Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-N6-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N6-dA, which is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N6-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N6-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N6-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER.DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[ Chemicals and radiation can damage DNA, resulting in structural alterations to the nitrogenous bases, deletions of genetic material, and breaks in chromosomes –4. The r–O6-methylguanine, a DNA lesion formed by methylating agents, partially blocks transcription elongation of human RNA polymerase and frequently directs insertion of a uridine nucleotide rather than a cytidine nucleotide into RNA . The first step in global NER, which distinguishes it from TCR, is DNA damage recognition that is accomplished in part by a protein complex in human cells called XPC/hR23B. The heterodimer binds to chemically modified bases in DNA that interrupt base pairing and distort the double helix, thus allowing global NER to remove a wide array of lesions from DNA , 19. FolTCR removes DNA damage from the transcription units of actively expressed genes; hence, this pathway, unlike global NER, operates on very specific regions of the genome , 23. TCRa]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph), both of which are known procarcinogens in humans (–trans and cis adducts. Among the adducts produced by B[a]P-diol epoxide and B[c]Ph-diol epoxide are (+)-trans-anti-B[a]P-N6-dA (B[a]P-N6-dA) and (+)-trans-anti-B[c]Ph-N6-dA (B[c]Ph-N6-dA) (A)  . PAHs arn humans –30. Certh-N6-dA) A 31). S. Sa]pyre) (A) 31–. The B[aobal NER . These sa]P-N6-dA and B[c]Ph-N6-dA adducts on transcription in primary human fibroblasts that are either DNA repair-proficient or lack global NER, TCR, or both. Either B[a]P-N6-dA or B[c]Ph-N6-dA was positioned on the transcribed strand of a reporter gene that encodes red fluorescent protein (RFP), specifically within the 5′-UTR of the gene's transcription unit. Human fibroblasts were transfected with the site-specifically modified DNA or an unmodified control. The quantity of RFP mRNA and the production of functional RFP were monitored over time. The results obtained for the site-specifically modified DNA were then compared with those of the corresponding, unmodified control DNA.An obvious implication of the existence of TCR is that DNA adducts that are typically refractory to global NER but pose blocks to transcription could be subject to TCR. The work reported here examined the effects of BP-N6-dA lesion (Templates suitable for escribed . In brieescribed . The 11-ectively B. T4 DNAA lesion .In vitro transcription reactions were performed using the HeLaScribe® nuclear extract in vitro transcription system (Promega) as the source of hRNAPII and other essential transcription factors (m HEPES (pH 7.9), 100 mm KCl, 0.2 mm EDTA, 0.5 mm DTT, 20% glycerol), 400 μm ATP, 400 μm GTP, 400 μm UTP, 16 μm [α-32P]CTP (∼25 Ci/mmol), and 8 units of HeLa nuclear extract. The mixture was incubated at 30 °C and quenched at an appropriate time with HeLaScribe® kit stop solution (0.3 m Tris-HCl (pH 7.4 at 25 °C), 0.3 m sodium acetate, 0.5% SDS, 2 mm EDTA, 3 μg/ml tRNA). RNA was isolated by extraction with phenol/chloroform/isoamyl alcohol  followed by ethanol precipitation. The RNA was resuspended in nuclease-free water, mixed with an equal volume of loading dye , denatured at 90 °C for 10 min, and resolved with 7% denaturing PAGE using 8 m urea at 2,000 V for ∼3.5 h. The gel was dried and exposed to a BAS-IP MS 2040 E multipurpose standard storage phosphor screen . The screen was scanned using an FLA Typhoon 9000 Imager . The transcripts were quantified using band densitometry analysis in Fiji  was transformed with plasmid WLZG-I-BsiWI-R. Log phase cultures of the transformed bacteria were superinfected with M13 helper phage VCSM13  at a multiplicity of infection greater than 10:1 phage/bacteria. Infected cultures were grown overnight at 37 °C in 2× YT medium ). Bacteria were pelleted, and bacteriophage that contained single-stranded DNA were recovered by polyethylene glycol precipitation. Single-stranded, circular DNA was recovered from helper phage by phenol extraction.Site-specific, modified vectors were synthesized by using a gapped duplex method  that inv38–m DTT for 4 h at 37 °C. Linear pWLZG-I-Insert-R was mixed with single-stranded, closed circular WLZG-I-BsiWI-R at a molar ratio of 1:5 to form the gapped duplex. The DNA mixture was denatured by the addition of 1 m NaOH to a final concentration of 0.3 m and incubated at room temperature for 15 min. The mixture was neutralized with 3 m MOPS free acid to reach a final concentration of 0.4 m MOPS. NaCl and EDTA were added to final concentrations of 0.75 m and 1 mm, respectively, and buffered phenol was added to 10% of the final volume. The reaction was mixed rapidly with a micropipette to form a phenol emulsion that was cycled between 0 and 65 °C at 90 s/cycle for 45 min to foster association between the transcribed strand of linear pWLZG-I-Insert-R and the single-stranded, closed circular WLZG-BsiWI-R. This resulted in the formation of duplex DNA with an 11-nucleotide gap on the transcribed strand of the vector. The gapped duplex DNA was purified by eliminating excess single-stranded DNA using benzoylated naphthoylated DEAE-cellulose (Sigma-Aldrich) followed by phenol/chloroform extraction (A modified oscillating phenol reassociation technique was used to generate gapped duplex DNA . In brietraction .a]P-N6-dA or B[c]Ph-N6-dA (44–m ATP. The resulting oligomer was ligated into the gapped duplex using a 3:1 molar ratio of oligomer to gapped duplex DNA for 16 h at 16 °C in the presence of T4 DNA ligase (Bayou Biolabs) and 1 mm ATP. The mixture was extracted three times with 1% Triton X-114 equilibrated with 10 mm Tris-HCl, 1 mm Na2EDTA (pH 7.8 at 25 °C) (TE), precipitated by centrifugation, and resuspended in TE. The resulting DNA was purified by CsCl gradient ultracentrifugation (1.01 g/ml water) in the presence of ethidium bromide (0.4 mg/ml). The final product, WLZG-I-B[a]P-R or WLZG-I-B[c]Ph-R, was a covalently closed circular plasmid containing the DNA sequence of the control vector (WLZG-I-BsiWI-R) with a single modified base. The presence of the damaged base was verified by resistance to digestion with restriction endonucleases (Thermo Fisher Scientific).A DNA oligomer 5′-CTCGTACGCTC-3′ containing either BP-N6-dA adduct (32P]phosphate via an exchange reaction by incubating them with 83 μm [γ-32P]ATP (∼3 Ci/mol) and 100 μm ADP in the presence of T4 polynucleotide kinase. The products were resolved using 7% PAGE in the presence of 8 m urea. Complete template ligation should have resulted in oligomers that were 187, 191, 950, and 958 bases in length following I-PpoI digestion, whereas incomplete ligation should have resulted in additional oligomers 110 and 118 bases in length. As shown in Fig. 3A, bands in the vicinity of 110 and 118 bases were absent following I-PpoI digestion, both for the template modified with B[a]P-N6-dA and for unmodified control DNA. These results show that template ligation went to completion, at least within the assay's limits of detection, and that no nicks were present.Following template synthesis, it was essential to ensure that complete ligation had occurred; nicks in the template's backbone could impede transcription, confounding the results sought for the B[A adduct . To testa]P-N6-dA adduct following template synthesis. To do this, the site-specific B[a]P-N6-dA was positioned within a BsiWI restriction site, making the region insensitive to cutting by BsiWI relative to a control template lacking the adduct. Unmodified, control template or template containing B[a]P-N6-dA was digested with BsiWI. The resulting oligomers were labeled with [32P]phosphate and resolved using denaturing PAGE as described earlier. BsiWI digestion of the DNA template lacking the B[a]P-N6-dA adduct should result in three DNA fragments 65, 69, and 1076 bases in length following denaturation. Bands ∼65 and 69 bases in length were present following digestion of the unmodified, control DNA as predicted . Note that the larger bands are not shown on the gel. In contrast, BsiWI digestion of the