Human adenovirus has evolved to infect and replicate in terminally differentiated human epithelial cells, predominantly those within the airway, the gut, or the eye. To overcome the block to viral DNA replication present in these cells, the virus expresses the Early 1A proteins (E1A). These immediate early proteins drive cells into S-phase and induce expression of all other viral early genes. During infection, several E1A isoforms are expressed with proteins of 289, 243, 217, 171, and 55 residues being present for human adenovirus type 5. Here we examine the contribution that the two largest E1A isoforms make to the viral life cycle in growth-arrested normal human fibroblasts. Viruses that express E1A289R were found to replicate better than those that do not express this isoform. Importantly, induction of several viral genes was delayed in a virus expressing E1A243R, with several viral structural proteins undetectable by western blot. We also highlight the changes in E1A isoforms detected during the course of viral infection. Furthermore, we show that viral DNA replication occurs more efficiently, leading to higher number of viral genomes in cells infected with viruses that express E1A289R. Finally, induction of S-phase specific genes differs between viruses expressing different E1A isoforms, with those having E1A289R leading to, generally, earlier activation of these genes. Overall, we provide an overview of adenovirus replication using modern molecular biology approaches and further insights into the contribution that E1A isoforms make to the life cycle of human adenovirus in arrested human fibroblasts. The late genes constitute primarily viral structural proteins. The first gene expressed upon viral infection is the immediate early gene called Early Region 1A (E1A). The HAdV5 E1A gene encodes for two major proteins of 289 and 243 residues (R) that are expressed immediately after infection. These proteins arise from differential splicing of the same transcript and differ only by the presence of an internal sequence of 46 amino acids in the larger protein. At a later point in infection, splicing produces three additional mRNA species, which encode for proteins of 217, 171 and 55 amino acids [Human adenoviruses (HAdV) are a family of small, non-enveloped viruses with linear double-stranded DNA genomes of ~35 kilobase pairs. The viral genome is sub-divided into regions termed early and late, depending on when their transcription commences. Early genes are expressed in the initial stages of the infection, and late genes are expressed only after viral genome has begun to replicate . The priHAdV5 E1A is a potent transcriptional regulator, yet it lacks the ability to directly bind to DNA. To overcome this limitation, E1A uses specific DNA-bound transcription factors to recruit itself to chromatin –13. ThisThe CR3 domain of HAdV5 E1A289R consists of residues 139–204, containing a C4 zinc finger domain that likely forms a defined structure and several sub-regions that are required for proper transcriptional activation and promoter targeting , 16 243Rdl520) or E1A289R (pm975) compare to a virus expressing all E1A isoforms (dl309) in viral growth, gene expression, and viral DNA replication. Here we show that viruses expressing E1A289R (dl309 and pm975) grow considerably better and faster than a virus lacking this isoform (dl520). Indeed, dl520 was never able to grow to the same degree as dl309 or pm975, suggesting an incomplete capacity by E1A243R to support virus replication. Viruses expressing E1A289R are able to drive viral gene and protein expression more efficiently, and are able to replicate their genomes faster and to greater numbers. Surprisingly, despite very different kinetics of infection and gene expression, induction of viral DNA replication occurred at similar times regardless of the E1A isoform present. We have also observed that the induction of cell cycle regulated genes occurs earlier when E1A289R is present. Our study provides further insights into how the different isoforms of E1A contribute to the viral life cycle and provides a modern view of the early events in viral infection under conditions that mimic the natural target of the virus, which are growth arrested and terminally differentiated cells.In the present study we re-evaluate the contributions of different E1A isoforms to viral infection in arrested human cells using modern molecular biology approaches. In order to better understand how the major E1A isoforms contribute to viral growth in arrested human cells, we set out to determine how viruses that express E1A243R  was applied to cells at a final concentration of 10μM 4 hours prior to the indicated harvest time without changing the media.IMR-90 cells (ATCC# CCL-186) were grown in Dulbecco’s Modified Eagle’s Medium (HyClone) supplemented with 10% fetal bovine serum (Invitrogen), streptomycin and penicillin (HyClone). To arrest IMR-90 cells, cells were grown to 100% confluence, and then incubated for an additional 72 hours to allow for complete growth arrest. All virus infections were carried out in serum-free media for 1 hour. Prior to infection, conditioned media from arrested IMR-90 cells was saved and was replaced back onto the cells after 1 hour incubation with the virus. All viruses used were grown in-house and were used at a multiplicity of infection (m.o.i.) of 5 for dl309, and m.o.i. of 10 for dl520 and pm975. One hour prior to fixation, cells were pulsed with EdU for 1 hour as per manufacturer’s specifications using the Click-It EdU labeling kit for microscopy (Life Technologies). After EdU labeling, cells were fixed in 3.7% formaldehyde, stained for EdU using the Click-It kit with AlexaFluor 488, and labelled for E1A using M73 monoclonal antibody and AlexaFluor-594 conjugated secondary antibody (Jackson Immunoresearch). Cells were visualized using LSM700 laser confocal microscope and ZEN software suite. Quantification was carried out using ImageJ software.IMR-90 cells were grown until 100% confluent on LabTek II 4-chamber slides (Thermo-Fisher). After becoming fully confluent, cells were incubated for a further 72 hours to achieve growth arrest. Infections were carried out as described above with m.o.i. of 5 for dl309, dl520, or pm975 at a variable m.o.i. to ensure equal E1A expression and at 8, 16, 24, and 72 hours after infections total cellular RNA was extracted using the TRIzol Reagent (Sigma) according to manufacturer’s instructions. 1.25 micrograms of total RNA was used in reverse-transcriptase reaction using SuperScript VILO reverse transcriptase (Invitrogen) according to the manufacturer’s guidelines using random hexanucleotides for priming. The cDNA was subsequently used for real-time expression analysis using the BioRad CFX96 real-time thermocycler. Fold changes in expression were determined by comparing expression levels of viral genes with dl309-infected cells at 24 hours after infection, while levels of cellular genes were compared to mock-infected cells. Analysis of expression data was carried out using the Pfaffl method [E1B, E2, E3, E4, and hexon were previously described [BLM, MCM4, and PCNA in [TTTTGAACCACCTACCCTTC, while the reverse primers were: 13S –CCACAGGTCCTCATATAGCAAA, and 12S –GGAGTCACAGCTATCCGTACTACT; for 11S and 10S the forward primer was common and the sequence is: GATCGAAGAGCCCGAGCA, while the reverse primers were the same as the reverse primers used for 13S and 12S, respectively. 9S splice product was detected with primers: TGATCGAAGAGGTCCTGTGTCT and TCAGGATAGCAGGCGCCA. Total E1A was detected with primers binding within exon 2, which is common to all E1A splice variants: TCCGGTCCTTCTAACACACC and GGCGTTTACAGCTCAAGTCC.IMR-90 cells were infected with escribed , and for PCNA in . For det7 copies per reaction down to 1.0 copy per reaction. The primers used were the same as those used for expression analysis of E1B region, the annealing temperature used was 60°C and 40 cycles were run.IMR-90 cells were lysed in lysis buffer  on ice for 10 minutes. Lysates were sonicated briefly in a Covaris M220 focused ultrasonicator to break-up cellular chromatin and subjected to digestion using Proteinase K (NEB) according to manufacturer’s specifications. Following digestion viral DNA was purified using GeneJET PCR Purification Kit (Thermo-Fisher). PCR reactions were carried out using SYBR Select Master Mix for CFX (Applied Biosystems) according to manufacturer’s directions using 2% of total purified DNA as template according to manufacturer’s instructions using a CFX96 Real Time PCR instrument (BioRad). Standard curve for absolute quantification was generated by serially diluting pXC1 plasmid containing the left end of HAdV5 genome starting with a concentration of 1.0x10dl309 [pm975 [dl520 [2, after which cells were bathed in conditioned media and were re-incubated at 37°C under 5% CO2. Virus titres were determined 24, 48, 72, 96 and 120 h after infection, and plaque assays were performed on 293 cells by serial dilution. Prior to harvest, images of cells were acquired using phase-contrast optics on a Fisher Micromaster microscope.Arrested IMR-90 cells were infected with HAdV5 dl309 , pm975 [9 [pm975 , or dl525 [dl520  viruses,dl309, and m.o.i. of 10 for dl520 and pm975. To ensure that equal levels of E1A proteins were expressed as compared to dl309, we determined E1A levels at 24 hours after infection by western blot using the M73 antibody and quantifying the respective E1A bands , dl520 (expressing E1A243R and E1A171R), or pm975 (expressing E1A289R and E1A217R). Viral titres were determined by plaque assays every 24 hours after initial infection starting at 24 hours and ending at 120 hours , replicated to the highest level. HAdV5 dl520, which does not express any E1A isoforms that contain CR3, showed consistently lower titres than either pm975 or dl309.Our previous work has shown that HAdV5 expressing different E1A isoforms  have different abilities to drive arrested IMR-90 cells into S-phase . Interesds inset . IMR-90 20 hours . Althougdl309 or pm975 showed phenotypic changes associated with CPE much sooner than dl520 infected cells, at approximately 72 hours after infection. CPE was not observed in dl520 cells until approximately 96 hours after infection. Complete CPE was observed only in pm975-infected cells at 120 hours, while