Establishment of persistent infection in memory B cells by murid herpesvirus-4 (MuHV-4) depends on the proliferation of latently infected germinal center B cells, for which T cell help is essential. Whether the virus is capable of modulating B-T helper cell interaction for its own benefit is still unknown. Here, we investigate if the MuHV-4 latency associated M2 protein, which assembles multiprotein complexes with B cell signaling proteins, plays a role. We observed that M2 led to the upregulation of adhesion and co-stimulatory molecules in transduced B cell lines. In an MHC-II restricted OVA peptide-specific system, M2 polarized to the B-T helper contact zone. Furthermore, it promoted B cell polarization, as demonstrated by the increased proximity of the B cell microtubule organizing center to the interface. Consistent with these data, M2 promoted the formation of B-T helper cell conjugates. In an in vitro competition assay, this translated into a competitive advantage, as T cells preferentially conjugated with M2-expressing B cells. However, expression of M2 alone in B cells was not sufficient to lead to T cell activation, as it only occurred in the presence of specific peptide. Taken together, these findings support that M2 promotes the formation of B-T helper cell conjugates. In an in vivo context this may confer a competitive advantage to the infected B cell in acquisition of T cell help and initiation of a germinal center reaction, hence host colonization. Gammaherpesviruses establish life-long persistent infections and are highly prevalent in the human population. Latent infection of circulating memory B cells is crucial to persistence and hence disease ontogeny. To access the memory B cell compartment gammaherpesviruses, such as Epstein-Barr virus (EBV) and murid herpesvirus-4 (MuHV-4), take advantage of germinal center (GC) reactions –7. In thH) cells scan for the highest affinity with particular antigen-presenting cells (APC). Such transient interactions occur in the border area between follicles and T cell zones and are mediated by adhesion molecules, resulting in the formation of B-TH cell conjugates. Upon peptide recognition, the formation of an organized signaling structure, the immunological synapse (IS), takes place [T cell help is critical for the initiation of a GC reaction in T cell-dependent immune responses. Before engaging in a cognate interaction with a B cell that will lead to its activation, proliferation and establishment of a GC , T helpees place . This pres place .+ T cells [FH) [MuHV-4 course of infection in the spleen has been recently characterized . The vir T cells , 15 or Tlls [FH) .H cell interactions, the M2 protein was chosen as a potential candidate. It is one of the few viral proteins that is expressed during the latency phase [120 and Tyr129), that are constitutively phosphorylated by Src family kinases [120 and Tyr129 residues mutated to phenylalanine) results in a delay in latency establishment [129 is critical for reactivation from latency and plasma cell differentiation [To investigate if MuHV-4 had the ability to modulate B-Tcy phase . It is acy phase –20. Contcy phase  where itcy phase –23. It c kinases , 19, tha kinases . Therefo kinases , 19. Fur kinases . IL-10 p kinases . In vivo kinases , 26, 27.lishment . More rentiation .H cell interaction. We observed that B cells expressing M2 are more prone to recruit T cell help, promoting the formation of B-TH cell conjugates. Furthermore, we showed that this function was dependent on the ITAM of M2, hence dependent on the ability to assemble multiprotein complexes with B cell signaling proteins. Overall, in vivo, this may confer a competitive advantage to the infected B cell in the recruitment of T cell help and initiation of a GC reaction, therefore host colonization.Here, we investigated if the latency associated protein M2 encoded by MuHV-4 is able to modulate B-TH cell interactions we started by analyzing the influence of the viral protein on the levels of selected B cell surface molecules, some of which have been shown to be involved in IS formation [In order to assess if M2 plays a role in B-Tormation , 29, 30.ormation , 19, 21.ormation , 29, 30,ormation , 29, 30 These results show that expression of M2 upregulates B cell co-stimulatory and adhesion molecules that participate in the formation of an IS. This function is dependent on tyrosine phosphorylation at the ITAM, hence related to the ability of M2 to assemble multiprotein complexes with B cell signaling proteins.H cells interact with each other forming conjugates. Upon cognate interaction and IS formation, cells undergo morphological and cytoskeletal changes, which result in the polarization of the microtubule organizing center (MTOC) [During T cell scanning that takes place in the border area between follicles and T cell zones, B and Tr (MTOC) –33, cellr (MTOC) , 34. Thir (MTOC) .H cell conjugates, we used a MHC-II restricted cell system in which mouse A20 B cells, pulsed with ovalbumin peptide (OVAp), were conjugated with primary mouse OVAp-specific TCR-transgenic CD4+ T cells purified from Balb/c DO11.10 mice [H cell interaction by expressing M2 and M2Y eGFP-tagged proteins. Furthermore, to address B cell polarization, we measured the B cell MTOC distance to the contact zone (H cells for 15min. Cells were then fixed and stained for pTyr (blue), and α-tubulin (red)  . Using tin (red) . Next, tin (red) . M2 expr B cells . M2Y alsH cellular interface in a MHC class II-restricted OVAp-specific manner, a process that is dependent on the protein phosphosites. Results also show that M2 promotes B cell polarization, as its expression increased MTOC proximity to the contact zone.We conclude that M2 polarizes to the B-TH cells interact with each other forming conjugates, a process that is mediated by adhesion molecules. Given that M2-expressing B cells showed increased levels of such molecules, we investigated if M2 had an impact in B-TH cell conjugate formation (H cells (loaded with DDAO live dye), in a 2:1 ratio, for 1, 3, 5 or 30 minutes. Conjugate formation was next assessed by flow cytometry, based on the percentage of CMFDA+DDAO+ events in the total DDAO+ population. H cells conjugated with M2Y-expressing B cells. In contrast, the integrity of the phosphosites resulted in a three-fold increase in conjugate formation with M2-expressing B cells. Up to 0.01μM M2 was able to promote conjugate formation. However, in the presence of high concentrations of OVAp, M2 did not influence conjugate formation. We conclude that M2 promotes conjugate formation in the absence or with low concentrations of OVAp. Such ability is dependent on the phosphosites responsible for binding to B cell signaling proteins.B and Tormation . We usedH cell interaction by confocal microscopy, including a marker for phosphorylated tyrosines (pTyr). This is indicative of signaling and formation of a functional immunological synapse but, in this particular case, can be also associated with constitutive phosphorylation of the tyrosine residues of M2, and the consequent phosphorylation of Vav1 and PLCγ2 [H cells were incubated in a 2:1 ratio, for 30 minutes. Prior to incubation, B cells were loaded with CMFDA live dye to allow their visualization. Cells were also stained for F-actin, to allow T cell, as well as B-TH cell interaction, visualization. Conjugate count was blind and based on B-TH cell contact and pTyr (purple) polarization to the contact zone  to inhibit protein secretion. As shown in the upper panel of H cells producing IFN-γ, compared to M2Y. Measurement of IFN-γ MFI of responding TH cells showed no significant differences between M2 and M2Y conditions  or M2Y-expressing (grey circles) B cells, that polarize IFN-γ to the contact zone  [H cell interaction, instead of impairing it. This is concomitant with the high prevalence of infection in GC B cells. Such positive effect does not exclude the possibility that, at some point during infection, MuHV-4 impairs IS formation as a mechanism of immune evasion.Modulation of T-APC interaction, and more specifically IS, by a viral protein has been previously described. Protein Nef from Human Immunodeficiency Virus type 1 (HIV-1) has been shown to impair IS formation –41, as as (KSHV) , and tyrs (KSHV) . In contH cell conjugate formation in vitro is in agreement with the delay in latency establishment observed in vivo with the recombinant M2Y virus [The inability of M2Y to promote B-T2Y virus . However2Y virus  may driv2Y virus .Despite the controversy regarding antigen dependence in the production of EBV-infected memory B cells, there is unequivocal evidence of the participation of EBV-infected B cells in the GC reaction , 2, as iH cell interaction, a process that is dependent on the modulation of B cell signaling. In vivo, this ability to attract T cell help in a competitive manner during the T cell scanning process, carried out in secondary lymphoid organs, may account for the selection of infected B cells for GC initiation, a crucial step in latency establishment, hence host colonization.In this study we propose that the latency-associated M2 protein competitively promotes B-TThe study accorded with the Portuguese official Veterinary Directorate (Portaria 1005/92), European Directive 2010/63/EU, and Federation of European Laboratory Animal Science Associations guidelines on laboratory animal welfare. It was approved by the Portuguese official veterinary department for welfare licensing  and by the IMM Animal Ethics Committee.+ T cells. Mice were bred and housed under specific pathogen-free conditions at Instituto de Medicina Molecular, Lisbon, Portugal. Mice were sacrificed by CO2 inhalation or cervical dislocation.8-week to 4-month old Balb/c TCR transgenic DO11.10 mice  were used for purification of CD4pEGPF-M2, pEGFP-M2Y plasmids have been described . To prodPrimary antibodies used for confocal microscopy were: anti-F-actin , anti-alpha tubulin , anti-pTyr-AF647 , anti-IFN-gamma . Secondary antibodies used for confocal microscopy were: anti-rat AF568 (Molecular Probes) and anti-mouse AF594 (Molecular Probes). Antibodies used for flow cytometry were: anti-TCR DO11.10 PE , anti-CD4 AF405 , anti-CD80 PB , anti-CD86 PE-Cy7 , anti-CD54 (ICAM1) APC , anti-CD40 PE-Cy7 , anti-CD48 APC , anti-MHC-II PB , anti-CD19 APC-H7 , anti-CD21 APC , anti-CD23 PE , anti-CD69 FITC , anti-CD95 PE , anti-IFN-γ PE . Primary antibodies used for western blot were: anti-EGFP (Clontech) and anti-actin . Secondary antibodies used for western blot were anti-rabbit  and anti-mouse (Jackson Immunoresearch) antibodies, conjugated with horse radish peroxidase (HRP).