DNA template containing a site-specific B[a]P-N6-dA adduct within the BsiWI restriction site resulted in no DNA fragments that were 65 and 69 bases in length, indicating that the adduct was present following template synthesis .In addition to ensuring complete vector ligation, it was imperative to test for the presence of the BCTP among the nucleotides needed for RNA synthesis. The HeLa nuclear extract was tested for activity by using a DNA template supplied by the manufacturer, which encodes a transcript 363 bases in length. Production of RNA approximating this length was observed only when the manufacturer's template was incubated with HeLa nuclear extract and rNTPs, demonstrating that the extract was indeed active (lanes 9–12).After template integrity was confirmed, transcription was examined In vitro transcription using undamaged, control DNA template, should result in the production of full-length, run-off RNA that is 384 bases in length when hRNAPII initiates at the +1 start site associated with the CMV promoter. In contrast, transcription using the DNA template with a B[a]P-N6-dA lesion positioned at the +317 site should result in RNA ∼317 bases in length if the adduct were to impede elongation. Transcription of the control template in which all necessary components of the reaction were present resulted in the production of RNA 380 bases in length, which approximates the length of expected run-off transcription (lane 4). Reactions in which one critical component was absent  resulted in no visible RNA approximating 380 bases in length (lanes 1–3). These results indicate that the synthesized template lacking B[a]P-N6-dA supports transcription by hRNAPII, resulting in a run-off transcript of the expected size. Note that synthesis of the larger transcripts in lanes with HeLa nuclear extract is a function of the extract itself.cription , lane 4.a]P-N6-dA adduct produced a single band of ∼320 bases, significantly smaller than that produced from the unmodified template and approximating the 317-base RNA that would result from hRNAPII stalling at B[a]P-N6-dA (lane 8). Note again that reactions lacking one critical component produced no RNA in the vicinity of 320 or 380 bases (lanes 5–7). These results clearly indicate that the B[a]P-N6-dA adduct poses a strong block to hRNAPII during elongation, suggesting that this lesion should be subject to TCR in cells.In contrast to the results obtained for the unmodified control template, transcription of the template containing a site-specifically modified BP-N6-dA , lane 8.a]P-N6-dA or B[c]Ph-N6-dA, primary human fibroblasts were transfected with site-specifically damaged vectors that lacked identifiable eukaryotic origins of replication (a]P-N6-dA or a B[c]Ph-N6-dA adduct was positioned on the transcribed strand of the RFP gene within the 5′-UTR (−/− human fibroblasts (GM05509) (−/− human fibroblasts (GM02993) (−/− human fibroblasts (GM01629)  , XPC−/− GM02993) , and CSBasts GM019 (62)  contained a single XhoI restriction site and a single BsiWI restriction site. The B[a]P-N6-dA adduct and the B[c]Ph-N6-dA adduct were positioned within the BsiWI site, making it refractory to cutting by the BsiWI restriction endonuclease. Hence, each vector should be sensitive to cutting with XhoI, but only the unmodified control vector should be sensitive to cutting with BsiWI as well. Each of the three vectors was cut with XhoI and BsiWI or with BsiWI alone. The results are presented in Prior to transfection experiments, the integrity of the DNA constructs was examined to ensure that the DNA adduct was intact following vector synthesis. Each of the three vectors .Characterization of the DNA construct containing the B[a]P-N6-dA and B[c]Ph-N6-dA lesions were plotted as a function of time for each cell line . However, the B[c]Ph-N6-dA adduct exerted a significantly adverse effect on transcription of the RFP gene in human cells proficient in DNA repair when compared with transcription of the unmodified, control vector (p < 0.02) (A). Furthermore, the B[a]P-N6-dA and B[c]Ph-N6-dA transcription time courses were also significantly different from one another in normal human fibroblasts, with less RFP mRNA resulting from the vector containing B[c]Ph-N6-dA when compared with B[a]P-N6-dA (p < 0.01). These results suggest that the B[a]P-N6-dA adduct is rapidly removed from DNA or bypassed during transcription in cells that are proficient in DNA repair. In contrast, the detrimental effects of the B[c]Ph-N6-dA adduct on transcription persist in normal cells, interfering with mRNA synthesis for at least 18 h following activation of gene expression in cells that are proficient in both global NER and TCR.In DNA repair-proficient normal human fibroblasts, the B[ < 0.02) A. Furthea]P-N6-dA and B[c]Ph-N6-dA exert severe, adverse effects on the production of RFP mRNA when compared with an undamaged control vector  (B). The quantity of RFP mRNA obtained from a vector containing B[c]Ph-N6-dA appeared to be slightly lower than that obtained from a vector containing B[a]P-N6-dA, although the difference was not statistically significant (p < 0.06). These data suggest that the XPA protein, which is involved in both global NER and TCR, is necessary to ameliorate the negative effects of both B[a]P-N6-dA and B[c]Ph-N6-dA on transcription. The data also show that a small amount of full-length mRNA is made via transcription of the template even in the absence of global NER and TCR.In primary human fibroblasts derived from patients with XP, complementation group A, that are deficient in both global NER and TCR, both B[ctively) B. The qua]P-N6-dA and B[c]Ph-N6-dA exert a significantly negative effect on the production of RFP mRNA compared with an undamaged control vector  (C). Importantly, there was no significant difference between the effects of B[a]P-N6-dA and B[c]Ph-N6-dA on RNA synthesis when the two damaged vectors were compared with one another (p < 0.4). For the B[c]Ph-N6-dA lesion, the RFP mRNA increases to levels comparable with that in normal cells, but the overall deleterious effect is still significant. These data indicate that the XPC protein, which is absent from these cells, assists significantly in negating the detrimental effects of B[a]P-N6-dA on transcription but has a nominal impact on the effect of B[c]Ph-N6-dA.In primary human fibroblasts derived from patients with XP, complementation group C, that are defective in global NER but proficient in TCR, both B[ctively) C. Importa]P-N6-dA adduct had no significant effect on transcription (p < 0.5), whereas the B[c]Ph-N6-dA adduct impeded transcription to a very large and significant extent (p < 0.01) (D). The transcript levels produced from vectors containing either B[a]P-N6-dA or B[c]Ph-N6-dA were also significantly different from one another (p < 0.01).In primary human fibroblasts derived from patients with CS, complementation group B, that are defective in TCR but proficient in global NER, the B[ < 0.01) D. The tra]P-N6-dA and B[c]Ph-N6-dA to reactivate the RFP gene? If TCR were acting on the B[c]Ph-N6-dA lesion, there should be significantly better recovery of RFP gene expression in normal human fibroblasts or XPC−/− cells in which TCR operates when compared with XPA−/− or CSB−/− cells in which TCR is defective. Comparisons for transcription of the vector containing a B[c]Ph-N6-dA lesion show that RFP mRNA quantities were significantly higher in XPC−/− cells compared with XPA−/− cells (p < 0.01) or compared with CSB−/− cells (p < 0.01). These comparisons show that TCR operates on B[c]Ph-N6-dA to a significant extent, a conclusion that is also borne out by the RFP that is made as shown below. In contrast, it is less clear that TCR operates efficiently on B[a]P-N6-dA. For RFP mRNA recovery from the vector containing B[a]P-N6-dA, XPC−/− cells were not significantly better at reactivating gene expression when compared with XPA−/− cells (p < 0.1). Although the results were not statistically significant by SMCV analysis, the quantity of RFP mRNA generated relative to the control at 18 h was clearly higher in XPC−/− cells (29.5 ± 10.4%) than in XPA−/− cells (12.0 ± 3.9%). This supports the notion that TCR does operate on the B[a]P-N6-dA adduct, but significantly observable differences are seen only at later time points.An important question emerges. Is TCR operating on B[a]P-N6-dA