dl309-infected cells showed almost complete CPE at this time point. Cells infected with dl520 displayed considerably delayed CPE and at 120 hours resembled those observed at 96 hours for dl309- or pm975-infected cells. These observations show that the different E1A isoforms contribute differentially to virus growth, leading to significant differences in viral titres. In particular, viruses that express E1A289R were found to replicate significantly faster as compared to dl520, which does not express E1A289R.To assess the phenotypic effects of viral infection on arrested IMR-90 cells we monitored the cytopathic effect (CPE) and cellular morphology during the infection . Uninfecdl309, dl520, or pm975  were never detectable in dl520-infected cells by western blot. Together, these observations demonstrate significant differences in the rates of viral infection of arrested IMR-90 cells.Levels of other viral proteins examined (E2A DNA-binding protein (DBP) and viral structural proteins) correlated closely with their respective mRNAs . The lev24 hours . Similardl309-infected arrested human fibroblasts , E1A289R (pm975), or E1A243R (dl520) replicate, express their genes and proteins, copy their DNA, and drive expression of select cell-cycle regulated genes in arrested lung fibroblasts.Human adenovirus expresses five different E1A mRNA species during the course of viral infection, with proteins of 289R, 243R, 217R, 171R, and 55R being present at some point during infection with HAdV5 , 37, 38 dl520, lacking E1A289R or E1A217R, replicated slower than dl309 and pm975. On average, this virus trailed by about 24 hours in terms of viral titres (dl520 never achieved titers as high as those observed with dl309 or pm975 (data not shown), suggesting a fundamental inability of E1A243R to fully support viral replication. Surprisingly, pm975, expressing only E1A289R, replicated slightly faster than dl309 virus that expresses all E1A isoforms, with the last time-point showing convergence between dl309 and pm975. This result is somewhat different from previous reports [pm975 behaved similarly to dl309 in several metrics used to assess how the virus is performing, such as gene expression and induction of CPE , or E1A243R (dl520)  were not detectable. Interestingly, the amount of E1A mRNA continued to climb throughout the course of infection  . E1A lev20 . E1A ion Figs , it is ndl520-infected cells as compared to those infected with dl309 or pm975 . Since most of our real-time data was analyzed during earlier time points , while k repair . It is p4 orf6/7 , which cdl520, lacking this isoform entirely. Importantly, replication of dl520 never reaches the same levels of viral yield as those observed for dl309 or pm975, suggesting a fundamental defect in the viral life cycle since initial virus input should not affect final virus output under ideal conditions (such as those seen in cell culture models as used here). Much remains to be discovered about the contribution that E1A289R makes to deregulation of cellular growth, but our studies have so far highlighted the differences in how this E1A isoform behaves compared to the others, providing greater understanding of deregulation of cellular growth by adenovirus.Recently, E1A289R has not been the primary focus of E1A research. However, our previous work , 16, 22


Xist (Xm-Xist) during preimplantation in mouse embryos is essential for establishing imprinted X chromosome inactivation. Nuclear transplantation (NT) studies using nuclei derived from non-growing (ng) and full-grown (fg) oocytes have indicated that maternal-specific repressive modifications are imposed on Xm-Xist during oogenesis, as well as on autosomal imprinted genes. Recent studies have revealed that histone H3 lysine 9 trimethylation (H3K9me3) enrichments on Xm-Xist promoter regions are involved in silencing at the preimplantation stages. However, whether H3K9me3 is imposed on Xm-Xist during oogenesis is not known. Here, we dissected the chromatin states in ng and fg oocytes and early preimplantation stage embryos. Chromatin immunoprecipitation experiments against H3K9me3 revealed that there was no significant enrichment within the Xm-Xist region during oogenesis. However, NT embryos with ng nuclei (ngNT) showed extensive Xm-Xist derepression and H3K9me3 hypomethylation of the promoter region at the 4-cell stage, which corresponds to the onset of paternal Xist expression. We also found that the chromatin state at the Xist genomic locus became markedly condensed as oocyte growth proceeded. Although the condensed Xm-Xist genomic locus relaxed during early preimplantation phases, the extent of the relaxation across Xm-Xist loci derived from normally developed oocytes was significantly smaller than those of paternal-Xist and ngNT-Xist genomic loci. Furthermore, Xm-Xist from 2-cell metaphase nuclei became derepressed following NT. We propose that chromatin condensation is associated with imprinted Xist repression and that skipping of the condensation step by NT leads to Xist activation during the early preimplantation phase.Repression of maternal  Summary: The analysis of chromatin state and H3K9me3 levels in mouse oocytes and early embryos provides insights into the dynamics of Xist repression and activation during early development and reprogramming. Xist) is essential for the initiation of X chromosome inactivation (XCI) in female mice and humans  expression is driven by the deposition of maternal Rnf12   is tightly protected by epigenetic factors. Using parthenogenetic embryos, which are composed of two maternal genomes, we previously demonstrated that histone 3 lysine 9 trimethylation (H3K9me3) is essential for Xm-Xist repression during early preimplantation phases  . Howevern phases .Xist loci of ng oocytes are in specifically permissive states for activation and that Xm-Xist imprints are established during oogenesis, as are those of autosomal imprinted genes. However, Xm-Xist silencing was observed in primordial germ cells .Using a nuclear transplantation (NT) technique, bi-maternal embryos were constructed from non-growing (ng) and fully grown (fg) oocytes . XCI in rm cells , suggestem cells . ConsideFig. S1), as described elsewhere , which facilitated chromatin analysis of many loci from small numbers of cells. We targeted 19 regions in the Xist genes containing promoter regions and a Gapdh promoter region as a negative control region for H3K9me3 modification. Our eChIP-qPCR system robustly correlated with the conventional method (no preamplification) . We therefore examined H3K9me3 in ng and fg oocytes. eChIP-qPCR in ng oocytes revealed that the H3K9me3 levels of the 19 Xist regions examined were markedly higher than that of the Gapdh promoter region  analysis showed that after fertilisation, global H3K9me3 was specifically imposed on the maternal genome . However, the dramatically low H3K9me3 levels in ESCs and sperm genomes were not observed at the 2- and 4-cell stages . These observations implied that the relaxed chromatin state characterised by low H3K9me3 levels at the 1-cell stage might be important for subsequent Xist expression in early preimplantation phases.Our findings showed that the l genome . Interesl genome   analysis for Xist RNA showed extensive Xist expression  and performed IF analysis against H3K9me3. We first examined whether the Xm-pression A, confirerial NT . Unexpecerial NT B. The sierial NT B, consiserial NT .Fig. 2.Kdm4a/b/c, writers: Suv39h1/2, Setdb1) at the 4-cell stage of ngNT and found that the expression levels varied among the ngNT, as well as between control group embryos . Although only Kdm4a was markedly reduced in most ngNT, it did not seem to affect H3K9me3. These results indicated that global ng genomic H3K9me3 following NT was comparable to that of the fg oocyte genome.We also examined the expression states of H3K9me3-associated genes  . As expected, H3K9me3 modifications at Xist major promoter regions in tetraploid ngNTs at the 4-cell stage declined dramatically (less than 15% of the control)  C. The A pression . Thus, iFig. S5A). However, H3K9me2 signals in ngNT were much lower than those in fgNT , although low levels of global H3K9me2 were only observed at the 1-cell stage, with no apparent differences at the 2- and 4-cell stages . Although the loss of H3K9me2 in the maternal genome did not affect Xm-Xist derepression  , indicating that the chromatin of the Xist genomic locus became condensed during oogenesis.Consistent with the above notion, DNA methylation levels were shown to dramatically change during oogenesis, and high levels of DNA methylation were observed only in fg oocyte genomes . We speceen loci A. Many seen loci . Howevereen loci . Therefoig. S6A) . The disXist genomic loci derived from paternal X chromosomes). At the 2-cell stage, the distance between loci in ngNT embryos was significantly larger than that of parthenogenetic and fgNT embryos, but smaller than that of androgenetic embryos , and androgenetic embryos  was robustly expressed at the 4-cell stage .Female B6D2F1 and C57BL/6N mice were purchased from Clea Japan, and oocytes were collected following standard methods. The ng and fg oocytes (C57BL/6N) were recovered from new-born (1- to 5-day-old) and adult (8- to 12-week-old) mice according to previous reports . For eCh2 and 5 μg/ml cytochalasin B (Sigma-Aldrich) (activation medium) and were cultured in KSOM (EMD Millipore). Serial NT of ng oocytes for immunofluorescence analysis was conducted as described  was prepared using a Nick Translation Kit (Abbott Laboratories) and Cy3-dUTP .RNA-FISH analysis was performed according to a previous report . In brieXist/Tsix regions and RP23-36C20: Slc16a2/Rnf12 regions) were purchased from Life Technologies. DNA probes of RP23-311P7 and RP23-36C20 were prepared using the Nick Translation Kit with Cy5-dUTP and Cy3-dUTP, respectively. The procedures were as previously reported. In brief, fixation  and permeabilisation (0.25% Triton X-100) were simultaneously conducted for 5 min at room temperature, and then the samples were plated onto glass slides. After RNaseA treatment, the samples were incubated in 0.2 N HCl containing 0.5% Triton X-100 on ice for 10 min. The images were obtained by LSM510 laser scanning confocal microscopy using a Plan-Apochromat 100×/1.46 Oil DIC objective (Carl