Saccharomyces cerevisiae, the mRNA export receptor Mex67 is recruited to mature nuclear transcripts to mediate mRNA export through the nuclear pore complex (NPC) to the cytoplasm. Mex67 binds transcripts through adaptor proteins such as the poly(A) binding protein Nab2. When a transcript reaches the cytoplasmic face of the NPC, the DEAD-box protein Dbp5 acts to induce a local structural change to release Nab2 and Mex67 in an essential process termed mRNP remodeling. It is unknown how certain proteins  are released during Dbp5-mediated mRNP remodeling, whereas others remain associated. Here, we demonstrate that Dbp5 associates in close proximity with Mex67 and Nab2 in a cellular complex. Further, fusion of Dbp5 to Nup159 anchors Dbp5 at the cytoplasmic face of the NPC and is sufficient for cell viability. Thus, we speculate that the essential role of Dbp5 in remodeling exporting mRNPs requires its localization to the NPC and is separable from other subcellular functions of Dbp5. This work supports a model where the diverse nuclear, cytoplasmic and NPC functions of Dbp5 in the mRNA lifecycle are not interdependent and that Dbp5 is locally recruited through complex protein-protein interactions to select regions of transcripts for specific removal of transport proteins at the NPC.In  An mRNA’s progress through the gene expression pathway is dictated by its subcellular localization and collection of proteins bound to it. At each stage in the mRNA lifecycle, members of the DEAD-box protein (Dbp) family displace specific proteins from the mRNA-protein complex (mRNP) by generating kinks in the local RNA structure, a process termed remodeling. Dbp5 is a uniquely multifunctional regulator of mRNPs, with roles in transcription, export from the nucleus, and translation. The most well understood of these functions is during mRNA export, where Dbp5 releases the transport proteins that mediate export through the nuclear pore complex (NPC). It was not known whether this function at the NPC is linked to Dbp5 actions in the nucleus, or how Dbp5 targets a specific protein for removal. Based on the results of a combination of genetic and cellular experiments, we speculate that Dbp5 remodels mRNPs at the NPC independently of its nuclear function, indicating separability of these functions. Furthermore, our data support a model wherein Dbp5 is locally recruited at the NPC through protein-protein interactions to select regions of transcripts for specific removal of transport proteins. These studies further elucidate the function of Dbp5, a regulator of mRNA fate during mRNA export and an important transition in the mRNA lifecycle. The nuclear envelope (NE) barrier of a eukaryotic cell separates the two major phases of messenger (m)RNA biogenesis and translation in the gene expression pathway, ensuring that only properly processed messages reach the cytoplasm for translation. In the nucleus, mRNA is generated and transformed into an export-competent macromolecular mRNA-protein particle (mRNP) through a series of carefully surveilled mRNA processing and protein packaging steps . Ea. Eamex67 mutants . Whereasmulation . In fact to examine the steady state co-localization of such complexes. Using the split Venus approach, two putative binding partners (or closely associating proteins in a complex) are fused to either the N-terminal (VN) or C-terminal domains (VC) of the Venus fluorophore. If the tagged proteins associate in close proximity, the Venus fluorophore is able to properly fold, and after a delay needed for chromophore maturation, fluorescence can be detected , th, th12], revisiae  and 55ms tentans . Convers tentans . Deletio tentans . To stabmodeling , and a dGFP-DBP5-nup159ΔNTD mutant cell lysate. We reasoned that this is a degradation product from post-lysis proteolysis of the Nup159 FG domain, which is commonly observed with FG nups. Live cell imaging of GFP-Dbp5-nup159ΔNTD and GFP-dbp5RR-nup159ΔNTD confirmed steady state localization for the fusion protein at the NE  for GFP-Dbp5, GFP-nup159ΔNTD and GFP-Dbp5-nup159ΔNTD. Nup82-GFP served as a control for stable association at the NPC, as previously shown [We first generated GFP-tagged alleles of the Dbp5-nup159ΔNTD fusions to confirm localization to the nuclear rim. GFP-Dbp5-nup159ΔNTD was detectable as a full-length protein from cell lysate . A smallt the NE  with no ly shown . As prevly shown ,69. Howemex67-5 mutants that also express wild-type copies of DBP5 and NUP159 from their endogenous loci. Although GFP-Dbp5 accumulated in the nucleus of mex67-5 mutants at the nonpermissive temperature, GFP-Dbp5-nup159ΔNTD exhibited nuclear rim localization with no observed redistribution to the nucleus , 9Δ dbp5Δ . FurtherTD alone , and GFPTD alone ,DBP5-nup159ΔNTD strain is viable, it does have a growth defect compared to strains expressing wild-type Dbp5 + RNA localization. Reflecting the growth defect in this strain, DBP5-nup159ΔNTD results in an accumulation of mRNA in the nucleus compared to wild-type cells at 23°C,  for Dbp5 and concluded that nuclear shuttling is not essential for mRNA export . The dis mutants . Indeed, nucleus , consiston mRNPs . This ags viable . We did R369G associates with Mex67 and Nab2, RNA is still required for the Dbp5-Mex67 and Dbp5-Nab2 association. We interpret this result to indicate that Mex67-Mtr2 and/or Nab2 adopt conformations permissive for association with Dbp5 when bound to RNA, and we predict that Mex67-Mtr2 and/or Nab2 interaction with RNA is required for binding to Dbp5. However, we have not been able to observe interactions between Dbp5, Mex67-Mtr2, Nab2, and Gle1 with recombinant proteins in the presence of RNA, suggesting that additional factors including unknown proteins or post-translational modifications are required for Dbp5 association with Nab2 and/or Mex67.Using a variety of approaches, we have also demonstrated that Dbp5 is recruited in proximity to Mex67 and Nab2 for interaction with RNA. Mex67 and Nab2 specifically co-precipitate with Dbp5 , whereas other RBPs do not . The assin vivo, not necessarily indicating a direct interaction. Additionally, the observed location of the Dbp5-VC Nab2-VN BiFC signal does not necessarily indicate the initial location of the Dbp5-Nab2 association. Due to the delay in fluorescence caused by chromophore maturation kinetics, it remains possible that the Dbp5-Nab2 association occurs first in the nucleus, and the Dbp5-VC Nab2-VN complex, stabilized by the complementation of the Venus fold, retains a steady-state localization at the NPC mediated by Dbp5 interaction with Nup159. Regardless, the viability of DBP5-nup159ΔNTD mutant suggests that nuclear association between Dbp5 and Nab2 or Mex67 is not essential and supports a model wherein these associations occur at the NPC. It is furthermore likely that the Dbp5-VC Nab2-VN fluorescent complementation signal overrepresents the native association due to the tendency of re-constituted fluorophores to remain associated, elongating the Nab2-Dbp5 association kinetics  in–Leu or YPD media at the indicated temperatures, and where indicated, shifted to 37°C for 30min, shifted to 42°C for 10min, or thiolutin (Enzo) was added for indicated time points. Concentration of thiolutin was based on [600 ~0.5) and grown for an additional 7hrs, and imaged. For live cell imaging, cultures were collected, re-suspended in synthetic complete media at the proper temperature, and imaged. For in situ hybridization, yeast were processed as described, hybridized with 1ng/μL Cy3-conjugated oligo d(T) or Alexa Flour 488-conjugated oligo d(T), stained with 0.1mg/mL DAPI to visualize the nucleus as described in [Yeast strains were grown to mid-log phase  in synthetic media, harvested, and lysed by bead beating (3x 2min) in lysis buffer (25 mM HEPES (pH 7.4), 150 mM KCl, 2 mM MgCl2, 0.1% NP-40, 1 mM PMSF, 1x cOmplete EDTA-free protease inhibitor (Roche)). Lysate was either treated with 0.5ug/uL Rnase A or water for 10min on benchtop, and incubated with polyclonal rabbit anti-GFP antibodies (ThermoFisher) and Protein A sepharose (GE) for 30min. Beads were washed 3x with lysis buffer, and bound protein was eluted in SDS loading buffer . Proteins were then resolved by SDS-PAGE and blotted using affinity-purified rabbit anti-Dbp5 (ASW42) [Immunoprecipitation was adapted from . 50mLs o (ASW42) , affinit (ASW42) , affinit (ASW42) , affinit (ASW42) , or affi (ASW42)  antibodi (ASW42)  and affi600 units of cell numbers were collected by centrifugation. Cells were re-suspended in SDS loading buffer, lysed by vortexing with glass beads, and boiled 5min. Lysates were then separated by SDS-PAGE and blotted using the above-described antibodies or anti-yPgk1 (Invitrogen). Alexa Flour 650-, 700-, or 800- conjugated secondary antibodies  were visualized with the Li-Cor Odyssey scanner .Whole-cell lysate immunoblotting was performed as in ). Cells norm = ((F(t)ROI−Fbkgd) / (F(t)cell- Fbkgd)) x ((F(i)ROI−Fbkgd) / (F(t)cell- Fbkgd)) where FROI is the signal of the ROI, Fcell is the signal from an adjacent cell (to control for photobleaching), Fbkgd is background signal, t is the time point, and i is the initial pre-bleach signal [Fluorescence recovery after photobleaching experiments were performed on wt strain expressing GFP fusion proteins. Cells were analyzed using an inverted confocal microscope  equipped with a 63×/1.40 NA Plan Apochromat oil lens (Carl Zeiss) at room temperature and photographed using a charge-coupled device camera . AxioVision version 3.0 SP software (Carl Zeiss) was used to acquire images. GFP signal was detected using a 488-nm excitation signal at 5% laser power. One prebleach and 20 post-bleach images were collected with two iterations averaged for each image. The bleach event was 15 iterations at 50% laser power with the indicated ROI. Normalized fluorescence intensity was calculated as in : F(t)norh signal . ImageJ S1 Fig600~0.5) in YPD, shifted to 37°C for 30min, processed for in situ hybridization with an Alexa Flour 488-labeled oligo d(T) probe, stained with DAPI, and imaged by wide-field fluorescence microscopy. Scale bar, 5μm. Images were adjusted identically. (C) WT and mutant GFP-Dbp5 constructs are expressed to similar levels. Mutant mex67-5 strains with GFP-dbp5 vectors were grown to mid-log phase (OD600~0.5) in minimal media and lysed in SDS loading buffer. Lysates were resolved by SDS-PAGE and immunoblotted using the indicated antibodies. (D) GFP-Dbp5 localization in wt strains. WT strains with the indicated vectors were grown to mid-log phase (OD600~0.5) in YPD, shifted to 37°C for 30min, and imaged by wide-field live-cell direct fluorescence microscopy. Scale bar, 5μm. This localization has been previously reported [mex67-5 mutants with GFP-dbp5 mutant vectors. Mutant strains with the indicated vectors were grown to mid-log phase (OD600~0.5) in YPD, shifted to 37°C for 30min, processed for in situ hybridization with a Cy3-labeled oligo d(T) probe, stained with DAPI, and imaged by wide-field fluorescence microscopy. Scale bar, 5μm. Images were adjusted identically.