+ T cells were isolated from the spleen of 8-week to 4-months old TCR transgenic DO11.10 mice using a mouse CD4+ T cell isolation kit (Miltenyi Biotec), according to manufacturer’s instructions. Purity was analyzed by flow cytometry and was above 90%. Cells were then stimulated with mouse T-activator CD3/CD28 Dynabeads® (Invitrogen), according to manufacturer’s instructions. After Dynabeads® removal, cells were cultured for a resting period of 5–8 days in RPMI 1640 supplemented as described above in addition to 50μM 2-mercaptoethanol, 100μM sodium pyruvate, 10mM HEPES and 60U/mL rmIL-2. For confocal microscopy experiments, by the end of the resting period, live cells were sorted based on FSC-A/SSC-A parameters. For production of viral particles used for A20 B cell transduction, HEK-293T cells were transiently transfected by the Calcium Phosphate Method with 20μg of pEQPAM3 and 20μg of pMSCV-M2-IRES-Zeo, pMSCV-M2Y-IRES-Zeo, pMSCV-eGFP-M2-IRES-Zeo or pMSCV-eGFP-M2Y-IRES-Zeo. After transduction, B cells were cultured for two weeks in RPMI supplemented with 400μg/mL of zeocin. In the case of eGFP-M2/M2Y constructs, GFP+ cells were sorted after transduction and prior to culture with zeocin. All transductions were performed in the presence of 8μg/mL of polybrene.HEK-293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 2mM glutamine, 100U/mL penicillin and streptomycin. A20 B cells were grown in RPMI 1640 supplemented as described above. In the case of zeocin-resistant stable cell lines, 4μg/mL of zeocin was added to the culture. Conjugates were incubated in RPMI 1640 supplemented with 5% fetal bovine serum, 2mM glutamine, 100u/mL penicillin and streptomycin and 10mM HEPES. In the case of the analysis by flow cytometry of intracellular production of IFN-γ the media was supplemented with 5% fetal bovine serum, 10mM HEPES, 60U/mL rmIL-2 (Peprotech) and 10μg/mL of Brefeldin A . Mouse CD4+ T cells were sorted on a BD FACSaria flow cytometer at the end of the resting period, based on FSC-A/SSC-A parameters. After sorting, cells were immediately used for conjugation experiments. A20 B cells transiently expressing eGPF-M2 or eGFP-M2Y were sorted on a BD FACSaria flow cytometer for eGFP expressing cells, prior to their use. Splenocyte single cell suspensions were prepared from spleens recovered at 14dpi. Red blood cells were lysed in hypotonic NH4Cl. Splenocyte suspensions or cell lines were incubated with Fc block prior to staining, washed with 1x PBS, and stained for 20min with primary antibodies. Cells were washed and incubated for 20min with streptavidin, when appropriate. All samples were resuspended in FACS buffer  and analyzed on LSR Fortessa (BD Biosciences) using DIVA software (BD Biosciences) for acquisition. For conjugation experiments, cells were analyzed in supplemented culture media, instead of FACS buffer. For IFN-γ intracellular measurements a FACSCalibur was used along with CellQuest software . For this experiment cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.2% triton X-100 prior to incubation with detecting antibody. All steps until incubation with PFA were performed in FACS buffer supplemented with BFA. T cells incubated for 5h with 100 ng/ml phorbol-12-myristate-13-acetate  and 2 μg/ml ionomycin (Sigma) were used as positive control. Data was analyzed using Flowjo (Tree Star).Live resting mouse CD4+ T cells were loaded with one of the following live dyes, as indicated for each experiment: CellTrackerTM Blue CMAC (7-amino-4-chloromethylcoumarin), CellTrackerTM Green CMFDA (5-chloromethylfluorescein diacetate), CellTraceTM Far-Red DDAO-SE, CellTrackerTM Orange CMTMR ((5-(and-6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine) from Molecular Probes, Invitrogen. B and T cells were added in the indicated ratios, centrifuged for 1min to promote conjugate formation, and incubated for a period of time, as is specified. For confocal microscopy experiments, after conjugation, cells were gently resuspended, added to poly-L-lysine-coated coverslips (BD Biosciences) and incubated for the indicated time at 37°C. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. Samples were incubated with primary antibodies for 1h, followed by 1h of incubation with a species-specific secondary antibody, when necessary. DNA was detected with DAPI (Invitrogen). Coverslips were mounted in Mowiol (Fluka) or Fluoromount-G (Southern Biotec) and examined using a Carl Zeiss LSM 510 confocal microscope, with a Plan-Apochromat objective of 63x (1.4 oil). Analysis was performed using AimImageBrowser (Zeiss LSM data server). For flow cytometry experiments, after conjugation, cells were gently resuspended and analyzed on a LSR Fortessa flow cytometer (BD Biosciences).A20 B cells, stably expressing M2, M2Y, eGFP, eGPF-M2 or eGFP-M2Y, were pulsed overnight, or at least 2h, with the indicated concentrations of OVAp  in RPMI. Afterwards, A20 B cells and/or mouse CD4H contact zone and signal quantifications were measured using AimImageBrowser . To evaluate the percentage of conjugates showing pTyr and/or IFN-γ polarization to the contact zone, only images with a minimum of three T cells were analyzed. Number of T cells conjugating was scored visually.Image quantification was performed in a blinded fashion, in a minimum of 45 randomly selected fields, from a total of three independent experiments. Distances of the MTOC to the center of the B-T+ T cells were processed as described above. T cells were loaded with 5μM Indo-1 AM (Invitrogen) [A20 B cells, loaded or not with OVAp, and mouse OVAp-specific CD4itrogen)  and conj+ T cells as described above and incubated for 20h. After the 20h incubation period, supernatant was recovered and stored at -20°C. IFN-γ production was quantified in the supernatants by sandwich ELISA using the commercial kit DuoSet® ELISA Development System (R&D systems), according to manufacturer’s instructions. 96-well plates were analyzed on an Infinite® M200 . T cells cultured for two to four days in the presence of 3μg/mL of anti-CD3 antibody were used as positive control of T cell activation. B cells cultured for 48h in the presence of 2.5μg/mL of anti-CD40 antibody and 5μg/mL of the F(ab’)2/F(ab) portion of an anti-mouse IgG antibody were used as positive control for B cell activation.A20 B cell lines were loaded, or not, with different concentrations of OVAp (aa 323–339) overnight. Cells were then conjugated with purified mouse CD4Statistical significance was evaluated with unpaired one-tailed t-test, one-way ANOVA or non-parametric Mann-Whitney U test, as appropriate, using GraphPad Prism software. ns indicates p>0.05; * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001.S1 Fig(A) M2/M2Y expression in total cellular lysates of the independent A20 B cell lines. eGFP or eGFP-M2/M2Y fusion proteins were detected on Western Blot with an anti-eGFP antibody. An anti-actin antibody was used to demonstrate that similar amounts of cellular lysates were used. An A20 B cell line expressing non-tagged M2 (lane 4) was used as a negative control. (B) Fold increase of the mean fluorescence intensities (MFI) of several surface molecules, normalized to eGFP A20 B cells. A20 B cell lines stably expressing eGFP, eGFP-M2 or eGFP-M2Y were stained with fluorescently labelled antibodies and the surface expression of the indicated molecules was analyzed on a LSR Fortessa flow cytometer. Bars represent the mean of eight independent experiments. Error bars represent standard error of the mean. Statistical significance was assessed with a one-tailed Students t-test.(TIF)Click here for additional data file.S2 Fig+ T cells at a 2:1 ratio. (A) Percentage of conjugates after 30min of incubation upon variation of the OVAp concentration. T cell populations were loaded with DDAO, to allow their discrimination. Results shown correspond to mean of three independent experiments. Statistical significance refers to comparison between M2 and M2Y conditions. (B) Percentage of conjugates per image after 30min of incubation, determined by confocal microscopy, upon variation of the OVAp concentration. Conjugate count was blind and based on B-TH contact and pTyr polarization to the contact zone. 15 to 35 images were taken per sample, for an equivalent number of analyzed T cells within each OVAp concentration. Only images with a minimum of three T cells were considered for analysis. Results are from one experiment. (C) Fold increase of the number of conjugates formed with eGFP-M2- (open bars) or eGFP-M2Y- (filled bars) expressing B cells relative to M2Y condition. eGFP-M2-expressing B cells, eGFP-M2Y-expressing B cells and CD4+ T cells were mixed at a 1:1:1 ratio and incubated for 30min. Prior to conjugation M2Y-expressing B and T cell populations were labeled with the live dyes CMTMR and DDAO, respectively, to allow their discrimination. Conjugate formation was analyzed on a LSR Fortessa flow cytometer as the percentage of eGFP+DDAO+ (M2) or eGFP+CMTMR+DDAO+ (M2Y) events in the total DDAO+ population. (D) Representative FACS plots for each OVAp concentration. Percentage of T cells conjugating with M2- or M2Y-expressing B cells is indicated in the respective quadrant. In flow cytometry experiments, error bars represent standard error of the mean. Statistical significance between groups was evaluated by a one-tailed unpaired Student’s t test. In confocal microscopy experiments, statistical significance of the difference between groups was evaluated by a Mann-Whitney U test.eGFP independent B cell lines were pulsed overnight, or not, with different concentrations of OVAp and incubated with OVAp-specific CD4(TIF)Click here for additional data file.S3 Fig+ T cells mobilizing calcium when conjugated with eGFP-M2-expressing (black bars), eGFP-M2Y-expressing (white bars) or eGFP-expressing (grey bars) B cells. eGFP independent B cell lines were pulsed overnight, or not, with different concentrations of OVAp and incubated with OVAp-specific CD4+ T cells for 5 min. Prior to conjugation T cells were loaded with Indo-I, a calcium indicator. Ionomycin was used as a positive control. Calcium fluxes were measured on a MoFlow cytometer for 21 minutes and were based on the 405/530 emission ratio over time. Graph shows results from one experiment. (B) Quantification of conjugates showing IFN-γ polarization to the contact zone per field. Prior to incubation B and T cells were labelled with CMFDA and CMAC live dyes, respectively. Cells were incubated for 2.5h, fixed and stained for IFN-γ and pTyr. Conjugates were evaluated by confocal microscopy based on B-TH contact and IFN-γ polarization. Only images with a minimum of three T cells were considered for analysis. Statistical significance of the difference between groups was evaluated by a Mann-Whitney U test.(A) Average of the percentage of CD4(TIF)Click here for additional data file.


