth compared with the WT plants, especially the double mutant abf3nac072 displayed the greatest ABA insensitivity . All of the above results indicate that NAC072 partly cooperates with ABF3 in the ABA response during the development of green cotyledons and axial root growth, but not during the germination process. However, it seems contradictory that there is cooperation between NAC072 and ABF3 with respect to physiological function, yet there is antagonism between these TFs in the mediation of ABA-inducible expression of genes such as RD29B. Therefore, NAC072 may perform a dual function in ABF3-mediated gene regulation of the ABA response.To examine this implication, ABA sensitivity was further assessed in Figure 2A), and NAC072 and ABF3 also interact with each other in the nucleus  promoter in WT plants. NAC072-OX/abf3 plants were obtained by crossing NAC072-OX transgenetic line with the abf3 mutant. For comparison, ABF3-OX/072 plants were produced by crossing ABF3-OX with the nac072 mutant. All homozygotes were determined by hygromycin resistance screening and tri-primer PCR after F2 generation segregation (data not shown). Accumulations of NAC072 or ABF3 in these plants were shown by western blot using antibodies against a Flag or c-Myc protein tag . Thus, ABA sensitivities of these plants were examined during the development of green cotyledon and axial root growth. Overexpression of NAC072 or ABF3 (in the NAC072-OX or ABF3-OX plants) resulted in slight growth inhibition and enhanced ABA sensitivity  resulted in less ABA sensitivity than in NAC072-OX or WT plants, and NAC072-OX/abf3 had less ABA sensitivity than abf3  showed further improved ABA sensitivity compared with ABF3-OX or WT plants  increased the transcription level of both RD29A and RD29B. In NAC072-OX/abf3 plants, RD29A and RD29B expressions were enhanced under control conditions, but little change was observed after ABA treatment . This suggests that, under ABA treatment, without ABF3, NAC072 cannot enhance the expression of RD29A or RD29B. That is NAC072 regulated the expression of RD29A and RD29B after ABA treatment need the ABF3 mediation. On the other hand, the loss of NAC072 function further contributed to ABF3-enhanced RD29B expression in ABF3-OX/nac072 plants, but there was no obvious change in RD29A expression . These findings add further confirmation that NAC072 is antagonistic to ABF3-mediated RD29B gene expression.To understand the crosstalk between NAC072 and ABF3 in ABA signaling, the regulations of NAC072 and ABF3 on RD29A and RD29B by ABF3 and/or NAC072 was further analyzed using GUS reporter systems. The 1200 bp region of the RD29A promoter and 600 bp region of the RD29B promoter were each fused with the GUS reporter gene. Either NAC072 or ABF3, driven by the 35S promoter, was co-expressed with each GUS construct in protoplasts. The result showed the RD29A promoter fusion with the GUS gene was more strongly induced by p35S::ABF3 together with p35S::NAC072 than by p35S::ABF3 or p35S::NAC072 alone , indicating that NAC072 cooperates with ABF3 to mediate the expression of the RD29A gene. A different regulation pattern was observed for RD29B expression. The single p35S::ABF3 expression resulted in a stronger transactivation of RD29B compared with p35S::NAC072 alone, or with p35S::ABF3 and p35S::NAC072 combined . These results further indicate that NAC072 acts antagonistically with ABF3 in regulating the expression of RD29B. Taken together, NAC072 may function as a cofactor, which not only cooperates with but also antagonizes ABF3-mediated regulation of the ABA response.The expression regulation of Arabidopsis NAC homologous genes . ABA sensitivity analysis indicated that NAC055 plays an important role during germination process, while NAC072 partly inhibited the expression of RD29B and RAB18, both of which are widely described as ABA signaling marker genes as their expressions are always induced by dehydration or ABA . Therefore, the ABF3-mediated ABA signaling pathway was chosen to illuminate the role of NAC072 in the ABA response. Subsequently, we uncovered four key points as follows: (1) the transcription level of NAC072 is partly regulated by ABF3; (2) NAC072 interacts and cooperates with ABF3 to regulate gene expression (including that of RD29A) in the ABA response; (3) NAC072 partly depends on ABF3 for its role in the ABA response during the development of cotyledon and main root growth; (4) NAC072 antagonizes the action of ABF3 to some extent in the regulation of RD29B expression.ABF3 is the key downstream regulator of ABA signaling and is important for ABA-mediated main root growth. Reprogramming the drought response by changing the timing or strength of expression of some drought-responsive genes was observed in plants with overexpressed ABF3 . ABA senNAC072/RD26 as well as NAC019 and NAC055/AtNAC3 is independent of ABF3 . Loss of ABF3 function reduced NAC072 transcript levels, but the expression of NAC072 could still be detected after ABA treatment . Hence, the expression of NAC072 seems partly dependent on ABF3.Transcriptome data from plants overexpressing ABF3 show that the expression of  of ABF3 . In accoFigure 2A). It seems likely, then, that the contradictory roles of NAC072 in ABA signaling might be resolved by an understanding of its relationship with ABF3. We assumed that NAC072 functions as a cofactor with ABF3 in the ABA signaling pathway. Accordingly, ABA sensitivity and ABA-inducible genes were analyzed in abf3, nac072, and abf3nac072 mutants . The results indicate that NAC072 cooperates with ABF3 in mediation of RD29A expression, but antagonizes RD29B expression. It is well known that both RD29A and RD29B rapidly respond to various stresses and ABA treatment (RD29B was directly and positively regulated by ABF3, while NAC072 may act as a repressor during this process. To further confirm this, NAC072 was overexpressed in WT and abf3 mutant plants. The expression of RD29B was enhanced in NAC072-OX/WT and NAC072-OX/abf3 plants under control conditions, but no change in expression occurred after ABA treatment. In contrast, overexpressed ABF3 rapidly increased RD29B transcription levels in control as well as ABA-treated plants. The combined loss of NAC072 function and ABF3 overexpression contributed to enhanced RD29B expression. Moreover, NAC072-OX/abf3 plants were more ABA insensitive than WT or NAC072-OX/WT plants, whereas ABF3-OX/nac072 was more ABA sensitive than WT or ABF3-OX/WT plants. In addition, NAC072 collaborates with ABF3 to regulate RD29A expression. Thus, we hypothesize that NAC072 and ABF3 may act as a “push-pull” device. Under certain conditions, NAC072 cooperates with ABF3-mediated gene expression, thus providing a “push” stimulus, while under other conditions it acts antagonistically to ABF3, giving a “pull” effect. How the device works would depend on specific conditions, a detailed understanding of which requires further investigation.ABF3, AREB2, and AREB1 can form homo- or heterodimers and have redundant function . Some NAreatment . In the XyL, XuL, and LL designed the research. XL, XlL, and YY performed research. XyL and XlL analyzed data. XyL and XuL wrote the article. The protein interaction experiments mainly performed for the revisions draft by XyL and ML.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.