(A and B) Mutants display a poly(A)+ export defect. Indicated mutant strains were grown to mid-log phase (ODreported . Images (TIF)Click here for additional data file.S2 Fig600~0.5) in YPD and imaged by wide-field live-cell direct fluorescence microscopy. Scale bar, 5μm. Images were adjusted identically to (A) Background fluorescence of haploid split-Venus tagged strains. Indicated strains were grown to mid-log phase (OD(TIF)Click here for additional data file.S3 Fig600~0.5) at 23°C, thiolutin was added to a final concentration of 5μg/mL for indicated time points, and cells were imaged by wide-field live-cell direct fluorescence microscopy. All Venus images were adjusted identically. Scale bar, 5μm. Note that the Dbp5-VC and Nab2-VN BiFC signal is weaker when cells are grown in minimal medium (D) Thiolutin disrupts mRNA export activity. An integrated DBP5-GFP strain was transformed with the nab2ΔRGG-mCherry mRNA export reporter. Cells were grown to mid-log phase (OD600~0.5) at 23°C, thiolutin was added to a final concentration of 5μg/mL for indicated time points, and cells were imaged by wide-field live-cell direct fluorescence microscopy. Scale bar, 5μm. (E) Using Nop1-mCherry signal as a guide for NE adjacent to the nucleolus, free-hand lines were drawn in Image J, and mean grey value was determined for background, nucleolus, and non-nucleolus regions. Only cells with crescent-shaped nucleoli were quantified.(A) Inversion of split Venus images from (TIF)Click here for additional data file.S4 FigDBP5-nup159ΔNTD has a temperature-sensitive growth defect. The indicated deletion strains  carrying the indicated vectors were grown to mid-log phase (OD600~0.5) in YPD at 23°C, fivefold serially diluted, and plated on YPD plates at the indicated temperature. (C) GFP tags do not affect growth of strains. The indicated strains were grown to mid-log phase (OD600~0.5) in YPD at 23°C, fivefold serially diluted, and plated on YPD plates at the indicated temperature. (D) Poly(A)+ localization in mutants. Indicated mutant strains were grown to mid-log phase (OD600~0.5) in YPD, shifted to 37°C for 30min, processed for in situ hybridization with a Cy3-labeled oligo d(T) probe, stained with DAPI, and imaged by wide-field fluorescence microscopy. Scale bar, 5μm. Images were adjusted identically. (E) Quantification of mRNA export defect of samples found in (D). At least eighty cells were scored for the accumulation of poly(A)+ signal in the nucleus from the indicated conditions.(A) GFP-Dbp5-nup159ΔNTD is expressed as a full-length protein. Lysates from indicated GFP-tagged strains were resolved by SDS-PAGE and immunoblotted using the indicated antibodies. Asterisks indicate full-length proteins visible with high exposure. Note that post-lysis degradation is common for unstructured FG-domain proteins. Note that full-length protein was not visible with the Dbp5 antibody. (B) (TIF)Click here for additional data file.S1 TableS. cerevisiae strains utilized in this study are listed. KANR denotes kanamycin resistance cassette.The genotype and source of (DOCX)Click here for additional data file.S2 TableTable provides the description and source of plasmids utilized in this study.(DOCX)Click here for additional data file.


Geminiviridae family, Begomovirus and Curtovirus comprise the two best characterized genera. Curtovirus and Old World begomovirus possess similar genome structures with six to seven open-reading frames (ORF). Among them, begomovirus and curtovirus V2 ORFs share the same location in the viral genome, encode proteins of similar size, but show extremely poor sequence homology between the genera. V2 from Beet curly top virus (BCTV), the model species for the Curtovirus genus, as it begomoviral counterpart, suppresses post-transcriptional gene silencing (PTGS) by impairing the RDR6/SGS3 pathway and localizes in the nucleus spanning from the perinuclear region to the cell periphery. By aminoacid sequence comparison we have identified that curtoviral and begomoviral V2 proteins shared two hydrophobic domains and a putative phosphorylation motif. These three domains are essential for BCTV V2 silencing suppression activity, for the proper nuclear localization of the protein and for systemic infection. The lack of suppression activity in the mutated versions of V2 is complemented by the impaired function of RDR6 in Nicotiana benthamiana but the ability of the viral mutants to produce a systemic infection is not recovered in gene silencing mutant backgrounds. We have also demonstrated that, as its begomoviral homolog, V2 from BCTV is able to induce systemic symptoms and necrosis associated with a hypersensitive response-like (HR-like) when expressed from Potato virus X vector in N. benthamiana, and that this pathogenicity activity does not dependent of its ability to supress PTGS.Geminiviruses are single-stranded DNA plant viruses with circular genomes packaged within geminate particles. Among the  Geminiviridae is divided into nine genera based on their genome features and biological properties , it has an enormously wide host range within dicot plants, including around 300 species in more than 40 plant families, as well as a broad geographical distribution that includes the Indian subcontinent, North and Central America and the Mediterranean region .All monopartite and Old World bipartite geminiviruses have similar genome structures, encoding 6 or 7 multifunctional proteins . In mostNicotiana benthamiana plants, when expressed from a Potato virus X (PVX)-derived vector  and post-transcriptional (PTGS) level . Additiod vector .Less is known about the function of curtovirus V2. Based on genome location and length, begomovirus and curtovirus V2 ORFs seem to be orthologous. However, their homology at the protein level, which is high within each genus, is extremely low. As its begomovirus counterpart, curtovirus V2 is needed for full systemic infection  and it fN. benthamiana when expressed from PVX, similar to that produced by the expression of its begomoviral equivalent. The functional analysis of the mutated versions of V2 indicated that a putative phosphorylation motif and two N-terminal hydrophobic domains conserved also in V2 from begomovirus, are required for the PTGS suppression activity, viral pathogenicity, V2 subcellular localization and for systemic infection of N. benthamiana and Arabidopsis thaliana plants. Collectively, these results suggest that begomovirus and curtovirus V2 proteins, in spite of their low sequence homology, have evolved to target the same cellular processes through similar mechanisms, providing a putative example of convergent protein evolution.Here we show that BCTV V2, besides functioning as a PTGS suppressor, also produces an HR-like cell death phenotype in Escherichia coli strains were carried out according to standard methods  ecotype and Nicotiana benthamiana. Plants were grown in chambers at 24°C in long-day conditions (16 h light/8 h dark) before and after agroinfiltration/infection. The Arabidopsis mutant lines used for geminiviral infection, rdr6–15 and the double mutant dcl2/4, were described elsewhere  and then subjected to qPCR analysis using primers described on N. benthamiana and A. thaliana samples, respectively . Proteins were stained by Coomassie blue and immunoblotted with anti-GFP mouse monoclonal antibody  and anti-mouse IgG whole molecule-peroxidase secondary antibody .For western blot analysis, 100 mg of leaf tissue per sample were used. Total protein was extracted with two volumes of extraction buffer . Samples were centrifuged 15 min at 4°C at 16000 N. benthamiana leaves were agroinfiltrated as previously described  in PVX-infected plants, conductivity (mS/cm) was measured using a conductivity meter Crison CM35 . Leaf discs were cut from infiltrated  or apical leaves (systemic tissue), washed for 30 min in 6 mL of deionized water, and then transferred to 6 mL of deionized water and the conductivity was measured every 24 h for a total of 3 days, starting at 6 dpi  or 8 dpi (systemic tissue). For BCTV infection in A. tumefaciens was transformed with binary vectors containing V2 mutants fused to GFP. N. benthamiana leaves were agroinfiltrated with cultures at OD600 0.5–1. For co-localization experiments Agrobacterium cultures containing V2-GFP constructs and the nuclear envelope marker AtSUN1–tagRFP  (see section “Introduction” for references). Despite sharing a similar function, the protein homology, which is highly conserved within each genus  a pch genus  and (ii)ch genus  that is ch genus . Sequencch genus . Sequencch genus  and seemch genus . On the ch genus . Interesch genus . A broadch genus , S2.To determine the functional relevance of the hydrophobic domains and the phosphorylation motifs in the PTGS suppression activity of BCTV V2, we generated point mutants in the mentioned domains . Non-polN. benthamiana wild-type leaves were co-agroinfiltrated as described in To evaluate the gene silencing suppressor activity of the V2 mutants, Arabidopsis thaliana, V2 from BCTV suppresses PTGS by interfering with the RDR6-dependent amplification pathway   . As expee plants . On the e plants . The rele plants , indicatthaliana .N. benthamiana of V2 from several begomoviruses using a Potato virus X (PVX)-derived vector, caused localized cell death in the infiltrated area and induced systemic symptoms and necrosis associated with a hypersensitive response-like (HR-like) phenotype  from a PVX-derived vector in G mutant . ConductG mutant . In apicG mutant . As prevG mutant , there wSymptoms intensification produced by PVX-recombinant viruses expressing other viral proteins has been related to a larger accumulation of genomic RNA from PVX . To ruleN. benthamiana. Leaves were agroinfiltrated, collected 2–3 days later and visualized using a confocal microscope. Wild-type GFP-V2 localized in the nucleus and in the cellular periphery and not significant differences were observed in the localization of the mutated V2 proteins  developed only mild leaf curling and chlorosis. Viruses containing the premature stop codon in V2 (V2stop), the mutations in the P1 phosphorylation motif (P1A and P1D) or in the hydrophobic domain H1 (H1GG) did not produce any detectable symptoms at 28 dpi  and DCL2 and DCL4 (double mutant dcl2-1/dcl4-2 or dcl2/4) with BCTV wild-type and V2 mutants unable to systemically infect N. benthamiana. A. thaliana Col-0 plants infected with BCTV were clearly symptomatic, whereas plants infected with any of the BCTV mutants did not develop symptoms (data not shown). The qPCR results from the infected plants indicated that, as it occurs in N. benthamiana, mutant virus in P1 phosphorylation site (P1A and P1D) or any of the two hydrophobic domains (H1GG and H2GG) were impaired in systemic infection of A. thaliana . Similar phenotypes are produced by the phosphomimic mutation of the P1 domain (P1D) suggesting that constitutive phosphorylation of the threonine 43 residue is not recovering the suppression activity, neither the ability to propagate the virus during the infection. In Old World begomovirus, this motif has been described to be involved in viral pathogenicity (The highly conserved protein kinase C (PKC) motif (P1) is required for the PTGS suppression activity of V2, as the replacement of the threonine residues of this motif impairs V2 ability to suppress GFP gene silencing in  pathway . Howevergenicity  but studN. benthamiana wild-type plants were infected with BCTV containing the H1GG or the H2GG V2 mutations, systemic infection was compromised although some differences were observed. While in wild-type N. benthamiana H1GG was fully impaired for systemic infection to a similar level than the null V2 mutant (V2stop), the virus