or B[c]Ph-N6-dA adduct within the 5′-UTR of the RFP gene's transcription unit. RFP activity following transfection was measured using FACS.Detection of full-length RFP mRNA in the transfection experiments suggests that RFP should be made even in cells in which the quantity of RFP mRNA was quite low. To test this, primary human fibroblasts of known DNA repair status were transfected with unmodified control vectors or those containing a B[a]P-N6-dA lesion was not significantly different from the RFP signal from a control vector (p = 0.99), consistent with the RFP mRNA data , also consistent with the RFP mRNA data, but the difference was not statistically significant for these data (p = 0.41). These results indicate that the B[c]Ph-N6-dA adduct is less well tolerated than B[a]P-N6-dA, even when global NER and TCR are operative.In normal human fibroblasts proficient in both global NER and TCR, the RFP signal generated from a vector containing a BP-N6-dA or B[c]Ph-N6-dA adduct were significantly lower than RFP from a control vector . These results indicate that the XPA protein is important in abrogating the impact of each of these adducts on transcription, more than likely by assisting in their removal via global NER and TCR. Interestingly, the RFP signal from a B[c]Ph-N6-dA vector was significantly lower than that from the B[a]P-N6-dA vector (p = 0.00011), suggesting that B[a]P-N6-dA is either more effectively bypassed by hRNAPII than B[c]Ph-N6-dA or potentially that B[a]P-N6-dA is slowly processed by additional repair pathways that do not require the XPA protein.In XPA−/− cells, which lack global NER but are proficient in TCR, the RFP signals obtained from vectors containing either a B[a]P-N6-dA or B[c]Ph-N6-dA adduct were both lower than the RFP signal obtained from the control vector ; however, only the B[c]Ph-N6-dA adduct produced results of statistical significance when compared with the control (p = 0.023).In XPC−/− cells that lack TCR, there is no significant effect of a B[a]P-N6-dA adduct on RFP fluorescence. In marked contrast, very little RFP signal results following transfection with a vector containing B[c]Ph-N6-dA (p = 1.2 × 10−8). In addition, the RFP signal resulting from the B[c]Ph-N6-dA-modified vector was significantly lower than that obtained from the B[a]P-N6-dA-modified vector. This suggests that the CSB protein is involved in diminishing the effects of B[c]Ph-N6-dA on transcription, and the effect is far more pronounced than that for B[a]P-N6-dA.In CSBa]P-N6-dA or B[c]Ph-N6-dA lesion, the differences in RFP fluorescence between cell types were elucidated. Note that protein recovery followed a similar pattern to that for RFP mRNA expression.To consider further the effect of TCR on recovery of transcription from templates with a B[c]Ph-N6-dA, significantly higher RFP recovery was observed in normal fibroblasts compared with XPA−/− cells that are deficient in global NER and TCR (p = 4.2 × 10−6) and CSB−/− cells that are only deficient in TCR (p = 0.0014). Indeed, RFP levels were greater in XPC−/− cells than in XPA−/− cells (p = 0.00010) and CSB−/− cells (p = 0.030), a reflection of the ability of XPC−/− cells to carry out TCR that is compromised in both XPA−/− cells and CSB−/− cells.For the vector containing B[a]P-N6-dA, significantly better recovery of RFP mRNA and RFP would be expected in XPC−/− cells in which TCR could operate on the lesion, especially in comparison with recovery in XPA−/− cells in which both NER and TCR are absent. Indeed, RFP fluorescence from cells transfected with the vector containing B[a]P-N6-dA was 2.6-fold higher in XPC−/− cells relative to XPA−/− cells, but the difference was not statistically significant (p = 0.34). However, the observed trend for these data is in the expected direction, again suggesting that repair pathways other than NER operate on B[a]P-N6-dA. This is in sharp contrast to recovery of RNA synthesis from a transcription unit containing B[c]Ph-N6-dA in which TCR appears to be the principal facilitator of its removal.For the vector containing BP-N6-dA and B[c]Ph-N6-dA, were examined using cell extracts and primary human fibroblasts. B[a]P-N6-dA poses a strong block to hRNAPII progression in vitro and is an excellent substrate for NER in vitro -2-acetylaminofluorene adduct, also escapes global NER and requires TCR for repair in SV40 transformed skin fibroblasts (In this study, the in vitro , 65. In in vitro , is a rein vitro . The resroblasts . These ra]P-N6-dA, where no altered transcripts were observed. However, in cells lacking functional XPA protein, which makes them deficient in NER, both globally and via TCR, a relatively small number of full-length transcripts was observed, but the mRNA contained significant base misincorporations, with adenosine or cytidine inserted at the position opposite B[a]P-N6-dA or adenosine and guanosine or cytidine inserted at the position opposite B[c]Ph-N6-dA. In addition deletions were seen. Indeed, base misincorporations and deletions in mRNA were also observed in other cell lines deficient in DNA repair for transcription of vectors containing B[a]P-N6-dA or B[c]Ph-N6-dA (In addition to its role in the recovery of gene expression following damage to the genome, DNA repair protects the integrity of the resulting transcripts by minimizing transcriptional mutagenesis. Qualitative, preliminary data from deep sequencing of transcripts recovered from the cell lines used in this study show that the full-length transcripts resulting from expression of the site-specifically modified RFP gene were mostly normal in human fibroblasts that are proficient in global NER and TCR, particularly for the adduct B[Ph-N6-dA .in vitro transcription data show that some bypass of B[c]Ph-N6-dA by hRNAPII does occur (–A plausible explanation for the generation of full-length transcripts is slow, error-free, or erroneous bypass of the lesion by hRNAPII, as previously documented in the case of cyclobutane pyrimidine dimers induced by ultraviolet radiation and oxidative DNA damage –69. In f–es occur . Alternaes occur . Anotheres occur , mismatces occur , or nucles occur . Finallyes occur –76.a]P-N6-dA and B[c]Ph-N6-dA on transcription in cells with varying DNA repair phenotypes. B[a]P-N6-dA is accessible to global NER, making TCR less relevant for its removal. In contrast, the B[c]Ph-N6-dA adduct adopts a conformation that allows it to escape detection by global NER, resulting in its persistence within the genome. However, when B[c]Ph-N6-dA is located on the transcribed strand within an expressed transcription unit, its ability to block RNA polymerase elongation results in TCR acting on the adduct. These results are consistent with the relatively high tumorigenic potential observed for B[c]Ph relative to B[a]P, since some adducts formed by the former PAH are more persistent in the genome.In The results reported here show that TCR ameliorates the deleterious effect of DNA adducts that block transcription, presumably by enhancing their removal from active genes even when they escape repair by global NER and perhaps other DNA pathways as well. Furthermore, the absence of global NER, TCR, or both results in decreased gene expression and alterations to the sequence of the mRNA produced. Hence, TCR protects transcription units from the dire consequences of transcription-blocking DNA adducts by reactivating gene expression and protecting the integrity of the resulting transcripts, including lesions that might be resistant to repair via other pathways.in vitro transcription reactions. L. C. prepared vector DNA for the studies in cells. C. B. supplied parent plasmids and collaborated on the design of the vectors used in this research. N. E. G. synthesized and contributed the site-specifically modified DNA. All authors approved the final version of the manuscript.A. N., J. A. B., and D. A. S. designed the study and wrote the paper. A. G. collaborated on the statistical analysis of the data. M. A. C. carried out