Drosophila and viral genes that antagonize it. We have recently shown that viral protein 1 (VP1) of Drosophila melanogaster Nora virus (DmelNV) suppresses Argonaute-2 (AGO2)-mediated target RNA cleavage (slicer activity) to antagonize antiviral RNAi. Here we show that viral AGO2 antagonists of divergent Nora-like viruses can have host specific activities. We have identified novel Nora-like viruses in wild-caught populations of D. immigrans (DimmNV) and D. subobscura (DsubNV) that are 36% and 26% divergent from DmelNV at the amino acid level. We show that DimmNV and DsubNV VP1 are unable to suppress RNAi in D. melanogaster S2 cells, whereas DmelNV VP1 potently suppresses RNAi in this host species. Moreover, we show that the RNAi suppressor activity of DimmNV VP1 is restricted to its natural host species, D. immigrans. Specifically, we find that DimmNV VP1 interacts with D. immigrans AGO2, but not with D. melanogaster AGO2, and that it suppresses slicer activity in embryo lysates from D. immigrans, but not in lysates from D. melanogaster. This species-specific interaction is reflected in the ability of DimmNV VP1 to enhance RNA production by a recombinant Sindbis virus in a host-specific manner. Our results emphasize the importance of analyzing viral RNAi suppressor activity in the relevant host species. We suggest that rapid co-evolution between RNA viruses and their hosts may result in host species-specific activities of RNAi suppressor proteins, and therefore that viral RNAi suppressors could be host-specificity factors.The ongoing conflict between viruses and their hosts can drive the co-evolution between host immune genes and viral suppressors of immunity. It has been suggested that an evolutionary ‘arms race’ may occur between rapidly evolving components of the antiviral RNAi pathway of  Viruses and their hosts can engage in an evolutionary arms race. Viruses may select for hosts with more effective immune responses, whereas the immune response of the host may select for viruses that evade the immune system. These viral counter-defenses may in turn drive adaptations in host immune genes. A potential outcome of this perpetual cycle is that the interaction between virus and host becomes more specific. In insects, the host antiviral RNAi machinery exerts strong evolutionary pressure that has led to the evolution of viral proteins that can antagonize the RNAi response. We have identified novel viruses that infect different fruit fly species and we show that the RNAi suppressor proteins of these viruses can be specific to their host. Furthermore, we show that these proteins can enhance virus replication in a host-specific manner. These results are in line with the hypothesis that virus-host co-evolution shapes the genomes of both virus and host. Moreover, our results suggest that RNAi suppressor proteins have the potential to determine host specificity of viruses. As obligate intracellular parasites, viruses modulate and exploit the host cellular environment for their replication. The host antiviral defense system restricts virus infections, and in turn, viruses dedicate a significant fraction of their coding capacity to produce factors that antagonize the antiviral immune response The RNA interference (RNAi) pathway is a major antiviral defense system in plants, arthropods, nematodes and fungi Dcr-2, R2D2, and AGO2 belong to the 3% fastest evolving genes in D. melanogaster and D. simulans and show very high rates of adaptive amino acid substitution with evidence for recent selective sweeps in multiple Drosophila species The ongoing arms race with viruses can impose a strong selective pressure on immune genes of the host Drosophila melanogaster (DmelNV) is a recently identified natural fruit fly pathogen, which contains a single-stranded positive-sense RNA genome and appears to fall within the order of PicornaviralesPicornavirales members, ORF 4 encodes capsid proteins Nora virus of DmelNV causes persistent infections in laboratory stocks as well as in wild caught flies. Persistent infections are thought to reflect a dynamic equilibrium between host defense responses and viral counter-defense mechanisms D. immigrans (DimmNV) and D. subobscura (DsubNV), and we use these viral genomes to study RNAi antagonism from an evolutionary perspective. We find that the RNAi suppressor activity of DimmNV VP1 appears to be restricted to its natural host species, whereas DmelNV VP1 does not display any evidence of host specificity. We conclude that co-evolution between Nora viruses and their Drosophila hosts can result in host species-specific antagonism of AGO2, and therefore that viral suppressors of RNAi are candidate host specificity determinants.Here we present two novel Nora-like viruses identified by metagenomic sequencing of wild populations of DrosophilaDrosophila species.RNAi genes evolve rapidly and adaptively in multiple species of Drosophila, we identified two novel Nora-like viruses in wild populations of D. immigrans (DimmNV) and D. subobscura (DsubNV). Following this observation, we took a metagenomic RNA-sequencing approach to recover near-complete viral genomes for both viruses from population samples of D. immigrans and D. subobscura collected in the United Kingdom. The viral sequences were 12,265 nt and 12,276 nt, respectively  and include all protein coding regions, a conserved CCTGGGSGGGGGTTA motif in their 5′ untranslated region, and a 3′ poly-A tract (D. melanogaster (DmelNV) Haematobia irritansChrysopa pallens and the moth Spodoptera exigua, or the more distantly related Nora-like virus described in the wasp Nasonia vitripennisDuring an exploratory RT-PCR survey of Nora virus prevalence in wild -A tract . These nOverall, DmelNV is more divergent from DimmNV than it is from DsubNV , but phylogenetic analysis based on the coat protein (VP4) suggests that DmelNV and DimmNV may be each other's closest relatives. The low genome-wide nonsynonymous to synonymous substitution ratio  estimated by PAML D. melanogaster and D. subobscura, or it may be a result of recombination in the history of these three viruses.Amino-acid divergence between the viruses varies substantially between genes . For exaD. melanogaster. To this end, we cloned the full-length (FL) VP1 sequences and N- and C-terminal deletion mutants thereof (ΔN and ΔC) as N-terminal fusions to the V5 epitope in an insect expression plasmid  reporter plasmids along with VP1 expression plasmids, and induced silencing of the Fluc reporter by soaking the cells in Fluc specific dsRNA. As reported earlier ΔC74, suppressed RNAi-mediated silencing of the Fluc reporter. In contrast, none of the DimmNV VP1 constructs efficiently suppressed silencing of the reporter  assays D. melanogaster and D. immigrans. Unfortunately, we were not successful in producing slicer competent lysates for D. subobscura. Moreover, members of the Drosophila obscura group encode multiple AGO2-like proteins of unknown function D. subobscura and DsubNV in subsequent analyses.The inability of DimmNV VP1 and DsubNV VP1 to suppress RNAi in 32P cap-labelled target RNA is induced by the addition of a target specific siRNA. Since the target RNA is radio-labelled at its 5′ cap, the 5′ cleavage product can be visualized by autoradiography after polyacrylamide gel electrophoresis. As expected, in both D. melanogaster and D. immigrans embryo lysates a specific cleavage product was observed after incubation with a target specific siRNA , was unable to do so  activity of a pre-assembled RISC in D. immigrans lysates, it is likely that DimmNV VP1 interacts with Dimm AGO2. We hypothesized that the inability of DimmNV VP1 to suppress RNAi in D. melanogaster may then be due to an inefficient interaction with Dmel AGO2. To test these hypotheses, we analyzed VP1 interactions with host and non-host AGO2 proteins by co-IPs. First, we co-expressed V5 epitope-tagged DmelNV VP1 or DimmNV VP1 with Dmel FLAG-AGO2 in S2 cells and immunopurified the VP1 proteins using V5 affinity beads. As controls, we analyzed IPs of cells transfected with empty vector. As observed above . As expected, the predicted protein domains of Dimm FLAG-AGO2 are similar to those of Dmel AGO2, suggesting that the overall protein structure of Dimm and Dmel AGO2 are alike. Overall amino acid identity is 56% (63% when excluding the poly-glutamine repeats), with a higher level of conservation in the PIWI domain (77% identity) than in the PAZ domain (45% identity). We thus analyzed the interaction of DmelNV VP1 or the DimmNV VP1 with Dimm FLAG-AGO2 in co-IP. Both DmelNV VP1 and DimmNV VP1 efficiently co-purified the Dimm AGO2 protein  or the 3′ untranslated region (3′ UTR) of the endogenous Dmel AGO2 transcript. To monitor AGO2 activity in these S2 cells we induced RNAi with the Rluc-specific RNA hairpin  recombinants expressing either DimmNV VP1, DmelNV VP1, or, as a control, GFP from a second subgenomic promoter  , our observations suggest that Nora virus is a member of a large family of widespread pathogens that infects multiple insect species.Nora virus is a positive-sense RNA virus that was recently identified in laboratory stocks of D. immigrans or D. subobscura contained sequences derived from the other Nora lineages , despite being initially collected as mixed samples of multiple Drosophila species. It is therefore possible that, as is the case for the purely vertically transmitted Sigma viruses, Nora viruses rarely move between hosts Although little is known regarding the natural host range of Nora viruses, it is worth noting that neither of our population samples of D. immigrans embryo lysates. In contrast, DimmNV VP1 was unable to suppress RNAi in D. melanogaster cells, did not interact with Dmel AGO2, and did not inhibit slicer activity in D. melanogaster embryo lysates. These results are consistent with a model in which adaption and co-evolution of DimmNV with its host resulted in a species-specific AGO2-VP1 interaction.Plant and insect viruses can suppress the antiviral RNAi pathway of their hosts via a variety of mechanisms Drosophila melanogaster or Arabidopsis thaliana, are often used to identify and characterize viral suppressors of RNAi, including those of viruses that naturally do not infect these hosts. Our observation that RNAi suppressor proteins may have species-specific activity suggests that it is important to take into account the correct evolutionary context in experiments aimed at the identification of viral suppressors of RNAi. For example, we note that we would not have detected RNAi suppressive activity in DimmNV, if we had solely relied on experiments in D. melanogaster.Our findings have important practical implications. Experimentally amenable model systems, such as D. immigrans and D. melanogaster embryo lysates. We suggest that there are two potential explanations for this. First, it may be that these viruses differ in natural host range; the broader-spectrum functionality of DmelNV VP1 across divergent hosts could be maintained by selection if DmelNV has a wider host range than DimmNV. In support of this hypothesis, although none of these three viruses was identified from the other host species, DmelNV (but not DimmNV) has been identified in wild Drosophila simulans . Second, if there is not a substantial trade-off associated with host-specialization and if DmelNV has colonized D. melanogaster quite recently, it could just be a matter of time until DmelNV loses its broad-spectrum VSR.In striking contrast to DimmNV, DmelNV VP1 did not show species-specific activity. It can engage in an interaction with both Dimm and Dmel AGO2 and, accordingly, it inhibited slicer activity in both D. melanogaster cells. This result suggests that the limited amino acid identity with Dmel AGO2 (∼63%) does not impede its ability to interact with Dmel Dicer-2 and R2D2 or other components of RISC and RISC-loading complexes. Thus, even though RNAi genes are rapidly evolving and show high rates of adaptive substitution, these results imply that this diversification has not impeded cross-species interactions of RNAi genes, even over the tens of millions of years that separate D. melanogaster and D. immigrans. This conservation of function may imply that the need for interaction between Dicer-2, R2D2, AGO2, and other RNAi pathway genes imposes a constraint on the evolution of these genes, and thus their opportunity to evolve in response to virus-mediated selection.We successfully reconstituted Dimm AGO2-based silencing in Together, our results suggest that rapid co-evolution between RNA viruses and their hosts may result in host species-specific activities of RNAi suppressor proteins. Moreover, our observation that DimmNV VP1 enhances viral RNA levels in a host-specific manner, suggest that viral RNAi suppressors are putative host-specificity