Zeiss).For DNA-FISH, BAC clones (RP23-311P7: http://rsb.info.nih.gov/ij/). Each nuclear radius, except for those of fg oocytes, used for distance normalisation was calculated using the DAPI-stained area measurement.Distance measurements were based on previous reports . BrieflyTable S1.We prepared 300 fg and ng oocytes and 50 tetraploid 4-cell embryos for the eChIP-qPCR assay. Zona pellucida-free fg oocytes, embryos and ng oocytes were suspended in PBS containing 0.5% Triton-X100, 0.5 mM DTT and protease inhibitor (PBS + lysis buffer) and incubated on ice for 30 min. The chromatin was incubated with 100 Gel U/μl micrococcal nuclease  (New England BioLabs) for 5 min at 37°C, after which the chromatin was extracted by centrifugation. After recovery of the supernatant, PBS + lysis buffer was added to the (invisible) pellet, and an additional treatment with 2000 Gel U/μl micrococcal nuclease  for 5 min at 37°C was performed. The chromatin was then incubated with an antibody against H3K9me3  conjugated with protein A (Dynabeads) overnight at 4°C. The pelleted beads were washed twice with both Buffer 1  and Buffer 2 . The pelleted beads were suspended in ChIP direct elution buffer (Nippon Gene) and incubated with proteinase K for 2 h at 37°C (Roche). The immunoprecipitated DNA was then purified using Agencourt AMpure XP beads (Beckman). The DNA was preamplified using the preamplification mix from the Single Cell-to-CT Kit (Ambion) and TaqMan probes according to the manufacturer’s instructions (except that the number of PCR cycles was changed to 20). The primers/probes used are listed in The antibodies for H3K9me2 , H3K9me3 , H3K27me3 , H3  and Hdac2  were used for immunofluorescence (IF) analysis. For IF against histone methylation, fixation by 2% paraformaldehyde and permeabilisation with 0.25% Triton X-100 were simultaneously conducted. Fixation was followed by permeabilisation for Hdac2 IF analysis. After fixation and permeabilisation the embryos were blocked in PBS containing 1% BSA (blocking buffer) for 1 h and incubated with antibodies overnight at 4°C. The first antibodies for H3K9me2 (1:500) and H3K27me3 (1:300), or H3 (1:500) and H3K9me3 (1:500), were simultaneously incubated in blocking buffer. Embryos were washed with PBS containing 0.1% polyvinyl alcohol (PBS-PVA) and then incubated for 1 h at room temperature with Alexa Fluor 488- or 546-conjugated anti-mouse or anti-rabbit IgG secondary antibodies . After the embryos were washed with PBS-PVA, the nuclei were stained with DAPI. The zona pellucida was removed prior to fixation and permeabilisation B. After supplementary Materials and Methods and Table S2.The qPCR analysis was conducted using TaqMan probes (Life Technologies). For further details, see the supplementary Materials and Methods.The culture of ESCs used for NT was according to a previous report . For fur


H5N6 avian influenza viruses (AIVs) may pose a potential human risk as suggested by the first documented naturally-acquired human H5N6 virus infection in 2014. Here, we report the first cases of fatal H5N6 avian influenza virus (AIV) infection in a domestic cat and wild birds. These cases followed human H5N6 infections in China and preceded an H5N6 outbreak in chickens. The extensive migration routes of wild birds may contribute to the geographic spread of H5N6 AIVs and pose a risk to humans and susceptible domesticated animals, and the H5N6 AIVs may spread from southern China to northern China by wild birds. Additional surveillance is required to better understand the threat of zoonotic transmission of AIVs. On May 7, 2014, China formally confirmed the first human infection with an H5N6 avian influenza virus (AIV)134The lung specimen from the deceased cat and all lung specimens from three deceased swan geese were positive for H5N6 AIV. No other influenza virus subtypes were detected. The viral genomes of each isolate were sequenced to assess the molecular and epidemiological characteristics of these H5N6 strains. The PB2, PB1, PA, HA, NP, NA, M, and NS segments of A/cat/Sichuan/SC18/2014 (H5N6) show 99.5%, 99.5%, 99.3%, 99.4%, 97.3%, 98.9%, 99.9%, and 99.6% nucleotide sequence identity to the corresponding segments of A/swan goose/Jilin/JL01/2014 (H5N6). Phylogenetic analysis of the HA gene sequences indicated that the HA segments of each isolate were derived from the Asian H5N1 clade 2.3.4.6 HA lineage . CollectWe next analyzed whether these H5N6 strains possessed key molecular features associated with increased virulence in mammals, mammalian transmissibility, and antiviral resistance . The H5N89In this study, we provide the first evidence of fatal H5N6 AIV infection in domestic cats and wild birds. The extensive migration routes of wild birds may contribute to the geographic spread of H5N6 AIVs and pose a risk to humans and susceptible domesticated animals. Based on our phylogenetic analysis, we speculated that the H5N6 AIVs may spread from southern China to northern China by wild birds. This hypothesis is supported by the recent outbreak of H5N6 AIV among chickens in Harbin city, Heilongjiang province, China, which affected 20,550 chickens of which 17,790 diedThe protocol of the study was conducted in accordance with guidelines of animal welfare of World Organization for Animal Health. All experimental protocol were approved by the Review Board Military Veterinary Research Institute of the Academy of Military Medical Sciences.Studies with H5N6 AIVs were conducted in a biosecurity level 3 laboratory approved by the Military Veterinary Research Institute of the Academy of Military Medical Sciences.During May–June 2014, seventy feces specimens from wild birds and one lung specimen from a dead domestic cat were collected in areas with close-proximity to the residence of the patient infected with H5N6 AIV in Nanchong city, Sichuan province, southwest China. The domestic cat is suspected of having succumbed to AIV infection based on flu-like clinical signs and necropsy findings. Additionally, lung specimens were collected following the sudden and unexplained deaths of three swan geese in Baicheng city, Jilin province, northeast China. No additional information was available. We screened all specimens by RT-PCR for evidence of influenza virus infection.We inoculated the allantoic cavities of 10-day-old specific pathogen-free embryonated chicken eggs with material from the H5N6 AIV-positive lung specimen from the domestic cat and one swan goose. After 60 hours incubation at 37 °C, we recovered two virus isolates, named A/cat/Sichuan/SC18/2014 (H5N6) and A/swan goose/Jilin/JL01/2014 (H5N6).Viral RNA was isolated from the allantoic fluid of inoculated eggs using the RNeasy Mini kit  according to the manufacturer’s protocol. Reverse transcription of viral RNA and subsequent PCR were performed using primers specific for each gene segment (sequences available upon request). PCR products were purified using the QIAquick PCR purification kit  according to the manufacturer’s protocol. Viral gene segments were sequenced by the Beijing Genomics Institute . The GenBank accession numbers of A/cat/Sichuan/SC18/2014 (H5N6) and A/swan goose/Jilin/JL01/2014 (H5N6) are KM873635 to KM873650, respectively.http://www.megasoftware.net/mega.php) with the neighbor-joining (NJ) method to calculate distance. Bootstrap values were estimated for 1,000 replicates.To investigate the molecular and epidemiological characteristics and to determine the profile of genetic diversity, phylogenetic trees were constructed using molecular evolutionary genetics analysis MEGA 6 .


Sequential prime-boost or co-administration of HIV vaccine candidates based on an adjuvanted clade B p24, RT, Nef, p17 fusion protein (F4/AS01) plus a non-replicating adenovirus 35 expressing clade A Gag, RT, Int and Nef (Ad35-GRIN) may lead to a unique immune profile, inducing both strong T-cell and antibody responses.E or F4/AS01B followed by Ad35-GRIN; Ad35-GRIN followed by two doses of F4/AS01B; or three co-administrations of Ad35-GRIN and F4/AS01B. T cell and antibody responses were measured.In a phase 1, double-blind, placebo-controlled trial, 146 healthy adult volunteers were randomized to one of four regimens: heterologous prime-boost with two doses of F4/AS01The vaccines were generally well-tolerated, and did not cause serious adverse events. The response rate, by IFN-γ ELISPOT, was greater when Ad35-GRIN was the priming vaccine and in the co-administration groups. F4/AS01 induced CD4+ T-cells expressing primarily CD40L and IL2 +/- TNF-α, while Ad35-GRIN induced predominantly CD8+ T-cells expressing IFN-γ +/- IL2 or TNF-α. Viral inhibition was induced after Ad35-GRIN vaccination, regardless of the regimen. Strong F4-specific antibody responses were induced. Immune responses persisted at least a year after the last vaccination. The complementary response profiles, characteristic of each vaccine, were both expressed after co-administration.Co-administration of an adjuvanted protein and an adenovirus vector showed an acceptable safety and reactogenicity profile and resulted in strong, multifunctional and complementary HIV-specific immune responses.NCT01264445ClinicalTrials.gov  Although an effective prophylactic HIV-1 vaccine is likely to require the induction of broad and potent Env-specific antibody responses, CD8+ T lymphocyte responses that control HIV replication and CD4+ T lymphocytes that help generate and maintain HIV-specific cellular and humoral responses may also be necessary. Many T-cell based vaccines assessed in humans induce responses that are skewed to either CD4+ or CD8+ T-cell responses –4. The iPhase 1 studies in Europe and the US, respectively, suggest that the F4 HIV vaccine  formulated with the AS01 adjuvant system has an acceptable safety and reactogenicity profile and induces robust CD4+ T-cell response and antibody responses in HIV-1-uninfected volunteers and HIV-1-infected patients  and thatThe study protocol was approved by the ethics committees of Kenyatta National Hospital, University of Nairobi, Uganda Virus Research Institute, University of Zambia and Emory University, and reviewed by the responsible regulatory authorities in each country. Each study participant provided written informed consent prior to undertaking any study procedures.Eligible adults were recruited at centers in Uganda, Kenya