The eukaryotic translation factor eEF1A assists replication of many RNA viruses by various mechanisms. Here we show that down-regulation of eEF1A restricts the expression of viral genomic RNA and the release of infectious virus, demonstrating a biological requirement for eEF1A in the respiratory syncytial virus (RSV) life cycle. The key proteins in the replicase/transcriptase complex of RSV; the nucleocapsid (N) protein, phosphoprotein (P) and matrix (M) protein, all associate with eEF1A in RSV infected cells, although N is the strongest binding partner. Using individually expressed proteins, N, but not P or M bound to eEF1A. This study demonstrates a novel interaction between eEF1A and the RSV replication complex, through binding to N protein, to facilitate genomic RNA synthesis and virus production. Human translation elongation factor eEF1A, is a subunit of the eukaryotic translation elongation 1 complex (eEF1) The virions of non-segmented negative-strand RNA viruses,  contain a virally-encoded RNA-dependent-RNA polymerase , which transcribes viral genomic RNA into discrete mRNAs, which encode viral proteins. The RdRp also synthesizes full-length positive-strand antigenome which is used as a template for the synthesis of new negative-strand genomes. A rhabdovirus from this order, vesicular stomatitis virus (VSV), forms two RdRp complexes; the transcriptase which incorporates eEF1A and a virus-encoded phosphoprotein (P), and the replicase which does not incorporate eEF1A, but rather the P and a virus-encoded nucleocapsid (N) protein Paramyxoviridae family, also from the mononegavirales order, and causes respiratory disease in infants, young children and the elderly worldwide Respiratory syncytial virus (RSV) is a member of the siRNA molecules targeting the eEF1A mRNA transcript  or a universal siRNA negative control  were transfected into HEK293T cells with Lipofectamine RNAiMAX according to the manufacturer's protocol (Life Technologies). An additional control of untreated cells was included. Cells were incubated with siRNA molecules at 37°C for 48 h to ensure maximal down-regulation of eEF1A prior to RSV infection. Three replicate cultures of untreated, siMM or sieEF1A siRNA-transfected cells were maintained in parallel with RSV-infected cultures for the infection period of a further 48 h. The efficiency of eEF1A down-regulation was quantified by western blot analysis and densitometry using ImageJ RSV  was propagated in human epithelial carcinoma cells (HEp-2a) and a stock for infection experiments was purified through a sucrose cushion (21) or random hexamer (Life Technologies) primers. qPCR was performed using dual labeled probe/primer sets (Sigma-Aldrich) specific for N mRNA or the N-P region of the RSV genome, and also cellular β-actin as an internal control , and 500 ng total RNA was used to generate cDNA with either oligo-dT control . mRNA anCell lysates were prepared in Pierce lysis buffer (Thermo Scientific) and total protein was quantified using a Bradford assay (Bio-Rad). Proteins were separated by SDS-PAGE, transferred to PVDF membranes and probed using rabbit anti-eEF1A polyclonal (Santa Cruz), goat anti-RSV polyclonal (Virostat) to detect RSV N, P and M, mouse anti-FLAG monoclonal (Sigma-Aldrich) to detect FLAG-tagged M protein, or mouse anti-β-tubulin as a loading control.in situ to proteins within fixed and permeabilized cells on a chamber slide. Then, secondary antibodies conjugated with oligonucleotides (PLA probe PLUS and PLA probe MINUS) were added. The two PLA probes ligate if they are in close proximity, and allow a polymerase-driven amplification reaction of fluorescent-labeled oligonucleotides. Images were taken using a confocal microscope and the number of positive foci quantified for 50 cells/reaction using the Duolink Imagetool software (Olink Biosciences). Antibodies to the human transcription initiation factor eIF3A were used as a negative control for proximity to RSV N.Uninfected (control) and RSV-infected (MOI 1 pfu/cell) A549 cells were fixed with cold acetone/methanol (1∶1) 24 h after infection and proximity assays performed using the Duolink II system and PLA probes (Olink Bioscience). Monoclonal primary antibodies specific for eEF1A (Santa Cruz) and either RSV N or P proteins (Abcam) were allowed to bind pTM plasmids expressing either RSV N or P (a gift from Peter Collins) were used to transfect BSR-T7 cells RSV-infected HEK293T and A549 cells, or cells transfected and expressing RSV N, P or M proteins were lysed in S100 buffer containing 0.3% Tween-20 and then incubated with Dynabeads M-270 Epoxy beads (Life Technologies) coupled to anti-eEF1A antibody (Santa Cruz) for 1 h at 4°C with rotation. Cell lysates were also incubated with uncoupled beads, or beads coupled to BSA or antibody to a control eIF3A (Santa Cruz). Beads were washed 3 times in PBS and the proteins were eluted by boiling in 1× SDS-PAGE loading buffer. Eluted proteins were separated by SDS-PAGE electrophoresis and western blot analysis was performed to detect immunoprecipitated eEF1A and RSV N, P and M proteins.Data were analysed for statistical differences using either paired Welch's t test from at least 3 independent experiments or measurements, or two way ANOVA.P<0.05) reduction of viral titres 48 h post-infection (p.i.) compared to cells either not transfected or transfected with siMM (We previously used siRNA to down-regulate eEF1A in HIV-1 infected cells to demonstrate that eEF1A is a component of the HIV-1 reverse transcriptase complex and an important factor in HIV-1 early replication ith siMM . While sith siMM  and 1C. ith siMM . AlthougP<0.05) detectable viral genomic RNA (P<0.05) in the ratio of N mRNA/genomic RNA was detected when eEF1A expression was reduced  were identified in infected cells, compared to ligation foci for RSV N or P with eIF3 (A proximity ligation assay (PLA) was performed to demonstrate the association of eEF1A with the N and P proteins of the RSV replication complex. RSV-infected A549 cells were fixed at 24 h p.i. and the PLA assay was performed using anti-eEF1A and anti-N, or anti-eEF1A and anti-P antibodies. As a control, anti-eEF1A antibody was replaced with anti-eIF3 antibody. A significant number of ligation foci for RSV N or P with eEF1A  is part of the translation elongation factor 1 complex that also contains valyl-tRNA and eEF1B, which itself is comprised of eEF1G, eEF1B and eEF1D. The role of eEF1B is to exchange GDP for GTP on eEF1A so that it can bind and deliver aminoacylated transfer RNA (aa-tRNA) to the elongating ribosome. eEF1A is an abundant and multifunction protein as it also plays a non-classical role in nucleocytopalsmic trafficking Here we focused on the role of eEF1A, rather than eEF1G, in RSV replication, as the overwhelming majority of published research suggests that eEF1A is the principal functional cellular partner involved in viral replication. We have previously shown an association between HIV-1 reverse transcriptase and eEF1G Our co-IP studies demonstrated that RSV N binds directly to eEF1A, and indirectly to P within the RNP. We cannot rule out the possibility that eEF1A binds directly to the viral polymerase (L), although the molecular tools to investigate this aspect are not readily available. We have also shown an indirect association between eEF1A and M. As the M protein is involved in virion assembly, this is not surprising. Indeed, the M protein has previously been co-precipitated with N Similarly to our observations here with RSV, down-regulation of the eEF1A plant homolog by gene silencing significantly reduced viral RNA levels and the spread of TBSV infection