factors.Drosophila populations were surveyed for the prevalence of Dmel Nora virus using RT-PCR , which resulted in the identification of Nora virus-like PCR products from D. immigrans and from members of the obscura group . A metagenomic approach was then used to obtain near-complete viral genomes. Flies were collected from elsewhere in the UK and samples were pooled by species for RNA extraction and Illumina double-stranded nuclease normalized RNA-sequencing. For D. subobscura, only male flies were used as females are difficult to distinguish morphologically from close relatives. RNA was extracted from each collection using a standard Trizol (Invitrogen) procedure, according to the manufacturer's instructions, and pooled in proportion to the number of contributing flies. In total, the two pools comprised 338 male D. subobscura  and 498 D. immigrans . Total RNA was provided to the Beijing Genomics Institute (Hong Kong) for normalization and 90-nt paired-end Illumina sequencing. Paired-end reads were quality trimmed using ConDeTri version 2 de novo using the Trinity transcriptome assembler with default settings  and 68,914 reads mapped to DsubNV . Consensus sequences have been submitted to GenBank under accession numbers KF242510 (DsubNV) and KF242511 (DimmNV).Wild Nasonia vitripennis Nora-like virus  for analysis. Two approaches to phylogenetic inference were used. First, MrBayes , pMT-Luc Renilla-hairpin The following constructs were described previously: all DmelNV VP1 constructs D. immigrans and D. subobscura was made using Promega MMLV-RT in the presence of Promega RNasin Plus according to manufacturer's instructions. Subsequently, DimmNV VP1 and DsubNV VP1 sequences were PCR amplified from D. immigrans and D. subobscura cDNA and cloned as full-length and deletion constructs downstream of the V5-His tag in pAc5-V5-His-Ntag (details available upon request).cDNA of D. immigrans AGO2 cDNA sequence (GenBank KF362118), including partial 5′ and 3′ UTRs, was PCR amplified using the primer pair 5′-TGCAGCAAAAATTAGAAGCAAA-3′ and 5′-AGCCGTACCTAGAACCAGCA-3′. The resulting PCR product was used as a template in a nested PCR using primer pair 5′-AGTTCTAGACCGCGGGAATGGGTAAAAAGAACAAGTTCAAACCA-3′ and 5′-AGTTCTAGACCGCGGGAAGCGCTGTGGCACAGCTTCCGC-3′. The nested PCR product was subsequently cloned into the pAFW vector using the SacII and SalI restriction sites. To fuse the DimmNV VP1ΔN295 protein to the C-terminus of the maltose binding protein (MBP), we PCR amplified the VP1 coding sequence from pAc5.1-Ntag-DimmNV VP1FL with primer pair 5′-AGTGGATCCCCAAAACTTCCAAGTGTACCTTCAAAG -3′ and 5′-GGTGTCGACTTAGTTTTGTTTATTTTTGTACCAATCGTTGG -3′. The DsubNV VP1ΔN281 sequence was amplified from pAc5.1-Ntag-DsubNV VP1FL with primer pair 5′-TGACGGATCCCCAAACAAACCTCTAAAACC -3′ and 5′-ACTGGTCGACTCATTGTTGCTGAGTTGATTTG -3′. The resulting PCR products were cloned into the pMal-C2X vector (New England Biolabs) using BamHI and SalI restriction sites.The in vitro transcription using T7 promoter-flanked PCR fragments as a template, as described previously 5′-TAATACGACTCACTATAGGGAGATACTATGGTGAAGAACGGGTCG-3′ and 5′-TAATACGACTCACTATAGGGAGAGAACATGTCCTCAATCTCCTCC-3′, or primer combination 5′-TAATACGACTCACTATAGGGAGAGCAACGTATTGAATCTTATT-3′ and 5′-TAATACGACTCACTATAGGGAGAAGAACAATATTTGGCGGACC-3′, respectively.Double-stranded RNA was generated by Drosophila S2 cells were performed as described 4 S2 cells were seeded per well in a 96-well plate. The seeded cells were co-transfected with 10 ng pMT-Fluc, 10 ng pMT-Rluc, 50 ng pRmHa-Renilla-hairpin, and 50 ng of expression plasmids encoding VP1 and/or AGO per well using Effectene transfection reagent (Qiagen). The pAc5-Ntag-DmelNV VP1Δ284 and pAc5-Ntag-DimmNV VP1ΔN295 plasmids were used for VP1 expression. For knockdown of endogenous AGO2, 5 ng of AGO2 dsRNA or control dsRNA was co-transfected along with reporter plasmids. Two days after transfection, the expression of the luciferase reporters and the Rluc hairpin was induced by the addition of 0.5 mM CuSO4 per well. The next day, cells were lysed and Fluc and Rluc activity was measured with the Dual luciferase reporter assay system (Promega) according to manufacturer's protocol.miRNA and RNAi sensor assays in 6 cells per well. The next day, cells were transfected with AGO2 and/or VP1 expression plasmids using Effectene transfection reagent (Qiagen). Expression plasmids encoding DmelNV VP1ΔN351, DmelNV VP1ΔN284, or DimmNV VP1ΔN295 were used for co-immunoprecipitation experiments, as indicated in the figure legends. Three days after transfection, cells were washed twice with PBS and subsequently resuspended in lysis buffer , 0.1% NP-40, 5 mM DTT) supplemented with protease inhibitor cocktail (Roche). After incubation on ice for 10 minutes, the samples were passed forty times through a 25-gauge needle, followed by incubation on ice for 10 minutes. Subsequently, cell lysates were centrifuged at 13,000 rpm for 30 minutes and a sample of the supernatant was taken to analyze the input for IP. To remove proteins that non-specifically bind to the IP beads, the remaining supernatant was incubated with Pierce protein G agarose at 4°C for 5 hours while mixing end-over-end. Next, the protein G agarose was separated from the supernatant by centrifugation, after which the supernatant was incubated overnight with anti-V5 agarose affinity gel (Invitrogen) at 4°C while mixing end-over-end. The next day, the anti-V5 agarose was separated from the supernatant by centrifugation, and a sample was taken from the supernatant. After the remaining supernatant was removed, the V5-agarose was washed three times with lysis buffer, and three times with either wash buffer 150  or wash buffer 200 . All wash steps were done with 40 to 60 times beads-volume of wash buffer. Subsequently, the beads were boiled in SDS sample buffer at 95°C for 10 minutes, followed by a brief centrifugation step to collect the beads at the bottom of the tube. The proteins in the supernatant were then separated on a SDS-PAGE gel, after which they were transferred onto a nitrocellulose membrane by western blot. Primary antibodies used for western blot detection were anti-FLAG-M2 , anti-V5 , anti-AGO2 , and anti-tubulin-alpha ; secondary antibodies were goat anti-mouse-IRdye680 , and goat anti-rabbit-IRdye800 . All western blots were scanned using an Odyssey infrared imager (LI-COR biosciences).For immunoprecipitations, S2 cells were seeded in 6-well plates at a density of 2×10ΔN295 and the pMal-C2X-DsubNV VP1ΔN281 plasmids were transformed into the Escherichia coli BL21 (DE3) strain. Subsequently, expression of recombinant protein was induced by addition of 0.2 mM IPTG. Protein expression was allowed to proceed overnight at 18°C. The next day, recombinant MBP-DimmNV VP1 and MBP-DsubNV VP1 were purified using amylose resin (New England Biolabs) according to the manufacturer's protocol. Purified protein was subsequently transferred to a dialysis membrane (molecular weight cut-off 12–14 kDa) and incubated overnight in dialysis buffer  at 4°C, followed by a second dialysis step for 5 hours at 4°C. The dialyzed protein solution was stored at −80°C in dialysis buffer containing 30% glycerol. Purification of MBP-DmelNV VP1ΔN284 has been described previously To purify recombinant VP1 as MBP fusion proteins, the pMal-C2X-DimmNV VP1D. immigrans isofemale line was established from flies collected in June 2012 in Edinburgh . D. immigrans was cultured similarly as D. melanogaster on standard media. Embryo lysates were generated from D. immigrans and from an RNAi-competent D. melanogaster laboratory control strain (w1118). In vitro target RNA cleavage assays in D. melanogaster embryo lysates were performed as described D. immigrans embryo lysate: the reaction contained 0.9 mM MgCl2 and was allowed to proceed for 5 hours at 25°C before RNA extraction. Suppressor activities of MBP-DmelNV VP1ΔN284, DsubNV VP1ΔN281, and MBP-DimmNV VP1ΔN295 proteins were analyzed in slicer assays.A new ΔN284 and DimmNV VP1ΔN295 were PCR amplified from the respective insect expression vectors using primers V5 Fw: AGTTCTAGAAACATGGGTAAGCCTATCC; Dmel VP1 Rv: GGTTCTAGATTAACATTGTTGTTTCTGCGAG; and Dimm VP1 Rv: TGACTCTAGATTAGTTTTGTTTATTTTTGTACC. PCR products were cloned into the XbaI site following the second subgenomic promoter of the pTE3'2J vector To produce recombinant Sindbis viruses, N-terminal V5 tagged DmelNV VP1Dicer-2 deficient C6/36 cells. The cells were cultures as described previously AACTCTGCCACAGATCAGCC; SINV NS4 Rv: GGGGCAGAAGGTTGCAGTAT) and Aedes Albopictus RpL5 for normalization .The replicative capacity of recombinant viruses was analyzed on Drosophila melanogaster w1118 and Drosophila immigrans) were grown on standard yeast/agar medium at 25°C on a 12-h light/dark cycle. Flies were cured of Wolbachia sp. by tetracycline treatments as described in 2-anesthetized and intrathoracical single injections of 50.6 nL, corresponding to 5,000 plaque forming units for each virus, were performed using a nanoinjector Nanoject II (Drummond Scientific Company) as described in Flies . RNase-free DNase I treatment (Roche) was performed according to manufacturer's instructions, followed by acid-phenol/chloroform (Life Technologies) inactivation. Total RNA was quantified using a ND-1000 NanoDrop spectrophotometer (Thermo Fisher Scientific). Reverse transcription was performed using SuperScript II Reverse Transcriptase with random hexamers as primers (Life Technologies) on 2 µg of total RNA. Quantitative PCR was performed with three technical replicates for each cDNA sample using FastStart SYBR Green Master (Rox) (Roche) on a ViiA7 Real-Time PCR instrument (Life Technologies). As negative controls, cDNA reactions without reverse transcriptase and PCR amplification without cDNA template were included. Oligonucleotide primers were as follows  Sindbis virus: SINV-NSP3_F, D. immigrans AGO2 cDNA sequence: KF362118; DsubNV consensus sequence: KF242510; DimmNV consensus sequence: KF242511; DmelNV: NC_007919.3; Nasonia vitripennis Nora-like virus: FJ790488; Haematobia irritans Nora-like virus: HO004689, HO000459, and HO000794; Transcriptome of Spodoptera exigua: GAOR01000957; Transcriptome of Chrysopa pallens: GAGF01018485.Figure S1Sequence alignment of Nora viruses from different Drosophila species. (A) The 5′ terminal sequence of D. immigrans Nora-like virus (DimmNV) and D. subobscura Nora-like virus (DsubNV) obtained by metagenomic RNA-sequencing was aligned to the first 70 nt of the 5′ UTR of D. melanogaster Nora virus . The DmelNV 5′ sequence had been determined by 5′RACE B) VP1 sequences of DmelNV, DsubNV, and DimmNV were aligned with Clustal Omega using default settings. Arrows indicate the first amino acid of the N-terminal deletion mutants (ΔN) and the last amino acid of the C-terminal deletion mutants (ΔC) that were used in this study.(TIF)Click here for additional data file.Figure S2DsubNV VP1 and DimmNV VP1 do not suppress RNAi in D. melanogaster. (A) Western blot analysis of V5-tagged full-length (FL) or N-terminal deletion (ΔN) constructs of DsubNV VP1 or DmelNV VP1. VP1 proteins were detected with anti-V5 (α-V5) antibody. Tubulin (α-tub) was used as a loading control. (B) dsRNA-induced RNAi sensor assay in D. melanogaster S2 cells. Firefly luciferase (Fluc) and Renilla luciferase (Rluc) reporter plasmids were co-transfected with plasmids encoding DmelNV VP1, DsubNV VP1, or a control plasmid (Vector). Two days after transfection, cells were soaked in medium containing Fluc dsRNA or control dsRNA. One day later, luciferase activities were measured and Fluc counts were normalized to Rluc counts and expressed as fold silencing relative to the corresponding control dsRNA treatment. (C) siRNA-induced RNAi sensor assay in S2 cells. The assay was done as described in panel B, except that siRNAs targeting Fluc (Fluc siRNA) or control siRNAs (Ctrl siRNA) were co-transfected with the plasmids instead of soaking the cells in dsRNA. Bars represent means and standard deviations of three independent biological replicates. One-way ANOVA followed by Dunnett's post hoc test was used to evaluate whether VP1 constructs significantly suppressed RNAi relative to the vector control (light gray bar). *** P<0.001; ns, not significant. (D) In vitro RNA cleavage (slicer) assays in lysates from D. melanogaster embryos. Radioactively cap-labelled target RNA was incubated in embryo lysate together with a non-specific control siRNA (lane 1) or a target specific siRNA (lanes 2–5). Target cleavage was determined either in the absence of recombinant protein (lane 2) or in the presence of 0.3 µM of MBP (lane 3), MBP-DmelNV VP1 (lane 4), or DsubNV VP1 (lane 5).(TIF)Click here for additional data file.Text S1Amino acid sequence alignment used for .(DOCX)Click here for additional data file.