and Zambia using informational seminars. The first screening was on 10 Jan 2011, the first enrolment was on 28 Feb 2011, the last enrolment was 13 Aug 2011, and the last follow-up was on 28 Feb 2013. Volunteers were healthy, aged 18–40 years, at lower risk for HIV infection with confirmed negative serology for HIV-1 and HIV-2 infection, willing to use an effective method of contraception, and testing negative for Ad35-specific neutralizing antibodies (EC90 titer <16). Women were not pregnant and not lactating. Volunteers with chronic medical disease, including hepatitis B or C infection, were excluded. Volunteer comprehension of the study was ascertained using an assessment of understanding tool with true/false questions. Volunteers were screened up to 42 days before vaccination , and followed for 64 weeks post first administration. Allocation schedules were computer generated. Investigators, volunteers, laboratory personnel and clinical monitors were blind to treatment assignments. Ongoing HIV risk assessments and prevention counseling were offered to volunteers during the trial. The study was a multi-center, double-blind, randomized, placebo-controlled phase 1 trial of heterologous prime-boost or co-administration 3-dose regimens . EnrolleB adjuvant is an Adjuvant System containing 50 μg 3-O-desacyl-4’-monophosphoryl lipid A (MPL), 50 μg QS-21  and liposomes. AS01E contained half the quantity of immunostimulants of AS01B. F4 was reconstituted in the AS01 liquid adjuvant immediately prior to vaccination and administered as 0.5mL containing 10 μg of F4/AS01 by intramuscular injection.The F4/AS01 HIV candidate vaccine consists of 10 μg of F4, a lyophilized recombinant fusion protein expressed in Escherichia coli and comprising 4 HIV-1 clade B antigens: p24 (BH10), RT (reverse transcriptase) (HXB2) mutated to remove the RT polymerase activity, Nef (Bru-Lai), and p17 (BH10). The vaccine antigen was prepared as a lyophilized pellet containing F4 in sucrose, ethylenediaminetetraacetic acid, arginine, polysorbate 80, and sodium sulfite in phosphate buffer. The F4 vaccine was manufactured according to the principles of Good Manufacturing by Practices (GMP) by GlaxoSmithKline Biologicals, Rixensart, Belgium. The AS012, Tween80 54 mg/L and 150mm NaCl in water for injection and administered as 1.0mL containing 2x1010 viral particles by intramuscular injection. The placebo was saline (NaCl 0.9%), produced and released by GSK Vaccines; this was given as 0.5mL or 1.0mL by intramuscular injection.The Ad35-GRIN is a recombinant replication-defective adenovirus serotype 35 containing HIV -1 subtype A gag, RT, integrase, and nef genes (abbreviated as GRIN). The genes were designed as a fusion product, and codon optimized for human cell expression and translation. Mutations were introduced into the sequence to abrogate functional activity. Ad35-GRIN vaccine was produced in HER96 cells by Transgene  according to the principles of GMP. The vaccine was prepared in formulation buffer composed of Tris 10 mM pH 8.5, Sucrose 342.3 g/L, 1mM MgClSafety and tolerability were assessed clinically and by routine laboratory tests. Volunteers recorded local and systemic reactogenicity on memory cards for 14 days post-vaccination, which were reviewed by clinicians. Data on spontaneously reported adverse events were collected during the vaccination period. Laboratory safety assessments, including full blood count and chemistries, were performed on the day of vaccination, 7 and 28 days after each vaccination, at week 36 and at end of study. Urinalysis was performed on the day of vaccination, 14 days after each vaccination and at end of study. Women were not vaccinated unless the urine pregnancy test was negative prior to each vaccination. Safety testing was done at the respective study center laboratories. All safety data were graded according to the Division of AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events (DAIDS AE Grading Table), Version 1.0, December 2004. Serious adverse events (SAEs) were defined in accordance with International Conference on Harmonization—Good Clinical Practice (ICH-GCP) guidelines. HIV-1/2 EIAs  were performed at weeks 4 or 12, 16, 20, 36, 48, and 64. Plasma from volunteers with reactive results was tested for HIV infection using the Abbott real time PCR kit (Abbott Molecular).B, while tested together with a DNA based vaccine with or without Imiquimod application, lens opacities were noted in a minipig model, however the relationship to F4/AS01B vaccination was not clear. No lens opacities were observed in a repeated toxicological study in rabbits. Despite a weak biological plausibility, the inconclusive nature of this preclinical observation led to further ophthalmological evaluations in all subsequent clinical trials investigating F4/AS01B, alone or in combination with other vaccine components. In the present study ophthalmologic examination with slit-lamp was performed at baseline and at the end of the study at two of the four study centers (48% of volunteers).During the preclinical assessment of F4/AS01nd F4/AS01 (M1.5 or M2 or M5) and 1 month after Ad35-GRIN administered alone (M1 or M5)  , 16. Sam1 or M5) . Periphe5 viable cells per well in quadruplicate with peptides at 1.5μg/mL representing the vaccine inserts as described previously [(PHA) at 10μg/mL and a mock stimulus (DMSO/medium) were also used as previously described [Cellular immunogenicity was assessed by IFN-γ ELISPOT as previously described . PBMC weeviously , 18. PepHIV-1-specific CD4+ and CD8+ T-cell responses were evaluated by intracellular cytokine staining (ICS) to assess the expression of interleukin-2 (IL-2), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and CD40-ligand (CD40L) using frozen PBMC isolated from venous blood . A viabi10 reduction in p24 content of CD8+ and CD4+ T-cell co-cultures, compared with infected CD4+ T cells alone. The threshold used for positive inhibition was determined from previous validation studies as reduction in measurable p24 production of >1.5 logs, the pre-vaccination response for the same virus must be negative  and the difference between the post-vaccination and pre-vaccination response should be ≥0.6 log10 inhibition.A VIA assay qualified for use in vaccine trials as described previously was used . VIA actImmunoglobulin G (IgG) antibody titers to F4, p17, p24, RT and Nef were analyzed using standard in-house enzyme-linked immunosorbent assays (ELISA) as previously described [Anti-Ad35 neutralization titers were measured using heat-inactivated serum samples at screening (the presence of pre-existing antibody to Ad35 was a criterion for exclusion), from 4 weeks after the first vaccination and 2 weeks after the second vaccination in a previously described, qualified cell-based assay . Anti-AdBlinded summary tables and listings of adverse events, including solicited reactogenicity events, were presented to an independent Safety Review Board (SRB). The SRB reviewed the blinded study data for the first 28 volunteers in groups A/B, for the first 14 volunteers in groups C and for the first 14 volunteers in group D at 2 weeks post month 1, and 2 weeks post month 4 vaccinations.Volunteers were randomized to vaccine or placebo in a 4:1 ratio, using a block size of 5, stratified according to site. The randomization schedule was prepared by statisticians at the data coordinating center, EMMES Corporation. The randomization list was sent to the site pharmacist of record for dispensing of vaccine and placebo assignments. Investigators at the study sites enrolled volunteers via an electronic enrollment system (administered by the data coordinating center), where allocation codes were assigned consecutively to eligible volunteers at the time of first vaccination.Study staff (with the exception of the pharmacist), volunteers and laboratories were blinded with respect to volunteer assignment between adjuvant groups A vs. B as well as to the allocation of active study vaccine or placebo within each group. There was no blinding between group schedules A/B, C and D. Volunteers in Groups C or D knew their specific group assignment and were blinded only with respect to the administration of vaccine or placebo.All study volunteers receiving at least one dose were included in the safety analyses. Two vaccine recipients that were confirmed by HIV RNA PCR testing to be HIV-infected during the study were excluded from the immunogenicity analysis, but included in the safety analysis until diagnosis. The proportions of volunteers with local and systemic reactogenicity for 14 days after each dose were summarized by treatment group. Frequency of reactogenicity and specific adverse events in the vaccine and placebo groups were compared by Fisher’s exact test. Immunogenicity results were summarized within each group at each time-point using descriptive statistics for continuous variables and percentages (with 95% CI) for categorical variables. The study was initially powered to demonstrate the non-inferiority of the immune response induced by F4/AS01E compared to F4/AS01B. The criterion used was the following: the upper limit of the 95% CI for the ratio of the magnitude of the CD40L+ CD4+ T cells expressing at least IL-2, between both groups at 2 weeks post second vaccination (Month1.5) should be below 1.5. Other exploratory comparisons were conducted between the vaccine groups.p value of less than 0.05 was considered to indicate statistical significance.Statistical analyses were performed with SAS software, version 9.2 . A two-sided From January 2011 through July 2011, 417 potential volunteers were screened for eligibility . Of 147 There were 190 protocol deviations in this study, mostly minor deviations involving protocol-specified study visit windows or schedule compliance or involving isolated inability to obtain complete collections of biological specimens . One volunteer was incorrectly administered vaccine and was analyzed with the vaccine group. One enrolled volunteer did not meet the eligibility requirements and was discontinued after the first vaccination. PBMCs were not collected at one of the clinical centers at Day 42. A symptom-directed physical exam was conducted at one of the clinical centers at the final visit, instead of protocol specified general physical exam, but general