E1Aand E2B  seem to be promisingtargets. Here, we propose an effective approach to downregulate the replicationof human species D adenoviruses by means of RNA interference. We generatedE1A expressing model cell lines enabling fast evaluation ofthe RNA interference potential. Small interfering RNAs complementary to theE1A mRNA sequences of human species D adenoviruses mediatesignificant suppression of the E1A expression in model cells.Furthermore, we observed a strong downregulation of replication of humanadenoviruses type D8 and D37 by small hairpin RNAs complementary to theE1A or E2B mRNA sequences in primary humanlimbal cells. We believe that our results will contribute to the development ofefficient anti-adenoviral therapy.Adenoviruses cause a wide variety of human infectious diseases. Adenoviralconjunctivitis and epidemic keratoconjunctivitis are commonly associated withhuman species D adenoviruses. Currently, there is no sufficient or appropriatetreatment to counteract these adenovirus infections. Thus, there is an urgentneed for new etiology-directed therapies with selective activity against humanadenoviruses. To address this problem, the adenoviral early genes  Adenoviral ocular infections are of great current concern in biomedicine due tothe wide prevalence and high rate of adenovirus infection episodes.Adenoviruses commonly cause human respiratory and gastrointestinal infections,adenoviral conjunctivitis (inflammation of the conjunctiva) and epidemickeratoconjunctivitis (combined inflammation of the cornea and conjunctiva). In RussCurrently, the limited efficacy of therapies for the adenovirus infection dictatesE2Bgene and E1A gene, which are involved in viral DNAreplication, seem to be potential targets for antiviral therapy [Human adenovirus early genes, such as the DNA polymerase  therapy -11.E1A gene products promote the G0 to S phase cell cycletransition. Interaction between E1A gene products and theretinoblastoma protein (pRb) activates the E2F1 transcription factor,triggering expression of the genes necessary for progression through the Sphase. This enables the adenovirus to replicate in the infected cell using thehost’s replication machinery. One of the functions of the E2F1transcription factor is transactivation of the p14/ARF protein that isaccompanied by induction of p53-dependent apoptosis. During viral infection,the 55 kDa E1B protein inactivates p53, thereby preventing induction ofapoptosis in the cell until the adenovirus replication cycle is completed[ompleted, 13.Small interfering RNAs (siRNAs), which are short 21–23 bp duplexes with2–3 overhanging nucleotides at 3’-ends capable of suppressing geneexpression at the post-transcriptional level, are commonly used in genefunction studies , 15. IntE1Aand E2B expression [We believe that this approach can be used for downregulation of the pression , 11 of HE1A gene expressiondownregulation in human species D adenoviruses types D8, D19, D36, and D37using siRNAs and shRNAs, as well as inhibition of HAdVs D8 and D37 replicationupon simultaneous downregulation of E1A and E2Bexpression using shRNAs.This paper presents the results of Cell cultureL-glutamine, 1 mM sodium pyruvate, andstreptomycin/penicillin at a concentration of 100 μg/mL and 100 U/mL,respectively, at 37°C under 5% CO2 atmosphere. Primary humanlimbal cells [L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 0.4 μMinsulin, 10 nM dexamethasone, and streptomycin/penicillin at a concentration of100 μg/ mL and 100 U/mL, respectively, at 37°C under 5%CO2.Human embryonic kidney cells HEK293 , human lal cells , 25 wereLentiviral vectors5 to 5 × 106were further used.Recombinant lentiviral vectors were constructed using standard geneticengineering techniques , 27. RecModel cell linesE1A gene of the human adenovirustype 36 (E1A-D36) were produced by transduction of A549 andH1299 cells with LeGO-iGT-Puroopt- based pseudolentiviral particlesE1A-LeGO-iGT containing an expression cassette“promoter–E1A gene of HAdVD36–IRES–dTomato marker gene –puromycinresistance gene.”Model cells expressing the  Cerulean fluorescent protein gene, and the blasticidin(BSD) resistance gene. 48 h post transduction, the cells were placed into aselective medium with 5 μg/mL blasticidin. Selection was conducted for 10days. H1299 shE1A cells were then analyzed by flow cytometry.In order to generate a H1299 shE1A model cell line, the initial H1299 cellswere transduced with lentiviral particles containing a sequence encoding shE1A,theLimb shE2B, Limb shE1A, and LimbshE2B/shE1A model cell lines were prepared bytransduction of primary human limbal cells with pseudolentiviral particlesencoding shE2B , 11 or ssiRNAsE1AmRNA sequences of HAdVs types 8, 19, 36, and 37. xpression. Tosuppress the target gene expression the following 21 bp siRNAs were synthesized: siE1A-1 ;siE1A-2, and siE1A-3 . siScr having no homology with knownviral mRNAs as well as human, mouse, and rat mRNAs  were used as a control. The siRNAswere designed using the Whitehead Institute siRNA Selection Program [We designed siRNAs complementary to different regions of the  Program .siRNA transfection4 cells per well, 1 day prior to the experiment and transfectedwith siRNAs at a concentration of 200 nM using a Lipofectamine® 2000Transfection Reagent (Life Technologies) according to the manufacturer’sprotocol.Cells in the exponential growth phase were seeded into 24-well plates, 3 ×10shRNAsWe constructed shE1A-LeGO-Cer/BSD and shScr- LeGO-Cer/BSD lentiviral vectorsencoding shRNAs: shE1A, which corresponds to siE1A-1  and a control shScr . In this work, we also used lentiviral vectors shE2B-LeGO-G  andshScr-LeGO-G, which were described previously in , 11.Flow cytometryThe cell fluorescence intensity was measured by an Epics 4XL flow cytometer. Obtained results were analyzed with WinMDI software,version 2.8Real-time PCRE1A-D36 mRNA levels wereassessed in real-time PCR using specific primers : sense sequence5’-GCATCCAGAGCCATTTGAGC-3’; antisense sequence5’-TTAGGGTCGTCATCATGGGC-3’. Resulting values for each sample werenormalized to the β-actin housekeeping gene expression. The β-actinexpression level was quantified using the following primers: sense sequence5’-ATGGATGATGATATCGCCGC- 3’and antisense sequence5’-CTTCTGACCCATGCCCAC- 3’. Real-time PCR was performed in 96-wellplates using a MiniOpticon system  and an iQ SYBR Green Supermixreagent (Bio- Rad) in accordance with the manufacturer’s recommendations.PCR products were analyzed using the MiniOpticon system software (Bio-Rad).Total RNA was isolated from cell cultures using a TRIzol® reagent (LifeTechnologies) according to the manufacturer’s protocol. Reversetranscription was carried out using ImProm-II™ Reverse Transcriptase. Quantification of the human adenovirus genome copy numberHAdV D8 (ATCC® VR-1085AS/RB™ ATCC® VR- 1085AS/RB™) andHAdV D37 (ATCC® VR-929™) were purchased from