A major challenge in HIFU mediated Image Guided Drug Delivery (IGDD) is developing accurate means to implement real-time drug delivery control, and to optimize intratumoral drug distribution. To facilitate clinical translation, objectives of this study were to: 1) develop echogenic E-LTSL, a low temperature sensitive liposome co-loaded with an US contrast agent  and doxorubicin, 2) determine stability of contrast agent encapsulation and characterize doxorubicin release from E-LTSL and 3) investigate the ability of E-LTSL to report on real-time doxorubicin distribution with Ultrasound (US)-guided hyperthermia.in vivo in a mouse xenograft model of human prostate cancer.E-LTSL was loaded passively with PFP using an innovative 1-step sonoporation method and actively loaded with doxorubicin. Doxorubicin release and PFP imageability from E-LTSL in phantoms was quantified by fluorescence spectroscopy, ultrasound imaging and transmission electron microscopy (TEM) in combination with mild hyperthermia (40-42°C). Ultrasound imageability of E-LTSL was determined vs. size increase and drug release kinetics of E-LTSL demonstrated no difference with control. Doxorubicin release in physiological buffer was <5% in 1 hr at baseline (25°C) and body temperatures (37°C), vs. >99% release with hyperthermia (~41°C). Intensity of observed ultrasound image with respect to temperature in the range of 31-40°C correlated strongly to the formation of gas bubbles in E-LTSL, and stabilized to a fixed intensity at the transition temperature. After the transition temperature of E-LTSL reached, the US intensity increased again similar to Dox release. Synthesized E-LTSLs were imageable in vivo and were stable in aqueous environment, with no visual evidence of particle aggregation after 48 hr storage at 4 °C. In conclusion, an US imageable heat sensitive liposome formulation co-loaded with doxorubicin and an US contrast agent was developed. Stability, imageability, and US monitoring of contrast agent and Dox release suggest that US-guided drug delivery from E-LTSL may assist physicians in real-time tumor drug delivery mapping. In vivo US-guided HIFU in combination with E-LTSL to demonstrate enhanced tumor drug distribution in C26 colon cancer model is currently in progress. This technology has potential for clinical translation.TEM images confirmed that the PFP emulsion formation is contained within LTSL. Phantom study clearly showed that only E-LTSLs are echogenic. Temperature


As a demonstration of the technique, a clearer signal of fluorescent protein-tagged phototropin, a blue-light photoreceptor localized at the chloroplast periphery, was visualized in planta.Chloroplast, an organelle facilitating photosynthesis, exhibits strong autofluorescence, which is an undesired background signal that restricts imaging experiments with exogenous fluorophore in plants. In this study, the autofluorescence was characterized  It is well known that chloroplasts exhibit very strong autofluorescence in red, with a peak of approximately 680 nm . A majorn planta –4. Howevin planta.In this study, autofluorescence of chloroplast is characterized by using confocal laser microscopy, and it is revealed that time-gated fluorescence imaging technique completely eliminates the autofluorescence in fluorescence imaging experiments using exogenous fluorophore Marchantia polymorpha, accession Takaragaike-1 (Tak-1), and transgenic liverworts, Citrine line (FM4) [−2 s−1 continuous white light [Arabidopsis thaliana was grown in soil for 3 weeks at approximately 100 μmol m−2 s−1 under a 16-h/8-h light/dark cycle at 23°C in a growth chamber, and the leaves were detached for observation [Male thalli of the liverwort ne (FM4)  and Mpphne (FM4)  with 1% te light ,7. For ote light . Arabidoervation .Agrobacterium-mediated transformation of M. polymorpha was used [Citrine gene to fuse at the 3′-terminus of the target gene, and it is adapted to the gateway cloning system . The coding sequence  of the MpPHOT gene (accession number: AB938188) was amplified from the cDNA library of M. polymorpha by polymerase chain reaction (PCR) with the following primers: 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGATGCCCTCCACGGAT-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCATATTCATCAAATGAGGCG-3′. The amplified DNA fragments were cloned into a pDONR207 plasmid by BP reaction . The resulting plasmid (pDONR207-MpPHOT) was mixed with pMpGWB106, and the LR reaction  was performed to generate pMpGWB106-MpPHOT, which contains a fusion gene for Mpphot-Citrine.To construct a gene for Citrine-fused Mpphot (Mpphot-Citrine), the binary vector pMpGWB106 for was used ,10. The Agrobacterium-mediated transformation of M. polymorpha was performed by the AgarTrap method using sporelings [M. polymorpha (T1) were cultivated for approximately 1 month, and then gemmae (G1) were obtained. After assessing sex by PCR [orelings . The resx by PCR , a male TM) and a highly flexible pulsed white-light laser  was used, according to the instructions of the manufacture . As commonly used, the Leica Application Suite X (LAS X) was used as a software platform, the objective lens used was the HC PL APO CS 63 × 1.20 WATER, scan speed was 400 Hz (400 lines/s) at a resolution at 512 × 512 pixels, and normal acquisition of the hybrid detector (standard mode of HyDTM) was used. The emission spectra of chloroplast autofluorescence were measured with 470-nm excitation laser from the white-light laser, and xyλ scanning was performed using the hybrid detector at 478–739 nm wavelength region. Excitation spectra of chloroplast autofluorescence were measured with an emission at 680–700 nm, and xyΛ scanning was performed with the white-light laser at 470–626 nm wavelength region. To scan spectra, neither line nor frame averaging was used. To capture the fluorescent images, the hybrid detector was used at the indicated green–yellow wavelength region, and the conventional photomultiplier tube (PMT) was used at the indicated red wavelength region. For imaging at green and yellow wavelength regions, 488-nm laser  and 514-nm laser  were employed, respectively. The laser power after passing through the objective lens was measured by using a power meter 1918-R  with a silicon detector 918D-SL-OD1 (detector active area: 1 cm2) . To capture bright field images, PMT for transmittance was used (Scan-BF mode of PMT trans in LAS X software). For the capture of the images, line and frame averages used were four and two times, respectively.Confocal laser microscopy (Leica TCS SP8X) equipped with a hybrid detector  was reduced by 17% -tagged phototropin (phot), a blue-light photoreceptor localized at the chloroplast outer envelope membrane, was analyzed in y 3.6 ns , and onlue light ,15. Recethaliana . However“Mpphot” . A previ“Mpphot” . In the lymorpha . Confocain planta is difficult when only using selections of emission and excitation wavelengths. In addition, it was revealed that the time-gated fluorescence imaging technique completely eliminates chloroplast autofluorescence. Time-gated fluorescence imaging thereby facilitates clearer fluorescence imaging of Mpphot-Citrine on the chloroplast periphery under a normal physiological condition. In addition to application in plants, the rejection of autofluorescence using time-gated fluorescence imaging may be a powerful technique for fluorescence imaging with fluorescent protein, fluorescent dye or immunofluorescence in various organisms and cell types, which exhibit natural and/or artificial autofluorescence.The present study characterized chloroplast autofluorescence in view of fluorescence imaging study using confocal laser microscopy and suggested that complete elimination of the autofluorescence S1 Fig(a) Effects of time-gated fluorescence imaging technique at the green wavelength region (495–535 nm) using 488-nm laser. Light intensities are shown as relative intensity that was calculated by dividing the mean intensities of gate-off experiments. All experiments were performed thrice (with three different samples); bars represent standard deviations. (b) Representative images of chloroplast autofluorescence time gating at the green wavelength region. G (OFF), chloroplast autofluorescence at the green wavelength region; G (ON), chloroplast autofluorescence time gating (gate-on time: 0.3–12.0 ns) at the green wavelength region; R, chloroplast autofluorescence at the red wavelength region (648–709 nm); BF, bright field. Scale bar, 10 μm.(PDF)Click here for additional data file.S2 Fig(a) Representative images of time gating of chloroplast autofluorescence at the yellow wavelength region (520–561 nm) using a 514-nm laser. (b) Representative images of time gating of chloroplast autofluorescence at the green wavelength region (495–535 nm) using 488-nm laser. Time gating of chloroplast autofluorescence was performed at 0.3–12.0 ns as gate-on time. Chloroplast autofluorescence at the red wavelength region (648–709 nm) is shown as controls. Scale bar, 10 μm.(PDF)Click here for additional data file.S3 Fig(a) Representative images of time-gated reduction of Citrine fluorescence intensity at the yellow wavelength region (520–561 nm) using a 514-nm laser. To clearly determine Citrine fluorescence, rhizoid cells were used, in which any mature chloroplast is not developed. Scale bar, 10 μm. (b) Light intensities are shown as relative intensity that was calculated by dividing the mean intensities of gate-off experiments. All experiments were performed thrice (with three different samples).(PDF)Click here for additional data file.