exams were performed once the deviation was identified. No protocol deviations were recorded as resulting in adverse events. The interpretation of the data presented here is not affected by the protocol deviations.No vaccine-related serious adverse events occurred in any group . All placebo recipients were grouped together for analysis. Overall, the vaccines were well tolerated in all groups. There were no statistically significant differences in moderate or worse local or systemic reactogenicity events among treatment and placebo groups. Injection site reactions were common after both F4/AS01 and Ad35-GRIN . Pain anHIV infection occurred in two vaccine recipients who received the co-administered vaccines (Group D), after receiving all vaccinations. One volunteer reported a new sexual partner of unknown HIV status; the other reported no risk factors. Both remain clinically well with CD4+T-cell counts >500 without antiretroviral treatment.IFN-γ ELISPOT responses were detected in all groups: The co-administration group had higher responses to any antigen than the Ad35GRIN-F4/AS01 sequential group; both had higher responses than the F4/AS01-Ad35-GRIN groups , regardless of adjuvant concentration . In grouAfter the third vaccination at M5, overall the most frequent ELISPOT responses were to subtype A GRIN peptides, GRIN-RT was the most frequently recognized with 43, 35, 91 and 92% response rates in Groups A-D . The nexE adjuvant compared to AS01B could not be formally demonstrated due to a subset of missing samples.The magnitude of the ICS responses against F4  and GRIN  over all time points for CD4+ and CD8+ T cells is shown in Figs F4/AS01 alone induced marginal CD8+ T-cell responses, as observed in groups A and B at M1.5 and M2 . OverallIn groups A and B, vaccination began with F4/AS01, which induced mainly CD4+ T-cells expressing 1 or 2 cytokines . These cnd and 3rd co-administrations.Viral Inhibition Activity (VIA) was assessed in a subset of randomly selected volunteers (10 vaccinees and 2 placebo) from Groups B-D at baseline and 4 weeks after vaccination . None ofThe VIA response rates across Groups B-D were not significantly different from each other, but with the small group size, there was insufficient power for the comparison. Subtype B (IIIB) and subtype A (U455) were the most frequently inhibited viruses; for these 2 viruses, the onset of virus inhibition was clearly associated with the administration of the Ad35-GRIN . VIA wasF4 specific IgG binding antibodies were detected in 100% of individuals after the second vaccination in groups A, B and D . In Ground and 3rd co-administration (M2 and M5 respectively) was 6/26 (23%) and 12/25 (48%) with GMT of 39 and 58 respectively among the positive responders. At M16 in Group D, 10/26 (38%) had a GMT of 35 among the positive responders. One volunteer in Group A/B who received placebo had a low Ad35 neutralization titer at M5 which was EC90; 19.7, just above the assay cut-off of 16.The percentage and titers of antibodies that neutralized the Ad35 vector were similar in all groups: the response rates to one Ad35-GRIN administration were 13, 14, 15, and 15% in groups A-D respectively at 4 weeks, with a geometric mean titer (GMT) of the positive responders of 18, 38, 28 and 35 respectively . Four weOverall the combination of adjuvanted F4/AS01 and Ad35-GRIN was well-tolerated with an acceptable safety and reactogenicity profile. Both local and systemic reactogenicity were common; they differed little amongst regimens, were self-limited, and were comparable to reactogenicity reported with these products individually , 9. TherAdjuvanted F4/AS01 and Ad35-GRIN regimens induced high ELISPOT response rates, balanced CD4+ and CD8+ T-cell responses which persisted over time, and high titers of F4 binding antibodies. The vaccine regimens assessed in this trial were designed to induce multifunctional CD4+ and CD8+ T-cell responses to deal with viral escape and inhibit viral activity across clades. The regimens tested in this trial appeared to induce responses that were additive when compared with each individual component tested alone –9.There is a reasonable consensus that both humoral and T-cell responses will be required for an effective HIV vaccine –25. The ELISPOT response rates in Group C (Ad35-GRIN + 2 F4/AS01) and Group D (3 co-administrations) were generally higher than those reported at peak immune response time points with other vaccine regimens tested in phase 1/2 trials and efficacy trials , 38–40. Multifunctional T-cell responses are present in HIV controllers, and such responses may be important for immune control of HIV. Balanced CD4+ and CD8+ T-cell responses were elicited by these vaccines, with profiles differing across regimens. F4/AS01 induced high levels of F4-specific CD4+ T-cells with cross-reactivity against Clade A and Ad35-GRIN induced CD4+ T-cells against Clade A with weak cross-reactivity against Clade B. Ad35-GRIN administered once in each of Groups A-C induced high levels of GRIN-specific CD8+ T-cells; a higher magnitude of CD8+ responses was maintained up to one year after three co-administrations in group D. The immune marker, CD40L is known as a costimulatory ligand required for T cell help and a marker for activated antigen-specific T cells –44. PrevIn this study, Ad35-GRIN was shown to be a better prime than boost i.e. higher CD8+ T cells frequency and higher polyfunctionality of CD4+ T cells. Adenoviral vectors appear to be good for priming immune responses as observed in HVTN 078 trial where NYVAC and Ad5 were evaluated. NYVAC was shown to be a better boost than prime for T-cell response as also seen in non-human primate studies , 14.The response rates as detected by flow cytometry were generally greater than response rates detected by ELISPOT, the main reason for this is because the ELISPOT is based on IFN-γ production only whereas ICS detects responses to IFN-γ, Il-2 and TNF-α. Responses to F4/AS01 induce CD4+ T cells expressing mainly IL-2 and Ad35 induces polyfunctional CD4+ T cells and CD8+ T cells that express mainly IFN-γ. Thus differences between ELISPOT and ICS are more marked at 1M and 1.5M post F4/AS01B prime particularly for CD4-specific responses. When Ad35-GRIN is used as a prime or in the co-administration group D, the profile is more polyfunctional and therefore the proportion of T-cells (including CD4 and CD8) expressing IFN-γ increases and the difference in percent responses as detected by ICS and ELISPOT is less distinct.cytotoxicity of CD8+ T cells, were not prioritized in this study due to the limited blood volume.Anti-HIV inhibitory capacity is considered to be an important attribute of CD8+ T-cells and such activities may help control HIV-viral load during acute and chronic infection particularly in long-term non-progressors , 46–48. Antibodies against F4-protein were detected in 100% of individuals in all groups. The high response rate and titers were driven by the F4/AS01 administration; Ad35-GRIN has previously been shown to induce only modest anti-Gag responses , 9. The In a collaborative study, we carefully studied the seroprevalence of Ad5, Ad26 and Ad35 in the populations targeted for the current study . The Ad3Ad35 vectors also have a different serotype, cellular receptor and innate and immune signaling mechanisms compared to Ad5 . In contIt would have been valuable to compare the impressive systemic T cell responses with those of mucosal cells such as α4γ7+ expressing CD4+ T cells. However, this study did not obtain biopsy samples hence did not evaluate T cell subsets that are targeted by the HIV virus. Future studies with these vaccines should evaluate target T cell subsets in humans as described recently . AnotherPlacebo recipients were included to provide an appropriate control group for evaluation of vaccine safety and tolerability, particularly local and systemic reactogenicity . We feltOverall the combination of adjuvanted F4 and Ad35 GRIN induced balanced CD4+ and CD8+ T-cell responses with multifunctional profiles which persisted over time and high titers of F4 binding antibodies. The vaccine regimens assessed in this trial were designed to induce multifunctional CD4+ and CD8+ T-cell responses to deal with viral escape and inhibit viral activity across clades. Next generation immunogens may further improve the quality and breadth of T-cell responses across clades and should be incorporated into regimens such as those assessed in this trial: using low-seroprevalent adenoviral vectors and adjuvanted proteins in co-administration, ideally combined with Env immunogens with the capacity to induce neutralizing and/or non-neutralizing antibodies.S1 CONSORT ChecklistIAVI B002 study CONSORT checklist of information.(DOC)Click here for additional data file.S1 Fig6 PBMC on a half-log scale. Panel A shows individual F4-specific responses across groups A-D: clade B p24, RT, Nef and p17 peptide pools and Panel B shows individual GRIN-specific responses across groups A-D: clade A Gag, RT, Int and Nef peptide pools. Gray dots: response below the cut-off to any of the 8 peptide pools; red circles: response above the cut-off to any of the 8 peptide pools. For the vaccine groups, the overlaid box plot summarizes the overall responses . Baseline (BL) and placebo (Pbo) groups were combined for M5.IFN-γ ELISpot responses to individual peptide pools 4 weeks after last vaccine (M5). The y-axis shows the SFC/10(DOCX)Click here for additional data file.S2 FigB administrations (M2) and after Ad35-GRIN (M5), Group C; 4 weeks after Ad35-GRIN (M1) and 4 weeks after two F4/AS01B administrations (M5) and Group D; 4 weeks after each co-administration of F4/AS01B and Ad35-GRIN .VIA activity across groups B-D (vaccinees only). The mean log viral inhibition +/- standard deviation for IIIB and U455 viruses is shown at baseline (M0) for each of groups B-D and Group B; 4 weeks after two F4/AS01(DOCX)Click here for additional data file.S1 ProtocolIAVI B002 Protocol V4.0. A Phase I double-blinded, placebo controlled, randomized trial in HIV-uninfected, healthy adult volunteers to evaluate the safety and immunogenicity of F4Co adjuvanted AS01B or AS01E administered with Ad35-GRIN.(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 TextAdditional safety data.(DOCX)Click here for additional data file.