theAmerican-Type-Culture-Collection (ATCC). Replication of species D humanadenoviruses was evaluated 6 days post infection. For this, HAdV D8 and D37infected cells were harvested, and total DNA was isolated using a QIAamp DNAMini Kit  according to the manufacturer’srecommendations. Quantitative PCR was performed as described in [Statistical data processingp < 0.05 was considered statistically significant.All data are presented as a mean ± standard deviation (SD). Thestatistical significance was determined using the unpaired two sample t-testand GraphPad Prism software 6 . The value ofE1AgeneDerivation of model cell lines expressing the HAdV D36 E1A-D36) were obtained in orderto analyze the functional activity of synthetic siRNAs and lentiviral vectorsdirecting the synthesis of shRNAs in transduced cells.Model cell lines A549 E1A and H1299 E1A expressing the E1A gene of humanspecies D adenovirus type 36 containing the expression cassette “promoter– target E1A-D36gene–IRES–dTomato fluorescent protein gene–puromycinresistance gene.” The marker gene encoding the dTomato fluorescentprotein and the target E1A gene are separated by the IRESsequence . IRES allows for the synthesis ofseveral proteins from a single mRNA in eukaryotic cells. Thus, the target andmarker genes are expressed with a comparable efficiency[E1A expression level via measuring dTomato fluorescence by flow cytometry.The poptosis . E1Aexpficiency. This enFig. 1B andFig. 1C.The efficiency of the introduced genes, expression was evaluated by flowcytometry detection of dTomato fluorescence and real-time PCR. The flowcytometry data for evaluation of the transduction and subsequent transgeneexpression efficiency are shown inThe number of cells of the A549 E1A and H1299 E1A model lines in which dTomatoprotein fluorescence was registered amounted to 44 and 85% of the total cellpopulation, respectively, compared to a control (nontransduced A549 and H1299line cells).dTomatomarker gene with a high efficiency.These findings indicate that the produced A549 E1A and H1299 E1A transgenicmodel cells express the introduced target gene and dTomato expression level may becaused by individual properties of the cells. The different number oflentiviral provirus molecules is integrated in the genome of transgenic cells.The viral DNA integrates into different regions of the genome, which mayprovide varying transgene expression levels. Despite the similarity of themorphology and origin of A549 and H1299 cells, differences between these twolines may be one of the causes. The E1A gene expression isknown to result in p53-dependent apoptosis. H1299 cells are p53-deficient.Hence, the likelihood of apoptosis in H1299 E1A cells is significantly lowerthan in A549 E1A cells. It may be assumed that A549 cells are transduced withthe same efficiency as H1299 cells, but p53-dependent apoptosis occurs in mostof the transduced A549 E1A cells, enabling detection of dTomato fluorescence ina smaller number of A549 E1A cells compared to H1299E1A model cells.According to the obtained results, the number of fluorescent cells in A549 E1Aand H1299 E1A model lines is significantly different. The heterogeneity of A549E1A and H1299 E1A cells in the E1A-D36 geneexpressionsiRNA-mediated downregulation of  E1A-D36gene–IRES–dTomato marker gene–puromycin resistance gene” enables rapid assessment of thesilencing activity of siRNAs and the lentiviral vectors directing the synthesisof shRNAs (precursors of siRNAs).The structure of the expression cassette “promoter–E1A-D36 mRNA, the cellstops producing both the E1A-D36 gene products and the dTomatofluorescent protein, which can be quantified by flow cytometry.Because of degradation of a mRNA common to both genes due to the inhibitoryactivity of interfering RNAs targeting E1A gene expression results in induction ofapoptosis, the experiment was performed as follows: siRNAs complementary tovarious regions of the E1A-D36 mRNA were transfected into A549and H1299 cells; after 24 h, the cells were transduced with pseudolentiviralparticles containing an expression cassette “promoterSFFV–E1A-D36 gene–IRES–dTomato fluorescentprotein gene–puromycin resistance gene.” The efficiency of siRNAswas evaluated by flow cytometry and real-time PCR 48 and 96 h post transduction.Since the E1AD36 mRNA causeda significant decrease in the dTomato expression level.In the population of A549 E1A cells transfected withsiE1A-1, siE1A-2, and siE1A-3, the mean fluorescence intensity (MFI) of dTomatowas decreased by 25, 49, and 53% after 48 h and by 55, 61, and 58% after 96 h,respectively, compared to control A549 E1A cells transfected with siScr. AsiRNA biological activity assay showed that MFI of the dTomato fluorescentprotein in the H1299 E1A model cell population was decreased by 18, 60, and 17%under the action of siE1A-1, siE1A-2, and siE1A-3 as early as after 48 h and by18, 44, and 56% after 96 h, respectively, compared to control H1299 E1A cellstransfected with siScr. All the presented data were obtained in threeindependent experiments (p < 0.05).The activity of siRNAs complementary to the E1A-D36 mRNA expression level inmodel cells was assessed 48 and 96 h post transduction with pseudolentiviralparticles encoding the target gene.The E1A-D36 mRNA expression level in A549 E1A cells transfectedwith siE1A-1, siE1A-2, and siE1A-3 was reduced by 58, 83, and 63% and 69, 88, and72% 48 h and 96 h post transduction, respectively, compared to control cells.The E1A-D36 mRNA level in H1299 E1A model cells transfectedwith siE1A-1, siE1A-2, and siE1A-3 was reduced by 28, 71, and 46% and 50, 69,and 47% 48 h and 96 h post transduction, respectively, compared to controlcells.Results obtained by flow cytometry are consistent with the real-time PCR data.The effect of siRNAs on the A reduction in the mean fluorescence intensity of the dTomato marker proteinoccurs more slowly and less effectively than suppression of target geneexpression at the mRNA level. These data may be explained by the fact that thefluorescent protein is quite stable, and its half-life is about 72 h.E1A-D36gene expression was mostly affected by siE1A-2. The siRNA silencingactivity depends on several factors. These include the secondary structure of atarget mRNA, siRNA sequence uniqueness, and stability and the thermodynamicasymmetry of siRNA duplexes. The secondary structure of a target mRNA or theproteins bound to it are known to be capable of hindering access for siRNAs[According to the flow cytometry and real-time PCR data, the r siRNAs, 32.E1A-D36 gene expression by alentiviral vector encoding shRNADownregulation of E1A-D36 expression) andcontrol siScr. Lentiviral vectors ensure integration of a sequence encodingshRNA into the cell genome, providing long-term suppression of the target geneexpression. These lentiviral vectors were constructed based on the Le-GO-Cerulean/BSDcarrying the Cerulean