DNA methylation in mammals is an epigenetic mark necessary for normal embryogenesis. During development active loss of methylation occurs in the male pronucleus during the first cell cycle after fertilisation. This is accompanied by major chromatin remodelling and generates a marked asymmetry between the paternal and maternal genomes. The mechanism(s) by which this is achieved implicate, among others, base excision repair (BER) components and more recently a major role for TET3 hydroxylase. To investigate these methylation dynamics further we have analysed DNA methylation and hydroxymethylation in fertilised mouse oocytes by indirect immunofluorescence (IF) and evaluated the relative contribution of different candidate factors for active demethylation in knock-out zygotes by three-dimensional imaging and IF semi-quantification.We find two distinct phases of loss of paternal methylation in the zygote, one prior to and another coincident with, but not dependent on, DNA replication. TET3-mediated hydroxymethylation is limited to the replication associated second phase of demethylation. Analysis of cytosine deaminase (AID) null fertilised oocytes revealed a role for this enzyme in the second phase of loss of paternal methylation, which is independent from hydroxymethylation. Investigation into the possible repair pathways involved supports a role for AID-mediated cytosine deamination with subsequent U-G mismatch long-patch BER by UNG2 while no evidence could be found for an involvement of TDG.There are two observable phases of DNA demethylation in the mouse zygote, before and coincident with DNA replication. TET3 is only involved in the second phase of loss of methylation. Cytosine deamination and long-patch BER mediated by UNG2 appear to independently contribute to this second phase of active demethylation. Further work will be necessary to elucidate the mechanism(s) involved in the first phase of active demethylation that will potentially involve activities required for early sperm chromatin remodelling. To date, the most clear examples of active DNA demethylation take place during the very early steps of mammalian development, namely in the zygote where the paternal genome undergoes a massive wave of loss of 5-methylcytosine (5mC) right after fertilisation [DNA methylation is an important epigenetic mark involved in gene silencing, X chromosome and transposon inactivation, genomic imprinting, and chromosome stability. DNA methylation is subject to reprogramming during development, involving both demethylation (active and passive) and lisation -4.Within 1 h of fertilisation, the paternal genome goes through major chromatin remodelling, loses protamines and is re-packaged by maternal nucleosomal histones, forming the paternal pronucleus ,6. Post-in vitro[The methyl-CpG-binding domain protein 2 (MBD2) was reported to possess direct demethylase activity , but thein vitro. Howeverin vitro.In vitro assays have shown AID has 5mC deaminase activity, resulting in thymine and, therefore, T-G mismatches in DNA, which can be effectively repaired through the BER pathway [An alternative to direct removal of 5mC by a DNA glycosylase is enzymatic deamination of 5mC to thymine, followed by T-G mismatch specific BER that replaces thymine with cytosine . Two cla pathway . Cytosin pathway ). Recent pathway ,30-32. M pathway ,33, a me pathway , suggestWe have previously characterised the dynamics of DNA methylation loss during the first cell cycle in the mouse by indirect immunofluorescence ,35. SincE. coli, negative DNA supercoiling, and hence generation of single-stranded DNA, enhances AID mutagenesis [We have established a protocol that allows us to reproducibly semi-quantify the staining levels of both DNA methylation and hydroxymethylation in pre-implantation embryos  and o system . Moreoveo system . These po system . Oocyteso system  which prAs a whole, our results support a role for both 5mC hydroxymethylation and cytosine deamination, followed by LP BER, as demethylation mechanisms in the mouse zygote. Both these mechanisms seem to contribute independently to a decrease in paternal DNA methylation coincident, but not reliant, on DNA replication. Furthermore, an initial loss of paternal DNA methylation, prior to S-phase, seems to take place, for which none of the activities investigated in this work seem to supply an explanation. It is likely that this early demethylation is related to the need for major chromatin remodelling of the sperm within the first hours post fertilisation and may involve other pathways of DNA repair. NER has been suggested as a candidate for DNA demethylation , remOur analysis shows that there are two observable phases of active DNA demethylation in the mouse zygote, before and coincident with DNA replication. We further show that TET3 seems to be involved only in the second phase of loss of methylation. Cytosine deamination and uracil BER seem to also independently contribute to this second phase of active demethylation. Together these findings allow us to conclude that demethylation is achieved by at least two parallel mechanisms which may or may not be partially redundant. Our results highlight the dynamic nature of DNA demethylation, with two apparent distinct stages (Phase I and Phase II). The major increase in 5hmC (Phase II) seems to be uncoupled from the initial loss of DNA methylation (Phase I) which was not previously acknowledged. Still, further work will be necessary to elucidate the mechanism(s) involved in the first phase of demethylation, likely to involve activities required for early chromatin remodelling on fertilisation and perhaps other types of DNA repair.To our knowledge, this is the first report of possible involvement of LP BER in DNA demethylation, opening new avenues of investigation not formerly considered.All experimental procedures were approved by the Animal Welfare, Experimentation and Ethics Committee (AWEEC) at the Babraham Institute and were performed under licenses by the Home Office (UK) in accordance with the Animals (Scientific Procedures) Act 1986.Fertilised mouse oocytes were collected on the day of plugging from naturally mated inbred C57BL/6 J (B6) mice supplied from breeding colonies in the Biological Support Unit at the Babraham Institute. Mechanistic investigation into the loss of DNA methylation was made possible through the generation of conditional deletion of key activities, Dnmt3a  and TDG Individual zygote pronuclear staging was performed as previously described . The celC57BL/6 N Tac ES cells (TaconicArtemis) were targeted with a vector introducing LoxP sites around exon 5 of Tet3 RefSeq NM_183138.2 (sequence: CCGGACCTGTGCTTGCCAAGGCAAAGACCCTAACACCTGCGGTGCCTCCTTCTCCTTCGGCTGTTCCTGGAGCATGTACTTCAACGGCTGCAAATATGCTCGGAGCAAGACGCCACGAAAGTTCCGCCTCACGGGAGACAATCCGAAGGAG) which encodes residues required for chelation of Fe(II) and is upstream of exons containing other key catalytic residues . ExpressAntibody staining of DNA methylation  and hydroxymethylation  was performed as previously described . Image aFluorescence semi-quantification analysis  was performed as follows, 3D reconstruction of confocal image stacks was performed using Volocity 5.5 (Improvision), after which regions of interest (ROIs) were defined around each pronucleus and total voxel count signal intensity (SUM) for each channel was computed. Examples of each of these steps can be found in Additional file 2D: Two-dimensional; 3D: Three-dimensional; 5caC: 5-carboxylcytosine; 5fC: 5-formylcytosine; 5hmC: 5-hydroxymethylcytosine; 5mC: 5-methylcytosine; AID: Activation-induced deaminase; ANOVA: Analysis of variance; APE1: Apurinic-apyrimidinic endonuclease 1; B6: C57BL/6 J mouse strain; BER: Base excision repair; C: Cytosine; DNA: Deoxyribonucleic acid; ES cells: Embryonic stem cells; Dnmt3a: DNA (cytosine-5)-methyltransferase 3a; Dnmt3b: DNA (cytosine-5)-methyltransferase 3b; G: Guanosine; HCl: Hydrochloric acid; HR: Homologous recombination; IF: Immunofluorescence; iPS cells: induced pluripotent stem cells; LP: Long-patch; MAT KO: Maternal knock-out; MBD2: Methyl-CpG binding Domain 2; MBD4: Methyl-CpG binding Domain 4; NER: Nucleotide excision repair; NHEJ: Non-homologous end joining; PARP1: poly-ADP-ribose polymerase 1; PCNA: Proliferating cell nuclear antigen; PGCs: Primordial germ cells; ROIs: Regions of interest; SP: Short-patch; T: Thymine; TDG: Thymine DNA glycosylase; TET3: Ten-eleven translocation 3; U: Uracil; UNG2: Uracil-DNA glycosylase 2; XRCC1: X-ray cross-complementing group 1.The authors declare that they have no competing interests.FS, WD and WR generated the main idea of the work and developed the study design, both conceptually and methodologically. WD organised and collected the samples. FS acquired and analysed the data. FS, WD and WR contributed to analysis and interpretation of the data. CR contributed materials. JP and HB were responsible for mouse genotyping of TET3 and TDG strains, respectively. FS, WD and WR wrote the manuscript. FS, WD, JP, HB, CR and WR made comments, suggested appropriate modifications and made corrections. All authors read and approved the final manuscript.Wolf Reik and Wendy Dean are senior authors.3D reconstruction of confocal image stacks and total fluorescence semi-quantification. Volocity 5.5 (Improvision) was used for 3D rendering and signal semi-quantification of each individual embryo Z-stack. (A) Screen-shot