Large cholinergic synaptic terminals known as C-boutons densely innervate the soma and proximal dendrites of motoneurons that are prone to neurodegeneration in amyotrophic lateral sclerosis (ALS). Studies using the Cu/Zn-superoxide dismutase (SOD1) mouse model of ALS have generated conflicting data regarding C-bouton alterations exhibited during ALS pathogenesis. In the present work, a longitudinal study combining immunohistochemistry, biochemical approaches and extra- and intra-cellular electrophysiological recordings revealed that the whole spinal cholinergic system is modified in the SOD1 mouse model of ALS compared to wild type (WT) mice as early as the second postnatal week. In WT motoneurons, both C-bouton terminals and associated M2 postsynaptic receptors presented a complex age-related dynamic that appeared completely disrupted in SOD1 motoneurons. Indeed, parallel to C-bouton morphological alterations, analysis of confocal images revealed a clustering process of M2 receptors during WT motoneuron development and maturation that was absent in SOD1 motoneurons. Our data demonstrated for the first time that the lamina X cholinergic interneurons, the neuronal source of C-boutons, are over-abundant in high lumbar segments in SOD1 mice and are subject to neurodegeneration in the SOD1 animal model. Finally, we showed that early C-bouton system alterations have no physiological impact on the cholinergic neuromodulation of newborn motoneurons. Altogether, these data suggest a complete reconfiguration of the spinal cholinergic system in SOD1 spinal networks that could be part of the compensatory mechanisms established during spinal development. Synapses converging onto the soma and the highly branched dendritic tree of motoneurons (Mns) have been divided into five different types: S, T, M, F and C –4. C-terGiven the importance of cholinergic neuromodulation in spinal locomotor networks, numerous studies have investigated C-bouton morphology in physiological and pathological conditions ,12. ThesAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that affects spinal motoneurons (Mns) and cortical motor neurons. In ALS patients and rodent models of the disease, it has been shown that Mns innervating fast-twitch muscles (fast Mns) are much more vulnerable to neurodegenerative processes than those connecting slow-twitch muscle fibers (slow Mns) ,16. InteIn the present work, by combining immunohistochemistry, extra- and intracellular electrophysiology and biochemistry, we revealed major reorganizations of the spinal cholinergic system in SOD1 mice compared to wild-type (WT) animals as early as the second postnatal week. Our data identified that (1) C-bouton evolution exhibits different temporal profiles in WT and SOD1 Mns, (2) M2 receptors present a complex dynamic in C-bouton PSM in WT Mns that is completely disrupted in SOD1 Mns, (3) lamina X cholinergic interneurons are more abundant in high lumbar segments in SOD1 mice compared to WT, (4) these neurons are subjected to neurodegeneration in the SOD1 ALS model and (5) early C-bouton alterations have no physiological impact on cholinergic neuromodulation of newborn Mns.G93A)1Gur/J from The Jackson Laboratory, Bar Habor, ME, USA) were bred with non-transgenic B6SJLF1/J females . Male offspring expressing the mutant SOD1-G93A gene (hereafter referred to as SOD1 mice) and age-matched male WT littermates were used. Mice were genotyped from genomic DNA purified from tail biopsies by PCR using the following primers: 5’ CATCAGCCCTAATCCATCTGA 3’ (forward), 5’ CGCGACTAACAATCAAAGTGA 3’ (reverse). All of the experiments and analyses presented here were performed blind to the genotype of the animals.This study was carried out in strict accordance with the recommendations of the European Committee Council Directive. The protocol was approved by the local ethics committee of the University of Bordeaux (permit number 5012031A). High-expressor hemizygous G93A SOD1 male mice , P10, P21, P40, P75 and P100  were aneFor the cholinergic interneuron counting experiments, a biotinylated anti-goat  secondary antibody was used, and ChAT-positive neurons were visualized with successive incubations with streptavidin , 3,3’-diaminobenzidine (DAB) and hydrogen peroxide solutions.Images were acquired on a Leica SP5 confocal microscope using a 63x oil immersion lens and analyzed with FIJI (ImageJ) plugins developed at the laboratory. For each animal, three different slides of lumbar spinal cord sections were randomly selected and processed for immunohistochemistry. Images of three randomly sampled lumbar spinal cord sections were then acquired for each slide. To analyze M2 receptor expression in the PSM adjacent to C-boutons, a sum projection along the z-axis was performed on both the ChAT and M2 channels. Constant-width bands (2.83 μm) of both channels centered on the Mn contour were extracted from this projection. Variations in the intensity of these two bands were measured along their length with the FIJI plot profile tool. ROIs containing C-boutons in the ChAT channel were automatically detected and applied to the M2 channel after an expansion of 1 μm. M2 labeling area and intensity under C-boutons were then computed. The M2 intensity under C-bouton was normalized by the mean M2 intensity computed in the Mn membrane devoid of C-boutons.ChAT/DAB immunostained slices from P10, P40 and P100 WT and SOD1 mice were scanned with a Nanozoomer (Nanozoomer 2.0 HT Hamamatsu C9600-12). Lamina X cholinergic interneurons located in an area 80 μm around the central canal were manually counted in individual slices with the NDP viewer software (Hamamatsu). To count neurons in a similar spinal region in each age-matched animal group, the intermediolateral neurons (IML) were used as anatomical landmarks. IML are cholinergic neurons, thereby appearing in ChAT/DAB labeling, which express a well-defined anatomical distribution throughout spinal segments. Indeed, IML extend from the first thoracic segment (T1) through to the second lumbar segment (L2). The measured lumbar segment lengths were 450, 950 and 1000 μm in P10, P40 and P100 mice, respectively. The individual slice thickness was 30 μm, and cholinergic lamina X interneurons were counted in 15 slices at P10 and 33 slices at P40-P100 more rostral from the last slice containing IML and in 50 slices at P10 and 100 slices at P40-P100 more caudal from the last slice containing IML. With this method, cholinergic lamina X neurons could be accurately counted from the L2 to the L5 cord segments.M2 muscarinic receptor protein expression was assessed in spinal cord tissue from at least 4 P1, P10, P21, P40, P75 and P100 SOD1 mice and 4 WT littermates using a previously described protocol [2 2.5, MgSO4 1.3, NaH2PO4 0.58, NaHCO3 25 and glucose 10, with a pH of 7.4 when bubbled with 95% O2 + 5% CO2 at room temperature (24–26°C). Motor outputs were recorded extracellularly from the lumbar ventral roots using glass suction electrodes. In each in vitro spinal cord preparation, motor outputs from the right and left lumbar 2  and one L5 ventral root were simultaneously recorded to investigate both the bilateral segmental alternation and the flexor/extensor activity [11 WT and 8 SOD1 mice aged P1-P3 were anesthetized using isoflurane until reflexes were lost and were decapitated. The spinal cord was then dissected free and placed ventral side up in a recording chamber. All dissections and recording procedures were performed under continuous perfusion with artificial cerebrospinal fluid (aCSF) containing the following (in mM): NaCl 130, KCl 3, CaClactivity ,23. Neur2, 7 mM MgCl2, 1.15 mM NaH2PO4, 26 mM NaHCO3, 11 mM glucose, and 205 mM sucrose. The saline was bubbled with 95% O2, 5% CO2. Transverse slices (350 μm) of the spinal lumbar enlargement were cut with a vibroslicer and then transferred in a holding chamber. Slices were allowed to recover in oxygenated aCSF  L-glutathione) for at least 1 hour at 30°C. The slices were transferred to a second holding chamber containing the same aCSF at 30°C but without sodium pyruvate and (reduced) L-glutathione before recordings. Whole-cell current-clamp recordings from Mns, identified by their relatively large size in lamina IX, were made under visual control with a Multiclamp 700B amplifier. Recording glass microelectrodes (4–7 MΩ) were filled with the following: 120 mM K-gluconate, 20 mM KCl, 0.1 mM MgCl2, 1 mM EGTA, 10 mM HEPES, 0.1 mM CaCl2, 0.1 mM GTP, 0.2 mM cAMP, 0.1 mM leupeptin, 77 mM d-mannitol, and 3 mM Na2-ATP, with a pH of 7.3. A bipolar tungsten electrode was used to stimulate axons crossing through the ventral commissure. The polysynaptic transmission was decreased using a high cation-containing aCSF with 7.5 mM CaCl2 and 8 mM MgSO4 in order to record monosynaptic excitatory postsynaptic potentials (EPSPs). Strychnine (1 μM), Gabazine (1 μM) and DNQX (5 μM) were added throughout the experiments to block glycinergic, GABAA and AMPA/kainate receptor activation, respectively. Oxotremorine (500 μM) was bath applied on the slices. All of the experiments were performed at room temperature (25°C).In 5 deeply anesthetized P7-P11 WT and 12 SOD1 mice, a laminectomy was performed to remove the spinal cord in an ice-cold sucrose-based saline solution containing the following: 2 mM KCl, 0.5 mM CaClt-test. Two-way analyses of variance (ANOVAs) with Sidak’s multiple comparison tests were performed to evaluate drug/age and mouse genotype effects. All data are expressed as the means ± SEM. Asterisks in the figures and tables indicate positive significances (p<0.05). For C-bouton parameter analysis . The other statistical analyses were conducted using the GraphPad Prism software (GraphPad Prism). Comparisons between two conditions were tested using Student’s In the first part of this study, we examined the evolution of C-bouton terminals juxtaposed to Mns throughout the life span of WT and SOD1 mice. For this purpose, ChAT-immunolabeling was performed in the lumbar spinal cords from male SOD1 mice and WT littermates at key developmental stages and over the course of the disease, in P1, P10, P21, P40, P75 and P100 animals  but are Altogether, these data show early alterations in the developing pattern of C-boutons juxtaposed to lumbar Mns and a loss of the most distal of these cholinergic synapses in presymptomatic stages (P100) in the SOD1 mice.Anatomical studies have shown accumulations of M2 receptors in the PSM of C-boutons  and elecTo go one step further, analysis of M2 receptor accumulation under C-boutons during normal development and ALS pathogenesis was performed using ChAT and M2 receptor double immunofluorescence staining Figs , Table 1Strikingly, this dynamic process is absent in SOD1 Mns. The histograms in Altogether, these data identify a complex dynamic of M2 receptors in the PSM of C-boutons during development and maturation of Mns that is impaired in SOD1 animals.A growing body of