fluorescentprotein gene and blasticidin resistance gene. The shScr-LeGO-Cerulean/BSDvector encoding a hairpin structure having no homology with known viral mRNAsas well as rat, mouse, and human mRNAs was used as a control to demonstrate theabsence of a nonspecific activity of shRNAs.We constructed lentiviral vectors encoding shRNAs corresponding to siE1A-2.p < 0.05). The expression level of theE1A target gene was down by 57, 77, and 80% under the actionof shE1A after 3, 6, and 10 days, respectively, compared to the control.Then, H1299 shE1A model cells were transduced with E1A-LeGO-iGTpseudolentiviral particles. The biological activity of shE1A was evaluated 3,6, and 10 days post transduction using real-time PCR in three independentexperiments  were transduced with shE2B-LeGO-Gand shE1A-LeGO-Cerulean/BSD lentiviralvectors encoding shE2B and shE1A, respectively. Limbal cells transduced withpseudolentiviral particles carrying shScr  were used as a control.We evaluated the ability of shRNAs targeting mRNAs of the Fig. 4D).Cells of the derived lines were infected with HAdV D8 and HAdV D37, which causeepidemic keratoconjunctivitis, at a multiplicity of infection of 20fluorescence- forming units (FFU)/cell. The cells were cultured for 6 days postinfection, which is sufficient to complete the full replication cycle of ahuman adenovirus. Six days post infection, the efficiency of shRNAs wasassessed via qPCR to detect HAdV D8 and HAdV D37 genomes. We observed asignificant downregulation of human adenovirus replication in primary humanlimbal cells. The copy number of HAdV D8 and HAdV D37 genomes was reduced by 59and 58% under the action of shE2V, 37 and 30% under the action of shE1A, and 73and 60% under the simultaneous action of shE2B and shE1A, respectively,compared to control cells[We suppose that downregulation of expression of human species D adenoviralearly genes results in premature termination of the adenovirus replicationcycle. This explains the significant reduction in the adenovirus genome copynumber. Nevertheless, we have not achieved a complete suppression of viralreplication. This may be associated with the high multiplicity of infection (20FFU/cell), the sample analysis time ),as welin vitro model system of adenovirus ocularinfection and demonstrated the high efficiency of shRNAs targeted against earlygenes of human spicies D adenoviruses.In the experiment, we used primary human limbal cells derived from the humancornea, which is affected in epidemic keratoconjunctivitis. We also used HAdVsD8 and D37 that are the main causative agents of the disease. Thus, wedeveloped an E1A gene of HAdVs D8, D19, D36, andD37, as well as lentiviral vectors that direct the synthesis of an analogousshRNA in cells, we produced model cell lines whose genome contains theexpression cassette “promoter–E1A-D36gene–IRES– dTomato marker gene–puromycin resistance gene.” These model cell lines enable rapidevaluation of the efficiency of interfering RNAs complementary to differentregions of the target gene mRNA.We developed an approach to efficiently suppress replication of human species Dadenoviruses via RNA interference. In order to evaluate the silencing activityof siRNAs specific to the The high efficiency of these vectors in downregulation of human species Dadenoviruses D8 and D37 replication was shown in primary human limbal cells.The simultaneous action of shE1A and shE2B led to a decrease in the adenovirusgenome copy number by 70%, on average.We believe that our findings will be helpful for the design and development ofnovel medicinal agents against human diseases caused by adenoviruses.


KRAS point mutations observed in NSCLC necessitates a multiplex approach to efficient mutation detection in circulating DNA. Here we report the design and optimisation of three discriminatory ddPCR multiplex assays investigating nine different KRAS mutations using PrimePCR™ ddPCR™ Mutation Assays and the Bio-Rad QX100 system. Together these mutations account for 95% of the nucleotide changes found in KRAS in human cancer. Multiplex reactions were optimised on genomic DNA extracted from KRAS mutant cell lines and tested on DNA extracted from fixed tumour tissue from a cohort of lung cancer patients without prior knowledge of the specific KRAS genotype. The multiplex ddPCR assays had a limit of detection of better than 1 mutant KRAS molecule in 2,000 wild-type KRAS molecules, which compared favourably with a limit of detection of 1 in 50 for next generation sequencing and 1 in 10 for Sanger sequencing. Multiplex ddPCR assays thus provide a highly efficient methodology to identify KRAS mutations in lung adenocarcinoma.Droplet digital PCR (ddPCR) can be used to detect low frequency mutations in oncogene-driven lung cancer. The range of  RAS family GTPases and EGFR . Knowleectively , particuectively . The pret cohort . Obtainit cohort . Droplett cohort ,10 that KRAS hotspot mutations by ddPCR has been limited by the variety of potential alleles within adjacent loci, although some KRAS mutations occur more commonly in cancer than others  in advanced colorectal cancer [KRAS mutations, nor has a multiplex tool been used in lung cancer.Detection of s Tables  and 2. Tl cancer  but not KRAS mutations and to demonstrate the application of these assays to patient-derived material using the Bio-Rad QX100 system.We set out to design multiplex digital PCR assays which would accurately identify nine different KRAS c.35G>T , c.35G>A , c.35G>C , c.34G>A , c.34G>C , c.34G>T , c.37G>T , c.38G>A , c.183A>C , WT for c.35G>T , WT for c.35G>A , WT for c.35G>C , WT for c.34G>A , WT for c.34G>C , WT for c.34G>T , WT for c.37G>T , WT for c.38G>A  and WT for c.183A>C  20x PrimePCR™ ddPCR™ Mutation Assays (containing both primers and ddPCR probes) were purchased from Bio-Rad  and used in this study.Each digital PCR probe is an oligonucleotide specific to the region of interest with a 5’ fluorophore and a 3’ quencher. The fluorophore is either HEX for probes specific to wild-type sequences or FAM for mutant probes. KRAS G12V, lung), NCI-H358 , A549 , SK-LU-1 , A427 , HCT-116  and NCI-H1975 . All cell lines were provided directly by Cancer Research UK Cell Services with the exception of HCT-116 . Cell gDNA was extracted using the DNeasy™ Blood and Tissue Kit  as per manufacturer’s instructions. All DNA used in subsequent digital PCR reactions, except synthetic oligonucleotides, was quantified on a QX100 ddPCR system using human RNase P TaqMan® Copy Number Reference Assay . 