showing an XYZ view of a representative Z-stack. (B) Screen-shot showing the 3D rendering of the same Z-stack. (C) Screen-shot showing the regions of interest (ROIs) defined around each of the objects inside the sample, paternal pronucleus (red), maternal pronucleus (green) and polar body (blue). (D) Screen-shot showing the protocol to define the ROIs and subsequent computation of several measurements, including Sum signal intensity for each of the channels, used as a measure of the total signal for 5hmC (Channel: 2) and 5mC (Channel: 3).Click here for fileFluorescence semi-quantification protocol validation data. (A) Four independent samples of B6xB6 generated fertilised oocytes between PN3 and PN5 were evaluated using the optimized protocol for simultaneous staining of 5mC and 5hmC, 3D image acquisition and semi-quantification. Statistical analysis (ANOVA) shows no significant differences can be found between the four replicates. (B) Mouse embryonic stem cells (E14) were cultured in both serum and 2i conditions [nditions  and analClick here for fileReplication inhibition does not affect DNA methylation or hydroxymethylation paternal/maternal ratios in the zygote. Two independent replicates of at least 15 B6xB6 early fertilised oocytes were collected and incubated in M16 medium (M-7292-SIGMA) supplemented with either 2.5 μL/mL DMSO (control) or 2.5 μL/mL Aphidicolin (A4487-SIGMA) and cultured  for 5 h. For replication analysis both groups (control-DMSO and Aphidicolin) were then transferred to a fresh same composition medium drop, to which 20 μM EdU  was added, for a further 1 h (detection according to the manufacturer’s instructions). (A) Representative images of control (DMSO) and replication inhibited (Aphidicolin) embryos. Single optical slices. EdU-green; 5hmC-red; 5mC-white. Scale bar 25 μm. f, female pronucleus; m, male pronucleus; pb, polar body. (B) Comparison of the changes of DNA methylation and hydroxymethylation between control (DMSO) and replication inhibited (Aphidicolin) mid-late zygotes. Box-and-whisker plots of the total immunofluorescence signal (3D imaging semi-quantification) ratio between the paternal and maternal pronuclei  for 5mC and 5hmC, respectively, showing no significant difference (unpaired t test) in the levels of methylation  or hydroxymethylation  in Aphidicolin-treated zygotes compared to controls (DMSO).Click here for fileAID is expressed in mouse oocytes and localises to the pronuclei. Wild-type controls (B6xB6) and AID null (AID KO x (C57Bl/6JxCBA)-F1) zygotes were stained with an antibody against AID  and DAPI. The control zygotes show a typical predominantly cytoplasmic localisation of AID protein, as has been described for B-cells (reviewed in [iewed in ), but thClick here for fileAID null zygotes show no significant 5mC difference during Phase I of paternal demethylation. Comparison of the changes of DNA methylation and hydroxymethylation between wild-type control (B6xB6) and AID null (AIDxAID) Phase I (PN1-PN2) and Phase II (PN3-PN5) zygotes. Box-and-whisker plots of the total immunofluorescence signal (3D imaging semi-quantification) ratio between the paternal and maternal pronuclei  for 5mC and 5hmC, respectively, showing, on the left, a very significant increase in the levels of paternal DNA methylation in Phase II but no significant difference in Phase I fertilised oocytes. No significant differences could be found in the levels of hydroxymethylation in AID null compared to wild-type in either Phase I or Phase II zygotes (on the right). ****, ns .Click here for fileAID-mediated deamination and BER SP and LP pathways. Diagram of the SP and LP BER pathways for AID-mediated DNA deamination. AID has been shown in vitro and in E. coli to be capable of deaminating 5-methylcytosine (5mC), generating T-G mismatches and thus directly removing methylation from DNA (left hand-side), although the preferred substrate is cytosine, very efficiently generating U-G mismatches but having no direct effect on DNA methylation loss (right hand-side). After removal of the mismatched base (black circles) by a DNA glycosylase (TDG in the case of a T-G mismatch and UNG2 in the case of an U-G mismatch) and incision by apurinic/apyrimidinic endonuclease (APE1), BER may proceed by the SP repair or by the LP repair. SP BER replaces a single nucleotide by polymerase β and the newly synthesized DNA sealed by DNA ligase III/X-ray cross-complementing group 1 (XRCC1) heterodimer. LP BER inserts two to 13 nucleotides by concordant action of polymerase δ, proliferating cell nuclear antigen (PCNA), flap endonuclease 1 and DNA ligase I. In this case, any methylated cytosines adjacent to the generated U-G mismatch would be replaced by new cytosines and, if not subsequently de novo methylated, the original methylated state would be lost resulting in demethylation. Poly ADP Ribose Polymerase 1 (PARP1), which binds to and is activated by DNA strand breaks, has been implicated in LP repair promoting the rapid recruitment of PAR-binding proteins to the site of DNA damage, which is important for efficient damage repair (modified from [ied from ).Click here for fileNo evidence for Dnmt3a mediated de novo DNA methylation in the paternal pronucleus. (A) Representative 2D projections of Z-stack images of control (B6xB6) and Dmnt3a maternally deleted oocytes fertilised by control sperm (Dnmt3a MAT KOxB6) late pronuclear stage embryos (PN3) simultaneously stained for DNA methylation (5mC- red) and hydroxymethylation (5hmC-green) showing no difference in both paternal loss of methylation and acquisition of hydroxymethylation, but a very obvious lack of maternal DNA methylation. Inset, merge with DNA staining (YOYO1) - blue. Scale bar 25 μm. f, female pronucleus; m, male pronucleus; pb, polar body. (B) Comparison of the changes of DNA methylation and hydroxymethylation between control (B6xB6), Dmnt3a maternally deleted (Dnmt3a MAT KOxB6) and AID null (AIDxB6) mid-late (PN3-PN5) zygotes. Box-and-whisker plots of the total paternal  pronucleus indirect immunofluorescence signal (3D imaging semi-quantification) for 5mC and 5hmC show no significant difference in either the levels of paternal DNA methylation (5mC) in Dnmt3a MAT KO fertilised oocytes relative to controls, on the left, or of hydroxymethylation (5hmC), on the right. ****; ns .Click here for file


Leprosy inflammatory episodes [type 1 (T1R) and type 2 (T2R) reactions] represent themajor cause of irreversible nerve damage. Leprosy serology is known to be influencedby the patient's bacterial index (BI) with higher positivity in multibacillarypatients (MB) and specific multidrug therapy (MDT) reduces antibody production. Thisstudy evaluated by ELISA antibody responses to leprosy Infectious Disease ResearchInstitute diagnostic-1 (LID-1) fusion protein and phenolic glycolipid I (PGL-I) in100 paired serum samples of 50 MB patients collected in the presence/absence ofreactions and in nonreactional patients before/after MDT. Patients who presented T2Rhad a median BI of 3+, while MB patients with T1R and nonreactional patients hadmedian BI of 2.5+ (p > 0.05). Anti-LID-1 and anti-PGL-I antibodies declined inpatients diagnosed during T1R (p < 0.05). Anti-LID-1 levels waned in MB with T2Rat diagnosis and nonreactional MB patients (p < 0.05). Higher anti-LID-1 levelswere seen in patients with T2R at diagnosis  and in patients with T2R during MDT. In MB patients, high and persistent anti-LID-1antibody levels might be a useful tool for clinicians to predict which patients aremore susceptible to develop leprosy T2R. Mycobacterium leprae. Although the disease affects the skin andperipheral nerves, it can present with a wide array of pathologies and clinicalmanifestations depending upon the patient's immune response (M. leprae characterised by aT-helper (Th)1 type response with interferon-gamma secretion that results in low bacillaryload, few skin lesions and low or absent antibody production. The lepromatous (LL) pole ischaracterised by low or absent M. leprae-specific CMI, but vigorousantibody production, high bacillary loads and multiple disseminated skin lesions. Theintermediary borderline forms  may show immunological changes towards either pole of the spectrum  and type 2 (T2R)reactions. Because they can cause irreversible nerve damage, leprosy reactions representthe major cause of permanent