evidence suggests that ALS is not just a motoneuron disease, but that other cellular subtypes are also prone to neurodegeneration in spinal networks during disease progression. C-boutons have been shown to originate from a subclass of V0 interneurons located in lamina X around the central canal ,10. In tTo address this question, in a first series of experiments, lamina X ChIns were visualized using ChAT/DAB staining  and counG93A motoneurons [This enrichment was no more present in SOD1 P100 mice, suggesting a loss of lamina X ChIns at this stage. Because the death-mediating protease, caspase-3, is activated in SOD1oneurons ,28, we iin vitro spinal cord preparations from P1-P3 WT and SOD1 newborn mice , partly supported by M2 receptor activation in lumbar Mns . These mAltogether these data suggest that the early alterations of the C-bouton system do not impact the cholinergic neuromodulatory influences on newborn Mns in SOD1 spinal networks.Previous studies have shown that C-boutons contacting motoneurons are subject to morphological changes in physiological and pathophysiological conditions ,14,17–20Rodents are classically considered as adults at P60-75 . StrikinRecent studies have identified specific markers of fast and slow MNs, the matrix metalloproteinase 9 (MMP9) and the estrogen-related receptor β (ERR β), respectively ,41. As fin vivo imaging of axonal branches has revealed that in individual motor pools, MNs could be divided into two different subpopulations: MNs that exhibit compensatory growth and Mns characterized by axonal dieback [In addition to these MN type adaptations, neurodegenerative processes appear to be heterogeneous within MN subpopulations in the SOD1 mouse model of ALS. Indeed,  dieback . As thes dieback .In this context, we think that co-immunostaining experiments with MMP-9 and ERR beta, would not have allowed us to consider that we have specifically discriminated the cholinergic modulation from that of fast MNs to slow MNs throughout ALS pathogenesis.in vitro spinal cord preparation but alters locomotor task modulation of muscle activity [The assumption that cortical motor neurons and spinal motoneurons are the only cellular subtypes to degenerate in ALS has often been questioned. A loss of Renshaw cells has been reported in SOD1 spinal networks . The preactivity . These rin vitro spinal cord preparations from WT and SOD1G93A mice were identical [In vivo patch-clamp recordings could be an alternative, but control over the Mn extracellular medium is limited compared to in vitro conditions. However, it would be of particular interest to investigate acetylcholine effects on mature WT and SOD1 Mns to further investigate the muscarinic influences received by these neurons and to determine whether this neuromodulatory system is a key contributor to ALS pathogenesis.Conflicting reports have been made concerning the age-related morphological changes of C-boutons juxtaposed to SOD1 Mns –20. The dentical  despite dentical ,49. Altodentical . It has dentical –52. In v


Sporadic emergence of the highly pathogenic avian influenza (HPAI) H5N1 virus infection in humans is a serious concern because of the potential for a pandemic. Conventional or quantitative RT-PCR is the standard laboratory test to detect viral influenza infections. However, this technology requires well-equipped laboratories and highly trained personnel. A rapid, sensitive, and specific alternative screening method is needed.By a luminescence-linked enzyme immunoassay, we have developed a H5N1 HPAI virus detection kit using anti-H5 hemagglutinin monoclonal antibodies in combination with the detection of a universal NP antigen of the type A influenza virus. The process takes 15 minutes by use of the fully automated luminescence analyzer, POCube.We tested this H5/A kit using 19 clinical specimens from 13 patients stored in Vietnam who were infected with clade 1.1 or clade 2.3.4 H5N1 HPAI virus. Approximately 80% of clinical specimens were H5-positive using the POCube system, whereas only 10% of the H5-positive samples were detected as influenza A-positive by an immunochromatography-based rapid diagnostic kit.This novel H5/A kit using POCube is served as a rapid and sensitive screening test for H5N1 HPAI virus infection in humans. Orthomyxovirus family, whose genome is composed of eight segments of negative-sense RNA encoding 12 proteins. Two major glycoproteins, hemagglutinin (HA) and neuraminidase (NA), are located on the viral envelope, and 16 HA subtypes and 9 NA subtypes of avian influenza A have been identified on the basis of their antigenicities [Influenza viruses belong to the nicities . The hignicities . This haThe 2003 and 2004 outbreaks of the highly pathogenic avian influenza (HPAI) virus of subtype H5N1 that occurred in poultry and wild birds were genetically traced back to the H5N1 HPAI virus that caused the first outbreak in Hong Kong in 1997, and also those of 2001 and 2002 ,7, the fThe H5N1 HPAI virus has evolved into many phylogenetically distinct clades and subclades, and these diverse lineages have been largely geographically separated since 2005 . During PCR-based molecular tests are one of the most sensitive ways to detect the influenza virus, and conventional and real-time RT-PCR methods have been developed to diagnose H5N1 HPAI virus infections in humans -16. HoweThe POCube is a fully automated and compact immunological analyzer developed by Toyobo Co. Ltd, which is intended to support the Point-of-Care Testing (POCT) system in clinics. The POCube analyzer is relatively small (280 × 310 × 275 mm), easy to operate, and rapidly measures antigen-Ab complexes by the sensitive detection of luminescence  kit, which is a commercially available rapid influenza diagnostic test (RIDT). This immunochromatography (IC)-based kit is meant to diagnose infection by all type A influenza viruses, including H5N1. However, because this kit uses an NP-specific antibody, it cannot discriminate H5N1 from other subtypes.50) using Madin–Darby Canine Kidney (MDCK) cells and by quantitative real-time RT-PCR as described previously [The A/Vietnam/1194/2004 (NIBRG-14) virus, which has a modified HA gene and an NA gene derived from the HPAI A/Vietnam/1194/2004 (H5N1) virus within the backbone of six other internal genes of A/Puerto Rico/8/34 (PR8), as well as other modified H5N1 viruses, A/Indonesia/5/2005 (Indo5/PR-8-RG2), A/turkey/Turkey/1/2005 (NIBRG-23), and A/Anhui/01/2005 (Anhui01/PR8-RG5), were provided by the National Institute for Biological Standards and Controls . The following subtypes of influenza A viruses were also used to evaluate the specificity of the POCube H5/A kit and were obtained from the Influenza Virus Research Center of the National Institute of Infectious Diseases (NIID), Tokyo, Japan: A/duck/Alberta/35/76 (H1N1), A/duck/Germany/1215/73 (H2N3), A/duck/Ukraine/1/63 (H3N8), A/duck/Czechoslovakia/56 (H4N6), A/turkey/Massachusetts/3740/65 (H6N2), A/duck/Hong Kong/301/78 (H7N1), A/turkey/Ontario/6118/68 (H8N4), A/turkey/Wisconsin/1/66 (H9N2), A/chicken/Germany/N/49 (H10N7), A/duck/England/56 (H11N6), A/duck/Alberta/60/76 (H12N5), A/gull/Maryland/704/77 (H13N6), A/mallard/Gurjev/263/82 (H14N5), and A/duck/Australia/341/83 (H15N8). All viruses were propagated in the allantoic cavity of 10-day-old embryonated chicken eggs. The virus titer and RNA copy number of the matrix gene were determined by the 50% tissue culture infective dose , packed into a cooling box and sent to the National Influenza Center, National Institute of Hygiene and Epidemiology (NIHE) for H5N1 HPAI virus detection by conventional RT-PCR. Clinical specimens  from Southern Vietnam collected in 2009 and 2012 were diluted in about 2 ml of VTM and sent to the National Influenza Center, Pasteur Institute in Ho Chi Minh City (PI-HCMC) for H5N1 HPAI virus detection by real-time RT-PCR. After a part of clinical specimen was used for RNA extraction, the remaining volume was aliquoted into cryotubes and maintained at −80°C until further analysis. The collection of clinical samples was approved by the institutional review boards of NIHE and PI-HCMC. All nasopharyngeal aspirates from patients, including seasonal and non-influenza, were collected after obtaining patient’s written informed consent.Throat swabs or tracheal aspirates were collected at the point of admission from patients with clinical H5N1 virus infections in Northern Vietnam from 2007 to 2010. Clinical specimens were diluted in about 2 ml of viral transport medium (VTM) consisting of MEM medium supplemented with 1% of 2.92% L-glutamine, 1% of Penicillin- Streptomycin , 1.5% of 37.5% NaHCODifferent combinations of anti-H5 HA mAbs  and a pa50 and quantitative real-time RT-PCR targeting the universally conserved matrix gene of influenza A. These viruses were serially 10-fold diluted and the luminescence counts were measured. Then, the virus TCID50 titers at each dilution were calculated and plotted as shown in Figure 1–103 TCID50.The H5/A kit contains two sets of mAb mixtures to detect H5 HA simultaneously with NP antigens which are common to all influenza A viruses. This combination is important for the definitive diagnosis of H5 HPAI virus infection. When a clinical specimen is loaded into a reservoir tank in the cartridge, which is placed in the machine along with two reaction vessels, an automatic diagnosis for H5N1 HPAI virus infection is provided  including strains recently endemic in Japan (mostly H1N1pdm or type B), indicating that the kit is highly specific to H5 HA (data not shown). Furthermore, we tested clinical swab specimens taken from 15 seasonal influenza and 30 non-influenza patients and found no false positives of H5. Thus, the POCube system offers a simple, safe, and rapid diagnostic system, and is expected to be highly sensitive compared with existing commercial kits that use immunochromatography (IC) technology.We tested clinical specimens (throat swabs) from nine patients infected with the H5N1 HPAI virus (identification confirmed by molecular diagnosis using RT-PCR) in NIHE during 2007 to 2010 (clade 2.3.4) and compared the detection rate of our H5/A kit by POCube with that of the commercially available rapid influenza diagnostic test (RIDT),  in 2009 and from three patients (K–M) in 2012 Table . These sWhen samples are H5-positive but type A-negative, such as was the case for patient M , the test needs to be repeated or confirmed by genetic analysis. This may occur when the level of virus antigen is at the threshold of detection, such as was the case for sample No. 2 of patient A. Again, the commercially available RIDT showed poor sensitivity, with only one of nine samples testing positive for type A.Table H5 HA HPAI virus detection using POCube and the H5/A kit is a highly sensitive and simple rapid diagnostic system that can be completed within 15 minutes. During the preparation of this manuscript, Sakurai et al. reported a 10–100-fold more sensitive detection of H5 HA using fluorescent beads and an improved IC method ; howeverThe POCube system using the H5/A kit is useful for the rapid detection of H5N1 HPAI virus infections in humans in hospitals and other clinical settings where technical resources are limited.50: 50% tissue culture infectious dose; VTM: Viral transport medium.HA: Hemmagglutinin; NA: Neuraminidase; NP: Nucleoprotein; HPAI: Highly pathogenic avian influenza; ALP: Alkaline phosphatase; RIDT: Rapid influenza diagnostic test; TCIDKN and SM are research staffs in Toyobo Co.Ltd. and collaborated with YYY, KO and TK in NIID (a government research institute) for this study, which was formally supported by a grant from the Health Science Foundation of the Ministry of Health, Labor and Welfare of Japan (KHC1218). The patent of H5/A kit was filed by Toyobo Co. Ltd and NIID. This may not cause any competing non-financial interests were they to become public after the publication of this manuscript.All other authors declare no potential conflicts of interest.YTY, KN, SM and KT determined the protocol for the assay. HLKN, MTQL, GTD, LTN prepared clinical specimens and helped YTY and KT for the analysis. MKI, HT and HT participated in the design of the study and helped to draft the manuscript. KO, SI and MT helped to analyze the results and finalize the manuscript. All authors read and approved the final manuscript.YTY worked in NIID for this study and recently moved to the Department of Medical Technology, School of Human Sciences, Tokyo University of Technology, 5-23-22 Nishi-Kamata, Ohta-ku, Tokyo 144–8535, Japan.