1 μL of eluate was added to a digital PCR reaction containing 10 μL ddPCR Supermix for probes (Bio-Rad) and 1 μL of TaqMan Copy Number Reference Assay, human, RNase P (Life Technologies) on a total volume of 20 μL. The reaction was partitioned into ~14,000 droplets per sample in a QX-100 droplet generator according to manufacturer’s instructions. Emulsified PCR reactions were run on a 96 well plate  on a G-Storm GS4 thermal cycler  incubating the plates at 95°C for 10 min followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec, followed by 10 min incubation at 98°C. The temperature ramp increment was 2.5°C/sec for all steps. Plates were read on a Bio-Rad QX-100 droplet reader using QuantaSoft v1.4.0.99 software (Bio-Rad). At least one negative control wells with no DNA were included in every run. The amount of amplifiable RNase P DNA was quantified from the concentration provided by the software. After quantification, cell line DNA was digested using Hind 3-HF™ restriction enzyme  at 37°C for 1 hour.Digital PCR assays were optimised using cell line genomic DNA (gDNA) or synthetic oligonucleotides as appropriate. Cell lines used for this study were authenticated using STR profiling by the Cell Services facility at the Cancer Research UK London Research Institute and the Institute of Cancer Research. These included NCI-H727 , relevant primer probe assays, DNA (variable volume) and nuclease-free water . Multiple wells were used if required to analyse the necessary DNA eluate. At least 500 pg equivalent of DNA eluate was analysed in each multiplex assay for each patient. 20 μL of PCR reaction mixture was transferred to a sample well in a disposable droplet generator cassette (Bio-Rad). 70 μL of droplet generation oil (Bio-Rad) was then loaded into the oil well for each channel and the cassette loaded into a QX100 Droplet Generator (Bio-Rad). The droplets were then transferred to a 96-well PCR plate. Emulsified PCR reactions were run on a G-Storm GS4 thermal cycler incubating the plates at 95°C for 10 min followed by 40 cycles of 94°C for 30 sec and 54°C for 60 sec, followed by 10 min incubation at 98°C. The temperature ramp increment was 2.5°C/sec for all steps. Plates were read on a Bio-Rad QX-100 droplet reader using QuantaSoft v1.4.0.99 software from Bio-Rad. At least one negative control well with no DNA was included in every run. Each well was then read using the QX100 Droplet Reader (Bio-Rad) and droplets analysed for emission in the HEX or FAM wavelengths. Two-dimensional amplitude plots displaying measured HEX and FAM amplitude for each droplet show four main droplet populations; droplets containing no amplified DNA, droplets with only mutant DNA and high FAM amplitude, droplets with only wild-type DNA and high HEX amplitude and droplets with both wild-type and mutant amplified DNA and high FAM and HEX amplitude. If multiple copies of wild-type and mutant DNA are contained in the same droplet, droplets with higher FAM and HEX amplitude will be read. These droplets have been excluded from any analyses throughout this study.To assess the mutant allele fraction, the concentration of mutant DNA (copies of mutant DNA per droplet) was estimated from the Poisson distribution. Number of mutant copies per droplet Mmu = -ln (1-(nmu/n)), where nmu = number of droplets positive for mutant FAM probe and n = total number of droplets. The DNA concentration in the reaction was estimated as follows: MDNAconc = -ln (1-(nDNAcon/n)), where nDNAconc = number of droplets positive for mutant FAM probe and/or wild-type HEX probe and n = total number of droplets. The mutant allele fraction = Mmu/ MDNAconc. The measured mutant allele frequency describes the mutant allele fraction expressed as a percentage.Formalin fixed paraffin embedded (FFPE) tumour samples from NSCLC patients surplus to clinical care were collected with written patient consent at The Royal Marsden NHS Foundation Trust. FFPE tissue DNA was extracted (after macrodissection if required to ensure >10% tumour content) using the Qiagen DNA FFPE Tissue Kit (Qiagen) as per manufacturer’s instructions. The eluted DNA was stored at -20°C.All patients provided written consent to this study , which incorporated surplus somatic DNA from the Cancer Research UK Stratified Medicine Programme .5’-TATTATAAGGCCTGCTGAAAATG-3’) and reverse (5’-TTGGATCATATTCGTCCACAA-3’) primers, 10 μL Amplitaq Gold® PCR Master Mix (Life Technologies) and 3 ng of template FFPE—derived DNA or 5ng gDNA. PCR conditions were as per manufacturer’s recommendations. Purified PCR product then underwent a further PCR step using chain-terminating dideoxynucleotides  and subsequent DNA sequence was read on an automated sequencer.The region of interest was amplified using 10 μM forward (Sequencing libraries were prepared with the Ion AmpliSeq™ Cancer Hotspot Panel v2 (Life Technologies) using the Ion AmpliSeq Library Preparation protocol with 3-5ng of DNA, according to manufacturer’s instructions. Following barcoding, libraries were quantified using qPCR and diluted to 100 pM. Libraries were templated with the Ion OneTouch2 system (Life Technologies) and sequenced on a PI chip using the Ion PI OT2 200 Kit (Life Technologies), 520 flows and an average amplicon length of 112 bases to a mean depth of x2721307. The sequencing resulted in 45272–11767856 reads per sample.www.1000genomes.org) were identified as possible somatic mutations. The data was cross referenced against the Cosmic database v70 (cancer.sanger.ac.uk) to identify possible hotspot mutations.Ion torrent Variant caller v4.0-r73742 with no Hotspot region and configuration “Germ Line Low Stringency” was used for calling variants. Read counts for all positions were computed using pileup (SAMtools v1.1 ) and thi2 = 1-(SSreg/SStot) where SSreg refers to the sum of the squares of distances to best-fit linear regression line and SStot refers to the sum of the squares of vertical distances from the null hypothesis  using GraphPad Prism version 6.0a .Linear regression was performed using the formula rKRAS wild-type gDNA were analysed with each multiplex. The limit of detection was set at 0.05% to reflect our measured results with spiked KRAS mutant gDNA species . Wild-type probe assays for each mutation were tested in combination with varying concentrations of mutant probe assays, giving rise to mutant droplet populations of varying FAM amplitude . This population was not observed when each mutant DNA species was tested in duplex with the G12D probe (left second panel). A similar second mutant population was observed with the G12V mutant probe and all three mutant DNA species as compared with each mutant DNA in duplex (left lower panels). This second population, particularly as seen with the G12D mutant probe, may fall at the expected FAM amplitude of a different KRAS mutation in the multiplex assay and lead to false positive mutation detection. To further explore this, FFPE tissue DNA from an individual known to have a KRAS 12/13 mutation (F124), as verified by Cobas® testing  was analysed  and A549 (KRAS G12S) gDNA were spiked into KRAS wild-type gDNA (NCI-H1975) and tested in the appropriate KRAS multiplex assay. The limit of detection for KRAS G12C mutant DNA in multiplex A was 0.03% and for KRAS