The long term effects of fingolimod, an oral treatment for relapsing-remitting (RR) multiple sclerosis (MS), on blood circulating B and T cell subtypes in MS patients are not completely understood. This study describes for the first time the longitudinal effects of fingolimod treatment on B and T cell subtypes. Furthermore, expression of surface molecules involved in antigen presentation and costimulation during fingolimod treatment are assessed in MS patients in a 12 month follow-up study.Using flow cytometry, B and T cell subtypes, and their expression of antigen presentation, costimulation and migration markers were measured during a 12 month follow-up in the peripheral blood of MS patients. Data of fingolimod-treated MS patients (n = 49) were compared to those from treatment-naive (n = 47) and interferon-treated (n = 27) MS patients.In the B cell population, we observed a decrease in the proportion of non class-switched and class-switched memory B cells (p<0.001), both implicated in MS pathogenesis, while the proportion of naive B cells was increased during fingolimod treatment in the peripheral blood (PB) of MS patients (p<0.05). The remaining T cell population, in contrast, showed elevated proportions of memory conventional and regulatory T cells (p<0.01) and declined proportions of naive conventional and regulatory cells (p<0.05). These naive T cell subtypes are main drivers of MS pathogenesis. B cell expression of CD80 and CD86 and programmed death (PD) -1 expression on circulating follicular helper T cells was increased during fingolimod follow-up (p<0.05) pointing to a potentially compensatory mechanism of the remaining circulating lymphocyte subtypes that could provide additional help during normal immune responses.MS patients treated with fingolimod showed a change in PB lymphocyte subtype proportions and expression of functional molecules on T and B cells, suggesting an association with the therapeutic efficacy of fingolimod. The role of other peripheral blood (PB) immune cells in MS pathogenesis, such as naive B cells (CD19+IgD+CD27-), double negative B cells (CD19+IgD-CD27-) and follicular helper T cells , is still unclear. B and T cells interact via surface molecules e.g. human leukocyte antigen (HLA)-DR/DP/DQ, CD80 and CD86 on B cells and programmed death (PD) -1 on T cells. Furthermore, migration of B and T cells is partly mediated via chemokine (C-X-C motif) receptor 5 (CXCR5) A complex interplay between T and B cells drives the disease course of multiple sclerosis (MS). Thereby, non class-switched  MS Limited information is available concerning the effects of fingolimod on different T and B cell subtypes and on the interplay between these lymphocyte populations in the PB of MS patients PB was collected from MS patients in both the Orbis Medical Center  and Rehabilitation and MS-center . For PB collection in the Orbis Medical Center, written informed consent was obtained from all participants after approval by the Medical ethical Committee Atrium-Orbis-Zuyd (12-N-56). Furthermore, PB was collected by the Rehabilitation and MS-center in Overpelt after written informed consent from all participants and approval by the UZ Leuven and Hasselt University Commissions of Medical Ethics (S54362 and S54363). A total of 123 MS patients were involved in the study, including 47 treatment-naive MS patients, 27 MS patients on interferon-β (IFN-β) treatment (together referred to as controls) and 49 MS patients on fingolimod treatment (0.5 mg/day). All MS patients were diagnosed according to the revised McDonald criteria Lympholyte; Cedarlane Laboratories, SanBio B.V., Uden, the Netherlands). PBMC (0.5×106 cells) were stained using anti-human CD19 PerCP-Cy5.5 and CD4 APC to discriminate between B and T cells, respectively . B cell subpopulations and surface molecules were defined using following anti-human antibodies: IgD APC-Cy7, CD27 PE-Cy7, HLA-DR/DP/DQ (major histocompatibility complex (MHC)-II) FITC, CD80 PE and CD86 PE-CF594 . Following anti-human monoclonal antibodies were used for T cell analysis: CD45RA APC-H7, CD45RO PE-CF594, CXCR5 Alexa Fluor 488 and PD-1 PE-Cy7 , CD25 PerCP-Cy5.5 and CD127 PE . Following isotype controls were used: mouse IgG1 PerCP-Cy5.5, IgG1 PE, IgG1 Pe-Cy7, IgG2aκ PE-CF594, IgG2bκ APC-H7, IgG1 APC, IgG2aκ FITC, IgG1κ PE-CF594, IgG1 Pe-Cy7, IgG2aκ APC-H7 and rat IgG2b Alexa Fluor 488 . All flow cytometric analyses were performed on a FACSAriaII flow cytometer and analyzed with FACS Diva software (BD Biosciences).PB was collected in heparin-coated tubes  and PB mononuclear cells (PBMC) were isolated using high density centrifugation  and SAS 9.3 software. Appropriate One-way ANOVA analysis was used with Dunn's multiple comparison post-hoc test for comparison of treatment controls and baseline fingolimod-treated patients after normality check (Kolmogorov-Smirnov). A mixed model was used for data analysis of treatment follow-up compared to baseline fingolimod. A p-value of <0.05 was considered statistically significant.In total, PB of 49 fingolimod-treated MS patients was collected at different time points up to 12 months of treatment. The cohort of fingolimod-treated MS patients was compared at baseline with 47 treatment-naive and 27 IFN-β treated MS patients (together referred to as controls). Fingolimod-treated MS patients at baseline and controls were comparable in terms of age, gender distribution and median EDSS score . Further+ B cell and CD4+ T cell numbers were decreased at 1 m and reached a steady state at 3 m  of fingolimod treatment compared with baseline and controls for the total duration of the study (12 m) p<0.001; . Furtherp<0.001; . Similarp<0.001; .During immune responses, B cells produce antibodies after maturation into plasma cells, function as antigen presenting cells, provide costimulation for T cells and play a role in immune memory. In MS, memory B cells and plasma cells may contribute to the pathogenesis by production of autoantibodies and cytokines +IgD+CD27+) and class-switched memory B cells (CD19+IgD-CD27+) were significantly decreased in the peripheral B cell population from 3 m until end of follow-up  was significantly increased within the B cell population at 1 m up to 12 m  made up about 50% of the remaining peripheral B cells and the proportion of these cells was increased after 3 m fingolimod until end of follow-up  and are important to provide costimulation to T cells via the surface molecules CD80 and CD86 + B cells was assessed using flow cytometric analysis. The percentage of HLA-DR/DP/DQ+ B cells (data not shown) and the expression of HLA-DR/DP/DQ (MFI) on B cells was significantly decreased after 3 m and 1 m of fingolimod treatment, respectively, in comparison with baseline  are considered to be main players in maintaining a normal immune response and exert autoreactivity in autoimmune diseases like MS +CD25hiCD127lo) are essential for immune homeostasis and were shown to be functionally impaired in MS pathogenesis Conventional T cells , memory (CD45RA-CD45RO+) and transitional (CD45RA+CD45RO+) cells within both Tconv and Treg populations were assessed using flow cytometry. For the Tconv population, the proportion of naive cells was decreased in fingolimod-treated patients at all timepoints measured, when compared with baseline , both in the regulatory and conventional T cell population, remained stable throughout the 12 month follow-up period. Of note, baseline levels of the fingolimod-treated groups differed significantly compared with treatment-naive patients for Tconv and Treg  have the capacity to recirculate in secondary lymphoid organs where they can interact with B cells and influence the germinal center response Circulating CXCR5FH (CD4+CD25-CD127+CXCR5+PD-1+) remained stable within the CD4+ population during fingolimod treatment  while the naive B cell proportion increases + B cells was decreased in the PB during fingolimod treatment, which could be beneficial for MS pathogenesis since less antigen presentation occurs. However, this effect could also be attributed to a change in B cell subtype proportions in the PB.B cells are important antigen presenting cells and recent evidence from mice studies has indicated that fingolimod can influence antigen handling Next to changes in B cells proportions, fingolimod treatment led to a decrease in the proportion of peripheral naive Tconv and an increase in the memory Tconv. Our results are in agreement with previous studies showing that effector memory T cells (TEM), lacking expression of C-C chemokine receptor type 7 (CCR7), were increased in the PB of fingolimod-treated MS patients Furthermore, the homeostasis and function of Treg is disturbed in MS FH are important for a normal germinal center response FH cells. Recent evidence indicated that circulating TFH cells consist of a CCR7loPD-1hi subpopulation with an effector phenotype. Therefore these cells could be less responsive to fingolimod as observed for memory Treg and Tconv cells As circulating TFH cells was observed. The percentage of CD86+ B cells was increased as well during fingolimod treatment, which could be attributed to a change in B cell subtype distribution. Expression of CXCR5 on TFH cells was unchanged during treatment, indicative of normal migration of these cells from the marginal zone to the follicles in the lymph nodes. Considering the beneficial clinical effects of fingolimod, we hypothesize that this increase in B and T cell costimulation and no change in migration capacity is a rescue mechanism to augment functionality of the remaining B and T cells, thereby warranting normal immunity. An additional proof of normal immune function is that vaccine specific production of IgM and IgG towards influenza A and B in fingolimod-treated individuals was not impaired when compared to levels in healthy controls Although fingolimod causes entrapment of lymphocytes in the lymph nodes, expression of surface molecules involved in costimulation was increased, which could point to a gain of functionality of the remaining circulating lymphocyte subtypes, although functional assessment is needed using both in vitro and animal studies to confirm this argument. During fingolimod treatment expression of CD86 and CD80 costimulatory molecules on B cells was increased and furthermore, an increase in PD-1 expression on T+ and natural killer (NK) cells were not assessed. Further limitations of the study are the lack of functional data, although we provide evidence that during fingolimod treatment expression of functionally relevant markers on the remaining B and T cell subtypes in the peripheral blood of MS patients can change.Due to technical limitations and low cell numbers available for analysis, CD8To conclude, this study shows that fingolimod induces compositional changes of B and T cell subtypes that are potentially implicated in MS pathogenesis and may explain the therapeutic efficacy of the treatment. While altered surface expression of functional molecules on B and T cells during fingolimod treatment suggests that normal immune function may prevail, functional evidence for this has still to be provided during future research. With this descriptive study we provide additional longitudinal immunological proof for the diverse mechanisms of action of fingolimod in MS patients.Figure S1Proportional changes of B cell subtypes during fingolimod treatment in MS patients. Proportional composition of (A) naive B cells, (B) non class-switched memory B cells, (C) class-switched memory B cells and (D) double negative B cells within the CD19+ B cell population of treatment-naive, IFN-β and fingolimod-treated MS patients.(TIF)Click here for additional data file.Figure S2Compositional changes of T cell subtypes during fingolimod treatment in MS patients. Proportional changes of (A) naive conventional T cells, (B) memory conventional T cells, (C) transitional conventional T cells, (D) naive regulatory T cells, (E) memory regulatory T cells and (F) transitional regulatory T cells. Changes are depicted as percentage within the CD4+ T cell population and measured in treatment-naive, IFN- β and fingolimod-treated MS patients.(TIF)Click here for additional data file.Table S1Mean percentages of different B and T cell subtypes.(DOCX)Click here for additional data file.Table S2Mean fluorescence intensity and percentage positive cells of different surface markers on B and T cells.(DOCX)Click here for additional data file.


