The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface receptor molecules. High concentrations of three of its putative proinflammatory ligands, S100A8/A9 complex , S100A8, and S100A12, are found in rheumatoid arthritis (RA) serum and synovial fluid. In contrast, soluble RAGE (sRAGE) may prevent proinflammatory effects by acting as a decoy. This study evaluated the serum levels of S100A9, S100A8, S100A12 and sRAGE in RA patients, to determine their relationship to inflammation and joint and vascular damage.Serum sRAGE, S100A9, S100A8 and S100A12 levels from 138 patients with established RA and 44 healthy controls were measured by ELISA and compared by unpaired t test. In RA patients, associations with disease activity and severity variables were analyzed by simple and multiple linear regressions.Serum S100A9, S100A8 and S100A12 levels were correlated in RA patients. S100A9 levels were associated with body mass index (BMI), and with serum levels of S100A8 and S100A12. S100A8 levels were associated with serum levels of S100A9, presence of anti-citrullinated peptide antibodies (ACPA), and rheumatoid factor (RF). S100A12 levels were associated with presence of ACPA, history of diabetes, and serum S100A9 levels. sRAGE levels were negatively associated with serum levels of C-reactive protein (CRP) and high-density lipoprotein (HDL), history of vasculitis, and the presence of the RAGE 82Ser polymorphism.sRAGE and S100 proteins were associated not just with RA inflammation and autoantibody production, but also with classical vascular risk factors for end-organ damage. Consistent with its role as a RAGE decoy molecule, sRAGE had the opposite effects to S100 proteins in that S100 proteins were associated with autoantibodies and vascular risk, whereas sRAGE was associated with protection against joint and vascular damage. These data suggest that RAGE activity influences co-development of joint and vascular disease in rheumatoid arthritis patients. Rheumatoid arthritis (RA) is a chronic inflammatory disease that leads to bone and cartilage destruction and extra-articular complications, including atherosclerotic vascular disease and premature mortality . The recRAGE gene (RAGE 82Ser) occurs more frequently in RA patients than in healthy controls /833 × SCr (mmol/l) × 0.85 for females . Hand raHigh resolution human leukocyte antigen (HLA)-DRB1 genotyping was carried out on buffy coat DNA using PCR and sequence-specific oligonucleotide probes. PCR-based restriction fragment length polymorphism (RFLP) analysis was used to delineate the RAGE Gly82Ser and protein tyrosine phosphatase, non-receptor type 22 (PTPN22) Cys1858Thr polymorphisms as described ,48. SharData were analyzed using STATA 9.1 . The variables included age, sex, BMI, current and previous smoking status, RF, ACPA, history of CV events, fasting glucose, homocysteine, cholesterol and triglyceride, ESR, CRP, HDL, LDL, creatinine, CrCl, systolic and diastolic blood pressure, history of diabetes or elevated blood sugar level, history of hyperlipidemia or elevated cholesterol, HLA-DRB1 genotype, Sharp erosion score, Sharp joint space narrowing score, RAGE Gly82Ser polymorphism, history of hypertension or elevated blood pressure, metabolic syndrome (modified AHA standard), serum S100A9, S100A8, S100A12 and sRAGE. Before further analysis, each variable was examined for normal distribution by histogram and box plot. If a variable was not normally distributed, it was transformed (either logarithmic base e or square root transformation) before further analysis. Results are reported as mean ± standard deviation (SD).P < 0.1 using this method were then subjected to multiple linear regression (MLR) analysis. An interaction and residual analysis was also performed on the MLR data. P values < 0.05 (two-tailed) were considered statistically significant.Unpaired t tests compared the serum levels of S100A9, S100A8, S100A12 and sRAGE between RA patients and healthy controls. Simple linear regression analysis was used to evaluate the relationship between a variable and the serum concentration of sRAGE or S100 proteins. Variables with We studied 138 patients with RA  and 44 healthy controls  with neither RA nor CV disease. The RA patients were characterized for RA clinical variables, CV risk factors, and RA complications such as vasculitis, radiographic changes, and CV events (Table P < 0.001). The S100A9 levels detected in patient sera with an anti-S100A9 antibody that detected S100A9, and S100A9 complexed with S100A8, were some 100-fold lower than those reported in other studies [Serum levels of S100A9, S100A8 and S100A12 in patients with RA (n = 138) were increased relative to serum levels in healthy controls . Serum levels of S100A9 in MLR model analysis were positively associated with body mass index, and with serum levels of S100A8 and S100A12 (Table P < 0.05).We analyzed the cohort of 138 RA patients for associations between serum levels of S100A9, S100A8, S100A12 and sRAGE with RA clinical variables, CV risk factors, and with complications such as vasculitis, radiographic changes, and CV events. In simple linear regression analysis, we found that serum levels of S100A9 in RA patients were positively associated with the presence of the PTPN22 Cys1858Thr genetic polymorphism, serum levels of S100A12, and serum levels of S100A8 (Table P < 0.05). The serum levels of S100A8 in MLR analysis were positively associated with RF, ACPA and serum levels of S100A9 (Table P < 0.05).In simple linear regression analysis, serum levels of S100A8 in RA patients were positively associated with ACPA, RF, carriage of HLA-DRB1*0401, RAGE 82S and of the RA-associated HLA-DR shared epitope , serum lP < 0.05). The serum levels of S100A12 in RA patients in MLR analysis were positively associated with ACPA, a history of diabetes, and serum levels of S100A9 (Table P < 0.05).In simple linear regression analysis, the serum levels of S100A12 in RA patients were positively associated with ACPA, RF, history of diabetes, and serum levels of S100A8 and S100A9 (Table P < 0.05). MLR analysis of sRAGE levels confirmed the negative associations with RAGE 82Ser, history of vasculitis, and with serum levels of CRP and HDL (Table P < 0.05).Simple linear regression analysis showed that serum sRAGE levels in RA patients were negatively associated with current smoking, family history of CV disease, history of vasculitis, diastolic blood pressure, RF, carriage of RAGE 82Ser, and serum levels of CRP and S100A8 (Table We found associations of sRAGE and S100 proteins with clinical inflammatory factors, complications, and CV risk factors in established RA patients. S100 A8, A9 and A12 were all elevated in serum from patients with established RA relative to healthy controls, and their levels were correlated in RA patients. By contrast, serum sRAGE levels did not differ in healthy controls and patients with established RA on treatment. Although a previous study reported reduced levels of sRAGE in RA compared to healthy control sera , it seemDespite the generally negative association of sRAGE with vascular risk factors, the single factor that showed a reverse trend in multivariate models was serum HDL. Serum HDL levels were also negatively associated with serum sRAGE among diabetic subjects with CV disease . In contThe positive association of S100A9 with S100A8 and S100A12 suggests that these proteins may be co-regulated in RA. This is supported by a previous study of S100 proteins in RA patients , and is In patients with Kawasaki disease, or with chronic hyperglycemia, serum levels of S100A12 were inversely associated with serum levels of sRAGE ,66. AlthOur analysis indicates that sRAGE and S100 proteins are associated not only with RA inflammation and autoantibody production, but also with the recruitment of classical vascular risk factors to end-organ damage. This association with vascular risk supports previous reports of low sRAGE and high S100A8/A9 and S100A12 levels in patients with type 1 and type 2 diabetes, and essential hypertension ,66-68. TRAGE gene affect splicing of the C-truncated, endogenously secreted form of the receptor, or susceptibility to cell surface RAGE cleavage by matrix metalloproteinases [Finally, we observed a novel association of low sRAGE levels with presence of the RAGE 82Ser polymorphism, which is found more frequently in RA patients ,19. It iteinases , thus alSeveral studies have been published which demonstrate the role of sRAGE, S100A8/A9 and S100A12 in the long-term development of vascular disease. These include a negative association between sRAGE levels and coronary artery disease in non-diabetic men , predictsRAGE and S100 proteins were associated with RA inflammatory factors and autoantibody production, and with the recruitment of classical vascular risk factors to end-organ damage. Consistent with its role as a RAGE decoy molecule, sRAGE had opposing effects to S100A12 and S100A8 in RA. Our data suggest that RAGE may mediate a key pathway coordinating conventional risk factors in the inflammatory RA setting for co-development of joint and vascular disease. Prospective studies will be of interest to determine the utility of these proteins as prognostic biomarkers of joint and vascular damage.ACPA: anti-citrullinated peptide antibodies; ACR: American College of Rheumatology; AGE: advanced glycation end product; BMI: body-mass index; CrCl: creatinine clearance; CRP: C-reactive protein; CT: computed tomography; CV: cardiovascular; ECG: electrocardiogram; ESR: erythrocyte sedimentation rate; HDL: high-density lipoprotein; HMGB1: high mobility group box chromosomal protein; HR: hazard ratio; LDL: low density lipoprotein; MI: myocardial infarction; PCR: polymerase chain reaction; RA: rheumatoid arthritis; RAGE: receptor for advanced glycation end products; RF: rheumatoid factor; SCr: serum creatinine; TG: triglyceride; TIA: transient ischemic attack; TNF: tumor necrosis factor; VLDL: very-low-density lipoprotein.The authors declare that they have no competing interests.YC, CG, MB and RT were involved in conception, design, acquisition, analysis and interpretation of data. WY and CG carried out S100A8, S100A9 and S00A12 assays. YC, CG, MB and RT wrote the manuscript. All authors read and approved the final manuscript.