G12S DNA in multiplex B was 0.045% (KRAS mutant gDNA in multiplexes A and B (r2 = 0.9992 and 0.0998 respectively), demonstrating the linearity of mutation detection in the multiplex assays. Mutant allele frequency showed little intra-well variability for either multiplex assay and high reproducibility between two different operators on three alternate days for a range of allele frequencies  or oligonucleotides and FFPE tissue DNA  or A549 (KRAS G12S) gDNA spiked into KRAS wild-type DNA (NCI-H1975) were simultaneously analysed using digital PCR and Sanger sequencing. KRAS G12C mutant DNA was detectable using multiplex A down to a mutant allele frequency of 0.2%, whereas the mutant peak on the chromatogram is only visible at a mutant allele frequency of 31.5% and not below 10%  but investigators were blinded to the specific KRAS genotype. At least one KRAS mutation was detected in each case and two cases had two detectable KRAS clones (KRAS wild-type DNA (NCI-H1975) gDNA to create three samples containing a major and minor KRAS clone; C1 is a combination of NCI-H358 (KRAS G12C) and A549 (KRAS G12S) gDNA, C2 is a combination of NCI-H358 (KRAS G12C) and A427 (KRAS G12D) gDNA and C3 is a combination of A427 (KRAS G12D) and A549 (KRAS G12S) gDNA. All KRAS species were detectable using the KRAS multiplex assays, including the minor clones down to a mutant allele frequency of 0.3% measured for the KRAS G12D mutant DNA in sample C3  to analyse the KRAS 12 and 13 loci. Next generation sequencing could not detect KRAS mutant allele frequencies below 4% in tissue DNA and none of the minor clones below 2% were detectable in the biclonal gDNA samples in either of the duplicate library preparations  may therefore be a false positive result. The low frequency KRAS G12C mutation in S010, however, is a G>T change and therefore much more likely to be a true result.Sub-clonal mutations can also be detected using this ddPCR approach to observed . The KRAKRAS molecule in 50 wild-type KRAS molecules. The KRAS G12S minor clone in the C1 gDNA sample that was measured at 1.9% on multiplex digital PCR, for example, was not detectable on NGS in either of two independent library preparations. Sanger sequencing, in comparison, has a limit of detection of only 1 in 10 and over a range of mutant allele frequencies, a sensitivity of 20%. Digital PCR maintains a very high specificity despite this increased sensitivity and is approaching the 100% specificity seen with NGS [KRAS multiplex ddPCR is reflected by the correlation observed with the mutant allele frequency detected by Ion Torrent ProtonTM sequencing, an established method of determining this variable. In addition, we have demonstrated the robustness of the multiplex assays across a range of mutant allele frequencies both within the same experiment and between operators on three non-consecutive days.We have demonstrated that the multiplex assays are more sensitive than Sanger and next generation sequencing and highly specific. The assays have linearity down to a limit of detection of 0.03–0.045%. Due to the false positive signal seen from FFPE tissue on NGS below mutant allele frequencies of 1.5%, the limwith NGS . The accKRAS genotypes in patient-derived material. Multiplexing of assays reduces the amount of clinical material required for testing, which will be particularly important in settings where the proportion of mutant relative to wild-type DNA is likely to be low, such as with circulating free tumour DNA from plasma samples. The three multiplexed assays described here between them will identify the nucleotide changes in 95% of the cancer cases where the KRAS gene is mutated.We have described the combination and optimization of commercially available digital PCR assays to develop novel multiplex assays that allow the accurate detection and discrimination of S1 FigKRAS G12V, G12C and G12D mutant DNA tested with 562.5 nM primers and 312.5 nM G12D FAM probe in one well (top left panel) or in three separate wells (i.e. one well G12V DNA with G12D probe assay etc.). Wells with only one type of KRAS mutant DNA are merged (left second panel) to compare with the single well containing all DNA species. KRAS G12V, G12C and G12D mutant DNA tested with 225 nM primers and 125 nM G12V FAM probe in one well (left third panel) or in three separate wells . A second droplet population with lower FAM amplitude is detected when more than one species of KRAS mutant DNA is present (red dashed box). Right panels: KRAS 12/13 FFPE tissue DNA sample (F124) tested with multiplex assay (top right) and then 3 separate duplex assays as indicated. The same scale is used for all panels (HEX amplitude up to 7000 and FAM amplitude up to 11000).Left panels: (EPS)Click here for additional data file.S2 FigKRAS mutations  with different KRAS mutation variants . More cross-reactivity, indicated by a droplet population near the empty droplets with relatively low FAM and HEX amplitude and a population near the true wild-type population (examples in red dashed square in top right panel), was seen between probes and DNA for the same nucleotide position in exon 2 of KRAS. Each panel shows FAM amplitude of up to 10000 and HEX amplitude between 1000 and 6000.Duplex assays for different (EPS)Click here for additional data file.S3 FigKRAS mutations  with different KRAS mutation variants . More cross-reactivity, indicated by a droplet population near the empty droplets with relatively low FAM and HEX amplitude and a population near the true wild-type population (examples in red dashed square in top right panel), was seen between probes and DNA for the same nucleotide position in exon 2 of KRAS. Key: X—omitted panel of duplex assay with correct probe and KRAS mutation variant as higher magnification. Each panel shows FAM amplitude of up to 3000 and HEX amplitude between 1000 and 6000.Duplex assays for different (EPS)Click here for additional data file.S4 FigG13C populations are indicated by a red dashed square, G12C populations by a blue dashed square and G12V droplet populations are indicated by a yellow dashed square. Multiplex 1 (top left panel) is an assay combination of 900 nM primers and 500 nM G13C probe, 450 nM primers and 250 nM G12C probe and 225 nM primers and 125 nM G12V probe, with 900 nM primers and 500 nM WT for G12C probe and 450 nM primers and 250 nM WT for G13C probe. Multiplex 2 (centre left) uses the same FAM assay concentrations as in Multiplex 1 with 450 nM primers and 250 nM WT for G12C probe and 900 nM primers and 500 nM WT for G13C probe. Multiplex 3 (centre right) is a combination of 450 nM primers and 250 nM G13C probe and 900 nM primers and 500 nM G12C probe with the same G12V and WT G12C and G13C assays as in Multiplex 1. Multiple 4 uses the same FAM assays concentrations as Multiplex 3 with the WT for G12C and G13C assay concentrations as in Multiplex 2. Each panel shows a FAM amplitude up to 18000 and a HEX amplitude up to 12000.