containing the H2GG mutation showed a slightly more, although not statistically significant, viral DNA accumulation. This difference among both mutated viruses was more noticeable when A. thaliana plants impaired in gene silencing were infected. In wild-type plants infected with the H2GG mutant, the amount of viral DNA is only 12.6 ± 5.1% of the viral DNA accumulated in plants infected with the wild-type virus. However, in rdr6 and dcl2/4 mutants the quantity of H2GG viral DNA represented 28.3 ± 9.6% and 34.3 ± 9.2%, respectively. This indicated that the failure of H2GG to produce a systemic infection is partially recovered by the inhibition of PTGS antiviral activity. Altogether, these results indicated that the inability of these mutants to systemically infect the plant is not due to the lack of PTGS suppression activity. Therefore, other V2 function required for the viral movement is altered by those mutations. Interestingly, mutations in these domains, also provoked changes in the subcellular localization of V2 protein. As begomoviral V2, BCTV V2 protein localizes in the nucleoplasm and in the cell periphery, mainly associated to ER. Additionally, accumulation in the Cajal body  located in the N-terminus, that are presented in all geminiviral V2 proteins. Replacement of two of the non-polar residues of those domains, dramatically affect the PTGS suppression activity of V2 and, as in the case of the P1 mutation, the activity is fully recovered when RDR6 activity is impaired. When jal body , BioRxivjal body , BioRxivjal body  but do njal body . In begojal body . Mutatiovia” a PVX-derived vector provoked the induction of local and systemic necrosis in the N. benthamiana leaves. Interestingly, BCTV infection in A. thaliana or N. benthamiana plants does not produce cell-death phenotype on systemically infected plants, maybe due to the limited number of cells that harbor its infection. Transient or permanent expression of V2 from a 35S CaMV promoter in N. benthamiana or A. thaliana, should increase the number of cells expressing V2 and bypass the tissue specificity. However, in none of the occasions in which BCTV V2 was expressed from those type of constructs a necrotic reaction was observed (Hypersensitive response (HR) is an active defense reaction derived from the activation of defense-related pathways, which lead to cell death (CD) . The fact that V2 ORF is present in Old World begomovirus and curtovirus, could suggest that this gene was already present in a common ancestor of both viral genera. However, the high level of conservation at the protein level among the species of each genus, but not between genera, and the absence of ORF V2 homologs in the genome of New World begomoviruses, argue against this hypothesis and suggest that V2 from curtovirus and begomovirus could have evolved independently.All datasets generated for this study are included in the article/AL, BR-R, and TR-D have performed the experiments, analyzed the data, and participated in the writing and critical reading of the manuscript. LC have provided technical assistance in the experimental procedures. ER-N has collaborated in some experiments and participated in the critical reading of the manuscript. EB and AC have planned and directed the experimental design of the work, have done the writing and the critical lecture of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To explore the feasibility of using Saccadic Vector Optokinetic Perimetry (SVOP) to differentiate glaucomatous and healthy eyes. A prospective case–control study was performed using a convenience sample recruited from a single university glaucoma clinic and a group of healthy controls. SVOP and standard automated perimetry (SAP) was performed with testing order randomised. The reference standard was a diagnosis of glaucoma based a comprehensive ophthalmic examination and abnormality on standard automated perimetry (SAP). The index test was SVOP. 31 patients with glaucoma and 24 healthy subjects were included. Mean SAP mean deviation (MD) in those with glaucoma was − 8.7 ± 7.4 dB, with mean SAP and SVOP sensitivities of 23.3 ± 0.9 dB and 22.1 ± 4.3 dB respectively. Participants with glaucoma were significantly older. On average, SAP sensitivity was 1.2 ± 1.4 dB higher than SVOP (95% limits of agreement = − 1.6 to 4.0 dB). SVOP sensitivity had good ability to differentiate healthy and glaucomatous eyes with a 95% CI for area under the curve (AUC) of 0.84 to 0.96, similar to the performance of SAP sensitivity . For 80% specificity, SVOP had a 95% CI sensitivity of 75.7% to 94.8% compared to 77.8% to 96.0% for SAP. SVOP took considerably longer to perform (514 ± 54 s compared to 267 ± 76 s for SAP). Eye tracking perimetry may be useful for detection of glaucoma but further studies are needed to evaluate SVOP within its intended sphere of use, using an appropriate design and independent reference standard. The test uses a white static stimulus displayed against a white background, with the contrast of the stimulus varied according to a staircase strategy to determine Differential Light Sensitivity (DLS). SAP has become the ‘gold standard’ perimetric test, yet patients often find it difficult to perform. Although patients accept that visual field testing is important, they find it more demanding than other common glaucoma tests and a qualitative investigation discovered a perception among patients that multiple tests were needed to become comfortable and to gain an accurate representation of their vision3. In reality, visual field testing is often performed less frequently, with a multicentre review of glaucoma clinics in England showing most patients have only one visual field test per year4. Similar findings were reported in the US, where even in an academic medical centre, patients had an average of only 1.24 visual tests per year5. Despite the guidance highlighting the importance of perimetry, there is evidence that the frequency of visual field testing has decreased, perhaps due to overreliance on imaging. A review of over 150,000 patients with glaucoma, found the odds of having an automated visual field test decreased by 44% from 2001 to 2009, whilst over the same time period there was a 147% increased odds of undergoing computer-based optic disc imaging6. Although many factors, particularly organisational and resource constraints, may affect the frequency of visual field testing, the perception that SAP is difficult to perform and unpopular with patients, may contribute to the discrepancy between recommended and observed frequencies of testing.SAP is also subject to considerable test–retest variability with the result that patients may need multiple tests to confidently identify change. Although research on the optimal frequency of clinical tests is limited, it has been suggested that to detect patients progressing at a rapid rate (≥ − 2 dB per year), 3 visual field tests should be conducted per year for the first 2 years of follow up, with more frequent testing required to detect slower rates of change13. Eye trackers have long been used to monitor fixation during visual field assessment, but eye tracking may also be useful for determining whether a stimulus has been seen. We have previously developed a suprathreshold eye tracking perimeter for use in children known as saccadic vector optokinetic perimetry (SVOP)11. SVOP was inspired by the work of Damato and colleagues, who in 1989 described a method of computerized perimetry using a moving fixation target presented on a computer monitor15. Patients were required to position a cursor over a fixation target using a mouse, and stimuli were presented when the cursor was in the correct position. By moving the fixation target, and by using the preceding stimulus as a fixation spot for the next stimulus, a large area of visual field could be tested using a standard computer monitor. A similar approach is used by the more recent Melbourne Rapid Fields test, an FDA approved application that allows testing of 30° of field using the 9.7-inch screen of a tablet computer16.Over the last decade several groups have explored the possibility of performing perimetry by tracking eye movements20. Eye trackers can also determine the position of the eyes relative to the screen to automatically adjust the size and position of stimuli allowing the patient to move their head during testing. Therefore, eye tracking perimetry may provide a more comfortable experience for patients that is likely to be particularly beneficial for those that struggle to maintain fixation for a prolonged period or have difficulty pressing a button to register a response.Damato’s perimeter and the Melbourne Rapid Fields require responses to stimuli to be registered by pressing a response button, similar to SAP; however, eye tracking could be used to determine if a stimulus is seen, by detecting eye movements towards the stimulus that occur within a prespecified time of stimuli presentation22. Tailor et al. reported many children were unable to complete testing and agreement with confrontational and Goldmann perimetry was moderate to poor21. In contrast, Simkin and colleagues reported a higher proportion of children were able to successfully complete SVOP compared to Goldmann perimetry, with SVOP significantly faster22. The differences in results may be due to the inherent difficulty of assessing visual fields in children or due to differences in characteristics of those included in the studies. As adults also report difficulty performing conventional perimetry, we have modified SVOP to develop a threshold version of the test. An advantage of evaluating the test in adults is it is easier to establish a reliable comparison to SAP, and in a previous investigation we found good correlation between threshold sensitivity values obtained with SVOP and SAP and demonstrated SVOP to have good repeatability19. Most patients found SVOP comfortable and almost three quarter preferred SVOP compared to standard testing.The suprathreshold version of SVOP for use in children showed mixed results23; however, the study provides useful data supporting further larger scale evaluation of SVOP.The aim of this study was to explore the feasibility of using SVOP to differentiate eyes with glaucoma from healthy controls and compare performance to SAP. Like many evaluations of diagnostic devices, the study was limited by a case–control design and so results may not be generalisable to other settings or groups, and it would be premature to recommend use of eye tracking perimeters in clinical practiceThis was a prospective case–control study including patients with glaucoma and healthy volunteers. A two-gate design was used, with patients with glaucoma recruited from the glaucoma clinic at the Princess Alexandra Eye Pavilion, Edinburgh, UK, and healthy participants recruited using the Scottish Health Research Register (SHARE), a national register of volunteers interested in participating in research. Patients attending the glaucoma clinic were invited to participate by their treating clinician. All participants provided written informed consent prior to enrolment and all study methods were prospectively approved by the South-East Scotland Research Ethics Committee (reference 13/SS/0045). The study adhered to the tenets of the Declaration of Helsinki.Patients attending the glaucoma clinic underwent a comprehensive ophthalmic