II atom in the title compound, [Ni(C24H43P2)(NO3)], adopts a distorted square-planar geometry with the P atoms in a trans arrangement. The compound contains a twofold rotational axis with the nitrate group offset from this axis, except for an O atom of the nitrate group, generating two positions of 50% occupancy for the other atoms of the nitrate group.The Ni DOI: 10.1107/S1600536808032376/at2643Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


A high level of virus circulation and introduction of an antigenic drift variant in a susceptible population contributed to the spread of resistant virus. In Europe, the 2007–08 winter season was dominated by influenza virus A (H1N1) circulation through week 7, followed by influenza B virus from week 8 onward. Oseltamivir-resistant influenza viruses A (H1N1) (ORVs) with H275Y mutation in the neuraminidase emerged independently of drug use. By country, the proportion of ORVs ranged from 0% to 68%, with the highest proportion in Norway. The average weighted prevalence of ORVs across Europe increased gradually over time, from near 0 in week 40 of 2007 to 56% in week 19 of 2008 (mean 20%). Neuraminidase genes of ORVs possessing the H275Y substitution formed a homogeneous subgroup closely related to, but distinguishable from, those of oseltamivir-sensitive influenza viruses A (H1N1). Minor variants of ORVs emerged independently, indicating multiclonal ORVs. Overall, the clinical effect of ORVs in Europe, measured by influenza-like illness or acute respiratory infection, was unremarkable and consistent with normal seasonal activity. Infection with influenza viruses A (H1N1), A (H3N2), or B causes substantial human illness and excess deaths each year ; by contBefore the introduction of NAIs in 1999, and until 2007, <1% of viruses tested from unselected surveillance studies in a number of countries demonstrated natural resistance to NAIs  (ORVs) in Europe caused by a H275Y (H274Y in N2 numbering) amino acid substitution in the neuraminidase (NA) of these viruses (The European Influenza Surveillance Scheme (EISS) actively monitored influenza activity from week 40 (October 1–7) of 2007 through week 19 (May 5–11) of 2008. EISS covers all 27 European Union countries plus Croatia, Norway, Serbia, Switzerland, Turkey, and Ukraine. In each country each week, 1 or several networks of sentinel general practitioners (GPs) reported rates of consultation for influenza-like illness (ILI) or acute respiratory infection (ARI)  for virus detection and characterization by using a variety of genetic or phenotypic methods (50) of virus isolates was determined by using fluorescent or chemiluminescent enzyme assays  project at a single laboratory in London  or directly by individual NICs by using methods described previously (www.eiss.org) was used to collect antiviral susceptibility data and linked patient demographic and clinical data  were first removed. Minor outliers were defined as values lying between the upper quartile (UQ) + 1.5 × interquartile region (IQR) and UQ + 3 × IQR; major outliers were defined as values lying above UQ + 3 × IQR, based on analysis of all viruses in a particular subtype over a particular winter season.We performed all statistical analyses by using the software package R version 2.8.0  gene sequences used maximum parsimony . Sequences of ORVs and oseltamivir-sensitive influenza A (H1N1) viruses (OSVs) were chosen as representative of influenza viruses A (H1N1) isolated during the 2007–08 influenza season  in different European countries and a few from other regions of the world and were compared with those of a few influenza viruses A (H1N1) isolated before the 2007–08 season, including sporadically isolated ORVs. GenBank accession numbers are listed in the The 2007–08 influenza season in Europe was initially dominated by influenza viruses A . Influenza viruses B  became dominant in week 8 . Of the 2 = 0.32 for the linear regression fit.The first countries in Europe where influenza viruses A started to circulate continuously were France, Spain, Switzerland, and the United Kingdom in week 40. Spatial analysis of the timing of the first week of continuous detection of influenza viruses A across Europe (n = 30 countries) showed a west-to-east pattern: estimated parameter for longitude was 0.261 weeks per degree longitude , and for latitude –0.108 weeks per degree latitude , with R50) and sequencing, 601 by phenotypic assay alone, and 1,268 by sequencing alone. Of the 2,949 viruses tested, 712 (24%) were oseltamivir resistant either by presence of the H275Y substitution (n = 548) or an IC50 >100 nmol/L for oseltamivir (n = 463) (50 <100 nmol/L) and the presence of H275 (n = 781) and between resistant phenotype (IC50 >100 nmol/L) and the presence of Y275 (n = 299). OSVs  had a median IC50 of 1.7 nmol/L for oseltamivir (range 0.1 nmol/L–23.2 nmol/L) and only 9 minor outliers (thresholds IC50 >12.0 nmol/L and <53.1 nmol/L) were identified. ORVs (n = 463) had a median IC50 of 653 nmol/L . None of the 429 phenotypically characterized ORVs showed evidence of resistance to zanamivir , and only 17 minor outliers (thresholds IC50 >8.5 nmol/L and <27.5 nmol/L) were identified. None of 237 ORVs tested for M2I sensitivity had any of the common resistance substitutions in the M2 protein.The estimated number of influenza viruses A (H1N1) among all detected influenza viruses A  was 10,291 following extrapolation from the proportion of 96% influenza viruses A (H1N1) among all 5,984 subtyped influenza viruses A. Of the 10,291 influenza viruses A (H1N1), 2,949 (29%) were tested for antiviral susceptibility, 1,080 by both phenotypic assay (ICn = 463) . CorrelaORVs were detected in 22 of the 30 countries for which susceptibility data were available, with Norway having the highest proportion of ORVs . Modelin2 = 0.36 for the linear regression fit. The average delay between the first week of continuous detection of influenza virus A and continuous detection of ORV was 5.7 weeks .The earliest detection of ORVs was in France and the United Kingdom in week 46 and in Norway in week 47. Countries where continuous detection of ORVs first began included Norway in week 47, France in week 49, the United Kingdom in week 51, and the Netherlands in week 52. Spatial analysis of the timing of the first week of continuous ORV detection across Europe (n = 14 countries) showed a west-to-east trend pattern: estimated parameter for longitude was 0.156 weeks per degree longitude , and for latitude 0.007 weeks per degree latitude , with RModeling showed a gradual increase for Europe in prevalence of ORVs over time, from close to 0 in week 40 to ≈56% in week 19 . This ovPhylogenetic comparisons of HA and NA genes showed that the sequences of most recent European influenza viruses A (H1N1) fell within clade 2B, represented by A/Brisbane/59/2007, the recently recommended vaccine virus for 2008–09 . The NA 50 values >100 nmol/L for NAI drugs , in concordance with results from worldwide surveillance  with a single amino acid substitution H275Y in the NA, which caused a several hundred-fold selective reduction in susceptibility to oseltamivir, emerged and were sustained in circulation in Europe during 2007–08, despite low antivirual drug use . Before This emergence of oseltamivir-resistant influenza virus A (H1N1) in Europe coincided with the dominant circulation of this virus subtype during the 2007–08 winter in Europe and the emergence of a new drift variant, A/Brisbane/59/2007 . In 6 coSporadically occurring A/New Caledonia/20/99-like ORVs with H275Y were detected during the 2006–07 season in the United Kingdom and United States but did not become epidemiologically important. Indeed, the genetic background plays a role in retaining the replication efficiency and pathogenicity of recombinant influenza viruses A (H5N1) and A (H1N1) after introduction of tyrosine at position 275  indicates the independent parallel emergence of multiple resistant variants. This is emphasized by small distinct clusters of closely related ORVs in Japan that are related to European OSVs, whereas only a few of the Japanese ORVs belonged to the large European ORVs cluster (The combined effect of the relatively high level of circulation of influenza viruses A (H1N1) in Europe; the introduction of a new antigenic drift variant in a susceptible population, partly related to the lack of substantial influenza virus A (H1N1) circulation since the 2000–01 season; and the uncompromised transmissibility of the ORVs contributed to the epidemiologic success of the ORVs during the 2007–08 season. This phenomenon shows clearly that continuation of antiviral susceptibility monitoring and increasing capacity for timely response are essential (Fitted curves to the proportion oseltamivir-resistant viruses among influenza viruses A (H1N1) tested for resistance (both sentinel and nonsentinel) for all countries for which data were available for inclusion in modeling the European trend see  and 5. TGenBank accession numbers of hemagglutinin and neuraminidase sequences used in the phylogeneticAnalyses.Statistical Analysis of Temporal Trends of Resistant Influenza A (H1N1) Viruses, Europe.


F5 (G1691A), F2 (G20210A) and MTHFR , in 469 healthy blood donors from São Miguel Island (Azores). We also analysed the CYP2C9  and VKORC1 (G1639A) variants in fifty-eight individuals with predisposition to thrombosis (possessing at least one variation in F5 or F2 genes and one in MTHFR) to evaluate their warfarin drug response genetic profiles.The Azorean population presents the highest standardized mortality rate for cardiovascular diseases (CVD) when compared to mainland Portugal and other populations. Since thrombosis is a common cause of CVD, we assessed four polymorphisms in three thrombotic risk genes – 2, p < 0.001). The combined analysis of these variants identified twenty-two different genetic profiles . Complete homozygosity for all wild-type alleles (GG GG CC AA) was present in 11.7%, being GG GG CT AA (22.4%) the most frequent profile. The results also demonstrated that 12.4% (58 out of 469) of São Miguel islanders have increased genetic predisposition to thrombosis. Subsequently, we evaluated these individuals for their warfarin response genetic profiles. The data showed that seven out of fifty-eight individuals are poor metabolizers (two with CYP2C9*2/*2 and five with CYP2C9*2/*3 genotypes). VKORC1 polymorphism analysis identified twelve individuals (20.7%) with AA genotype, who probably will require lower doses of warfarin. The joint analysis of CYP2C9 and VKORC1 revealed that 79.3% (46 out of 58) of the individuals carry at least one polymorphism in these genes. Within these, twenty-five individuals (43.1%) need intermediate and/or low doses of warfarin, if treatment is started.Among the 469 individuals, the data showed that thrombotic risk allele frequencies – 1691A (4.9%), 20210A (1.8%), 677T (41.7%) and 1298C (24.8%) – were similar to other Caucasians, but significantly different from mainland Portuguese (χThe present study demonstrated, for the first time, that São Miguel, and possibly the Azores population, shows significant differences on allele frequencies of thrombotic risk factors when compared to mainland Portugal. This research constitutes a primary approach for future studies on CVD, as well as for the implementation of warfarin dosing protocols using the patient's genotypic information. F5; 1q23), resulting in resistance to activated protein C (APC). In the coagulation biochemical pathway, APC prevents blood clots from growing extensively large, by inactivating coagulation factor V (encoded by F5). The G1691A variation may increase the risk for thrombosis by 8 and 91 in heterozygous and homozygous states, respectively [et al. [F2; 11p11-q12) represents the second most important inherited thrombotic risk factor. A single base substitution (G20210A) in the F2 3'-untranslated region causes elevated plasma prothrombin levels. The same authors reported a 2.8-fold increase risk for venous thrombosis in heterozygous and homozygous variant individuals [MTHFR; 1p36.3), includes two variants – C677T and A1298C [et al. [et al. [Cardiovascular diseases (CVD) are complex disorders that include dysfunctional conditions of the heart, arteries and veins. Within these diseases, thrombosis is the third most common cardiovascular event worldwide . It can tion G202A in the CYP2C9 (Cytochrome P450 2C9) and VKORC1 (Vitamin K epoxide reductase complex subunit 1) genes account