physical disabilities and deformities . T1R is M. leprae genome more than a decade ago, more than 200protein antigens have been evaluated in immunological assays (M. leprae for the diagnosis or prognosisof leprosy reactions.The detection of IgM antibodies against phenolic glycolipid I (PGL-I) represents the mostevaluated serologic assay for leprosy, with levels correlating with bacillary loads suchthat levels rise across the TT to LL spectrum . Althougl assays , 2010. Tl assays . We therM. leprae-specificserum antibody responses. To consider the impact of both MDT and the immunosuppressivetreatments of leprosy reactions, which respectively has been shown to reduce antibodylevels, MB patients were stratified according to the type of leprosy reaction (T1R and T2R)and the time of occurrence of reactions (at diagnosis or during MDT). Our data indicatethat responses to LID-1 are highest in patients presenting with T2R and that thepersistence of anti-LID-1 antibodies during treatment indicates patients more susceptibleto develop T2R.Previous studies have shown that IgM and IgG leprosy serology is influenced by thepatient's bacterial index (BI) with higher positivity towards MB disease . MoreovePatients - This study was approved by the Research Ethical Committee ofthe Clinics Hospital, Federal University of Goiás (UFG), Goiânia, state of Goiás, and bythe Brazilian Research Ethics Commission, with all participants signing an informedconsent before enrolment. A retrospective analytical study was conducted with 50patients that were recruited at the time of leprosy diagnosis and monitored during MDTfor the development of leprosy reactions at the main regional outpatient clinic.Newly diagnosed, untreated MB leprosy patients  were recruited as presenting with or withoutreaction, then fully characterised according to Ridley and Jopling criteria consideringclinical, bacilloscopic and histopathology analyses . PatientAntigen-specific antibody detection - Serum IgM antibodies toM.leprae PGL-I were detected by ELISA. Briefly, polysorp 96-well plates (NuncMaxisorp) were coated with 0.01 μg/mL natural trisaccharide-phenyl conjugated to bovineserum albumin (NT-P-BSA), the trisaccharide synthetic analog of PGL-I kindly provided byDr Fujiwara, Nara University, Japan, and blocked with phosphate-buffered saline (PBS)-1%BSA. Serum samples diluted 1/300 in 1% BSA were added to duplicate wells of eitherNT-P-BSA or BSA-coated plates. After incubation for 1 h at 37ºC and washing withPBS-Tween, horseradish peroxidase-conjugated to anti-human IgM  was then added. After incubation for 1 h at 37ºC and further washes,3,3′,5,5′-tetramethylbenzidine peroxidase colour substrate (Sigma) was added for eachwell. The colour reactions of the entire plate were stopped with 2.5N-H2SO4. The optical density (OD) was read at 450 nm using aMultiskan Ex microplate reader . The results were expressed asmean absorbance of the duplicates. The final OD value of each serum sample wascalculated by subtracting the OD value of wells coated only with BSA from the OD valueof the test wells coated with NT-P-BSA. The cut-off was defined as OD > 0.250 inaccordance with 2SO4. The corrected OD of each well at 450 nm was readusing a Multiskan Ex microplate reader. Based on previous data, the threshold forpositive responses was calculated as 2x standard deviation of the OD of sera fromhealthy endemic controls, such that samples with OD > 0.3 were considered positive(Serum IgG antibodies to the di-fusion protein LID-1 were detected by ELISA. Polysorp96-well plates (Nunc Maxisorp) were coated with 2 μg/mL of LID-1 at 4ºC overnight andblocked with PBST with 1% BSA for 1 h at room temperature (RT). Serum samples diluted1/200 in 0.1% PBS-BSA were added in duplicates and incubated for 2 h at RT. Plates werewashed and incubated with 100 μL of peroxidase-conjugated with anti-human IgG (Sigma)diluted to 1/5,000 in PBST, 0.1% BSA. After washings, reactions were developed withperoxidase colour substrate  and quenched by the addition of1N-Hpositive.Statistical analyses - GraphPad Prism v.5 was used for the calculationof the median and mean values of OD and for graphics. Statistical significance wasassessed by Kruskal-Wallis one way analysis of variance for comparison of multiplegroups and Mann-Whitney U for comparison between two groups. Resultswere considered statistically significant when p values < 0.05 were obtained.Ethics - This study was approved by the Ethical Committee of theClinics Hospital/UFG (protocol 456.226). All participants were informed about the studyaims and the procedures involved, then included only after signing the Informed ConsentForm in accordance with Resolution 196/1996 of the National Health Council.Patient demographics at time of initial diagnosis - The study group wascomposed by 50 MB patients with ages ranging from 17-79 years (median = 47 years) ofwhom the majority was male . Although the patients presenting with T2R were predominantly LL, theBIs of MB reactional patients and reaction-free MB patients were similar (p >0.05).was male .Stratifwas male .Within was male . TheonlAntibody responses at time of initial diagnosis - As expected, at thetime of diagnosis, the vast majority of MB patients presented with positive anti-LID-1and anti-PGL-I responses . The rate of seropositivity amongnonreactional MB patients was 75% (9/12) for anti-LID-1 and 67% (8/12) for anti-PGL-Iantibodies , D. In cAntibody responses during MDT - The rate of seropositivity againstLID-1 among patients who did not have reactions was 75% (9 of 12). Although 25% (3 of12) were still seropositive after completing MDT, the magnitude of anti-LID-1 responseof the seropositive patients was lower relative to before the commencement of MDT . Anti-PGL-I responses of these patientsbefore and during reactions were similar subsequently developed them during MDT (8 developed T1R and 5 developed T2R). For MBpatients who developed T1R during MDT (n = 8), 50% patients were seropositive to LID-1at time of diagnosis and at time of reaction  (p > 0.0 similar (p > 0.0>0.05).Similarly, 80% of this subset were seropositive against PGL-I at the time of diagnosisand although this was reduced to 40% at the time of T2R, this change was not significant(> 0.05).All MB patients who developed T2R during the MDT were seropositive to LID-1 at the timeof diagnosis and no significant reduction in seropositivity was observed at the time ofT2R  (p >0.05nificant (p> 0.05= 0.020), but not when compared to patients who developedT1R . All seven patients that were diagnosedduring a T2R episode were anti-LID-1 positive and three out of them were anti-PGL-Ipositive. Compared to anti-PGL-I positivity, a higher number of anti-LID-1 positives wasseen in patients with T1R at diagnosis and with T1R during MDT, however thesedifferences were not statistically significant (p = 0.083 and p = 0.533 respectively).For the 18 patients diagnosed during a T1R episode, 14 were anti-LID-1 positive and ninewere anti-PGL-I positive. Eight out of nine PGL-I positives were LID-1 positives.M. leprae and up to now there is no laboratory marker or predictorof these events. The key findings of this study were that anti-LID-1 IgG levels werehighest in MB patients first diagnosed during a T2R and that anti-LID-1 levels persistedin patients that developed reactions during MDT. These results indicate that high andpersistent levels of anti-LID-1 may be associated with the occurrence of T2R, regardlessof whether the episode occurs at diagnosis or during MDT.Leprosy reactions can be a major complication for leprosy patients and are a major causeof permanent disability and incapacities. Reactions are acute immune inflammatoryepisodes that are characterised by dysregulated and exacerbated immune responses toRecent studies have addressed potential applications of anti-LID-1 serology. A rapidtest using PGL-I mimetic, ND-O and LID-1 antigens as a single fusion complex (ND-O-LID)impregnated on a nitrocellulose membrane has been developed as a simple and fast systemthat can be used with a minimal amount of training to provide an objective diagnosis ofMB leprosy . The decConfirming previous reports, our examination of anti-PGL-I responses among MB patientswho developed T1R or T2R did not reveal detectable differences compared to nonreactionalMB patients . The dec M. leprae duringleprosy