examination, including best-corrected visual acuity, slit lamp biomicroscopy, intraocular pressure (IOP) measurement using Goldmann applanation tonometry, gonioscopy and dilated fundoscopy. The reference standard was a diagnosis of glaucoma made by the treating glaucoma consultant based on the comprehensive ophthalmic examination and all patients with glaucoma were required to have a glaucomatous visual field defect on SAP using the Humphrey Field Analyzer (HFA) SITA Fast 24-2 test . The treating glaucoma consultant did not have access to the SVOP test results. Patients with non-glaucomatous ocular or non-ocular conditions, such as neurological disease, that might affect the visual field were excluded. Healthy participants were required to have no previous history of significant eye disease, no known history of visual field defect and no neurological conditions that might affect the visual field.Carl Zeiss Meditec, Dublin, CA) with the 24-2 test pattern and the SITA Fast algorithm. The index test, SVOP, was performed at the same visit using a threshold SVOP research device, described in detail in previous publications19. All patients completed SAP and SVOP in both eyes, with the order of testing randomised. A random group of participants then performed repeat SAP and SVOP in one eye, randomly chosen for healthy subjects and with the worse affected eye selected in those with glaucoma. Visual field tests were reviewed for reliability and artefacts. SAP tests with ≥ 15% false positives or ≥ 20% fixation losses were considered unreliable and excluded. False negative rates were not considered due to evidence indicating false negative rates are more strongly indicative of glaucoma severity than attentiveness24. SVOP does not provide information about false positives or fixation losses as it does not require the patient to maintain fixation on a single point or register a response with a button. Inherent to the test, SVOP accounts for fixation by not presenting a further stimulus until fixation is achieved.All participants had SAP using a Humphrey Field Analyzer (HFA) 750i  screen  and a 60 Hz eye tracker Participants were seated in front of an LCD screen with their eyes aligned with the screen’s centre at an initial distance of 55 cm Fig. . Written26. Saccades larger than 5° take only approximately 20 to 30 ms, with an additional 2 ms for each additional degree.During testing participants were instructed to look towards any peripheral stimulus seen. The eye tracker evaluated gaze responses to the stimuli and software determined whether the stimulus had been seen based on the direction and amplitude of the gaze response. Whether or not the stimulus was seen was determined based on the direction and amplitude of the change in eye position relative to the stimulus and the point of fixation. The start of a fixation change was defined as the start point of a greater than 50 pixels change in gaze and the end location was defined by the point at which 5 consecutive gaze data samples were separated by a distanced of < 50 pixels after the detection of a fixation change. Stimuli were equivalent to Goldmann size III and each stimulus was presented for 200 ms using coordinates equivalent to the SAP 24–2 test pattern. As the eye tracker provides data on eye location, the size and position of stimuli were able to be automatically adjusted to compensate for changes head position during testing. A stimulus duration of 200 ms was selected as it is the same duration as HFA and previous work has shown visual processing speed and speed of the saccade response is sufficiently fast to reach a stimulus within this time period2) and stimulus luminance levels. To minimize the risk of variability in luminance affecting results, uniformity of the display screen was assessed using a luminance meter . Luminance values were examined for different grey-scale levels to ensure LCD RGB values corresponded to stimulus luminance used by SAP. For example, a stimulus of 20 dB (41.9 Cd/m2) was found to be equivalent to an LCD RGB value of 136, 136, 136. Stimuli luminance replicated the luminance values corresponding to 14 to 40 dB with SAP, with the background to stimuli luminance ratio also replicated. The LCD display was unable to display stimuli with luminance < 14 dB due to the maximum intensity being limited by the maximum luminance of the LCD backlight.Grey-scale level colours were produced by the LCD screen by setting red, green and blue (RGB) levels to equal each other and luminance was varied by adjusting RGB levels. The maximum level of luminance was obtained by setting the RGB level to 255, 255, 255 and the minimum to 0, 0, 0. Screen calibration was performed using a Look-Up Table to pair grey levels of each pixel to the corresponding required background (10 Cd/mThresholds were obtained using a 4–2 bracketing strategy and began by testing four seed locations (one in each quadrant), which were then used to set the starting stimulus luminance levels for neighbouring locations which in-turn were used to calculate the remaining starting luminance levels. The SVOP stimulus intensity and background intensity values were matched in luminance to those of SAP to allow direct comparison. Version 2.0 of the SVOP threshold software was used for all participants.The relationship between SAP and SVOP visual field sensitivity was examined using scatter plots and pointwise linear regression. Histograms were also constructed to survey the distribution of differences in sensitivity between SAP and SVOP for each visual field test location. All results were transposed to right eye format. Bland–Altman plots were used to compare results from SAP and SVOP and determine 95% limits of agreement.27. A ROC curve is a plot of sensitivity versus 1-specificity across threshold values, showing the intrinsic capacity of the test to discriminate diseased and nondiseased status28. The area under the ROC curve (AUC) was used to summarise the diagnostic accuracy. To account for differences in age between participants with glaucoma and controls, the covariate effect of age was adjusted for using the ROC generalised linear regression method described by Pepe et al.28 and first described for evaluation of glaucoma diagnostic tests by Medeiros et al.29 Results were reported with age set at the sample mean.Receiver Operating Characteristic (ROC) curves were constructed to assess the ability of SVOP and SAP to differentiate participants with glaucoma from healthy controls29. All statistical analyses were performed with commercially available software . The α level (type I error) was set at 0.05.ROC regression used a 1000 repetition bootstrap technique to estimate 95% confidence intervals. As measurements from both eyes of the same subject are likely to be correlated, the cluster of data for each participants was used as the unit of resampling when calculating confidence intervals61 eyes of 31 participants with glaucoma and 47 eyes of 24 healthy participants were included in the study. 26 of 55 participants (47.3%) were female. There was no difference in gender between healthy and glaucomatous participants, however those with glaucoma were significantly older (P < 0.001). Demographic and clinical details of all participants are shown in Table 2 = 0.0002, P = 0.886)30.Mean SAP mean deviation (MD) in glaucomatous eyes was − 8.7 ± 7.4 dB, with mean SAP and SVOP sensitivities of 23.3 ± 0.9 dB and 22.1 ± 4.3 dB respectively. There was strong correlation between mean SAP and SVOP sensitivities   of 0.98  and a mean difference between repeat measures of 0.37 ± 0.91 dB (95% limits of agreement of − 1.41 to 2.16 dB). In comparison, SAP had concordance correlation coefficient of 0.99  and a mean difference between repeat measures of 0.13 ± 0.87 dB (95% limits of agreement of − 1.56 to 1.83 dB).Pointwise analysis showed the average (± SD) difference in SAP and SVOP sensitivities ranged from a low of 0 ± 3.28 dB to a high of 3.35 ± 5.00 dB Fig. A, with cSVOP mean sensitivity had good ability to differentiate healthy and glaucomatous eyes included in the study with an AUC of 0.90 (95% CI 0.84 to 0.96), which was not significantly different to the performance of SAP mean sensitivity   but older participants were slower at SAP . Each decade of increasing age was associated with 49 s slower SAP test time . SAP also took longer in those with worse disease severity, on average taking 12 s longer for every 1 dB worse average SAP sensitivity . In multivariable analysis, disease severity remained signficantly associated with SAP test duration (P < 0.001), whereas age did not (P = 0.092). The duration of SVOP was not influenced by average sensitivity (P = 0.697) meaning the difference in test times between SAP and SVOP was greater in healthy participants compared to those with glaucoma  respectively. There was no significant relationship between age and test time for SVOP .20. The lower sensitivity in the present study likely reflects the difference in reference standard, with McTrusty and colleagues evaluating the ability to detect and abnormal visual field rather than the ability to differentiate glaucomatous and healthy individuals. In addition, McTrusty and colleagues defined an abnormal SVOP based on masked grading rather than the summary index of average sensitivity and did not perform ROC analysis.McTrusty and colleagues previously evaluated the ability of threshold SVOP to identify normal versus abnormal SAP, reporting a sensitivity of 98% and specificity of 78%The good performance of SVOP likely reflects a high level of agreement with SAP. On average, DLS measured by SAP was 1.16 dB higher than DLS measured by SVOP, with 95% limits of agreement of − 1.64 to 3.96 dB  separated by < 50 pixels were required to define an endpoint of change in gaze, meaning the maximum time available for an eye movement between first appearance of a stimulus and fixation on the stimulus was only 200–80 = 120 ms. This was deemed sufficient time for a saccade to each the stimulus but insufficient for smooth pursuit.9. Performing any form of automated perimetry in patients with abnormal eye movements is lilkely to be challenging, however, eye tracking perimetry may be particularly unrelaible in such individuals. As no participants in the current study had eye movement disorders we were not able to evaluate their effect on SVOP relaiblity but this is likely to be a limitation of the test.Using an ealier suprathreshold version of SVOP, Tailor and colleagues highlighted that patients with abnormal saccades may have artefactual non-specific visual field defectsIn summary, this study provides evidence that eye tracking perimetry using SVOP may be useful for aiding glaucoma detection, with performance in this case control study, similar to conventional automated perimetry. The eye tracking perimeter has the advantage of not requiring the patient to place their chin on a rest or press a button when a stimulus is seen and in previous studies has been found to be preferred by patients, however, a major limitation was that SVOP took considerably longer to perform. The study was limited by a case–control design, small sample size, and imperfect reference standard, but nevertheless the results show that eye tracking perimetry is feasbile and provide a foundation for potential further studies examining performance of eye tracking perimetry in a clinical pathway.