for approximately 50–60% of drug dosing variability [CYP2C9, the most frequent variants are: CYP2C9*1 , CYP2C9*2 (Arg144Cys) and CYP2C9*3 (Ile359Leu). These last two are associated with decreased metabolic efficiency of the CYP2C9 enzyme and increased risk of bleeding when administrated initial dosages of warfarin [VKORC1 gene results in decreased mRNA transcription and increased sensitivity to warfarin inhibition of hepatic synthesis of functional vitamin K-dependent coagulation factors [et al. [VKORC1 polymorphisms and classified individuals according to warfarin dose requirements into distinct groups: high (GG), intermediate (GA) and low (AA).The genetic characterization of the individual's potential risk for developing thrombosis and response to drugs is essential in clinical follow-ups, as well as for medical prescription. Warfarin is one of the most widely used anticoagulant drug, which requires a thorough risk-benefit analysis since the dose prescribed should avoid hemorrhagic complications and achieves suppression of thrombosis . The admiability ,14. Cytoiability . Althougwarfarin . A G>A v factors . Rieder  [et al.  studied F5, F2 and MTHFR – involved in blood clot formation. Moreover, in the individuals with predisposition to thrombosis, we evaluated the pharmacogenetic profile for warfarin, by characterizing CYP2C9 and VKORC1 polymorphisms. This information is important for the implementation of warfarin pre-prescription genotyping with the goal of performing individualized treatment.The Azores population presents the highest standardized mortality rate caused by CVD when compared to mainland Portugal   and otheF5 (G1691A), F2 (G20210A) and MTHFR . After evaluating the genetic variants in the aforementioned genes, we selected a subset of individuals who met one of the following criteria: 1. one variant in F5 and one in MTHFR ; 2. one variant in F2 and one in MTHFR  and 3. one variant in F5, F2 and MTHFR . These criteria were chosen to restrict the sample to individuals who would have a higher genetic risk for a thrombotic event, and hence fifty-eight individuals were analysed for CYP2C9 and VKORC1 polymorphisms.The population sample comprised a group of 469 São Miguel Island blood donors from the anonymized Azorean DNA bank located in the main Hospital of Azores archipelago, Portugal . São MigF5 and MTHFR polymorphisms . The genotyping protocol for the detection of the F5 polymorphism and MTHFR variants was adapted from Kumar et al. [et al. [ms Table  were evar et al.  and Skib [et al. , respectF2, CYP2C9 and VKORC1 , F2 (G20210A) and MTHFR  polymorphisms were assessed in 469 healthy individuals from São Miguel Island, Azores  was present at 9.8% . We did not detect 1691AA individuals. The results on the F2 analysis show an allele frequency of 1.8% for the 20210A variant. Although this polymorphism is also uncommon in other populations, one homozygous variant individual (0.2%) and fifteen heterozygous (3.2%) were observed in the present study. The frequency of the variant alleles in MTHFR (677T and 1298C) was 41.7 and 24.8%, respectively. Overall, 17.9% are 677TT , and 6.0% present the 1298CC genotype .Allele and genotype frequencies of F5, F2 and MTHFR (order in genotype profile) were observed in São Miguel's population . No heterozygous or homozygous profiles for all four variants were observed. Almawi et al. [F5 G1691A or F2 G20210A with MTHFR 677TT genotype, respectively, enhancing the risk for deep vein thrombosis (DVT). Based on this criterion, fifteen  and two individuals  have higher risk for DVT development  to 4.9% (São Miguel Island). This trend is not evident for F2 G20210A, where the values present a lower range, from 0.3% for African-American to 2.6% for Spanish. African populations showed the lowest frequency for MTHFR variants. Taken together, the results demonstrate that, in São Miguel, the frequencies for all polymorphisms were similar to those found in Caucasians.The comparison of allele frequencies of the thrombotic genetic risk factors – F5 or F2 genes and one in MTHFR, we studied CYP2C9 and VKORC1 gene polymorphisms. The allele frequencies for CYP2C9*2 and CYP2C9*3 were 15.5 and 10.3%, respectively (Table VKORC1 gene (G1639A), we identified twenty (34.5%) wild-type individuals, twenty-six (44.8%) heterozygous and twelve (20.7%) homozygous variant, corresponding to an allele frequency of 43.1% for 1639A  individuals that probably will require high doses of warfarin , and eight (13.8%) will need lower doses . The remaining seventeen individuals (29.3%) present one of the following genotypes: *1*1-AA→nine individuals, *1*2-GA→four, *1*3-GA→three and *2*3-GG→one, where all of them will possibly necessitate intermediate doses. In summary, twenty-five individuals (43.1%) will need intermediate and/or low doses of warfarin, if treatment is started.The majority of individuals with a genetic predisposition to thrombosis  carry at least one polymorphism in the genes involved in the metabolism of warfarin  are more frequent and combined with a genetic predisposition further enhance the risk of a thrombotic event. In the present study, the results obtained from 469 São Miguel healthy individuals demonstrate that approximately 5% have the uese, 1% , we did r et al. , this vaal, 2.5% .MTHFR C677T variant, which has a relatively high frequency throughout the world and is considered a low risk factor for vascular diseases [MTHFR 677T shows an allelic frequency of 41.7%, very similar to Europeans . Family-based investigation tends to have biased relative risk estimation, because oversampling of affected individuals is normally present. Therefore, the genetic characterization of disease variants in the general population is important to correct this potential bias. Consequently, the present research will aid the clinical follow-ups of patients with recurrent thrombotic events.The joint analysis of all variants demonstrated that 12.4% of the total sample (58 out of 469) has increased predisposition to thrombosis development. In addition, comparison of allele frequencies for all thrombotic risk factors between São Miguel and mainland Portugal revealed statistically significant differences  -, indicating that individuals with this genotype require the highest warfarin maintenance doses [In today's medicine, the association between pharmacogenetics and drug therapy represents an important tool to treat or control complex diseases. Drug treatment is based on the assumption that a particular dose will yield a predefined blood concentration in circulation and, thereby, establish the desired therapeutic effect . Recent d VKORC1 . Here, wce doses .CYP2C9*2 and CYP2C9*3 alleles demonstrate a close similarity with different European populations [CYP2C9*1/*2 and CYP2C9*1/*3) represent a middle range of metabolic activity – intermediate metabolizers, IMs [CYP2C9*2 and CYP2C9*3 variants have less activity and metabolize warfarin more slowly. Individuals with one or two of these variations – poor metabolizers (PMs) – have an enhanced response to warfarin, as well as an increased risk of bleeding when beginning and during treatment if not genotyped previously [CYP2C9*2/*2 and five with CYP2C9*2/*3 genotypes; Table CYP2C9 PMs may exhibit different pharmacokinetics than wild-type individuals if they are medicated with warfarin. Consequently, these individuals possibly will require non-conventional doses of drugs that need CYP2C9 enzyme for biotransformation [Considering São Miguel islanders with genetic predisposition to thrombosis, the data of ulations -44. Indiers, IMs ,44. The ormation .VKORC1, a target of warfarin inhibition, may explain 23% of this drug dosage variability [VKORC1 promoter, potentially resulting in decreased transcription of the gene product. In general, the results suggest that 65.5% of the fifty-eight individuals with predisposition to thrombosis will require intermediate and/or low doses of warfarin, if treatment is started. Personalized medicine relies on data based on the human genome and disease biology characterization for the development of preventive, diagnostic, and therapeutic strategies that target the underlying determinants of disease in populations with specific molecular profiles [Polymorphisms within iability . Rieder iability  divided profiles . For thiThe identification of hereditary risk factors, with appropriate clinical evaluation will allow the informed patient and physician to work together for effective management of thrombosis and prevention of subsequent thrombotic events. The present study demonstrated, for the first time, that São Miguel, and possibly the Azores population, shows significant differences on allele frequencies of thrombotic genetic risk factors when compared to mainland Portugal. This research constitutes a primary approach for future studies on CVD, as well as for the implementation of warfarin dosing protocols using the patient's genotypic information.CYP2C9: Cytochrome P450 2C9 gene; DVT: Deep vein thrombosis; EMs: Extensive metabolizers; F2: Prothrombin gene; F5: Factor V Leiden gene; IMs: Intermediate metabolizers; MTHFR: Methylenetetrahydrofolate reductase gene; OR: Odds Ratio; PCR-RFLP: Polymerase chain reaction – restriction fragment length polymorphism; PMs: Poor metabolizers; SNP: Single nucleotide polymorphism; tHcy: Total homocysteine; VKORC1: Vitamin K epoxide reductase complex subunit 1 gene; VTE: Venous thromboembolism.ADRs: Adverse drug reactions; APC: Activated protein C; CI: Confidence interval; CVD: Cardiovascular diseases; The authors declare that they have no competing interests.MTHFR variants and PRP participated in the analysis. LMV designed the study, provided scientific orientation and revised the manuscript. All authors read and approved the final manuscript.CCB and TP, contributed equally, by performing the experiments, statistical analysis and drafting the manuscript. RC genotyped


Tomato bushy stunt virus (TBSV) replication in a yeast model host. Additional in vitro assays with whole cell extracts prepared from yeast strains expressing the eEF1A mutants demonstrated several functions for eEF1A in TBSV replication: facilitating the recruitment of the viral RNA template into the replicase complex; the assembly of the viral replicase complex; and enhancement of the minus-strand synthesis by promoting the initiation step. These roles for eEF1A are separate from its canonical role in host and viral protein translation, emphasizing critical functions for this abundant cellular protein during TBSV replication.Replication of plus-strand RNA viruses depends on host factors that are recruited into viral replicase complexes. Previous studies showed that eukaryotic translation elongation factor (eEF1A) is one of the resident host proteins in the highly purified tombusvirus replicase complex. Using a random library of eEF1A mutants, we identified one mutant that decreased and three mutants that increased  Tomato bushy stunt virus (TBSV) replication in a yeast model host. Based on a random library of eEF1A mutants, we identified eEF1A mutants that either decreased or increased TBSV replication. In vitro studies revealed that eEF1A facilitated the recruitment of the viral RNA template for replication and the assembly of the viral replicase complex, as well as eEF1A enhanced viral RNA synthesis in vitro. Altogether, this study demonstrates that eEF1A has several functions during TBSV replication.Plus-stranded RNA viruses are important pathogens of plants, animals and humans. They replicate in the infected cells by assembling viral replicase complexes consisting of viral- and host-coded proteins. In this paper, we show that the eukaryotic translation elongation factor (eEF1A), which is one of the resident host proteins in the highly purified tombusvirus replicase complex, is important for  Genome-wide screens for host factors affecting RNA virus infections have led to the identification of several hundreds host proteins in recent years Tomato bushy stunt virus (TBSV) and other tombusviruses are model plant RNA viruses with 4.8 kb genomic (g)RNA coding for two replication proteins, termed p33 and p92pol, and three proteins involved in cell-to-cell movement, encapsidation, and suppression of gene silencing Saccharomyces cerevisiae) expressing p33 and p92pol replication proteins can efficiently replicate a short TBSV-derived replicon (rep)RNA One of the major advantages of studying TBSV replication is the availability of genomic and proteomic datasets on