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/362/prepub


Some of the Supporting information was erroneously omitted from the publication. Please view the corrected Supporting Information in its entirety here.File S1Supporting Information. Figure S1. Flow chart for microarray analysis. Affymetrix GeneChip Human Genome U133 plus 2.0 arrays were applied for all analysis. The analysis was performed with GeneSpring GX 12.5 following the layout on this flow chart. The details are shown in the Methods section. Insignificant or unannotated probe data was eliminated at each step. The number on the right side shows the number of the remaining probe sets or genes at each step. The 384 selected genes were used for further analyses in Figure 1 and Table S1 in File S1. Figure S2. Gene regulation by pitavastatin in HUVECs and the aortae of Apo-E-deficient mice. (A) HUVECs were treated with 1 µM pitavastatin for the indicated time. (B) ApoE deficient mice were orally administered pitavastatin twice daily at 3 mg/kg/treatment for 12 weeks before sacrifice. Total RNA was isolated and determined by real-time quantitative PCR, as described in Methods. The sequences of applied primers are shown in Table S2B in File S1. Vertical lines indicate the S.D. , * P<0.01, ** P<0.001, compared with the control sample, Dunnett's test in A and Student's t test in B. Figure S3. Histological examination of atherosclerotic regions in the aortic sinus. Eight-week-old ApoE deficient mice (n  =  12 for each group) were treated twice daily for 12 weeks with vehicle alone (-) or pitavastatin at 3 mg/kg/treatment (+). The heart and aorta were removed rapidly and fixed and embedded in paraffin for Victoria blue-HE staining as described in Methods. Victoria blue-hematoxylin-eosin staining (A) revealed the atherosclerotic lesions (arrow). (B) shows the total plasma cholesterol and triglyceride levels. Note that pitavastatin reduced the plaque area without changing the plasma cholesterol and triglyceride levels. The vertical lines indicate the SEM (n  =  12), * P<0.001 compared with the control sample, Student's t test. n.s. indicates not significant. Figure S4. Identification of theMEF2A, MEF2C, KLF2andKLF4antibodies. HUVECs were incubated with 1 µM pitavastatin for 4 hours. Before pitavastatin treatment, as described in the Methods section, cells were transfected with siRNA to KLF2 (A), KLF4 (B), MEF2A (C) and MEF2C (D).  The whole cell fraction was prepared for the Western blotting experiment to detect KLF2, MEF2A and MEF2C. Beta-actin was used as the internal control. (B) HUVECs were incubated with 1 µM pitavastatin for 24 hours. The nuclear extract fraction was prepared for further Western blotting to detect KLF4. Nucleoporin p62 was used as an internal control. NS; Non silencing for negative control. Note that loss of band detection was observable only by treatment specific siRNA, which demonstrates the specificity of new antibodies. N2212, Y6929, Y0841, Y1740 are clone numbers for each antibody. Figure S5. Triple knock down of MEF2A, Cand DreducesKLF2and KLF4. HUVECs were incubated with 1 µM pitavastatin for 4 hours. Before the treatment, as described in the Methods section, cells were transfected with siRNA to MEF2A, -2C and -2D. A whole cell fraction was prepared for a further Western blotting experiment to detect KLF2, and a nuclear extract fraction was prepared for KLF4. Alpha Tubulin and Nucleoporin p62 were used as the internal controls for the whole cell lysate and nuclear extract fractions, respectively. NS; Non silencing for negative control. Figure S6. Localization of theMEF2Cbinding sites in HUVECs. HUVECs were incubated with DMSO [statin(-)]or 1 µM pitavastatin for 4 hours. (A) Chromatin immunoprecipitation was performed using an MEF2C antibody, followed by deep sequencing. 4,878 MEF2C binding sites were detected in the control [pitavastatin (−)]. 42% of the MEF2C binding sites were located between the TSS and 3′UTR of the genes, while the remaining of 58% were found in intergenic regions. (B) Co-localization of MEF2C binding sites and H3K27ac in control [pitavastatin (−)] HUVECs. 25,477 binding sites were detected using anti-H3K27ac antibody ChIP-seq analysis in the control [pitavastatin (−)] HUVECs. Among them, 798 binding sites displayed co-localization of H3K27ac and MEF2C. (C) Distribution of the MEF2C binding sites in the pitavastatin-treated HUVECs. 13,030 MEF2C binding sites were detected in the treated HUVECs; 40% of them were located between the TSS and 3′UTR of the genes, while 60% were located in intergenic regions. Figure S7. ChIP-qPCR with an anti-MEF2Cantibody against theKLF4gene. MEF2C Binding sites detected in the KLF4 upstream region  were quantitatively evaluated by ChIP-qPCR. The Kb –147 KLF4 region was used as the negative control. The sequences of the primers are shown in Table S2C in File S1. Vertical lines indicate the S.D. (n  =  3), *P<0.01, compared with the kb −147 KLF4, statin (−), ‡P<0.01 compared with the kb −98 or kb-148 KLF4, statin (−), Student's t test. Figure S8. The specificity of the Pol II antibody. A newly-developed monoclonal antibody (Pd75C9) was used to perform ELISA with RNA polymerase II C-terminal domain peptides containing different phosphorylation patterns. (A) The list of peptides used for ELISA. Phosphorylated amino acids are indicated in red. C-terminal repeat of RNA Polymerase II is comprises from 25 to 52 tandem copies of the consensus repeat heptad Y1S2P3T4S5P6S7 (Also shown in the right panel of figure A). And antibodies for a variety of phosphorylated CTD were raised using 19 AA peptides including phosphorylated Serine as is depicted. (B) ELISA. Microtiter plates coated with the peptides (the sequence shown in A) were incubated with 3-fold dilutions of the monoclonal antibody (starting from a 1:27 dilution of a hybridoma culture supernatant). After incubation with a peroxidase-conjugated secondary antibody and washing, the colorimetric signal of tetramethylbenzidine was detected by measuring the absorbance at 405 nm (Abs) using a plate reader. Although Pd75C9 was generated using the Pd peptide to immunize mice, this clone reacted with all of the phospho-serine containing peptides, with preference for those containing phospho-S5 and phosphor-S2, but reacted only slightly with the unphosphorylated peptides. Figure S9. Sequence of the PCR product generated in the quantitative 3C assay. To validate the quantitative 3C assay, PCR products were directly sequenced and identified. The chromatogram shows the sequence of the target analysis (the fragments including KLF4 and 148 kb upstream from the TSS of KLF4) under pitavastatin treatment. As expected, the sequence of the TSS and that of the kb −148 enhancer were directly connected by a Hind III recognition sequence, and the measurement of the amount of this PCR product stands for the frequency of chromatin interaction. Figure S10. The sensitivity and specificity of the probes. To validate the probes, BAC clone DNA (RP11-80F13 and RP11-359L14) was tested for correct chromosomal location at 9q31.2 and the absence of other signals by 2D-FISH, respectively. The following datafrom (A) to (K) is the detailed information on probe K and (L) to (Q) is the detailed information of probe M. (A) Probe design for the 2D-FISH analysis of the target region on human Chr.9q31.2. The numbers in the middle indicate the location on Chr.9 using the hg19 build program. Probe K includes the KLF4 gene. Three-color 2D-FISH was carried out by a combination of a labeled Probe K and human Chr.9 arm-specific painting probes . (B)The p arm of Chr.9 is represented in purple. (C) The q arm of Chr.9 represented in green. (D) Probe K is represented in red. (E) Nuclear DNA was counterstained with DAPI  and is shown in blue. The merged image with all of the colors is shown in (F). Probe K in the interphase is shown in Figures G to K. All of the combinations of the labeled Probe K and human Chr.9 p and q arm-specific painting probes were the same as in B-F. The white arrows indicate the representative signals of Probe K in the interphase (K). (L) Probe design for the 2D-FISH analysis of the target region on human Chr.9q31.2. The numbers in the middle indicate the location on Chr.9 using the hg19 build program. Probe M includes the MEF2C binding region, which is located 148 kb upstream from the TSS of KLF4. (M) The q arm of Chr.9 is represented in red. (N)The p arm of Chr.9 is represented in purple. (O) Probe M is represented in green. (P) Nuclear DNA was counterstained with DAPI  and is represented in blue. The merged image with all of the colors is shown in (Q). The white arrows indicate the representative signals of Probe M in the metaphase. The signals in the interphase are shown in the other nuclei. It was confirmed that the signal of the BAC clone was clearly detected on the Chr.9q region and absent from the other chromosomes. Figure S11. Statin effect onKLF4 expression. HUVECs were treated with pitavastatin at a concentration ranging from 0.01 to 10 µM for 12 hours. Total RNA was isolated and determined by Real-time quantitative PCR, as described in the Methods section. The vertical lines indicate the S.D. (n  =  3), * P<0.001, compared with the sample of 0 µM, Dunnett's test. Table S1. Table S2.(PDF)Click here for additional data file.