Tomato yellow leaf curl virus (TYLCV) genome requires the Cajal body in Nicotiana benthamiana cells. Methylation of the viral genome is countered by a virus-encoded protein, V2, which interacts with the central RdDM component AGO4, interfering with its binding to the viral DNA; Cajal body localization of the V2-AGO4 interaction is necessary for the viral protein to exert this function. Taken together, our results draw a long sought-after functional connection between RdDM, the Cajal body, and antiviral DNA methylation, paving the way for a deeper understanding of DNA methylation and antiviral defences in plants.In plants, establishment of de novo DNA methylation is regulated by the RNA-directed DNA methylation (RdDM) pathway. RdDM machinery is known to concentrate in the Cajal body, but the biological significance of this localization has remained elusive. Here, we show that the antiviral methylation of the  DNA methylation in cytosine residues is a conserved epigenetic mark essential for protecting the eukaryotic genome against invading nucleic acids, namely viruses and transposable elements. In plants, establishment of de novo DNA methylation is believed to be regulated by the RNA-directed DNA methylation (RdDM) pathway. The canonical RdDM pathway requires the activity of two plant-specific RNA polymerase II-related enzymes, Pol IV and Pol V, and leads to cytosine methylation in a sequence-specific manner. In brief, the current understanding of RdDM is as follows: Pol IV generates RNA transcripts subsequently converted to double-stranded RNA (dsRNA) by RDR2 , and theGeminiviruses are a family of plant viruses with circular single-stranded (ss) DNA genomes infecting multiple crops and causing dramatic yield losses worldwide. The geminiviral genome replicates in the nucleus of the infected cell by using the host DNA replication machinery, made available following the viral re-programming of the cell cycle , the essential virus-encoded protein V2 has been shown to suppress DNA methylation  to identify plant interactors of this viral protein in the context of the infection (N. benthamiana (NbAGO4-1 and NbAGO4-2) as associated to V2-GFP   , the natAGO4a-d) . All founfection . SlAGO4ae assays . Our resN. benthamiana, since the V2 null mutant accumulates to very low levels and produces no noticeable symptoms in systemic infections , which converts the second codon (encoding tryptophan) to a stop codon , making fections .AGO4 could partially complement the lack of V2 during the TYLCV infection. For this purpose, we employed virus-induced gene silencing (VIGS) to silence both NbAGO4-1 and NbAGO4-2. VIGS efficiently knocked-down both NbAGO4 orthologues, but did not affect accumulation of the transcript of the close homologue NbAGO6 , but dramatically improved performance of the V2 null mutant virus (3.23-fold), suggesting that one of the main roles of V2 during the viral infection is the suppression of AGO4 function  to measure DNA methylation of the intergenic region (IR) of the viral genome, which presents the highest methylation levels during the infection . As showAGO4 silencing. Strikingly, AGO4 silencing resulted in a ~30% decrease in the percentage of methylated cytosines in all contexts  methylation of the viral DNA at least partially depends on AGO4 function and (b) V2 can at least partially counter AGO4-dependent methylation of the viral DNA.We then compared the percentage of cytosine methylation in the intergenic region in local infections with the V2 null mutant TYLCV in basal conditions or upon contexts , indicatN. benthamiana, which require longer timespans and involve viral cell-to-cell and long-distance movement, methylation of the wild-type viral genome could be detected at ~8–23% in all contexts  brings the methylation of the V2 null mutant genome back to wild type-like levels, again supporting the idea that AGO4-dependent methylation of the viral genome occurs during the infection and is partially countered by V2. Notably, the detected decrease in methylation correlates with the enhanced viral accumulation in the AGO4-silenced plants , a subunit of Pol V, in the Cajal body, which was then suggested to be a center for the assembly of AGO4/NRPE1/siRNA complexes, enabling RdDM at target loci . Howeverrillarin , upon trthamiana . Of notethamiana . Analysithamiana .In order to evaluate the relevance of the Cajal body localization of the V2-AGO4 interaction, we took advantage of the fact that a GFP-V2 fusion protein, as opposed to the previously mentioned V2-GFP, does not localize to the Cajal body  and 2, ocoilin on the formation of Cajal bodies has been previously demonstrated  was found to interact with AGO4 in N. benthamiana, leading to enhanced viral accumulation and a reduction in viral DNA methylation , the full-length coding sequence of AGO4 genes was amplified using cDNA as template. NbAGO4-1, NbAGO4-2, SlAGO4a, SlAGO4b, SlAGO4d, and SlWRKY75 were cloned into pENTR/D-TOPO (Invitrogen) following the manufacturer’s instructions. The binary plasmids to express RFP and 3xHA N-terminal fusions were generated by Gateway cloning the AGO4 coding sequence into pGWB555 and pGWB515, respectively  and then Gateway-cloned into the pBiFC-2in1-CN vector . pDONR207-Fibrillarin  were grown in a controlled growth chamber under long day conditions  at 25℃.Agrobacterium tumefaciens strain GV3101, with the exception of pBINTRA6, which was transformed into A. tumefaciens strain C58c1. A. tumefaciens clones carrying the constructs of interest were liquid-cultured in LB with appropriate antibiotics at 28°C overnight. Bacterial cultures were then centrifuged at 4000 g for 10 min and resuspended in the infiltration buffer  and adjusted to an OD600 = 0.5. Next, the bacterial suspensions were incubated in the buffer at room temperature and in the dark for 2–4 hr and then infiltrated into 3- to 4-week-old N. benthamiana plants. For co-expression experiments, the different Agrobacterium suspensions were mixed at 1:1 ratio before infiltration.All binary plasmids were transformed into 1-116aa) and anti-Actin (Agrisera AS13 2640) antibodies, respectively.Protein extraction and co-immunoprecipitation assays were performed as described in Protein extracts were immunoprecipitated with GFP-Trap beads , and analyzed by western blot with anti-GFP  and anti-HA (12CA5)  antibodies.N. benthamiana leaves transiently expressing 3xFLAG-NbAGO4-1 were collected, ground in liquid nitrogen and homogenized in 6x (w:v) extraction buffer ) at 4°C with rotation for 30 min. The extract was subjected to centrifugation  at 4°C. 5 μg anti-FLAG antibody  per gram of tissue were added to the supernatant in a new tube and incubated at 4°C overnight. The next day, 20 μl of slurry Protein G beads (Invitrogen) per gram of tissue were added and subjected to a further incubation for 2 hr with rotation at 4°C. After incubation, Protein G beads were washed three times in 3x (v:v) homogenate wash buffer . The quality of purification was examined by SDS-PAGE followed by immunoblotting.For 3xFLAG-NbAGO4-1 IP followed by vsiRNA extraction, 6 grams of A. tumefaciens harboring V2-N-luc or C-luc-NbAGO4-1/2, C-luc-SlAGO4a/b, or C-luc-SlWRKY75 at OD600 = 0.5 were mixed at 1:1 ratio. Three different combinations of A. tumefaciens were infiltrated on the same N. benthamiana leaf. 1 mM luciferin (in H2O) was infiltrated into the inoculated leaves 2 days after Agrobacterium infiltration. A low-light cooled CCD imaging apparatus (NightShade LB985 with IndiGO software) was used to capture and analyze the luciferase signal at 2 dpi.Split-luciferase complementation imaging assays were performed as described . Equal vN. benthamiana epidermal cells was performed on a Leica TCS SP8 point scanning confocal microscope using the pre-set sequential scan settings for GFP , RFP , and CFP . Confocal imaging for co-localization of Fibrillarin-RFP  and for CFP .For bimolecular fluorescence complementation (BiFC) analyses, nstructs  and CFP-NbAGO4-1 cDNA (from nt 1920 to 2281) and a 389 bp fragment of Nbcoilin cDNA (from nt 2222 to 2610) were amplified using specific primers shown in NbAGO4 and TRV-Nbcoilin. VIGS assays were performed as described in A. tumefaciens carrying pBINTRA6 and TRV-NbAGO4 or TRV-EV were mixed and inoculated into 18-day-old N. benthamiana plants. Two weeks later, fully expanded young leaves were infiltrated with A. tumefaciens carrying the TYLCV infectious clone and samples were collected at 4 days post-inoculation (dpi) to detect viral accumulation. For TYLCV systemic infection assays, A. tumefaciens carrying pBINTRA6 and TRV-NbAGO4 or TRV-EV (empty vector) and the TYLCV infectious clone were mixed and inoculated into 18-day-old N. benthamiana plants. The three most apical leaves of each plant were collected at 3 weeks post-inoculation (wpi) to detect viral accumulation. TYLCV systemic infection assays in Nbcoilin-silenced plants were performed as previously described for NbAGO4-silenced plants.The vectors used for virus-induced gene silencing (VIGS) were pBINTRA6  and pTRVN. benthamiana leaves  using the CTAB method . RNA was reverse-transcribed into cDNA by using the iScriptTM cDNA Synthesis Kit (Bio-Rad #1708890) according to the manufacturer’s instructions. NbTubulin was used as reference gene ; amplified fragments were cloned into the pMD18-T vector by TA ligation and sequenced (>15 clones per experiment). Cytosine methylation analysis was performed with Kismeth (http://katahdin.mssm.edu/kismeth/revpage.pl) , and 500 ng of purified DNA was subjected to bisulfite treatment using EpiTect Plus DNA Bisulfite Kit  according to the manufacturer’s handbook. The selected fragment  of the bisulfite-treated DNA was amplified by PCR (Fw: page.pl) . Values Agrobacterium clone carrying the binary vector to express 3xFLAG-NbAGO4-1 was co-infiltrated with those carrying the TYLCV or TYLCV-V2null infectious constructs in N. benthamiana leaves, and tissues were collected at 2 dpi. RNA immunoprecipitation (RIP) assays were performed as previously described , and 1% Protease Inhibitor Cocktail (Sigma)). The samples were then centrifuged at 16,000 g for 20 min. The extract was subjected to immunoprecipitation with FLAG antibody  bound to Dynabeads Protein G (Invitrogen) for 2 hr with gentle rotation at 4°C. After incubation, beads were washed three times in lysis buffer.The escribed . In brie3) at 60°C for 15 min. After reverse crosslinking, 4 µl EDTA 0.5 M, 8 µl Tris and 40 µg proteinase K (Invitrogen) were added to each sample, which was incubated at 42°C for 1 hr.Immunocomplexes were eluted with 200 µl of Elution buffer  at 37°C for 1 hr and then reverse-transcribed with Superscript III ﬁrst-strand synthesis system (Thermo Fisher Scientiﬁc) with random hexamers. qPCR was used to determine the relative enrichment for each sample, which was