virus-host interactions pol, the heat shock protein 70 chaperones , glyceraldehyde-3-phosphate dehydrogenase , pyruvate decarboxylase (Pdc1p), Cdc34p ubiquitin conjugating enzyme Additional global approaches based on the yeast proteome microarray (protein array) have led to the identification of over 100 host proteins that interact with viral RNA or the viral replication proteins TEF1 and TEF2), whereas animals and plants have 2–7 genes and several isoforms of eEF1A. (ii) eEF1A provides essential functions for cell viability and mutations could have pleiotropic effects on protein translation, actin bundling and apoptosis. (iii) eEF1A is a very abundant protein that constitutes 1–5% of total cellular proteins, making it difficult to completely remove eEF1A from biochemical assays using cell extracts. (iv) eEF1A is also required for the translation of viral proteins in infected cells, making it difficult to separate its effect on translation versus replication, processes that are interdependent.eEF1A is a highly abundant cellular protein with a role in delivering aminoacyl-tRNA to the elongating ribosome in a GTP-dependent manner. Many additional functions have been ascribed to eEF1A including quality control of newly produced proteins, ubiquitin-dependent protein degradation, and organization of the actin cytoskeleton Turnip yellow mosaic virus (TYMV) Tobacco mosaic virus (TMV) and Turnip mosaic virus (+)RNA The first evidence that translation elongation factors, such as EF-Tu and EF-Ts, play a role in (+)RNA virus replication was obtained with bacteriophage Qbeta in vitroThe actual biochemical functions provided by eEF1A for (+)RNA virus replication are currently poorly understood. In case of WNV, eEF1A is co-localized with the WNV replicase in the infected cells and mutations in the WNV (+)RNA within the mapped eEF1A binding site have led to decreased minus-strand synthesis pol replication proteins eEF1A has been shown to interact with the components of the tombusvirus replicase, including the 3′-UTR of the repRNA, as well as the p33 and p92in vitro approaches. The obtained data support the model that eEF1A plays several roles during TBSV replication, including facilitating the assembly of the viral replicase complex. Moreover, using in vitro replication assays, we demonstrate that eEF1A enhances minus-strand synthesis via stimulating the initiation step of the viral RNA-dependent RNA polymerase. Since eEF1A is also associated with several other viral replication proteins or binds to viral RNAs, it is possible that the uncovered functions of eEF1A might be utilized by other RNA viruses during their replication as well.In this paper, we characterized the functions of eEF1A in TBSV replication based on identification of functional eEF1A mutants in yeast as well as using TEF1/TEF2) were deleted from the chromosome, while the wt or mutated eEF1A was expressed from plasmids. Importantly, a given eEF1A mutant is the only source of eEF1A in the yeast cells used. Using the high-throughput assay, we identified one yeast strain (N21) expressing an eEF1A mutant that supported reduced TBSV repRNA replication, while the other three strains with eEF1A mutants  showed increased level of repRNA accumulation -stranded repRNA and (+)-stranded progeny Since eEF1A is part of the tombusvirus replicase complex in vitro TBSV repRNA replication more efficiently than the replicase with the wt eEF1A. In contrast, CFE from N21 yeast supported TBSV repRNA replication to similar extent as the CFE containing wt eEF1A  or (−)-strand synthesis, we analyzed the replication products under non-denaturing versus denaturing conditions (32P-labeled (−)RNA product hybridized with the (+)RNA] increased ∼3-fold in case of C42, C53 and C62 mutants , which is unlike the E. coli-expressed TBSV or CNV p92pol RdRp, does not need the yeast cell-free extract to be functional in vitroin vitro to produce the complementary (−)RNA product RNA synthesis by a viral RdRp.To test directly if eEF1A could stimulate RNA synthesis by the viral RdRp, we chose the in vitro RdRp assay was due to binding of eEF1A to the (+)RNA template and/or to the TCV RdRp protein. Pre-incubation of the purified wt eEF1A with the TCV RdRp prior to the RdRp assay led to a ∼5-fold increase in in vitro (−)RNA synthesis (Since it is known that eEF1A can bind to the 3′-UTR of TBSV (+)RNA as well as to the tombusvirus replication proteins de novo initiation of RNA synthesis by the TCV RdRp de novo initiation step in the RdRp assay.To test if eEF1A stimulates the rate of initiation of (−)RNA synthesis, we analyzed the amount of abortive RNA products, which are generated during de novo initiation, we analyzed the amount of 3′-terminal extension (3′-TEX) RNA products, which are generated from an internal primer by the TCV RdRp RNA synthesis.To test if eEF1A stimulates the rate of RNA synthesis in the absence of TCV RdRp  [55]. AdTCV RdRp , suggestTo further test the function of eEF1A in TBSV replication, we used chemical inhibitors of eEF1A, including Didemnin B (DB) and Gamendazole (GM). DB inhibits the activity of GTP-bound eEF1A during translation by binding to a pocket in eEF1A involved in the interaction with the aminoacylated tRNA and the nucleotide exchange factor eEF1Balpha in vitro assembly/replication assay, also based on CFE containing endogenous eEF1A 32P-labeled UTP) to the membrane fraction of the CFE to allow for RNA synthesis by the pre-assembled replicase complex  and, second, biotin-labeled viral (+)repRNA was added. After short incubation in the absence or presence of various amount of DB, we performed affinity-purification of the viral RNA. Phosphoimaging revealed that eEF1A was co-purified with the viral RNA and the amount of protein co-purified with the viral (+)repRNA was inhibited by increasing amount of DB in the assay , and template recruitment to intracellular membranes he assay . Altoget(+)RNA virus replicases contain viral- and host-coded components, which likely provide many yet undefined functions to facilitate robust virus replication in infected cells. Translation factors, such as eEF1A, are among the most common host factors recruited for (+)RNA virus replication. eEF1A is an integral component of several viral replicases, including the highly purified tombusvirus replicase complex. Since eEF1A is an essential G protein involved in translation elongation, it is difficult to obtain evidence for its direct involvement in virus replication in living cells. Indeed, down-regulation of eEF1A in cells has led not only to decreased TBSV repRNA accumulation, but also reduced p33 levels pol, but likely via directly altering viral replication and affecting the activity of the viral replicase. On the other hand, N21 mutant of eEF1A resulted in decreased TBSV RNA accumulation and also led to reduction in the level of p33 replication protein. This is reminiscent of the previously characterized GDP-binding mutant T22S Based on the previous successful strategy of analyzing eEF1A mutants, here we generated ∼6,000 random mutants covering the entire eEF1A sequence and found four mutants, which greatly affected TBSV repRNA accumulation in yeast . Among tpol in this in vitro assay , the role of eEF1A in TBSV replication must be separate from its role in protein translation. Altogether, these data strongly established that eEF1A is directly involved in TBSV replication, independent of the role of eEF1A in protein translation.In addition to this genetic evidence on the relevance of eEF1A in TBSV replication in yeast, we also obtained additional supporting data by showing that chemical inhibitors of eEF1A, such as DB and GM, strongly inhibited replication of TBSV repRNA in the cell-free replication assay . Since wThe identified eEF1A mutants were also useful to dissect the functions of eEF1A in TBSV replication. Based on a cell-free TBSV replication assay in CFE prepared from yeast expressing the C42, C53 or C62 mutants, we found that the minus-strand synthesis was enhanced by ∼3-fold, while the rate of plus-strand synthesis was proportionate with (−)RNA synthesis, resulting in ∼10-fold more (+) than (−)RNA products for wt and each mutant.in vitro by using a highly purified eEF1A and the recombinant TCV RdRp, which is closely homologous with the TBSV p92pol. Interestingly, it seems that eEF1A stimulates the RdRp activity directly, since pre-incubation of eEF1A and the RdRp prior to the RdRp assay led to the highest level of stimulation of (−)RNA synthesis repRNA in vitro experiments with purified tombusvirus replicase preparations confirmed the lack of inhibition of RNA synthesis by DB or GM RNA DB or GM  on pre-ain vitroThe inhibition of the tombusvirus replicase complex by DB or GM might come from the ability of these compounds to inhibit the template RNA recruitment step . If the de novo initiation for RNA synthesis. Indeed, the chaperone activity of eEF1A and its bacterial homolog EF-Tu has been shown before The presented data are also in agreement with the function of eEF1A as a chaperone of the viral RdRp. Binding between the eEF1A and RdRp might alter the structure of the RdRp that favors Overall, the current work demonstrates two major functions for eEF1A in TBSV replication : (i) stiSaccharomyces cerevisiae strain BY4741 (MATahis3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was obtained from Open Biosystems . Plasmid-borne TEF1/2 TKY strains  were published before Cucumber necrosis virus (CNV) p33 and the TBSV replicon RNA, called DI-72, was described earlier LYS2-based plasmid pRS317-Tet-His92, expressing CNV p92 under the control of Tetracycline-regulatable (Tet) promoter was constructed as follows: the Tet promoter sequence was obtained from pCM189-His92/Tet EcoRI and BamHI, and CNV p92 coding sequences from pGAD-His92 BamHI and PstI, followed by ligation into pRS317 vector treated with EcoRI and PstI. To generate mutations within TEF1 coding sequence by random mutagenesis, we constructed the TRP1-based plasmid pRS314-pTEF1-TEF1, which expressed TEF1 under the control of its native promoter. The TEF1 promoter sequence, the TEF1 coding region and the Cyc1 terminator sequences were amplified by PCR with the following primer pairs, #2764 (CCGCGAGCTCATAGCTTCAAAATGTTTCTAC)/#2765 (CCGCGGATCCGTAATTAAAACTTAGATTAGATTGC), #2768 (CCGCGGATCCAAAATGGGTAAAGAGAAGTCTC)/#1877 (CCGCCTCGAGTTATTTCTTAGCAGCCTTTTGAGCAGC), and #2769 CCGCCTCGAGGAGGGCCGCATCATGTAA/#2770 (CCGCGGTACCAGCTTGCAAATTAAAGCCTTC), respectively. This was followed by cloning the PCR products into pRS314 digested with SacI and KpnI.2, 0.3 mM MnCl2, 1 mM dCTP and dTTP each, 0.2 mM dGTP and dATP each, 0.2 µM of each primer, 20 pM of template DNA and 10 units of Taq polymerase in a 10 µl reaction volume in 10 aliquots. The PCR was performed for 30 cycles at 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min in a conventional thermal cycler. Three overlapping ∼300–500 bp N-, central- and C-terminal segments of the TEF1 gene were amplified separately by PCR using primer pairs: #2767 (GTTTCAGTTTCATTTTTCTTGTTC)/#2788 (GAGTCCATCTTGTTGACAG), #2787 (CATCAAGAACATGATTACTGGTAC)/#2790 (GACGTTACCTCTTCTGATTTC) and #2789 , respectively.The mutagenic PCR conditions were as follows: 50 mM KCl, 10 mM Tris (pH 8.3 at 25°C), 7 mM MgCl−) by shaking at 29°C overnight. To completely suppress TBSV replication before induction, 1 mg/ml Doxycycline was added to the media to inhibit the expression of p92. The plasmid pool carrying the randomly mutated TEF1 gene was introduced into the yeast cells already transformed with the two virus expression plasmids by in vivo gap repair mechanism via homologous recombination  was transformed together with overlapping PCR (20 µg) products carrying the TEF1 mutations created by random mutagenic PCR (see above). The transformed yeast cells were selected on SC media lacking uracil, tryptophan, leucine, histidine and lysine. The colonies were further streaked onto SC media plate lacking tryptophan, leucine, histidine and lysine (SC-TLHK−) with 0.1%  5-Fluoroorotic Acid (5-FOA) media to select against the URA3-based wild-type TEF1 plasmid repRNA transcript, 400 ng purified MBP-p33, 100 ng purified MBP-p92pol (both recombinant proteins were purified from E. coli), 30 mM HEPES-KOH, pH 7.4, 150 mM potassium acetate, 5 mM magnesium acetate, 0.13 M sorbitol, 0.4 µl actinomycin D (5 mg/ml), 2 µl of 150 mM creatine phosphate, 0.2 µl of 10 mg/ml creatine kinase, 0.2 µl of RNase inhibitor, 0.2 µl of 1 M dithiothreitol (DTT), 2 µl of 10 