MALT1 gene, involved in insulin and glycaemic pathways, and related to taurocholate levels in blood. Integrating the DNA methylome findings with T2D GWAS meta-analysis results reveals a strong enrichment for DMRs in T2D-susceptibility loci. We also detect signals specific to T2D-discordant twins in the GPR61 and PRKCB genes. These replicated T2D associations reflect both likely causal and consequential pathways of the disease. The analysis indicates how an integrated genomics and epigenomics approach, utilizing an MZ twin design, can provide pathogenic insights as well as potential drug targets and biomarkers for T2D and other complex traits.DNA methylation has a great potential for understanding the aetiology of common complex traits such as Type 2 diabetes (T2D). Here we perform genome-wide methylated DNA immunoprecipitation sequencing (MeDIP-seq) in whole-blood-derived DNA from 27 monozygotic twin pairs and follow up results with replication and integrated omics analyses. We identify predominately hypermethylated T2D-related differentially methylated regions (DMRs) and replicate the top signals in 42 unrelated T2D cases and 221 controls. The strongest signal is in the promoter of the  Type 2 diabetes (T2D) is a highly heterogeneous disease with a strong genetic component. Here the authors examine genome-wide methylation patterns in T2D-discordant, T2D-concordant and healthy concordant monozygotic twin pairs, and identify DNA methylation signals that may represent new biomarkers or drug targets for T2D. Type 2 diabetes (T2D) is a highly heterogeneous disease caused via a combination of genetic susceptibility and environmental exposures. Recent genome-wide association studies (GWAS) have identified at least 65 T2D loci, which explain only ~6% of disease susceptibility variance236In this study, we use high-throughput methylation immunoprecipitation sequencing  to obtain whole-blood DNA methylation profiles. MeDIP-seq enables a cost-effective genome-wide DNA methylome characterization and provides access into moderately dense CpG regions of the genome, such as CpG island ‘shores’ refs , 10 and P<0.05). We also assess the overlap between the suggestive T2D-DMRs with the most variable DNA methylome regions  detected by whole-genome bisulfite sequencing from 42 data sets across 30 human cell and tissue types defined by Ziller et al.P=0.003).MeDIP-seq data were generated at ~30 million paired-end reads of length 50 bp per individual, and mapped to the human genome using NovoalignALPPL2 (2q37.1 region), AHRR and F2RL3 within our data set. We selected previously identified smoking-associated 450k CpG sites in these genes, and extracted MeDIP-seq bins that overlap the smoking-associated CpG site. We find significant associations between smoking status and methylation levels at CpG sites in the ALPPL2 and AHRR genes (Bonferroni corrected P<0.05) and borderline uncorrected significance for the F2RL3 gene. These findings are reassuringly consistent with recent discoveries, despite the small numbers of smokers in our sample (11 out of 54) and the use of different technologies.To further validate our MeDIP-seq methylome analysis, we also assess the highly-replicated DNA methylation association with smoking status in the genes P<0.05), and a further 4 DMRs show the same direction of effect, but do not reach nominal significance.To validate our MeDIP-seq results with another platform, we targeted the top 24 DMRs from the more stringent FDR 10% set and 7 other suggestive T2D-DMRs in the same set of samples using the Illumina HumanMethylation450K Bead Chip . Of thesP<0.05).We then attempted replication of all 4,545 suggestive T2D-DMRs in an independent sample of 42 unrelated T2D cases and 221 UK controls, matched for age, body mass index (BMI) and sex with single-end MeDIP-sequencing profiles. Of the 4,545 DMRs, 3,939 (87%) show the same direction of association and 1,355 (30%) replicate with the same direction of association and with nominal significance  overlaps another T2D-DMR  or expression in adipose, lymphoblastoid cell lines , skin tissue  methylation patterns with 503 fasting blood metabolites profiled by mass spectrometryMALT1 methylation in both the discovery and replication samples . Second, we compare the 65 genes closest to the published GWAS T2D loci with our 3,597 T2D-DMR genes, and find highly significant overlap of genes (Fisher’s test P=0.004). Third, the ranking of DMR p values within the GWAS T2D loci in the genome is significantly higher than that for sets of randomly sampled Ensembl genes that had MeDIP-seq coverage  and THADA (thyroid adenoma-associated gene). The DNA methylation levels at these 12 T2D GWAS loci clustered the individuals in our study into two clear case–control groups , and furthermore show a twofold enrichment with 83 confirmed imprinted genes (Fisher’s test P=0.1). This includes genes previously linked to diabetes and obesity, for example PRDM16  with well-established imprinted loci from The Genomic Imprinting Website24% with we252627M16 refs , 30. We cis mQTL, we tested common SNPs within 50 kb of the T2D-DMRs and found an enrichment for genetic effects, suggesting that a number of the disease DMRs have a genetic contribution. Altogether, 397 out of the 4,545 T2D-DMRs possess a cis mQTL . For example, SNP rs10495903 associates with a DMR 8 kb from the 5′ end of THADA  and SNP rs2086675 associates with an intragenic ANKRD55 (Ankyrin Repeat Domain 55) DMR . We further assessed genetic trans associations for 757 replicated T2D-DMRs (replication linear regression P<0.01) in the replication samples. Of these 757 T2D-DMRs, 29 (3.8%) have trans mQTL , but none show significant evidence that methylation is the mediator of the genetic effect on disease using the causal inference testTo further investigate the genetic contribution to the T2D-DMRs, we attempted to identify methylation quantitative trait loci (mQTL), and thus associate methylation levels with genotype variation. For t-test P=3 × 10−7, FDR=10%) and 914  T2D giDMRs within the 17 discordant MZ twin pairs, which overlap 890 genes in total , and a further eight show the same direction of change. We also observe that three of the top four DMRs showed the same trend of association in skeletal muscle in another unrelated cohort of 30 cases and controls profiled with the Illumina 450k array. Altogether, 175 giDMRs (which mapped to 109 genes) overlap the 4,545 FDR 25% T2D-DMRs from the linear mixed model analyses. The MALT1 DMRs do not pass the FDR 25% giDMR threshold, but show modest giDMR effects consistent with the direction of T2D-DMR association in the primary linear model, suggesting that they were primarily independent of genetic influences . The top giDMR, which is also identified in the primary linear model analyses (discovery P=3.78 × 10−6 and replication P=0.01), is in the promoter of the GPR61 (G-protein coupled receptor 61) gene. Knockout studies of GPR61 in mice exhibit hyperphagia-induced obesity and higher plasma insulin levelsPRKCB  gene region, and another top giDMR is in the PRKCB TSS, and both are hypo-methylated in T2D cases. Increasing PRKCB levels caused by hyperglycaemia, are involved in insulin resistanceUsing our monozygotic discordant twin pair model, we are also able to search for epigenetic differences within genetically identical twins, and we name these regions ‘genetically independent DMRs (giDMRs). Unlike T2D-DMRs identified from the linear model, which reflect both genetic and environmental effects on T2D, giDMRs likely represent pure environmental effects, stochastic mechanisms or consequences of the disease itself. We find 2 . This suggests that differential blood cell types do not significantly confound the major T2D-methylation changes observed in our twin analysis.White blood cell (WBC) subtype proportions have been previously reported to associate with T2D38P=0.02), suggesting that medication is not a major confounder.To explore possible effects of T2D medication on our findings, we first tested for association between methylation level and medication at time of blood sampling. We find that 2 (0.04%) of 4,545 T2D-DMR are associated with medication (FDR<0.05). We then divided cases into those on and off medication at the time of blood sampling as a covariate. After adjusting for medication, both of these T2D-DMRs are still nominally associated with T2D status  and/or self-report of T2D in the questionnaire data. The healthy twin in discordant twins was required to have a serum fasting glucose level test of ≤5 mmol l−1. The replication sample set was also selected from the TwinsUK cohort and comprised 42 unrelated T2D cases and 221 controls matched for age, BMI and sex.All participants in the study provided written informed consent in accordance with the St Thomas’ Hospital local ethics research committee. Altogether, 27 pairs of MZ twins , for four cell types in our sample set: neutrophils, eosinophils, monocytes and lymphocytes.E-MTAB-3051.All sample preparations and MeDIP sequencing were performed by the BGI-Shenzhen, Shenzhen, China. Extracted DNA was fragmented using a Covaris sonication system and sequencing libraries were prepared from 5-μg fragmented genomic DNA. End repair, <A > base addition and adaptor ligation steps were performed using Illumina’s Paired-End DNA Sample Prep kit. Adaptor-ligated DNA was immunoprecipitated by anti-5mC using a commercial antibody (Diagenode) and MeDIP products were validated by quantitative PCR. MeDIP DNA was purified with ZYMO DNA Clean & Concentrator-5 columns and amplified using adaptor-mediated PCR. DNA fragments between 200 and 500 bp in size were gel-excised, and the amplification quality and quantity were evaluated by Agilent BioAnalyzer analysis. The libraries were subjected to highly parallel 50-bp paired-end sequencing on the Illumina GAII platform. All sequencing data passed initial quality checks for base composition (no exclusions) using FASTQC  of 25% , estimated by Benjamini–Hochberg (95) q valueDMRs and giDMRs were calculated using the MEDIPS RPM values. For T2D DMR estimation, the RPM values at each 500 bp bin were normalized to N  before the analysis. Using a linear mixed effects model, we regressed methylation levels at each 500-bp window on fixed-effect terms, which included disease status (at the same time as the visit for the blood and DNA draw), BMI, age (for the blood and DNA withdraw), sex and random-effect terms denoting family structure. giDMRs were characterized among 34 (17 pairs) discordant monozygotic twins and To investigate the potential effect of medication use and cell subtype bias, we used records of any diabetes medication use and cell subtype counts data in the discovery samples. At each of the 4,545 suggestive T2D-DMRs we tested the effect of medication record and cell subtype data of DNA methylation levels. We fit a linear mixed effects model—BMI, age, sex, medication use (or cell subtype counts) were incorporated as fixed-effects and we included a random-effect term denoting family structure.β, which represent the ratio of array intensity signal obtained from the methylated beads over the sum of methylated and unmethylated bead signals44Illumina Human Methylation 450K array data were used to validate the top T2D DMR results. The Illumina 450K array methylation values are reported as In the validation, we matched 20 probes from whole-blood Illumina 450K array for 45 individuals to the top 31 DMRs . When a We also tested the selected DMRs methylation profile in skeletal muscle biopsies from 24 T2D patients and 6 controls .P<0.05) were then tested in the replication sample set. The meta-analysis of discovery (n=36 IDs) and replication (n=24 IDs) samples provided the overall p value for the association between metabolite levels and DNA methylation profiles.Among the 54 MZ twin individuals, 36 had plasma and/or serum metabolites profiles. Altogether, 503 metabolites were measured using non-targeted technology gas chromatography–mass spectrometry and liquid chromatography–mass spectrometryGene expression results in adipose, skin and lymphoblastoid cell line tissue was extracted for 590 subjects from MuTHER studyGenotype data for the individuals in this study were obtained on a combination of Illumina platforms . The genotypes were called with the Illuminus calling algorithm (maximum posterior probability of 0.95). Imputation was performed using the IMPUTE software package (v2) using two reference panels: P0  and P1 . After imputation, SNPs were filtered for MAF of >5% and IMPUTE info value of >0.8 ref. .Cis methylation QTL at DMRs was analysed using SNPs within 50 kb of the region. For each DMR, the methylation values were normalized to N , and we then fitted a linear model, regressing the methylation levels on fixed-effect terms including genotype, age and gender. Multiple testing was corrected for by the Bonferroni correction. We further tested whether methylation acts as a mediator between genotype and phenotype. This was assessed using the causal inference test8Trans methylation QTLs at DMRs were analysed in the replication sample using 2.1M SNPs in the genome . For each DMR, the methylation values were normalized to N , and we performed association analyses by using linear regression implemented in PLINKPathway analysis was performed using two methods: Cytoscape v2.83 ref.  and GREAT.D.S., S.L.J., J.T.B. and J.W. conceived the study. W.Y. and J.T.B. led the analyses. K.S., I.E. and T.F. contributed to a part of the analyses. K.W., Y.X., F.G., H.W., Y.X., H.L., C.L.H. and L.Y. preformed sample selection, sequencing and contributed experimental or technical support. A.K.L., M.J.B., A.M., P.D., S.L.J. and M.M. contributed materials, reagents, technical support and discussion. W.Y., C.G.B. and J.T.B. prepared the manuscript, with contributions from K.W., K.S., A.K.L., M.J.B., S.L.J., M.I.M. and T.D.S. All authors read and approved the manuscript. J.T.B., J.W. and T.D.S. jointly supervised this work.How to cite this article: Yuan, W. et al. An integrated epigenomic analysis for type 2 diabetes susceptibility loci in monozygotic twins. Nat. Commun. 5:5719 doi: 10.1038/ncomms6719 (2014).Accession codes: MeDIP-seq data have been deposited in EMBL under the accession code E-MTAB-3051.Supplementary Figure 1Demography of studying twinsList of DMR which mapped to genes31 DMR genes validationMetabolites associate with MALT1 methylation DMRs in T2D GWAS lociT2D DMRs in other diseases genesDMR in known imprinted genesgiDMR which mapped to genesgiDMR DNA methylation difference in blood case-controls replication samplesPathway analysis for giDMR gene