calculated by normalizing the value of immunoprecipitated RNA to that of the input.Input and immunoprecipitated RNA were extracted with TRIzol reagent (Thermo Fisher Scientiﬁc) and resuspended in 200 µl or 20 µl ddHAgrobacterium clone carrying the binary vector to express 3xFLAG-NbAGO4-1 was co-infiltrated with those carrying the TYLCV or TYLCV-V2null infectious clones in N. benthamiana leaves, and tissues were collected at 2 dpi. Chromatin immunoprecipitation (ChIP) assays were performed as described (3) at 65°C for 15 min. After reverse crosslinking, 10 µl of 0.5 M EDTA, 20 µl of 1 M Tris pH 6.5 and 1 µl of proteinase K (Invitrogen) were added to each sample, which was incubated at 45°C for 2 hr. DNA was then purified using QIAquick PCR Purification Kit . The products were eluted into 200 µl of ddH2O, and analyzed by qPCR. The primers used in this experiment are listed in Actin are taken from The escribed . In brie32P-labeled probes for the intergenic region (IR) of the viral genome amplified by PCR  or oligonucleotides in PerfectHyb buffer (Sigma). Washed membranes were exposed to X-ray films at −80°C for 3 days.Small RNA (sRNA) extraction and northern blot were performed as described . Briefly In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Your study reports on a Geminivirus suppressor, V2, that counteracts AGO4-mediated methylation of viral DNA upon host plant infection through physical interaction with AGO4. AGO4 is central to the RNA-directed DNA methylation machinery that has been found to localize to so-called Cajal bodies. Your findings make Cajal bodies central hubs for antiviral defense. In addition, this work is important as it reveals that evolution of plant resistance mechanisms  is counteracted by the evolution of viral mechanisms suppressing host plant immunity, a phenomenon reported many times for bacterial, fungal or oomycete effectors, but less so from plant-virus interactions.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, including Thorsten Nürnberger as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Christian Hardtke as the Senior Editor.Thank you for submitting your article "A virus-encoded protein suppresses methylation of the viral genome in the Cajal body through its interaction with AGO4" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Your study provides a nice examples of how plant resistance mechanisms  is counteracted by the evolution of viral mechanisms suppressing host plant immunity, a phenomenon reported for bacterial, fungal or oomycete effectors, but less so from plant-virus interactions. This, together with the finding that Cajal bodies are central hubs for antiviral defense in plants is undoubtedly important for furthering our molecular mechanistic understanding of plant virus interactions.The reviewers feel, however, that your manuscript requires essential revisions that are listed below.Essential revisions:1) Major aspects described in your study have been already reported from this or related viral systems. These include the immunosuppressive role of geminiviral V2 proteins on RdDM , V2-AGO4 interaction , the requirement of Cajal body for anti-geminiviral role , AGO4 binding to DNA in RdDM , the cajal body localization of AGO4 and its role in RdDM . Please address these concerns carefully as advance over existing knowledge is an important criterion for publication in our journal.2) In the light of the above said it appears most desirable to provide additional mechanistic insight into how V2 brings about suppression of AGO4-mediated viral DNA methylation. This could be, for example, achieved by sequential ChIP analysis. Likewise, it is important to provide unambiguous evidence that the effect reported may not be brought about by V2-HDA6 interaction. This is best achieved by providing a V2 mutant that may still bind HDA6, but is unable to bind to AGO4 and to suppress antiviral defense.3) There is also concern about data variation in DNA methylation quantification. For example, the CG methylation percentages in Figure 4A are approximately 2% and 55% for 3dpi/TYLCV and 3dpi/TYLCV-V2null samples, respectively. In Figure 7B, the corresponding percentages are 8% and 22%. Great differences can also be found between and Figure 4C and Figure 7D (systemic infection) despite of the fact that plant materials used in Figure 4 and Figure 7 are comparable. This needs an explanation. Essential revisions:1) Major aspects described in your study have been already reported from this or related viral systems. These include the immunosuppressive role of geminiviral V2 proteins on RdDM , V2-AGO4 interaction , the requirement of Cajal body for anti-geminiviral role , AGO4 binding to DNA in RdDM , the cajal body localization of AGO4 and its role in RdDM . Please address these concerns carefully as advance over existing knowledge is an important criterion for publication in our journal.It is indeed known that AGO4 is a central component of the RdDM pathway, and that it localizes in the Cajal body, although the connection between RdDM and this subnuclear compartment has not been further functionally dissected. The function of the geminivirus V2 protein as a TGS suppressor has in fact been previously described, and its association with AGO4 recently shown in the case of the geminivirus Cotton leaf curl Multan virus . Nevertheless, the localization of V2 in the Cajal body and the specificity of its interaction with a RdDM regulator at this site have never been reported; the relevance of the Cajal body for anti-viral DNA methylation is, to our knowledge, also demonstrated here for the first time; additionally, our work provides insight into the molecular mechanism by which this viral protein interferes with the function of AGO4, showing that the interaction hampers the AGO4 association to the viral genome, probably through hindering the binding to the scaffold RNA . Therefore, we believe that this work not only sheds light on long sought-after connections between the Cajal body, RdDM, and anti-viral DNA methylation, but also furthers our understanding of the viral manipulation of the plant RdDM at the molecular level.2) In the light of the above said it appears most desirable to provide additional mechanistic insight into how V2 brings about suppression of AGO4-mediated viral DNA methylation. This could be, for example, achieved by sequential ChIP analysis.Following the reviewers advice, we have performed new experiments to try to obtain additional mechanistic insight into the suppression of AGO4 mediated by V2.Since V2 physically interacts with AGO4 and interferes with its binding to the viral genome, our hypothesis was that V2 likely covers a surface in the AGO4 proteins required for the interaction with an ‘anchoring’ element, e.g. NRPE1 or the PolV lncRNA transcript. Following with this idea, we have undertaken a double approach: (i) We have performed IP-MS experiments of tagged AGO4 in the presence of WT or a V2 null mutant TYLCV to identify differential interacting partners; (ii) We have performed RIP experiments with tagged AGO4 in the presence of WT or a V2 null mutant TYLCV to evaluate the capacity of AGO4 to bind virus-derived RNA in the presence or absence of V2. The results of these experiments are now included in Supplementary file 2 and Figure 5B/Figure 5—figure supplement 1.As seen in new Supplementary file 2, peptides corresponding the NRPE1 can be detected in the AGO4 immunoprecipitates, and no significant quantitative differences can be observed between samples with or without V2. Nevertheless, since V2 might be affecting only a subpopulation of the total AGO4 pool, these results do not rule out the possibility that the viral protein might be interfering with the interaction of AGO4 with NRPE1 or other components of the RdDM machinery.The RIP experiments presented in Figure 5B/Figure 5—figure supplement 1 show a decrease in the binding of AGO4 to viral RNAs corresponding to the IR (non-coding) or the V2 coding sequence in the presence of the V2 protein. Therefore, the interaction with V2 seems to interfere with the association of AGO4 to the viral scaffold RNA, hence impairing its binding to the viral DNA and the subsequent recruitment of DRM2 and methylation of the viral genome.Likewise, it is important to provide unambiguous evidence that the effect reported may not be brought about by V2-HDA6 interaction. This is best achieved by providing a V2 mutant that may still bind HDA6, but is unable to bind to AGO4 and to suppress antiviral defense.We agree with the reviewers in that having a mutant V2 version enabling the uncoupling of the interactions with HDA6 and AGO4 would be an ideal tool to dissect the relative contribution of these targets to anti-viral DNA methylation; unfortunately, such a hypothetical mutant version is not currently available. Nevertheless, we have performed co-localization experiments between HDA6 and V2, which clearly demonstrate that HDA6 is not accumulated in the Cajal body ; the BiFC results shown in Wang et al., 2018, Figure 1B, support this finding, given that the detected interaction between HDA6 and V2 occurs outside of the nucleus. Since the suppressive effect of V2 on viral DNA methylation detected in this work requires localization of the viral protein in the Cajal body , this effect is most likely the result of the interaction with Cajal body-localized AGO4, and not with HDA6. It is possible that both AGO4 and HDA6 are targeted by V2 and contribute to different steps of viral DNA methylation.3) There is also concern about data variation in DNA methylation quantification. For example, the CG methylation percentages in Figure 4A are approximately 2% and 55% for 3dpi/TYLCV and 3dpi/TYLCV-V2null samples, respectively. In Figure 7B, the corresponding percentages are 8% and 22%. Great differences can also be found between and Figure 4C and Figure 7D (systemic infection) despite of the fact that plant materials used in Figure 4 and Figure 7 are comparable. This needs an explanation.The level of DNA methylation of the viral genome is the final result of the combined influence of a number of factors, reflecting the status of the dynamic interaction between virus and host plant at the time of sampling. Intrinsic variation can be detected between different batches of this experiments; nevertheless, all biological replicates include the appropriate controls, with which comparisons are made.Please note that we used at least 6 independent plants for each group of samples in each biological replicate, and at least three biological replicates were performed in Figure 4C and Figure 7E. As shown in Supplementary file 1 , although the absolute values of DNA methylation from each biological replicate are different, the same trends in relative values consistently emerge, supporting the reliability of the results.