mM ATP, CTP, and GTP and 0.25 mM UTP and 0.1 µl of [32P]UTP 32P-labeled RNA products were separated by electrophoresis in a 5% polyacrylamide gel (PAGE) containing 0.5× Tris-borate-EDTA (TBE) buffer with 8 M urea. To detect the double-stranded RNA (dsRNA) in the cell-free replication assay, the 32P-labeled RNA samples were divided into two aliquotes: one half was loaded onto the gel without heat treatment in the presence of 25% formamide, while the other half was heat denatured at 85°C for 5 min in the presence of 50% formamide 32P-labeled RNA was performed at 37°C for 30 min in a buffer containing 5 mM sodium acetate (pH 4.5 at 25°C), 0.28 M NaCl, 4.5mM ZnSO4 and 40 U S1 nuclease (Boehringer).Whole cell yeast extract capable of supporting TBSV replication In vitro TBSV replication in the fractions was performed as described Fractionation of the whole cell extract was done according to E. coli were carried out as described earlier with modifications E. coli strain BL21 Rosetta (DE3). Protein expression was induced using isopropyl β-D-thiogalactopyranoside (IPTG) for 8 h at 16°C, then the cells were collected by centrifugation . The recombinant TCV p88C protein was purified on an amylose resin column (NEB), as described pol were purified as above, except 30 mM HEPES-KOH pH 7.4 was used instead of 20 mM Tris-Cl pH 8.0. eEF1A was purified from yeast as described Expression and purification of the recombinant TBSV p33 and p92 and TCV p88C replication proteins from 2, 10 mM KCl, 0.5 M NaCl, 0.1% Nonidet P-40 (NP-40), and 1% [V/V] yeast protease inhibitor cocktail (Ypic)] by glass beads using FastPrep Homogenizer . The yeast cell lysate was cleared by centrifugation at 500× g for 5 min at 4°C to remove unbroken cells and debris. The membrane fraction containing the viral replicase complex was collected by centrifugation at 21,000× g for 15 min at 4°C and then solubilized in 1 ml TG buffer with a buffer containing 1% NP-40, 5% SB3–10 [caprylyl sulfobetaine] (Sigma), 1% [V/V] Ypic via gentle rotation for 1 h min at 4°C. The solubilized membrane fraction was centrifuged at 21,000× g for 15 min at 4°C and the supernatant was incubated with 20 µl anti-FLAG M2-agarose affinity resin (Sigma) pre-equilibrated with 0.7 ml TG buffer. After 2 h of gentle rotation at 4°C, we washed the resin 5 times with TG buffer containing 1% NP-40, the resin-bound replicase complex was eluted in 100 µl elution buffer [50 mM Tris–HCl [pH 7.5], 10% glycerol, 15 mM MgCl2, 10 mM KCl, 0.05 M NaCl, 0.5% Nonidet P-40 (NP-40), 1% Ypic and 0.15 mg/ml Flag peptide (sigma)]. In vitro RdRp activity assay was performed by using DI-72 RI(−) RNA template transcribed in vitro by T7 transcription Yeast strains  were transformed with plasmids pESCHIS4-ADH-HF33/CUP1-DI-72 expressing 6XHis- and Flag-tagged CNV p33 and the TBSV DI-72 repRNA, and pRS317-Tet-His92, expressing CNV p92 under the control of Tet promoter 2, 1 mM DTT, 0.1 mM EDTA, 10% [vol/vol] glycerol, 10 U of RNase inhibitor, 10 nM 32P-labeled DI-72 (+) RNA probe and 0.5 µg purified eEF1A protein EMSA was performed in a 10 µl-reaction containing 20 mM HEPES [pH 7.6], 50 mM KCl, 2 mM MgCl35S methionine using Rabbit Reticulocyte Lysate (Promega) according to manufacturer's manual. The in vitro eEF1A translation product (10 µl) was pre-incubated in a 200 µl binding buffer  with 150 µM Didemnin B  or DMSO for 30 min at 30°C and then incubated with biotinylated DI-72(+) RNA-bound SA-PMPs for 1 h at 4°C. The SA-PMPs were collected in a magnetic stand and washed 5 times with the binding buffer, followed by elution with 30 µl SDS-PAGE sample buffer. The eluted protein samples were resolved by SDS-PAGE and then exposed to phosphorimager.For in vitro eEF1A-repRNA co-purification, DI-72(+) repRNA was biotin-labeled in standard T7 transcription reaction in the presence of 20 µM Biotin-16-UTP (Roche). After the T7 transcription, the unincorporated biotin-UTP was removed on a Bio-Rad mini gel filtration column. The biotinylated RNA was immobilized on a column containing Streptavidin MagneSphere Paramagnetic Particles (SA-PMPs). Briefly, a 30-µl suspension of SA-PMPs (Promega) was washed three times with 1 ml of water and re-suspended in 1× Phosphate Buffered Saline (PBS). Biotinylated DI-72(+) RNA (5 µg) was then added to the suspension of SA-PMPs, followed by 30 min incubation at 4°C with gentle rotation. The SA-PMPs were collected on the side of the tube in a magnetic stand and washed 3 times with 1× PBS buffer. eEF1A was translated in vitro and labeled with 2, 10 mM DTT, 1.0 mM each ATP, CTP, and GTP, 0.01 mM UTP plus 0.1 µl of [32P]UTP, 7 pmol template RNA, 2 pmol affinity-purified MBP-p88C. 20 pmol eEF1A was added to the reaction at the beginning or as indicated in the text and 32P-labeled RNA products were analyzed by electrophoresis in a 5% or 15% PAGE/8 M urea gel The TCV RdRp reactions were carried out as previously described for 2 h at 25°C in vitro reaction are indicated in the text. The cell-free TBSV replicase assay and the in vitro TBSV replicase assembly assay were performed according to pol and (+) repRNA were added to the cell-free reaction in the presence of 1.0 mM ATP and GTP in step 1. After incubation at 25°C for 1 h, the in vitro reactions were centrifuged 21,000× g at 4°C for 10 min. The supernatant containing extra p33, p92pol and repRNA, which were not bound to the membranes in the cell-free extract, was discarded, while the membrane pellet was re-suspended in a standard in vitro replicase assay buffer containing [32P]-UTP and ATP, CTP, and GTP, and incubated at 25°C for 3 h Purified Didemnin B (NSC 325319) was kindly provided by the Natural Products Branch, NCI , while Gamendazole was a generous gift from Dr. Tash . Both chemicals were dissolved in DMSO . The concentrations of chemical and time point of the addition of the chemicals to the in vitro RNA recruitment reaction was performed according to 32P-labeled DI-72 (+)repRNA were used and rCTP, rUTP, 32P-labeled UTP, and Actinomycin D were omitted from the reaction. As a negative control, a recruitment-deficient repRNA, termed C99-G mutant, was used  in vitro assay, followed by incubation on ice for 10 min. Samples were centrifuged at 35,000× g for 1h, and the pellet was washed with 1 ml reaction buffer, followed by centrifugation at 35,000× g for 10 min. The membrane-bound repRNA was extracted from the pellet by adding 0.1 ml stop buffer and 0.1 ml phenol/chloroform and vortexing, followed by isopropanol/ammonium acetate precipitation The TBSV viral RNA gets recruited to the membrane from the soluble fraction with the help of TBSV replication proteins and host factors present in the yeast CFE. The was used , lane 2 Figure S1tef1Δtef2Δ carried a plasmid that expressed one of the random eEF1A (TEF1) mutants from the native promoter. Each yeast strain also carried pESCHIS4-ADH-His33/CUP1-DI-72 and pRS317-Tet-His92 to induce TBSV repRNA replication as described in the M&M section. (B) Additional experiments on the effect of eEF1A mutations on TBSV repRNA accumulation in yeast. The yeast strain expressed only one form of eEF1A, as indicated. Top panel: Replication of the TBSV repRNA was measured by Northern blotting 24 h after initiation of TBSV replication. (C) The accumulation level of repRNA was normalized based on the rRNA . Panels (D), (E) and (F) show the accumulation of p92pol, p33 and eEF1A, respectively, estimated by Western blotting using anti-His and anti-eEF1A antibody. (G) SDS-PAGE analysis of total protein extract from the above yeast strains, after Coomassie blue-staining.Schematic presentation of the random mutagenesis strategy used to obtain 6,000 eEF1A mutants. (A) The yeast strain ((0.15 MB PDF)Click here for additional data file.Figure S2in vitro. (A) MBP-tagged TCV p88C (lacking the p28-overlapping domain from the N-terminus), MBP-TBSV p92, MBP-TBSV p92C (lacking the p33-overlapping domain from the N-terminus) and MBP-TBSV p33 or MBP (1 µg each) were separately immobilized on amylose beads, followed by incubation with a cytosolic extract prepared from yeast. The bound host proteins were eluted from the beads and were analyzed by 10% SDS-PAGE and detected via Western blotting using anti-eEF1A antibody (Top panel). The affinity-purified recombinant MBP-TCV p88C, MBP-TBSV p92, MBP-TBSV p92C, MBP-TBSV p33 and MBP were analyzed by 10% SDS-PAGE and Coomassie blue-staining (Bottom panel). (B) The effect of eEF1A mutations on binding to the viral p33 and p92 proteins in vitro. MBP-tagged p92, p33 or MBP were separately immobilized on amylose beads, followed by incubation with a cytosolic extract prepared from yeast expressing wt or mutated eEF1A. The bound eEF1A was eluted from the beads and were analyzed by 10% SDS-PAGE and detected via Western blotting using anti-eEF1A antibody (Top panel). The affinity-purified recombinant MBP-TBSV p92, MBP-TBSV p33 and MBP were analyzed by 10% SDS-PAGE and Coomassie blue-staining (Bottom panel). (C) The effect of eEF1A mutations on binding to the viral repRNA. CFE containing WT or mutated eEF1A was incubated with biotin-labeled DI-72(+) repRNA. Then the repRNA was captured with streptavidin-coated magnetic beads, followed by elution of the co-purified proteins from the beads. Western blot analysis shows the amount of co-purified eEF1A using anti-eEF1A antibody.Binding of eEF1A to TBSV and TCV replication proteins (0.12 MB PDF)Click here for additional data file.Figure S3Lack of inhibition of TBSV repRNA replication by Cyclohexamide in a cell-free TBSV replicase assay. The cell-free TBSV replicase assay was performed as described in (0.05 MB PDF)Click here for additional data file.Figure S4in vitro activity of the purified tombusvirus replicase by Didemnin B and Gamendazole. (A) The membrane-bound tombusvirus replicase in a yeast lysate was solubilized with Triton X-100/SB3-10 detergent, followed by purification on a FLAG-affinity column as described. The activity of the affinity-purified TBSV replicase was tested on the same amount of DI-72(−) RNA added to each sample. DB (panel on the left) and GM (panel on the right) were added in the following amounts: 0, 100, 150, 200, 250 µM for DB and 0, 25, 50, 100, 200 µM for GM. Denaturing PAGE analysis of the 32P-labeled RNA products obtained with the purified tombusvirus replicase is shown. Note that this replicase preparation is only capable of complementary RNA synthesis on the added template RNA, but incapable of supporting a full cycle of replication. (B) The effect of GM and DB on binding to the viral p33 and p92 proteins in vitro. MBP-tagged p92 and p33 were separately immobilized on amylose beads, followed by incubation in the presence of 0 or 100 µM GM or 150 µM DB with a cytosolic extract prepared from yeast expressing wt eEF1A. The bound eEF1A was eluted from the beads and were analyzed by 10% SDS-PAGE and detected via Western blotting using anti-eEF1A antibody (Top panel). The affinity-purified recombinant MBP-TBSV p92, MBP-TBSV p33 and MBP were analyzed by 10% SDS-PAGE and Coomassie blue-staining (Bottom panel).Lack of inhibition of the (0.09 MB PDF)Click here for additional data file.


The effect of charge modification of photoimmunoconjugates (PICs) on their biodistribution in a xenograft model of ovarian cancer was investigated. Chlorin(e6)c(e6) was attached site specifically to the F(ab')2 fragment of the murine monoclonal antibody OC125, directed against human ovarian cancer cells, via poly-1-lysine linkers carrying cationic or anionic charges. Preservation of immunoreactivity was checked by enzyme-linked immunosorbent assay (ELISA). PICs were radiolabelled with 125I and compared with non-specific rabbit IgG PICs after intraperitoneal (i.p.) injection into nude mice. Samples were taken from normal organs and tumour at 3 h and 24 h. Tumour to normal 125I ratios showed that the cationic OC125F(ab')2 PIC had the highest tumour selectivity. Ratios for c(e6) were uniformly higher than for 125I, indicating that c(e6) became separated from 125I. OC125F(ab')2 gave highest tissue values of 125I, followed by cationic OC125F(ab')2 PIC; other species were much lower. The amounts of c(e6) delivered per gram of tumour were much higher for cationic OC125F(ab')2 PIC than for other species. The results indicate that cationic charge stimulates the endocytosis and lysosomal degradation of the OC125F(ab')2-pl-c(e6) that has bound to the i.p. tumour. Positively charged PICs may have applications in the i.p. photoimmunotherapy of minimal residual ovarian cancer.