(EPS)Click here for additional data file.S5 FigG12S droplet populations are indicated by a red dashed square, G12D populations by a blue dashed square, and G13D populations by a yellow dashed square. Multiplex 1 (top left panel) is an assay combination of 675 nM primers and 375 nM G12S probe, 450 nM primers and 250 nM G12D probe and 225 nM primers and 125 nM G13D probe with 225 nM primers and 125 nM WT for G12C probe and 450 nM primers and 250 nM WT for G13D probe. Multiplex 2 (top middle panel) is a FAM assay combination of 225 nM primers and 125 nM G12S probe, 675 nM primers and 375 nM G12D probe and 450 nM primers and 250 nM G13D probe with the same WT for G12C and G13D assay concentrations as in Multiplex 1. Multiplex 3 (top right panel) is a combination of, 450 nM primers and 250 nM G12S probe, 675 nM primers and 375 nM G12D probe and the same FAM 13D assay concentration as in Multiplex 1. WT assay concentrations are 450 nM primers and 250 nM WT for G12C probe and 225 nM primers and 125 nM WT for G13D probe. Each panel shows a FAM amplitude up to 18000 and a HEX amplitude up to 9000.(EPS)Click here for additional data file.S6 FigG12R droplet populations are indicated by a red dashed square, G12A populations by a blue dashed square and Q61H populations by a yellow dashed square. Multiplex 1 (top left panel) is an assay combination of 675 nM primers and 375 nM G12R probe, 450 nM primers and 250 nM G12A probe and 900 nM primers and 500 nM Q61H probe with 450 nM primers and 250 nM WT for G12C probe and 900 nM primers and 500 nM WT for Q61H probe. Multiplexes 2–4 use the same WT assay concentrations. Multiplex 2 (centre left panel) is a FAM assay combination of 450 nM primers and 250 nM G12R probe and 675 nM primers and 375 nM G12A probe with the same FAM Q61H assay concentration as in Multiplex 1. Multiplex 3 (centre right panel) is an assay combination of 900 nM primers and 500 nM G12R probe, 225 nM primers and 125 nM G12A probe and 450 nM primers and 250 nM Q61H probe. Multiplex 4 (top right panel) is an assay combination of 450 nM primers and 250 nM G12A probe and 225 nM primers and 125 nM Q61H probe with the same FAM G12R assay as in Multiplex 3. Each panel shows a FAM amplitude up to 16000 and a HEX amplitude up to 11000.(EPS)Click here for additional data file.S7 FigKRAS mutation variants and analysed with multiplexes A, B and C. Multiplex A is an assay combination of G13C, G12C and G12V. Multiplex B is an assay combination of G12S, G12D and G13D. Multiplex C is an assay combination of G12R, G12A and Q61H. Panel A1 –multiplex A with KRAS G12V, C, D, S and R DNA; panel A2 –multiplex A with KRAS G12A DNA; panel A3- multiplex A with KRAS G13C and G13D DNA. Panel B1 –multiplex B with KRAS G12V, C, D, S and R DNA; panel B2 –multiplex B with KRAS G12A DNA; panel B3- multiplex B with KRAS G13C and G13D DNA. Panel C—multiplex C with KRAS G12V, C, D, S, R, A, 13C, 13D and Q61H DNA. Panels A1-3 and B1-3 show a FAM amplitude up to 18000 and an HEX amplitude up to 7000. Panel C has a FAM amplitude up to 18000 and a HEX amplitude up to 10000. Key: * = KRAS G12V mutant DNA, ¶ = G12D, ⌃ = G12C, ★ = G12S, ♦ = G12R, • = G12A, § = G13C, ■ = G13D; black- empty droplets, blue- mutant DNA FAM positive droplets, green- wild-type DNA HEX positive droplets, brown—wild-type and mutant DNA double positive droplets.Droplet populations seen with the addition of cell line DNA or oligonucleotides containing different (EPS)Click here for additional data file.S8 FigKRAS mutant G12C DNA spiked in against a KRAS wild-type DNA background; right panels: Multiplex B tested with decreasing amounts of KRAS mutant G12S DNA against a KRAS wild-type DNA background. Top panels: Correlation of amount of input KRAS mutant DNA with measured mutant allele frequency. Middle panels: Mutant allele frequency per well as compared with mean mutant allele frequency across all wells for a range of allele frequencies . Bottom panels: Mutant allele frequency measured on three non-adjacent days by two different operators across a range of amounts of spiked KRAS mutant DNA. Key: Triangle = Day 1, square = Day 2, circle = Day 3.Left panels: Multiplex A tested with decreasing amounts of (EPS)Click here for additional data file.S9 FigKRAS G12C mutant DNA samples at allele frequencies ranging from 0.2–31.5% analysed by digital PCR in KRAS multiplex assay A (upper panels) or Sanger sequencing (upper chromatograms). KRAS G12S mutant DNA samples at allele frequencies ranging from 0.1–17% analysed by digital PCR in KRAS multiplex assay B (lower panels) or Sanger sequencing (lower chromatograms). All panels in the upper row show a FAM amplitude up to 10000 and a HEX amplitude up to 6000. Panels in the lower row have a FAM amplitude up to 14000 and a HEX amplitude up to 11000. Key: black- empty droplets, blue- mutant DNA FAM positive droplets, green- wild-type DNA HEX positive droplets, brown—wild-type and mutant DNA double positive droplets; black arrow—mutant nucleotide not discernible from background signal on Sanger sequencing, red arrow—mutant thymine nucleotide detectable using Sanger sequencing, green arrow—mutant adenine nucleotide detectable using Sanger sequencing.(EPS)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 TableKey: F- former smoker, S- smoker, N- never smoker, Unk- unknown.(DOC)Click here for additional data file.


This study was conducted to investigate the ablative efficacy and safety of sequential intravesical gemcitabine and mitomycin C with mitomycin C alone in refractory non-muscle invasive bladder cancer (NMIBC).A total of 219 patients with refractory NMIBC were prospectively enrolled at tertiary academic center over period of Feb 2009-Jan 2012 & followed for next 3 yrs. They were randomly assigned to either of treatment arms: Gentamycin & mitomycin C (group A) or mitomycin C (group B). All patients underwent a 6-week induction regimen followed by a monthly maintenance regimen for one year if they responded to the induction course.In Group A 98 of 102 & in group B 94 of 96 patients completed the therapy and were evaluated for response while 6 patient left the therapy in between. The therapy was well tolerated in the rest of patients. In group‘A’ i.e Gentamycin & mitomycin C a total, 82 patients (83.67%) exhibited a complete response to intravesical therapy. In 12.2% (12) patients had biopsy proven recurrence (22±6.16 months). In group ‘B’ (mitomycin C), 63 (65.60%) patients exhibited a complete response to intravesical therapy, 22 patients (22.9%) showed a partial response. During follow-up, 16 patients (25.3%) developed recurrence within this period. (14.5 +/- 8.26 months).Chemoresection with sequential intravesical gemcitabine and mitomycin C administration may be a viable option for BCG refractory non-muscle invasive bladder cancer (NMIBC).