Vascular endothelial barrier function is maintained by cell-to-cell junctional proteins and contributes to vascular homeostasis. Various risk factors such as inflammation disrupt barrier function through down-regulation of these proteins and promote vascular diseases such as atherosclerosis. Previous studies have demonstrated that aged garlic extract (AGE) and its sulfur-containing constituents exert the protective effects against several vascular diseases such as atherosclerosis. In this study, we examined whether AGE and its sulfur-containing constituents improve the endothelial barrier dysfunction elicited by a pro-inflammatory cytokine, Tumor-necrosis factor-α (TNF-α), and explored their mode of action on TNF-α signaling pathway.Human umbilical vein endothelial cells (HUVECs) were treated with test substances in the presence of TNF-α for various time periods. The endothelial permeability was measured by using a transwell permeability assay. The localization of cell-to-cell junctional proteins and actin cytoskeletons were visualized by immunostaining. RhoA and Rac activities were assessed by using GTP-binding protein pulldown assay. Gene and protein expression levels of signaling molecules were analyzed by real-time PCR and western blotting, respectively.S-1-propenylcysteine (S1PC), reduced hyperpermeability elicited by TNF-α in HUVECs. In addition, S1PC inhibited TNF-α-induced production of myosin light chain (MLC) kinase and inactivation of MLC phosphatase through the suppression of the Rac and RhoA signaling pathways, respectively, which resulted in the dephosphorylation of MLC2, a key factor of actin remodeling. Moreover, S1PC inhibited the phosphorylation and activation of guanine nucleotide exchange factor-H1 (GEF-H1), a common upstream key molecule and activator of Rac and RhoA. These effects of S1PC were accompanied by its ability to prevent the disruption of junctional proteins on the cell–cell contact regions and the increase of actin stress fibers induced by TNF-α.We found that AGE and its major sulfur-containing constituent, The present study suggested that AGE and its major constituent, S1PC, improve endothelial barrier disruption through the protection of junctional proteins on plasma membrane.Video abstract Vascular endothelial cells maintain vascular homeostasis by exerting a barrier function on vascular walls, preventing the infiltration of circulating cells and proteins in blood from vascular lumen into tissues , 2. ChroCell–cell junctions play an important role in the maintenance of endothelial barrier function and are organized by tight junctions (TJs), adherens junctions (AJs) and desmosomes in cell–cell contact regions . Among TEndothelial barrier dysfunction triggered by inflammation is the first step of various vascular diseases. Tumor necrosis factor-α (TNF-α) is known to disrupt endothelial cell–cell junctions and increase vascular permeability , 10. TheS-1-propenylcysteine (S1PC), S-allylcysteine (SAC) and S-allylmercaptocysteine (SAMC) [Aged garlic extract (AGE) is a unique garlic preparation that is produced by aging of raw garlic in an aqueous ethanol solution for more than 10 months. AGE has been shown to exert various biological and pharmacological activities such as anti-inflammatory , 21, ante (SAMC) , 36, whie (SAMC) , 37, 38.e (SAMC) , 39, 40.In the present study, we examined whether AGE and its sulfur-containing constituents affect the barrier function of human vascular endothelial cells. We found that S1PC, a major sulfur-containing constituent of AGE, improved TNF-α-induced endothelial barrier dysfunction by inhibiting both RhoA and Rac pathways through the suppression of the common upstream molecule, GEF-H1, in the TNF-α signaling.Allium sativum L.) was sliced into pieces, which was followed by immersing in ethanol 20–50% (v/v) and aging for more than 10 months at room temperature. S1PC, SAC and SAMC were synthesized according to the previous methods [AGE was prepared according to the previous report . Briefly methods , 42, and886), anti-GEF-H1, anti-RhoA, anti-phospho-Cofilin (Ser3), anti-Cofilin, anti-phospho-MYPT1 (Thr696), anti-MYPT1, anti-phospho-MLC2 (Ser19), anti-MLC2, horseradish peroxidase (HRP)-conjugated rabbit IgG, HRP-conjugated mouse IgG and HRP-conjugated rat IgG from Cell Signaling Technology ; anti-VE-cadherin, anti-ZO-1 and Donkey anti-rabbit IgG conjugated to Alexa Fluor 594 from Thermo Fisher Scientific ; anti-Rac1/2/3, anti-phospho-ETS domain-containing transcription factor 1 (Elk1) (Ser383) and anti-Elk1 from Santa cruz Biotechnology ; anti-TNFR2 and anti-TNFR-associated factor 2 (TRAF2) from Funakoshi ; anti-phospho-ERK1/2 (Thr202/Tyr204) and anti-ERK1/2 from BioLegend ; anti-GEF-H1 and anti-β-actin peroxidase conjugate from MBL life science ; anti-Claudin-5 from Bioworld Technology ; anti-MLCK from Abcam ; anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) peroxidase conjugate from WAKO pure chemical industries ; IgG from rabbit serum from Merck Millipore ; acti-stain 488 phalloidin from Cytoskeleton . Human recombinant TNF-α and human plasma-derived thrombin were purchased from WAKO pure chemical industries and Sigma Aldrich , respectively. Y-27632 and PD98059 were obtained from Merck Millipore and Thermo Fisher Scientific, respectively.The following antibodies were used in this study: anti-TNFR1, anti-phospho-GEF-H1  were purchased from Lonza  and grown to be confluent in endothelial cell basal medium-2 (Lonza) containing 2% fetal bovine serum albumin (BSA) and supplements attached to the medium kit in the presence of 5% CO3 cells/well on transwell inserts and cultured in a receiver plate. After grown to be confluent, HUVECs were stimulated with 50 ng/mL TNF-α or 1 U/mL thrombin in the absence or presence of test substances  for 24 h. In the case of the study using the inhibitors of RhoA and Rac signaling molecules, HUVECs were pre-treated with Y-27632  or PD98059  for 1 h before the TNF-α stimulation. Then, FITC-dextran was added on each transwell insert and the extent of permeability was determined by measuring the fluorescence of each receiver plate solution. The fluorescence intensity was quantified with EnVision plate reader  at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.Endothelial permeability was measured by using in vitro vascular permeability assay kit (Merck Millipore). HUVECs were seeded at 1.5 × 10After seeding on 24-well plates, HUVECs were stimulated with TNF-α 50 ng/mL in the absence or presence of test substances  for 10, 15, 20, 30, 40 min, 1, 3 or 24 h. In the case of the study using the inhibitors of RhoA and Rac signaling molecules, HUVECs were pre-treated with 10 μM Y-27632 or 30 μM PD98059 for 1 h before the TNF-α stimulation. Then, whole protein was extracted using Radioimmunoprecipitation assay (RIPA) buffer  containing protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). After dilution with SDS sample buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 0.005% bromophenol blue, 175 mM dithiothreitol), 10 μg of total protein was separated by SDS-PAGE and transferred onto nitrocellulose membrane . The transferred proteins were incubated with antibodies and immunoreactive proteins were detected with ECL prime western blotting detection system  or Supersignal west femto maximum sensitivity substrate (Thermo Fisher Scientific). Chemiluminescent signals were visualized with ChemiDoc MP imaging system (Bio-Rad Laboratories) and quantified using ImageLab software (Bio-Rad Laboratories).HUVECs were seeded on 12-well plates and stimulated with TNF-α 50 ng/mL in the absence or presence of 300 μM S1PC for 30 min or 24 h. Then, cell membrane and cytoplasmic proteins were separately isolated by using Mem-PER™ plus membrane protein extraction kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. The isolated membrane and cytoplasmic proteins were analyzed by western blotting. The total proteins were stained by using Quick-coomassie brilliant blue staining kit  according to the manufacturer’s instructions in order to normalize the membrane and cytoplasmic protein levels.Immunoprecipitation was performed to investigate the interaction between GEF-H1 and Rho family proteins. HUVECs were seeded on 10 cm dishes and stimulated with TNF-α 50 ng/mL in the absence or presence of 300 μM S1PC for 1 h. Then, whole proteins were isolated using RIPA buffer containing protease and phosphatase inhibitor cocktail. Cell lysates were immunoprecipitated using GEF-H1 antibody and protein G magnetic beads (Merck Millipore). The magnetic beads were washed 5 times by RIPA buffer and boiled in SDS sample buffer at 95 °C for 5 min. Finally, the released proteins from beads were analyzed by western blotting.To investigate the effect of S1PC on cellular localization of the junctional proteins and F-actin, these proteins were visualized by immunofluorescent staining. HUVECs were seeded on 8-well slide chambers  and stimulated with TNF-α 50 ng/mL in the absence or presence of 300 μM S1PC for 24 h. Then, the cells were fixed in 4% paraformaldehyde , permeabilizated with 0.3% TritonX-100 (Sigma Aldrich) in phosphate buffered saline  and blocked with 3% BSA (Sigma Aldrich) in PBS followed by the incubation with primary antibodies in 3% BSA/PBS. Then, the cells were treated with an Alexa Fluor 594-conjugated secondary antibody to detect junctional proteins. F-actin was labeled by the incubation with acti-stain 488 phalloidin. Finally, the cells were mounted using Vectashield medium-containing 4′,6-diamidino-2-phenylindole (DAPI) . The fluorescent staining was visualized with Biorevo Bz9000 fluorescence microscope  and analyzed using Bz-II analyzer software (Keyence).2+ lysis/wash buffer (MLB) containing protease and phosphatase inhibitor cocktail, the cells were incubated with glutathione-agarose beads (Sigma Aldrich) for 30 min at 4 °C. Then, the supernatant collected from the incubation medium was added with Rho or Rac assay reagent containing glutathione-agarose beads bound to fusion proteins that include Rho or Rac binding domain of each effector protein, and then gently agitated. After centrifugation, the precipitated bead pellets were washed 3 times using MLB and boiled in SDS sample buffer. Finally, the supernatants were analyzed by western blotting.The effects of S1PC on the activities of Rho family proteins were evaluated by using RhoA and Rac activation assay kits (Merck Millipore). HUVECs were seeded on 10 cm dishes and stimulated with TNF-α 50 ng/mL in the absence or presence of 300 μM S1PC for 1 h. After being detached using MgMLCK forward, 5ʹ-AGCCCGCTCAATGCAGAAAA-3ʹ; human MLCK reverse, 5ʹ-AGCAGCACTTCCCTCCACAA-3ʹ; human β-Actin forward, 5ʹ-CGCGAGAAGATGACCCAGAT-3ʹ; human β-Actin reverse, 5ʹ-GGTGAGGATCTTCATGAGGTAGTC-3ʹ. The relative mRNA expression level of MLCK to β-Actin was calculated using the comparative CT (ΔΔCT) method.HUVECs were seeded on 48-well plates and stimulated with TNF-α 50 ng/mL in the absence or presence of 300 μM S1PC for 9 h. Then, total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific) and used for the synthesis of the first-strand cDNA by using Primescript RT reagent kit with gDNA Eraser . The quantitative real-time PCR was performed with Piko Real Real-time PCR system  using SYBR premix Ex Taq II (Takara). The primer sequences used for PCR were