Drosophila and previous in vitro studies of mammalian Atonal homolog 1  suggest an anti-oncogenic function for the Atonal group of proneural basic helix-loop-helix transcription factors. We asked whether mouse Atoh1 and human ATOH1 act as tumor suppressor genes in vivo. Genetic knockouts in mouse and molecular analyses in the mouse and in human cancer cell lines support a tumor suppressor function for ATOH1. ATOH1 antagonizes tumor formation and growth by regulating proliferation and apoptosis, likely via activation of the Jun N-terminal kinase signaling pathway. Furthermore, colorectal cancer and Merkel cell carcinoma patients show genetic and epigenetic ATOH1 loss-of-function mutations. Our data indicate that ATOH1 may be an early target for oncogenic mutations in tissues where it instructs cellular differentiation.Colon cancer accounts for more than 10% of all cancer deaths annually. Our genetic evidence from  Atonal homolog 1 (ATOH1), is highly conserved in flies, mice, and humans. We asked whether ATOH1 could be a pivotal factor in causing colon cancer in mice and humans. Our studies show that colon-specific loss of ATOH1 in mice is sufficient to trigger colon cancer and that the majority of human colon cancers also have an inactivated ATOH1. Reactivating ATOH1 in cultured human colon cancer cells causes these cells to stop dividing and to commit suicide. Since reactivation of this epigenetically silenced gene can be achieved using small chemical compounds, studying how ATOH1 acts may offer therapeutic avenues in the future.Like most cancers, colon cancer displays a loss of differentiation, and the stronger this property, the more aggressive the cancer. This suggests that the loss of the capacity to differentiate may be a critical and possibly early event during the formation of these tumors. The key gene instructing secretory cell fate differentiation in the epithelium of the colon, namely  A master regulator of differentiation in the colon is a tumor suppressor gene for colon cancer. Ato (CG7508) ortholog, ATOH1 (Ensembl accession number: ENSG00000172238), is essential for cell fate commitment of mechanoreceptive Merkel cells in the skin ) were cultured in McCoy medium supplemented with 10% FCS. Primary human keratinocytes were isolated and pooled from foreskins of three different donors (less than 6 y). Fourth passage cells were used in the experiments. The procedure has been approved by the ethical committee of the University of Leuven. Experiments performed adhered to the Declaration of Helsinki Principles. Keratinocytes were seeded in serum-free and growth factor–containing medium , which contains several growth factors  were calculated using the formula T2X = LN(2)/((LN(ntΔ)-LN(n0))/Δt) with Δt, time in culture, n0, number of seeded cells, and ntΔ the number of cells after ΔtA fixed amount of cells was seeded in standard culture conditions, and the number of cells was counted after 3 to 5 d. The doubling times , and one with eGFP alone (MCC14.2-GFP) as a negative control, under the control of a CMV promoter  in culture medium. A 2-ml layer of 0.35% agarose in culture medium was added on top of the 2-ml 0.6% layer. After 1 wk, 2 ml of 0.35% agarose in culture medium was added. Cells were cultured for 2 wk in standard conditions, and the number of colonies was analyzed under an inverted microscope with a 10× magnification. The experiment was done in triplicate.Cells were grown up to 40% confluency under standard conditions and were trypsinized. Cells were fixed and permeabilized using 70% ice-cold ethanol for 2 h. Cells were washed in PBS, and cells were stained for 30 min using 0.1% (v/v) Triton X-100 (Sigma) in PBS and 0.2 mg/ml DNase-free Rnase A (Sigma) and 20 μg/ml PI (Sigma). Cells were analyzed on a FACSCalibur cytometer (BectonDickinson). To analyze the cell cycle progression, cells were pulsed for 20 min with 10 μM BrdU (Sigma). Medium was then substituted with normal medium for 6 h. Cells were collected and fixed in 70% ice-cold ethanol. DNA was denatured using 2 M HCl for 20 min. Cells were stained using anti-BrdU and IgG-alexa555 and analyzed in a FACSCalibur (BD Bioscience).Annexin-V staining was performed using Annexin-V-biotin (Roche) and streptavidin-PercP (BD biosciences) as described by the manufacturer. Cells were imaged with the Leica TCS Sp2 confocal microscope, and images were analyzed and quantified using LCS software.3VO4, 20 μM PAO, and Complete Protease Inhibitor. The crude extract was separated by SDS-PAGE in a 4%–12% NuPAGE novex bis-tris gel (Invitrogen) and electroblotted onto Hybond-ECL membrane (Amersham). Antibodies were diluted in the appropriate concentrations in 5% BSA in TBS-tween20. The antibodies used are beta-actin , anti–cleaved caspase-3 , polyclonal antibody to caspase-9(active) (ALEXIS), chromogranin A+B (Abcam), cyclinA1 (Santa Cruz Biotechnology), pJNK , p27 (BD Bioscience), p21waf1 , PCNA , TRK (Santa Cruz Biotechnology), Atoh1 , NT3 (Santa Cruz Biotechnology), and Cleaved Notch . All western blot analyses are quantified in Lysates were obtained by scraping cells in medium and centrifuging the medium. The pellet was resuspended in lysis buffer containing PBS with 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 1% TritonX, 5 mM NaK252a diluted in DMSO, from VWR, and DMSO as control were used. Final concentrations ranged from 0 μM to 0.5 μM; when no concentration is mentioned, 0.33 μM was used. SAPK inhibitor II, from Calbiochem, was used at a concentration of 10 μM. We used inhibitor X as a γ-secretase inhibitor . Concentrations used are mentioned in the figure legend.ATOH1 primers CAGCCAGTGCAGGAGGAAAA and GAAAATTCCCCGTCGCTTCT, and the Hes1 primers GGACATTCTGGAAATGACAGTGAA and AGCGCAGCCGTCATCTG, according to the manufacturer's protocol. Quantitative RT-PCR was performed on a ABI prism 7000 (Applied Biosystems). The primers were designed using Primer Express (Applied Biosystems). The primers used are ATOH1: CAGCCAGTGCAGGAGGAAAA and GAAAATTCCCCGTCGCTTCT; Atoh1: GCTGTGCAAGCTGAAGGG and TCTTGTCGTTGTTGAAGG. Primers to check copy number of the ATOH1 locus: ATOH1 locus set 1: CCCCGGGAGCAATCTTG and GGGACCGAGGCGAAGTT; control locus set 1: TCTGGGACCTGAGCTAATGGA and GGCCATAATTAGGACCATGAAAGA; and ATOH1 locus set 2: GCCAGTGCAGGAGGAAAACA and GAAAATTCCCCGTCGCTTCT. Control locus set 2: GGGTTCAGCCTCAACTTGTATCC and CCCACCACCTGGCATCTCT.mRNA was amplified using the Superscript II One-Step RT-PCR system with Platinum Taq-100 reactions (Invitrogen) and the free (Ambion) following the manufacturer's protocol. cDNA was synthesized using random primers and SuperscriptII reverse transcription (Invitrogen) using the manufacturer's protocol. cDNA was amplified using gene-specific primers (concentration 5 μM) for the different human tyrosine kinases.RNA was diluted to 300 μg/μl and treated with TURBO DNA-Methylation of the DNA was detected using ApaI enzyme. DNA (2 μg) was dissolved in 50 μl of the appropriate buffer and 10 U of ApaI, and incubated at 30 °C overnight. A mirror condition was done in which the ApaI was exchanged by glycerol as a control for unspecific degradation. The resulting DNA was analyzed using two primer sets: one set spanning the restriction site (AATAAGACGTTGCAGAAGAG and TCGCAGAGCAAAAATTAAAGGGTGC) and another set next to the restriction site (CCCCGGGAGCATCTTGCAGCCA and TCGCAGAGCAAAAATTAAAGGGTGC).Pull-down of methylated DNA fragments (restricted using EcoRI) was performed using the Methylcollector kit (Active Motif), and bisulfite modification of DNA was done using the EZ DNA Methylation-Gold kit (Zymo Research) according to the manufacturers' protocols.We carried out array CGH using Code Linked Slides (AP Biotech) containing the 3,527 BAC clones from the Wellcome Trust Sanger Institute 1 Mb Clone Set, a gift from N. P. Carter (The Wellcome Trust Sanger Institute). Array CGH was performed as described .The ATOH1 open reading frame was amplified with PCR using AATAAGACGTTGCAGAAGAG and TCGCAGAGCAAAAATTAAAGGGTGC and AmpliTaq Gold DNA polymerase and the GeneAmp PCR System 2400 (Applied Biosystems). The PCR products were purified and sequenced in both directions on the ABI Prism BigDye (Terminator Cycle Sequencing Kit version 1.1) on an ABI PRISM 3100 Genetic Analyser (Applied Biosystems).Figure S1wtAtoh1 mice showed large GALT that were macroscopically counted as polyps; shown is 5× magnification of representative colonic GALTs. GALT indicated by an arrow.The colons of AOM-treated (726 KB PDF)Click here for additional data file.Figure S2minAPC background still have goblet cells, indicating that Atoh1 is still active.(A) Polyps in the minAPC; ΔintestineAtoh1 mice originate in Atoh1 mutant tissue as seen by the absence of goblet cells.(B) The polyps in the (4.70 MB PDF)Click here for additional data file.Figure S3wtAtoh1 (WT) crypts were distinguished from Atoh1-null crypts by the lack of the secretory goblet cells in the null crypts.AOM-treated colon sections with well-oriented crypts were used for BrdU and cleaved caspase-3 (c-Caspase 3) counting. The genotypes of the representative slides are indicated on the left side of the figure. The specific stain is identified at the top part of the figure. wtAtoh1 crypts in wtAtoh1 mice (A); and nondeleted wtAtoh1 (B) and Atoh1-null (C) in Atoh1Δintestine mice.(A–C) Hematoxylin and eosin (H & E) staining of wtAtoh1 mice (D) and nondeleted wtAtoh1 (E) and Atoh1-null (F) in ΔintestineAtoh1 mice.(D–F) Representative BrdU staining of normal-appearing crypts in wtAtoh1 mice (G); and nondeleted wtAtoh1 (H) and Atoh1-null (I) in ΔintestineAtoh1 mice. Arrows point to positive cells at the surface of the crypts. The images were captured at 20× magnification.(G–I) Representative cleaved caspase-3 staining of normal-appearing crypts in (4.96 MB PDF)Click here for additional data file.Figure S4ATOH1 mRNA transcripts in five independent MCC cell lines. The name of the cell line is indicated above each lane. RT-PCR was done with 100 ng of RNA under nonsaturating conditions.(A) (B) Doubling times in hours of the five MCC cell lines. Error bars indicate the standard deviation.ATOH1 in MCC1 and MCC14.2 cell lines. ATOH1 mRNA levels standardized to GADPH mRNA levels.(C) RT-qPCR for (760 KB PDF)Click here for additional data file.Figure S5ATOH1 genomic DNA (gDNA) over control locus is presented for each of the two primer sets as analyzed by qPCR. The next two columns indicate the classification of the deletion/duplication status of the ATOH1 locus. The mRNA expression is shown relative to control colon samples, with next to it, the classification of the expression. Clinical data are given in the third panel, namely cancer stage and metastasis. In the last panel, methylation of the ATOH1 locus is shown using three different methods.First column indicates patient numbers. Next, the relative ratios for (760 KB PDF)Click here for additional data file.Figure S6ATOH1 locus is shown with an arrow. The abnormalities were confirmed by dye swap experiments.Each bar represents the log2 of the value for affected individuals (ind) versus a control sample (reference) for each probe, ordered based on the probes' chromosomal location. The region between 131 Mb and 141 Mb is shaded. The location of the (4.55 MB PDF)Click here for additional data file.Figure S7ATOH1 locus. The white box indicate the ATOH1 ORF, and the gray box the position of the CpG island. The primers are indicated as arrows.(A) Schematic representation of ATOH1 locus using the ApaI methylation-sensitive restriction enzyme. PCR fragments generated using primers spanning the ApaI restriction site from ApaI restricted genomic DNA; presence of a band indicates methylation of the ATOH1 CpG island.(B) Detection of methylation at the ATOH1 CpG island.