We studied the incremental benefits associated with adding mericitabine  to PI plus PegIFN alfa-2a/RBV-based therapy in two double-blind randomized multicenter phase 2 trials . The primary endpoint in both trials was SVR, defined as HCV RNA <25 IU/mL 12 weeks after the end of treatment (SVR12). Overall, the addition of mericitabine to PI plus PegIFN alfa-2a/RBV therapy resulted in SVR12 rates of 60–70% in DYNAMO 1 and of 71–96% in DYNAMO 2. SVR12 rates were similar in patients infected with HCV genotype 1a and 1b in both trials. The placebo control arms in both studies were stopped because of high rates of virological failure. Numerically lower relapse rates were associated with longer treatment with mericitabine (24 versus 12 weeks), telaprevir-containing regimens, and regimens that included 48 weeks of PegIFN alfa-2a/RBV therapy. No mericitabine resistance mutations were identified in any patient in either trial. The addition of mericitabine did not add to the safety burden associated with either telaprevir or boceprevir-based regimens. These studies demonstrate increased SVR rates and reduced relapse rates in difficult-to-treat patients when a nucleoside polymerase inhibitor with intermediate antiviral potency is added to regimens containing a first-generation PI.Most patients with chronic hepatitis C virus (HCV) genotype 1 infection who have had a previous null response (<2-logTrial Registration: ClinicalTrials.gov NCT01482403 and ClinicalTrials.gov NCT01482390 Chronic hepatitis C virus (HCV) infection is estimated to affect approximately 180 million people worldwide and is a leading cause of hepatocellular carcinoma, cirrhosis, and liver-related death . The goaThe first direct-acting antiviral agents (DAAs) approved for treatment of chronic HCV infection were the serine protease inhibitors boceprevir (BOC) and telaprevir (TVR), both of which are only effective against HCV genotype 1 and must be used in combination with peginterferon alfa/ribavirin , 8. Comp10 decline in HCV RNA by week 12)  and two of whom experienced relapse [one G1a and one G1b]) were monitored for viral resistance.BOC resistance mutations (in NS3) were identified in seven of these patients: T54S in two patients in Arm A who relapsed (one G1a and one G1b), V55A in one patient in Arm B who relapsed (G1b), R155K in one patient in Arm B who relapsed (G1a), V36M-T54S in one patient in Arm B who experienced breakthrough (G1a), T54S/T-V170A in one patient in Arm A who relapsed (G1b), A156S-M175L in one patient in Arm A who relapsed (G1b). No MCB resistance mutations were detected. Pre-existing BOC resistance mutations (M175L or T54S/V55I) were identified in baseline samples from two patients (both of whom achieved an SVR). No pre-existing MCB resistance mutations were detected.In DYNAMO 2, two patients in Arm A who experienced relapse (one G1a and one G1b), one patient in Arm B who experienced breakthrough while on P/R (G1a), and six patients in Arm C who experienced breakthrough while on P/R (four G1a and two G1b) were monitored for viral resistance. TVR resistance mutations (in NS3) were identified in seven of these patients: R155K in two patients in Arm C who experienced breakthrough (G1a), V36M in one patient in Arm A who relapsed (G1b), T54S in one patient in Arm C who experienced breakthrough (G1b), V36A/V-T54A/T in one patient in Arm C who experienced breakthrough (G1b), V36M-R155K in one patient in Arm C who experienced breakthrough (G1a), and V36M-R155R/T in one patient in Arm A who relapsed (G1a). No MCB resistance mutations were detected. Pre-existing TVR resistance mutations (V36L or T54S) were identified in baseline samples from two patients (both of whom achieved an SVR). No pre-existing MCB mutations were detected.9/L. Her lowest platelet count (19 x 109/L) occurred on study day 133. She was hospitalized on four occasions for platelet transfusions. All treatment with study medications was discontinued on study day 168. The two incidences of anemia considered to be related to ribavirin occurred in a 71-year-old woman whose baseline hemoglobin concentration (135 g/dL) and creatinine clearance (85.8 mL/min) were both in the normal range. On study day 43, her hemoglobin concentration was 74 g/L, at which time she was hospitalized and received treatment with oral iron supplements, folic acid, and two units of packed red blood cells. Treatment with ribavirin was withheld for three days. The second episode of anemia occurred on study day 73 (hemoglobin concentration not reported), at which time all treatment with study drugs was discontinued. On study day 77, she received a second transfusion of two units of packed red blood cells. No deaths were reported during the study. High rates of grade ≥3 reductions in hemoglobin and neutrophils were reported for all three arms of the study; grade 3 platelet decreases were also common  are considerably higher than those previously reported for the approved first- and second-generation protease inhibitor-based triple regimens in patients with prior null response (31–46%) , 18, 26.in vitro data [In both DYNAMO 1 and DYNAMO 2, the highest SVR12 rates and lowest incidences of relapse were attained when 24 weeks of treatment with an MCB-containing, four-drug regimen was followed by 24 weeks of peginterferon alfa-2a/ribavirin (administered with BOC in DYNAMO 1). Consistent with tro data , 28, we tro data . The inctro data , 11, 30.tro data , 30. As tro data –12, 26, The higher SVR12 rates in DYNAMO 1 and DYNAMO 2 relative to prior studies of protease inhibitor-based triple therapy combinations most likely reflect a reduction in the incidence of protease inhibitor resistance with the incorporation of MCB into therapy. In particular, the rate of relapse across Arms A–C of DYNAMO 2 12 weeks after end of treatment (3%) appeared substantially lower than the 26% relapse rate at week 72 reported in the Phase 3 study of TVR plus peginterferon alfa/ribavirin among prior null responders –34. As eThe adverse event profiles of the MCB-containing four-drug regimens, including the rates of serious adverse events and discontinuations due to safety reasons, were similar to reports for BOC or TVR in combination with peginterferon/ribavirin, suggesting that MCB did not add to the safety burden –13, 30. The early termination of the control arms limits the strength of the conclusions that can be drawn from the DYNAMO studies. However, the SVR12 rates associated with the addition of MCB to BOC- and TVR-based regimens in these studies are remarkably better than the historically poor outcomes achieved with BOC- and TVR-based triple therapy in null responders, especially in patients with advanced liver disease , 16. TheThe treatment of chronic HCV infection is currently undergoing rapid change. At the time the DYNAMO 1 and DYNAMO 2 studies were designed, BOC- and TVR-containing triple regimens represented the standard of care for HCV genotype 1 infection ; howeverCollectively, the results of the DYNAMO 1 and DYNAMO 2 studies suggest that the addition of 24 weeks of treatment with a nucleoside analog inhibitor with intermediate antiviral potency to BOC- or TVR-based combinations improves SVR12 rates.S1 File(DOC)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.