The microRNA miR-101 is downregulated in several cancers, including bladder cancer. However, miR-101’s role in the invasion, metastasis, and chemosensitivity of bladder cancer cells remains unclear. This study was conducted to determine miR-101’s role on the lymphangiogenic molecule vascular endothelial growth factor C (VEGF-C) and their effects upon bladder cancer cell migration, invasion, and chemosensitivity to cisplatin.Two bladder cancer cell lines (T24 and 5637) and the tool cell line 293T were employed here. Bladder cancer cells were transfected with either a miR-101 overexpression vector or a scrambled-sequence lentivirus, both of which exhibited a high transfection efficiency. Non-transfection was used as a mock negative control. Wound healing and Transwell assays were performed to measure cell migration and invasiveness. A luciferase reporter assay was performed to validate miR-101’s interaction with VEGF-C’s 3′ untranslated region followed by RT-PCR and Western blot confirmation. An MTS assay was used to evaluate the cisplatin sensitivity of the cell lines.miR-101 overexpression significantly inhibited the migration and invasiveness while significantly enhancing cisplatin sensitivity. miR-101 negatively regulated VEGF-C protein expression, and VEGF-C overexpression rescued the effects of miR-101 overexpression, indicating that miR-101 negatively regulates VEGF-C protein expression post-transcriptionally. miR-101 and VEGF-C interference independently enhanced cisplatin cytotoxicity in bladder cancer cells.miR-101 suppresses VEGF-C expression, inhibits cell migration and invasion, and increases cisplatin sensitivity in bladder cancer cells. This study provides new insight into miR-101’s role in bladder cancer and shows miR-101’s promise as a potential molecular target for bladder cancer. Bladder cancer is the most common urinary tract malignancy, producing approximately 150,000 annual deaths worldwide , 3. DespMicroRNAs (miRNA) are phylogenetically-conserved small non-coding RNAs that negatively regulate targeted mRNA 3′ untranslated regions (3′UTR) in several cancers and have been increasingly identified as tumor suppressors or carcinogenic agents –13. MoreVEGF-C, a member of vascular endothelial growth factor (VEGF) family, is regarded as an important lymphangiogenic molecule and is known to increase the permeability of lymphatic vessels –27. In c2.The bladder cancer cell lines T24 and 5637 as well as the human embryonic kidney tool cell line 293T used in this study were purchased from the Type Culture Collection of the Chinese Academy of Sciences  and were maintained in RPMI-1640  and Dulbecco’s Modified Eagle Medium , respectively, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. All cell lines were incubated at 37- in humidified 5% COA fragment of miR-101 was generated by using the following primers: sense, 5′-CGGGTACCGGTAGTCCTTCACTTCATGGGGAG-3′ and antisense, 5′-CGGAATTCAAA- AAACCCAGCCACCTGTTTCAC-3′ and inserted into the GV209 vector with a green fluorescent protein (GFP) reporter gene within the AgeⅠand EcoRⅠrestriction sites. The aforementioned miR-101 plasmid, pHelper1.0, and pHelper2.0 were co-transfected into 293T cells, and the lentiviral particle-enriched supernatant was obtained 48 h later. A scrambled sequence (5′-TTCTCCGAACGTGTCACGT-3′), which had no homology with the human gene, was used as a scrambled negative control. The VEGF-C cDNA was amplified using the following primers: sense, 5′-TCCGCTCGAGATGCACTTGCTGG- GCTTCTTC-3′ and antisense, 5′-ATGGGGTACCGTGCTCATTTGTGGTCTTTTCC-3′ and cloned into the GV147 vector with a red fluorescent protein (RFP) reporter gene containing the restriction enzyme sites XhoⅠand Kpn I. Non-transfection was used as a mock negative control . All seq260/OD280 ratio greater than 1.8 used for cDNA synthesis. The reverse transcription of miR-101 and its specific amplification were performed using a miRNA qRT-PCR Detection Kit . U6 was used as an endogenous control. cDNA synthesis of VEGF-C was performed using a First-Strand cDNA Synthesis Kit , and the real-time quantitative PCR (RT-qPCR) reaction of VEGF-C was performed using qPCR Mix . GAPDH was used for VEGF-C template normalization. All primers were purchased from GeneCopeia. miR-101 and VEGF-C amplification were determined on a ROTOR-GENE 3000 platform. The target PCR threshold cycle (Ct) values were normalized by subtracting the U6 or GADPH Ct value, which provided the ΔCt value. Relative expression was calculated using the following equation: relative gene expression level = 2 -.Total RNA was isolated using Trizol reagent  according to the manufacturer’s instructions. Isolated RNA was measured using a Nanodrop 1000 spectrophotometer  with an ODRIPA lysis buffer containing 1 mM PMSF was added to the cells in six-well plates and then centrifuged for 10 min at 4-. The total protein content of the supernatant was determined by the Lowry method. The protein sample was diluted, heated for denaturation, and then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane , 5% skimmed milk powder was applied for 2 h at 37-. Then, 1:1000 dilutions of rabbit polyclonal primary antibody anti-VEGF-C  or anti-β-actin  was incubated with the membrane overnight. The next day, the membrane was rinsed in TBST three times and then incubated with a fluorescent secondary anti-rabbit antibody for 2 h. The membrane was scanned by an infrared imaging system (Odyssey), and the band intensity was detected by Odyssey 3.0 analysis software.TargetScan analysis was used to discover a seven-nucleotide complementary sequence between miR-101 and the VEGF-C 3′UTR. A wild type (wt) VEGF-C 3′UTR sequence including Xba1 restriction sites (5′-TCTAGAACGAACCGCCAGAAGGCTTGTGAGCCAGGATTTTCATATAGTGAAG- AAGTGTGTCGTTGTGTCCCTTCATATTGGAAAAGACCACAAATGAGCTAAGATTGTACTGTTTTCCAGTTCATCGATTTTCTATTATGGAAAACTGTGTTGCCACAGTAGAACTGTCTGTGAACAGAGAGACCCTTGTGGGTCCATGCTAACAAAGACAAATCTAGA-3′) in addition to a mutant type (mut) VEGF-C 3′UTR sequence with a completely different miR-101-binding sequence, were synthesized and each independently cloned into separate GV272 luciferase reporter vectors . The previously described miR-101-overexpressing or scrambled-sequence plasmid along with either a wt or mut VEGF-C 3′UTR plasmid were co-transfected into 293T cells using Lipofectamine 2000 reagent , creating four experimental groups. Then, 293T cell luciferase assays were performed to determine the interaction between miR-101 and the wt and mut VEGF-C 3′-UTR. Renilla and firefly luciferase activities were measured at 48 h post-transfection using the Dual-Glo Luciferase Reporter Assay System  according to the manufacturer’s protocols. The ratio of renilla-to-firefly luciferase value was recorded . Experim8 transducing units/ml) and polybrene (5 ug/ml). After 48 h, a trypsinized single cell suspension was prepared and seeded in six-well dishes (5×105/well) until the cells reached 80% confluence. Then, 1-ml sterile pipette tip was used to scrape a cross in the center (similar to a wound), rinsed with PBS three times, and the serum-free medium was replaced immediately. Cells were allowed to migrate for 24 h, and the scratches were carefully observed and photographed. Light microscopic and fluorescent images were captured with an Olympus IX71 microscope. The gap lengths were also calculated from the photomicrographs. Each cell line was measured in triplicate.The target cells, including T24 and 5637 cells, were transfected with recombinant lentivirus  with 8-μm polyester membrane filter pores, was used for both the invasion assay and migration assay. The difference between the invasion assay and the migration assay was that the invasion assay used coated Matrigel  in the upper chamber, which simulated the characteristics of the extracellular matrix, while the migration assay did not. A total of 2×103 cells/well (0.2 ml/well) for 24 h before use. The culture medium was replaced with fresh medium containing cisplatin with different concentrations for 72 h. Next, MTS (20 μl/well) was added to each well, and the absorbance at 490 nm of each well was recorded with a universal microplate reader  after 3 h. Dose-inhibition rate curves were plotted according to absorbance values. The IC50 value  was also calculated. The assay was repeated in triplicate.The cell suspension was seeded in 96-well culture plates at a density of 4×10t-test or one-way ANOVA. P<0.05 was deemed to be statistically significant.Results were expressed as means ± SDs. All data were analyzed using SPSS software version 21.0. Experiments were statistically analyzed by Student’s Due to previous findings of lower miR-101 expression in bladder cancer ,31, we cTo evaluate the molecular mechanism(s) of miR-101, TargetScan analysis found that VEGF-C is a potential target gene for miR-101, as there is a seven-nucleotide complementary sequence between miR-101 and the VEGF-C 3′UTR . Thus, aTo verify the role of VEGF-C in the bladder cancer cell lines, we performed rescue experimentation .. After miR-101 overexpression increased the sensitivity of both T24 and 5637 cells to cisplatin, with the IC50 value increasing from 4.06 ± 0.69 mg/l to 9.17 ± 1.24 mg/l and from 2.96 ± 0.46 mg/l to 5.56 ± 0.36 mg/l, respectively . In ordeAfter T24 cells were transfected with the miR-101 lentivirus for 48 h, they were then transiently transfected with either a VEGF-C overexpression plasmid, a VEGF-C control plasmid, or a VEGF-C shRNA plasmid. We found that VEGF-C overexpression partially recovered bladder cancer cell sensitivity to cisplatin compared the VEGF-C control with IC50 values of10.05 ± 1.05 mg/l and 3.14 ± 1.02 mg/l to 0.63 ± 0.16 mg/l, respectively . MoreoveEmerging evidence shows that miR-101 expression is lost in bladder cancer tissues and bladder cancer cell lines ,31. The The production and secretion of VEGFs—potent stimulators of angiogenesis that affect endothelial cell proliferation and motility as well as vascular permeability—has been commonly observed in several aggressive tumor types and significantly influences the prognosis of cancer patients , Althougin vitro in the absence of lymphangiogenesis. These findings suggest that VEGF-C acts through non-lymphangiogenic mechanism(s) in promoting bladder tumor progression. Further research is required to determine the downstream affector(s) of the VEGF-C-Flt4/VEGFR-3 axis in bladder cancer cells that promote bladder cancer cell mobility.With respect to VEGF-C’s role in malignancy, VEGF-C mRNA expression was initially detected in a mixed set of solid human tumors with VEGF-C’s role in lymphangiogenesis providing a possible mechanism for metastasis via newly-formed lymphatic vessels . Later sCisplatin chemotherapy remains the first-line treatment for locally advanced bladder cancer, so cisplatin resistance is a major clinical issue for bladder cancer patients . AlthougThere are two limitations to this study. First, this study shows that VEGF-C plays a key role in bladder cancer cell migration, invasion, and cisplatin chemosensitivity and is a direct target of miR-101. However, this fact does not preclude the possibility that miR-101 has other targets that have similar cellular functions to VEGF-C. Using the TargetScan database, we found 803 transcripts that are predicted to be targets of hsa-miR-101, including EZH2, BTBD3, NLK, and MITF. Further study is needed to determine whether these 803 putative targets are actual targets of miR-101 and whether any such protein targets are associated with bladder cancer cell migration, invasion, and cisplatin chemosensitivity. Second, previous work has shown that miR-101 is downregulated in TCC and that miR-101 inhibits TCC cell proliferation and colony formation through directly repressing the histone methyltransferase EZH2 . TherefoIn summary, we demonstrate miR-101 post-transcriptionally suppresses VEGF-C expression, inhibits bladder cancer cell migration and invasion, and increases cisplatin sensitivity. Although further research is needed to identify other relevant targets of miR-101, this study provides new insight into miR-101’s role in bladder cancer and shows miR-101’s promise as a potential molecular target for bladder cancer.S1 Data(DOC)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.