(C) Detection of methylation using methylation-sensitive PCR: upon bisulfite modification, presence of a band indicates methylation of the (159 KB PDF)Click here for additional data file.Figure S8(A) GST Pull-down assays using Atoh1 protein fused to GST (GST-Atoh1) and in vitro translated Dnmts .l[methyl-3H] methionine incorporated into an oligonucleotide substrate. GST-tagged embryonic ectoderm development protein (GST-EED) was used as a positive control.(B) A GST-fused Atoh1 protein was used to purify DNA methyltransferase activity from nuclear extracts. After incubation, the beads were washed and assayed for DNA methyltransferase activity read as c.p.m. of S-adenosyl-(C) Coimmunoprecipitation experiments shows that Atoh1 associated with Dnmt1. The 293T cells were transiently transfected in culture dishes (10-cm diameter) with 3 μg of HA-Atoh1 plasmid(D) Mapping of Atoh1 binding to Dnmt1. GST Pull-downs were performed with Dnmt1 fragments fused to GST and in vitro–translated Atoh1. The upper part is a schematic representation of the Dnmt1 sequences used.(1.04 MB PDF)Click here for additional data file.Figure S9wtAtoh1 crypt (dashed white line). The arrows indicate pJNK-positive cells.(A) Representative pJNK1/2 staining in Atoh1-null (dashed black line) crypts in ΔintestineAtoh1 mice.(B) pJNK-positive cells (arrows) in wild-type (dashed gray line) and Atoh1-null crypts in ΔintestineAtoh1 mice. Error bars indicate the standard error of the mean. No significant differences between genotypes were detected.(C) Bar graph showing the percentage of pJNK-positive cells in wild-type mice (white); and wild-type (gray) and (4.18 MB PDF)Click here for additional data file.Figure S10Atoh1 expression (MCC14.2-Atoh1.1a and MCC14.2-Atoh1.2a). No significant change in distribution throughout the cell cycle can be observed.(A) Cell cycle distribution of MCC14.2-derived cell lines without (MCC14.2 and MCC14.2-GFP) and with (B) Maximal projection image of AnnexinV staining (red) on cells transduced with lentiviral vectors expressing GFP (left panel) or Atoh1-IRES-GFP (right panel). GFP is in green.kip, c-myc, phospho-H3 and p21waf1 of lysates of MCC14.2 cells, MCC14.2-GFP and two MCC14.2 cell lines transduced with Atoh1-IRES-eGFP (MCC14.2-Atoh1.1a and MCC14.2-Atoh1.2a). The corresponding actin loading controls are shown under each blot.(C) Western blot analysis for CyclinA1, PCNA, p27(D–H) Quantification of expression levels of cyclinA1 (D), PCNA (E), p27 (F), c-myc (G), and phospho-histoneH3. (H) Representative blots are shown in (C).(2.90 MB PDF)Click here for additional data file.Figure S11Atoh1-IRES-eGFP (MCC14.2-Atoh1.1a and MCC14.2-Atoh1.2a). GADPH loading control is shown below.(A) RT-PCR for target of Notch signaling HES1 on mRNA isolated from MCC14.2 cells, MCC14.2 cells transduced with GFP, and two MCC14.2 cell lines transduced with (B) Western blot analysis for cleaved intracellular Notch (NICD). Different concentrations of presinilin inhibitor X were used; a plus sign (+) indicates a positive control for cleaved NICD.(C) Different concentrations of γ-secretase inhibitor (inhibitor X) on MCC14.2 do not influence the proliferation rate. First lane: 10 μM inhibitor X, second lane: DMSO control of previous lane, third lane: 1 μM inhibitor X, fourth lane: DMSO control of previous lane, and fifth lane: untreated.Atoh1-IRES-eGFP vectors (lane 3: MCC14.2-Atoh1.2a) and on untransfected HT29 cells (lane14: HT29), GFP-transfected HT29 cells (lane5: HT29-GFP), and HT29 cells transfected with the Atoh1-IRES-eGFP construct (lane 6: HT29-Atoh1).(D) RT-PCR for expression of 90 tyrosine kinases scored from undetectable (white) over orange (expression) to red (strong expression) on mRNA or on untransduced MCC14.2 cells (lane 1), GFP transduced MCC14.2 cell line (lane 2), and two independent MCC14.2-derived cell lines transduced with ATOH1 mRNA levels (compared to GADPH mRNA levels) in MCC cell lines. MCC1 (first lane: MCC cell line with endogenous high ATOH1 expression), MCC14.2 (lane 2), GFP-transduced MCC14.2 cell line (lane 3), and two independently made MCC14.2-derived cell lines transduced with Atoh1-IRES-GFP vectors .(E) RT-qPCR for (F) Western blot analysis for NTRK1 and Neurotrophin-3 (NT3) of untransduced MCC14.2 cells (lane 1), GFP-transduced MCC14.2 cell line (lane 2), and two independently made MCC14.2-derived cell lines transduced with Atoh1-IRES-GFP vectors ; actin loading controls are represented below each blot.(G and H) Quantifications of western blots in (F), normalized to actin.(2.04 MB PDF)Click here for additional data file.Figure S12The quantifications are performed with USI software. The signal for the protein of interest was standardized to its respective actin loading control. Antibody is stated above each graph, representative experiments are shown in main figures: (A) (365 KB PDF)Click here for additional data file.


Recombinant viruses labelled with fluorescent proteins are useful tools in molecular virology with multiple applications . We generated by homologous recombination three recombinant cytomegaloviruses carrying the enhanced yellow fluorescent protein (EYFP) fused with the viral proteins IE-2, ppUL32 (pp150), and ppUL83 (pp65). In growth kinetics, the three viruses behaved all like wild type, even at low multiplicity of infection (MOI). The expression of all three fusion proteins was detected, and their respective localizations were the same as for the unmodified proteins in wild-type virus–infected cells. We established the in vivo measurement of fluorescence intensity and used the recombinant viruses to measure inhibition of viral replication by neutralizing antibodies or antiviral substances. The use of these viruses in a pilot screen based on fluorescence intensity and high-content analysis identified cellular kinase inhibitors that block viral replication. In summary, these viruses with individually EYFP-tagged proteins will be useful to study antiviral substances and the dynamics of viral infection in cell culture. Betaherpesvirinae subfamily. With a genome size of approximately 235 kbp it contains one of the largest genomes among viruses. HCMV is known to have a narrow host range infecting only humans, where 40–90% of the global human population become seropositive after primary infection. Postnatal primary infection is usually asymptomatic in immune competent hosts and is followed by a lifelong persistence. In individuals with an immature or compromised immune system HCMV can cause considerable morbidity and mortality The human cytomegalovirus (HCMV) is a herpesvirus belonging to the The replication cycle of HCMV in the host can be divided into three different phases, the IE-phase, where mainly genes with regulatory functions are expressed, the early-phase for the expression of enzymatic proteins and the late-phase for expression of the structural components Aequorea victoria green fluorescent protein (GFP) for the labelling of viral proteins. The enhanced yellow fluorescent protein (EYFP) is a mutant of the avEGFP with an emission maximum of 530 nm instead of 509 nm for the EGFP Recombinant fluorescent viruses have been generated for other viruses to investigate the movement of viral particles inside the infected cell In this study we generated by recombineering  or 72 hpi (UL83-EYFP and UL32-EYFP). Fusion proteins were detected using specific antibodies directed against the respective viral protein or GFP. The IE2-EYFP fusion protein could be readily detected as a band of about 115 kDa, both by an IE1/2 specific serum  and a GFSince ppUL83 (pp65) and ppUL32 (pp150) represent major components of the virus particle we analyzed concentrated viral particles for the presence of EYFP-fusion proteins in the respective virus variants. Viral particles were standardized for equal amount of major capsid protein (MCP), as shown in We then analyzed, if the intracellular localization of the viral EYFP-fusion proteins corresponded to the situation in wild type infected cells. For this, we infected HFF cells with wild type virus and the three different recombinant viruses and performed immunofluorescence analyses at different time points post infection. For TB4-IE2-EYFP a nuclear localization was observed at all time points . As in wTo determine, whether the infection with these viruses could be quantified, we established the infection in the 96-well format and measured the fluorescence signal in live cells using a Cary Eclipse fluorescence spectrophotometer. First, we determined the best time-point for measurement and analyzed the dependence of the fluorescence signal on the MOI used for infection. We infected HFF cells with different MOI ranging from 0.01 to 10 and measured fluorescence intensity in arbitrary units. We obtained the best results 8 dpi, which was chosen for further measurements. As shown in R, a commercial hyperimmune globulin preparation, a neutralizing antibody directed against glycoprotein H (14-4B) and a non-neutralizing antibody (27–287) directed against glycoprotein B. As demonstrated in R and the neutralizing antibody was readily detected. The EC50 for FlebogammaR was determined at a dilution of 1/1024 and for anti-gH the EC50 was at a dilution of 1/512. These results were also confirmed by fluorescence microscopy. As control, after preincubation with different concentrations of the non neutralizing anti-gB antibody no reduction in fluorescence intensity was observed.To evaluate the use of these viruses for measuring virus inhibitors, we first used the TB4-IE2-EYFP to measure inhibition of virus entry by neutralizing antibodies. For these experiments we chose to infect cells with MOI of 0.1. Virus was preincubated with FlebogammaIn additional experiments we evaluated the effect of GCV on the replication of TB4-IE2-EYFP, TB4-UL83-EYFP and TB4-UL32-EYFP. These measurements were performed on a Tecan Safire 2, which allowed us to detect signals as early as 4 dpi. Using the three viruses we established EC50 values for GCV of 5.43±0.15 µM, 7.58±0.39 µM and 8.09±0.06 µM, respectively. These experiments demonstrate the use of these viruses to quantify infection and antiviral activities.To demonstrate the feasibility of antiviral screening with these viruses, we used a library of cellular kinase inhibitors. As before infections were performed in the microplate format and measured in the Tecan Safire 2 microplate reader at 4 dpi. Inhibitors were added at a concentration of 10 µM at the time of infection and replaced every second day. We first evaluated the toxicity of the compounds in HFF used in our experiments . In seveWhen we compared whether the three fluorescent viruses produced similar results we observed, that some compounds produced differential pattern of inhibition. Two compounds  showed a more pronounced inhibition of TB4-UL83-EYFP or TB4-UL32-EYFP compared to TB4-IE2-EYFP with a high viability of the non-infected cells, while others  had a more reduced cell viability (30–50%). These compounds could be involved in the regulation of effects selective for specific phases of the replicative cycle. The confirmation of these effects would require additional experiments at different concentrations, which were beyond the scope of this work.Finally, we analyzed the nucleocytoplasmic translocation of ppUL83-EYFP on the single cell level . For thiIn this study, we constructed three different recombinant HCMV containing fusions of EYFP to viral proteins of different gene expression kinetic classes. We could show that all recombinant viruses grew similar to wild-type and observed no disturbance of protein expression and intracellular localization. In consideration of the urgent need to find new antiviral agents for the treatment of patients, we established a new method to measure the effect of antivirals in intact cells rather than fixated cells or cell extracts.To generate the recombinant viruses we developed a vector, where EYFP was placed next to a kanamycin resistance marker, to be able to select for transfer. Both genes were amplified with primers containing sequences homologous to the desired insertion site. The