as follows: human To examine whether GEF-H1 is a key molecule of TNF-α-activated RhoA and Rac signaling pathways, we prepared GEF-H1 knockdown HUVECs with siRNA. HUVECs seeded on 12-well plates were transfected with 9.6 pmol control siRNA  or GEF-H1-targeting siRNA  using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. At 48 h after the transfection, the cells were stimulated with TNF-α 50 ng/mL for 15, 30 min, 1 or 24 h, and the levels of protein expressions were evaluated by western blotting.Cell proliferation assay was performed to investigate the effect of S1PC on the proliferation of HUVECs by using Cell counting kit-8 . Briefly, HUVECs seeded on 96-well plates were treated with 75–300 μM S1PC for 48 or 72 h, and each well of the plates was added with CCK-8 regent and incubated for 1 h. The colorimetric absorbance of the wells was measured at 450 nm with Multiskan FC microplate reader (Thermo Fisher Scientific).P values less than 0.05 were considered to be statistically significant.Data were expressed as means ± standard deviation (SD) after outlier data were detected and removed using Thompson's rejection test. Bonferroni’s or Dunnett’s multiple comparison test was performed to assess statistically significant differences between groups by using WinSTAT statistics software  or KyPlot software , respectively. Differences at We first evaluated the effect of AGE on endothelial permeability of HUVECs by using a transwell permeability assay. The permeability was evaluated by fluorescently measuring the transport of FITC-dextran from transwell inserts to the receiver plate wells through the monolayered HUVECs. Stimulation with TNF-α 50 ng/mL for 24 h increased cellular permeability in HUVECs. AGE prevented the increase in the transport of FITC-dextran induced by TNF-α in a concentration-dependent manner  at Sertraction , 48, 49.383 [We investigated whether the inhibitory effect of S1PC on the GEF-H1 phosphorylation affects the Rac signaling. In this signaling pathway, Rac promotes ERK phosphorylation by activating MEK, and then ERK phosphorylates Elk1 at Ser383 . Followi383 . We firsWe investigated the effects of Y-27632, a ROCK inhibitor, and PD98059, a MEK1 inhibitor, on the RhoA and Rac signaling pathways, respectively, and vascular permeability in HUVECs. Y-27632 reduced the phosphorylation of MYPT but not the phosphorylation of ERK1/2 induced by TNF-α  in the N-terminal domain that is essential to activate both RhoA and Rac [RhoA and Rac comprise a family of Rho GTPase and their activities are regulated by various GEFs. GEF-H1 has been known to activate both RhoA and Rac in response to the stimulation of TNF-α and thrombin , 63, 64. and Rac . Our stu and Rac –24.Apart from the role in the regulation of endothelial junctions, GEF-H1 is involved in several cellular events such as cell division , 66, 67,This study showed that S1PC, a sulfur-containing constituent in AGE, protected against the TNF-α-induced disruption of endothelial junctions in HUVECs by suppressing the GEF-H1 activation in the signaling pathway. Vascular endothelial TJs act as a primary barrier for maintaining the homeostasis and function of blood vessels and organs. Accordingly, the proper maintenance of barrier function by S1PC may help protect the blood vessels against risk factors of vascular and organ diseases. In addition, the protective effect of S1PC on vascular cells may explain in part the beneficial effect of AGE on the atherosclerotic patients in the clinical studies –24.Additional file 1: Figure S1. Effects of AGE and its major sulfur-constituents on TNF-α-induced hyperpermeability of HUVECs. a The concentration-dependent effect of AGE on TNF-α-induced hyperpermeability of HUVECs. Vascular permeability assay was conducted after the stimulation with TNF-α (50 ng/mL) in the presence or absence of AGE  for 24 h. Data are shown as mean ± SD, n = 3–4. Significant difference compared to the control group (##p < 0.01) or TNF-α-treated group (**p < 0.01) was determined by Dunnett’s multiple comparison test. b Chemical structures of SAC and SAMC. c Effects of major sulfur-containing constituents of AGE on TNF-α-induced hyperpermeability of HUVECs. Vascular permeability was conducted after the stimulation with TNF-α (50 ng/mL) in the presence or absence of sulfur-containing constituents of AGE  for 24 h. Data are shown as mean ± SD, n = 4. Significant difference compared to the control group (#p < 0.05) or TNF-α-treated group (*p < 0.05) was determined by Bonferroni’s multiple comparison test.Additional file 2: Figure S2. The concentration-dependent effect of S1PC on thrombin-induced hyperpermeability of HUVECs. Vascular permeability assay was conducted after the stimulation with thrombin (1 U/mL) in the presence or absence of S1PC  for 24 h. Data are shown as mean ± SD, n = 4. Significant difference compared to the control group (##p < 0.01) or TNF-α-treated group (**p < 0.01) was determined by Dunnett’s multiple comparison test.Additional file 3: Figure S3. Effects of S1PC on protein levels of TNF receptors and TRAF2 in HUVECs. a Effects of S1PC on the protein levels of TNFR1 and TNFR2 in HUVECs. The membrane and cytoplasmic proteins were extracted after the stimulation with TNF-α (50 ng/mL) in the presence or absence of S1PC (300 μM) for 24 h and analyzed by western blotting with indicated antibodies. Quantitative data are shown as mean ± SD, n = 3. b Effect of S1PC on the protein level of adaptor protein TRAF2 in HUVECs. The membrane and cytoplasmic proteins were extracted after the stimulation with TNF-α (50 ng/mL) in the presence or absence of S1PC (300 μM) for 30 min and analyzed by western blotting with indicated antibodies. Quantitative data are shown as mean ± SD, n = 4.Additional file 4: Figure S4. Effects of major sulfur-containing constituents of AGE on TNF-α-induced GEF-H1 phosphorylation in HUVECs. Cell lysates were obtained after the stimulation with TNF-α (50 ng/mL) in the presence or absence of sulfur-containing constituents of AGE  for 30 min and analyzed by western blotting with indicated antibodies. Quantitative data are shown as mean ± SD, n = 3. Significant difference compared to the control group (##p < 0.01) or TNF-α-treated group (*p < 0.05) was determined by Bonferroni’s multiple comparison test.Additional file 5: Figure S5. Effects of S1PC on the cell proliferation and morphology of HUVECs. a Effect of S1PC on the cell proliferation in HUVECs. Cell viability was measured with cell proliferation assay system after the treatment with S1PC  for 48 and 72 h. Data are shown as mean ± SD, n = 3. b Effect of S1PC on the cell morphology in HUVECs. Cell nuclei were stained with DAPI to assess normal cytokinesis after the treatment with S1PC (300 μM) for 24 h. Specific fluorescence: blue for nuclei stained with DAPI. Scale bar, 100 μm.Additional file 6: Figure S6. Effects of GEF-H1 knockdown on downstream signaling of TNF-α, RhoA and Rac, in HUVECs. Effects of GEF-H1 knockdown on TNF-α-induced MLC2 phosphorylation (a), MYPT phosphorylation (b), ERK1/2 phosphorylation (c) and MLCK protein expression (d) were examined in HUVECs. HUVECs were transfected with control siRNA (siControl) or GEF-H1-targeting siRNA (siGEF-H1). Cell lysates were obtained after the stimulation with TNF-α (50 ng/mL) for 24 h  or indicated periods , and analyzed by western blotting with indicated antibodies. Quantitative data are shown as mean ± SD, n = 3. Significant difference compared to non-treatment siControl group  or TNF-α-treated siControl group with the same treatment time  was determined by Bonferroni’s multiple comparison test.Additional file 7: Figure S7. Effects of ROCK inhibitor on downstream signaling of TNF-α, RhoA and Rac, in HUVECs. Effects of ROCK inhibitor on TNF-α-induced phosphorylation of MYPT (upper graph) and ERK1/2 (lower graph) were examined in HUVECs. After the pre-treatment with ROCK inhibitor (10 μM Y-27632) for 1 h, HUVECs were stimulated with TNF-α (50 ng/mL) in the presence or absence of S1PC (300 μM) for 10, 20, 40 or 60 min. Cell lysates were analyzed by western blotting with indicated antibodies. Quantitative data are shown as mean ± SD, n = 3. Significant difference compared to the control group  or TNF-α-treated group with the same treatment time  was determined by Bonferroni’s multiple comparison test.Additional file 8: Figure S8. Effects of MEK1 inhibitor on downstream signaling of TNF-α, RhoA and Rac, in HUVECs. Effects of MEK1 inhibitor on TNF-α-induced protein expression of MLCK (a) and MYPT phosphorylation (b) were examined in HUVECs. After the pre-treatment with MEK1 inhibitor (30 μM PD98059) for 1 h, HUVECs were stimulated with TNF-α (50 ng/mL) in the presence or absence of S1PC (300 μM) for 24 h (a) or indicated periods (b). Cell lysates were analyzed by western blotting with indicated antibodies. Quantitative data are shown as mean ± SD, n = 3. Significant difference compared to the control group  or TNF-α-treated group with the same treatment time  was determined by Bonferroni’s multiple comparison test.Additional file 9: Figure S9. Effects of ROCK and MEK1 inhibitors on TNF-α-induced hyperpermeability of HUVECs. After the pre-treatment with ROCK (10 μM Y-27632 (a)) or MEK1 inhibitor (30 μM PD98059 (b)) for 1 h, HUVECs were stimulated with TNF-α (50 ng/mL) in the presence or absence of S1PC (300 μM) for 24 h, and then vascular permeability assay was conducted. Data are shown as mean ± SD, n = 3–4. Significant difference compared to the control group  or TNF-α-treated group (*p < 0.05) was determined by Bonferroni’s multiple comparison test. N.S. indicates no statistical significance (p > 0.05).


Heart failure with preserved (HFpEF) and mid-range ejection fraction (HFmrEF) constitute two heart failure categories, representing about 50–70% of the total . For botJournal of Clinical Medicine, we present seven works  addressing different and relevant aspects of HFpEF and HFmrEF. In the first review, Bayés-Genís and collaborators focus on the non-left ventricular mechanisms that play a crucial role in the transition from pre-HFpEF to symptomatic HFpEF. In this work, the authors discuss the role of the atria, right heart cavities, kidneys, and systemic inflammation on this transition [In this Special Issue of the ansition . On the ansition . In thisansition .In this issue, we also have the opportunity to present five original articles addressing important clinical aspects in HFpEF. First, Rettl R. et al. aimed to evaluate the proportion of patients who participated in a prospective national registry that fulfills the PARAGON-HF trial’s eligibility criteria . These sIn another article, Santas et al. point out the prognostic importance of right-heart failure in HFpEF in 1355 patients consecutively discharged for decompensated HFpEF. These authors propose a right-ventricular failure staging system by combining the tricuspid annular plane systolic excursion to pulmonary artery systolic pressure ratio with functional tricuspid regurgitation severity . ConsideAnother work published in this issue focuses on the clinical relevance of iron deficiency in determining the functional capacity and quality of life of HFpEF patients. With this purpose in mind, Alcaide-Aldeano A et al. analyzed different surrogate biomarkers of iron deficiency in 447 HFpEF patients .