kanamycin gene was flanked by FRT-sites, which were then used by FLP recombinase to remove the resistance marker. As a result, EYFP was integrated into the viral genome and one FRT site along with flanking vector sequences of a total 88 bp remained after the marker excision. Previously we had used a similar strategy for re-insertion of a deleted gene into a deletion mutant of HCMV So far only few recombinant herpesviruses encoding fluorescent proteins have been described We first analyzed whether the recombinant viruses could be used for investigations of the dynamics of virus infection. For this the fusion proteins needed to be expressed and localized correctly. Since EYFP is a spectral variant derived from EGFP by a few point mutations we could use an anti-EGFP antibody to detect EYFP. By western blot experiments we could detect the fluorescent fusion partner in all recombinant viruses by using either a specific antibody or an antibody directed against GFP. In all cases a shift in protein size of about 26 kDa, the size of EYFP, was observed. Only in the case of ppUL83 a shorter specific band of about the size of native ppUL83 was observed. In principle, such a form could arise by incomplete synthesis or alternatively by post-translational processing. The latter case would generate free EYFP, which would be detrimental to the use of the virus for analysis of intracellular dynamics. However, the anti-EGFP antibody did not detect any free EYFP, favouring the first hypothesis, namely incomplete synthesis. In the next step, we analyzed the localization of the EYFP-fusion proteins. We compared both the intracellular localization in wild type and recombinant virus, and the fluorescence distribution arising from EYFP and the viral protein stained by specific antibodies. In all cases a close overlap was observed. IE2 showed a purely nuclear staining, as reported As application we explored the use of these recombinant viruses to measure the potency of different antiviral agents against the human cytomegalovirus. We could establish conditions suitable to measure fluorescence intensity non-destructively in live cells and could show that the relative fluorescence intensity is highly correlated to the amount of virus . Previous experiments using fluorescent HCMV required lysis of cells, which makes these measurements more labour-intensive To test different antiviral substances we first infected HFF and investigated the efficiency of ganciclovir to inhibit viral replication. Ganciclovir (GCV) is a potent antiviral agent which is used for treatment in patients with acute HCMV infection and disease R, as well as supernatants from hybridoma cell lines directed against the viral glycoproteins gH or gB, respectively. We could clearly measure a neutralising capacity of FlebogammaR and the monoclonal antibody 14-4B (anti-gH) To investigate a different mode of antiviral intervention, we also measured the capability of neutralizing antibodies to block the infection using our marked viruses. Since neutralizing antibodies act at the stage of virus attachment or entry we used the recombinant virus TB4-IE2-EYFP. For neutralization we used an approved hyperimmune serum, FlebogammaFinally, we performed a pilot screen using 80 kinase inhibitors. We observed strong inhibition with two compounds . All oligonucleotides are noted in 5′ to 3′ direction.5′-TGAGCCTGGCCATCGAGGCAGCCATCCAGGACCTGAGGAACAAGTCTCAGGGCCCCGGCCCCATGAATTCACTGATCAAGGA-3′, IE2-ko5-BAC 5′-CGGGGAATCACTATGTACAAGAGTCCATGTCTCTCTTTCCAGTTTTTCACCGTCGTGGAATGCCTTCGAATTC-3′, ul83-egfp-BAC 5′-ACGCCTTGCCCGGGCCATGCATCGCCTCGACGCCCAAAAAGCACCGAGGTGGCCCCGGCCCCATGAATTCACTGATCAAGGA-3′, UL83-ko5 5′-AGTGGACGTGGGTTTTTATAGAGTCGTCCTAAGCGCGTGCGGCGGGTGGCCGTCGTGGAATGCCTTCGAATTC-3′, ul32-egfpBAC 5′-CCGTGCAGAACATCCTCCAAAAGATCGAGAAGATTAAGAACACGGAGGAAGGCCCCGCGGCCATGAATTCACTGATCAAGGA-3′, ul32-ko5-gfp 5′CACTATCCGATGGTTTCATTAAAAAGTACGTCTGCGTGTGTGTTTCTTAACGTCGTGGAACTTCGAATTC-3′IE2-3gfpBAC SalI and NheI. The resulting plasmid, pSL-FRT-Kan-EYFP, contained the EYFP-gene adjacent to a kanamycin-resistance gene, which was flanked by FRT-sites to allow its removal by FLP-mediated recombination.To generate a suitable recombination vector, we excised the EYFP gene from pEYFP-C1  and cloned it into the vector pSL-FRT To generate linear fragments for recombination, we performed touchdown PCR to amplify the whole EYFP kanamycin-cassette. Primers included 50 nts sequence homologous to the intended insertion site. In addition, a GPGP linker peptide was encoded by the fusion primer between the last codon of the respective viral gene and the first codon of EYFP to ensure conformational uncoupling between the viral protein and the fused EYFP.DpnI to remove the template plasmid. The fragments were transformed into E. coli strain DH10B  containing the bacterial artificial chromosome TB4 (short for TB40E-BAC4) and plasmid pKD46 encoding the γ, β and exo genes of the Red recombinase system The PCR-products were purified  and digested with 2.Human foreskin fibroblasts (HFF) were cultivated in minimal essential medium (MEM)  supplemented with 10% fetal calf serum, 2 mM L-Glutamine , 100 U/ml Penicillin/100 µg/ml Streptomycin (Gibco/BRL). The cells were grown at 37°C with 95% humidity and 5% CO2 flasks and incubated for 2 weeks to let plaques develop. The successful reconstitution was monitored by fluorescence microscopy. Cell-free virus stocks were produced by ultracentrifugation of the supernatants of infected HFF.For reconstitution, DNA from recombinant BAC clones was purified by alkaline lysis  and transfected into HFF using the EasyjecT Optima® electroporation system  with the settings 200 V and 1500 µF. After electroporation cells were seeded into 75 cmEcoRV and separated by gel electrophoreses using a 0.6% agarose-gel. For blotting, the gel was washed for 20 min in 0.25 M HCl, rinsed in water followed by incubation for 30 min in 1.5 M NaCl and shortly washed with 0.5 M NaCl. The gel was then equilibrated in transfer buffer (1.5 M NaCl and 0.25 M NaOH) and nucleic acids transferred to a Hybond N nylon membrane  by capillary blot BAC-DNA was digested with the restriction enzyme 32P-dCTP. Radioactive hybridizations were performed in a hybridization incubator  essentially as described previously Probes for hybridization were generated by PCR amplification and subsequent agarose gel purification. Radioactive labelling was performed by random hexamer priming using Ready-to-go DNA labelling beads  and α4 HFF were seeded into a 24-well plate and infected with a multiplicity of infection (MOI) of 1, 0.1 or 0.01 for TB4-wt and all three different variants. After infection the plates were centrifuged for 30 min with 2000 rpm at room temperature and incubated for 3 h after the centrifugation. Thereafter, the cells were washed with PBS and the medium was changed. At day 1, 3, 5, 7 and 10 after infection supernatants were harvested and stored frozen in sucrose phosphate-buffer at −70°C. After the titration using HFF in 96-well plates the plaque forming units (pfu) per ml were determined. All titrations were done in triplicates in three independent experiments.For virus growth kinetics, 4×10HFF were infected with MOI of 1 for 72 hours in 6-well plates and then harvested by scraping the cells off in 500 µl PBS. Cell pellets were resuspended in 5xSDS-Buffer  and boiled at 95°C for 5 min. The cell-lysates were separated by using a 10% SDS-PAGE and blotted on a PVDF-Membrane. For detection rabbit serum directed against IE1+2 For the detection of major tegument proteins and fusion proteins in virus particles, supernatant from infected cells was clarified from cellular debris by low speed centrifugation  followed by ultracentrifugation  to concentrate virus particles. Virus stocks were adjusted for equal levels of major capsid protein by titration and immunoblot detection. For detection, rabbit serum directed against GFP , ppUL32 (XP1-5) 4 HFF were seeded on glass cover slips in 24-well plates and incubated overnight. Cells were infected with MOI of 0.5 and incubated for 24, 48 and 72 hours. For fixation cells were incubated in 4% PFA for 10 min and 0.1% Triton X-100 for 2 min followed by washing steps in PBS. Viral proteins were detected using specific primary monoclonal mouse antibodies against ppUL83 , ppUL32 (XP-1) or IE1–2 . As secondary antibody we used Alexa555-labelled polyclonal goat anti-mouse antibody (Invitrogen).For immunofluorescence analysis, 4×104 HFF per well in culture media (as described before) and incubated at 37°C over night. To measure the relation between multiplicity of infection and fluorescence intensity, we measured different MOI after 8 days of incubation. For this experiment the cells were infected with MOI of 10, 3, 1, 0.3, 0.1 and 0.01.To assess the usefulness of the TB4-IE2-EYFP, TB4-UL83-EYFP or TB4-UL32-EYFP virus variants for detection of antiviral activity, we performed infection experiments in 96-well plates. We seeded 1.7×10To test viral sensitivity for antiviral agents, cells were infected with TB4-IE2-EYFP, TB4-UL83-EYFP or TB4-UL32-EYFP at MOI of 0.1. Ganciclovir  was added to the infected cells using different concentrations. After 9 days of incubation at 37°C, cells were washed and incubated in PBS for the measurement. PBS gave the best results compared to medium without pH-indicator or normal medium. Relative fluorescence intensity was measured using a fluorescence spectrophotometer . Using excitation and emission slit width of 10 nm the emission was scanned from 500 nm to 600 nm at an extinction setting of 480 nm.To measure neutralization by antibodies, TB4-IE-EYFP (MOI of 0.1) was pre-incubated with Flebogamma , mouse monoclonal anti-gH (14-4B) 4 HFF per well in 96-well plates in culture media and incubated at 37°C over night. Cells were infected with fresh supernatant of TB4-IE2-EYFP, TB4-UL83-EYFP or TB4-UL32-EYFP. Virus supernatant was titrated in parallel and corresponded to a MOI of 0.3 to 1. As compounds we used a kinase inhibitor library ; each inhibitor was tested in triplicates. Inhibitors were added together with virus at a final concentration of 10 µM and were replaced every second day. For measurements cell culture medium was removed and cells were washed twice in PBS. Measurements were taken in a Safire 2  from the top at excitation wavelength 485 nm (20 nm bandwidth) and emission wavelength 525 nm (20 nm bandwidth) with an integration time of 40 µs and 10 reads per well. We used optimal gain, optimized Z-position and high sensitivity flash mode. Background was substracted from raw data and infected cells were normalized to 100%. Calculations and descriptive statistics were carried out in Excel. EC50 values were determined with the IC50 package in the R environment http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm071173.pdf). Cell viability was measured in parallel plates using the colorimetric WST-1 cell proliferation assay .For screening a compound library, we seeded 1.7×10The nucleocytoplasmic translocation of ppUL83-EYFP was analyzed in a Pathway 855 . Two hours before imaging live cells Hoechst 33342  was added at a final concentration of 1 µg/ml to allow delineation of the nuclei. A matrix of 3×3 images was taken for each well . To define regions of interest, the Hoechst stained nuclei where segmented. To define cytoplasmic localization, a second ring-shaped mask around a nucleus was defined starting 10 pixels away from the nucleus and reaching 10 pixels into the cytoplasm. The gap between the nuclear mask and the ring mask reduces nuclear fluorescence in the cytoplasmic region. The algorithm automatically prevents adjacent ring masks from overlapping. All image processing was performed using the BD-Attovision Software Version 1.6.1. To exclude background staining, we set a threshold for EYFP nuclear staining at an intensity of 450. Samples with less than 40 cells above this threshold were excluded from further analysis (