reaction has not been described; however, previous serological analyses haveindicated antigen-specific IgG levels reflect the bacterial load of the patient,similarly to what has been shown in IgM anti-PGL-I serology  these differences were not statisticallysignificant. Further studies on the correlation of PGL-I and LID-1 responses in leprosyreactional patients may validate these findings. It is possible that higher anti-LID-1responses compared to PGL-I may be, at least partially, due to the fact that LID-1 is afusion protein and responses to it represent the sum of serologic responses to twohighly immunogenic proteins ML0405 and ML2331.An additional clinical finding in our study was that MB patients who developed T2R wereyounger than nonreactional MB patients . A previous study that evaluated risk factors for the development of T2Rshowed a decreased risk in patients older than 40 years . For T1RThe significant drop in the antibody levels observed during treatment has suggested thepotential application of anti-PGL-I and anti-LID-1 serology in monitoring theeffectiveness of MDT . ExtendiIn an attempt to minimise potential confounding factors, the serologic reactivity of MBpatients was stratified according to the type of leprosy reaction (T1R or T2R) and timeof occurrence of reactions (at diagnosis or during MDT). These analyses allowed us tocompare serologic reactivity without the influence of MDT among patients who developedT1R or T2R at diagnosis and in nonreactional patients. We also compared theseroreactivity among different groups of patients under the effect of MDT testingpatients who developed T1R or T2R during treatment and nonreactional patients. Althoughwe have monitored 50 well-characterised patients, the complexities of leprosy clinicaloutcomes at both the time of presentation and during treatment resulted instratification of patients into small subgroups that somewhat limits interpretation.Regardless, our results indicate compelling differences associated with clinical outcomeand certainly support validation with larger sample numbers. Large-scale valuations ofnew treatment strategies such as uniform trial MDT designed to investigate the use of asingle treatment scheme with rifampicin, dapsone and clofazimine for six months for bothPB and MB patients  wouldapAnti-PGL-I antibody levels in MB patients who developed T1R/T2R at diagnosis weresimilar to the levels observed in reaction-free patients. Accordingly, a previous studyof our research group did not detect differences in anti-PGL-I IgM and IgG antibodies inpatients who developed T1R or T2R at diagnosis compared to patients who werereaction-free at diagnosis and to healthy endemic controls . FurtherResults of serology of paired serum samples in MB patients collected in the presence andabsence of T1R/T2R showed variable serological profiles with a declining trend inantibody levels after the reactional episode. Comparative analyses of seroreactivity inMB patients showed that high anti-LID-1 antibody levels indicated higher susceptibilityto develop T2R at diagnosis or during MDT and these results merit further examination inexpanded studies.


Plum pox virus (PPV) infection by replacing the five native siRNAs with two 210-bp fragments from the CP and the 3´NCR regions of the PPV genome. Deep sequencing analysis of the small RNA species produced by both constructs in planta has shown that phased processing of the syn-tasiRNAs is construct-specific. While in syn-tasiR-CP construct the processing was as predicted 21-nt phased in register with miR173-guided cleavage, the processing of syn-tasiR-3NCR is far from what was expected. A 22-nt species from the miR173-guided cleavage was a guide of two series of phased small RNAs, one of them in an exact 21-nt register, and the other one in a mixed of 21-/22-nt frame. In addition, both constructs produced abundant PPV-derived small RNAs in the absence of miR173 as a consequence of a strong sense post-transcriptional gene silencing induction. The antiviral effect of both constructs was also evaluated in the presence or absence of miR173 and showed that the impairment of PPV infection was not significantly higher when miR173 was present. The results show that syn-tasiRNAs processing depends on construct-specific factors that should be further studied before the so-called MIGS (miRNA-induced gene silencing) technology can be used reliably.The use of syn-tasiRNAs has been proposed as an RNA interference technique alternative to those previously described: hairpin based, virus induced gene silencing or artificial miRNAs. In this study we engineered the TAS1c locus to impair  The term RNA silencing describes an ensemble of regulatory pathways that share the key role played by diverse sets of endogenous small RNAs (sRNAs) of 21–24 nt in length . One strIn plants, primary products of some miRNA- and siRNA-directed cleavages serve as templates for RNA-dependent RNA polymerases (RDR), to generate double stranded RNA, which is processed into a second wave of siRNAs –9. Thesetrans-acting siRNAs (tasiRNAs) [Solanaceae and Fabaceae, but they are not well conserved between different plant species [Pentatricopeptide Repeat (PPR) genes [Some secondary siRNAs are 21-nt phased (phased siRNAs) as a result of successive DCL-catalyzed processings from the end of a dsRNA substrate originated by an RDR from an AGO-catalyzed cleaved RNA at a miRNA target site . Phased  species . TasiRNA species ,20 and hR) genes  or the nR) genes –26.TAS), comprising eight different loci have been identified in the Arabidopsis thaliana genome [TAS3 family, which generates tasiRNAs by the two-hit mechanism triggered by miR390 loaded in the specialized argonaute AGO7, is widely conserved in moss and higher plants, genes of TAS1/TAS2 families, whose primary transcripts are targeted by a single hit of the 22-nt-long version of miR173, are unique to Arabidopsis and closely related species [Four families of genes coding for tasiRNA precursors  able to silence endogenous genes, including FAD2 [PDS [CH42 [FT or TRY/CPC/ETC2 [MiR173-triggered production of tasiRNAs can be settled in heterologous plants, such as ing FAD2 , PDS [29DS [CH42  and FT oCPC/ETC2 . This geCPC/ETC2 .Plum pox virus (PPV), a positive strand RNA virus of the genus Potyvirus [TAS1c locus to produce syn-tasiRNAs targeting either the 3’ noncoding region (NCR) or the capsid protein (CP) coding region of PPV RNA. We used a transient agro-infiltration assay in Nicotiana benthamiana and Illumina deep-sequencing analysis, to assess the small RNA accumulation and the susceptibility to PPV infection of plants expressing the PPV-targeted syn-tasiRNA constructs alone or co-expressed with A. thaliana miR173 precursor.Artificial miRNAs (amiRNAs) has been shown as a valuable alternative to RNA silencing approaches based on the expression of large virus-derived sequences to generate antiviral resistance –35. In totyvirus . We engiN. benthamiana the miR173 required to trigger syn-tasiRNA formation from syn-tasiR-3NCR and syn-tasiR-CP, we generated a construct, MIR173, containing the AtmiR173 precursor (509 nt) [TAS1-like constructs designed to produce siRNAs targeting the different regions of the PPV genome were engineered. We generated two constructs that included the miR173 target site (22 nt) and two upstream nucleotides of TAS1a and c, immediately followed by 126 nt from either the 3’NCR nt 9529–9654) or the CP coding region (nt 9242–9367) of PPV. These constructs were cloned downstream a 35S promotor into pMDC32 yielding syn-tasiR-3NCR and syn-tasiR-CP . In orde–9654 or N. benthamiana leaves. Agrobacterium tumefaciens cells bearing either of these constructs were co-infiltrated with cells expressing MIR173 (A. tumefaciens bacteria by the plasmid they carry). As a control, syn-tasiRNA constructs were also infiltrated without the miR173-expressing plasmid. At three days post-agroinfiltration (3 dpa), small RNAs (sRNAs) were extracted from the infiltrated leaves. sRNA samples from leaves expressing syn-tasiR-3NCR and syn-tasiR-CP were pooled and subjected to deep sequencing analysis. Northern blot analysis verified that miR173 accumulated at detectable levels only in the N. benthamiana leaves infiltrated with MIR173  matching Agrobacterium genome, iii) matching common regions of agroinfiltrated pMDC32-derived plasmids, iv) matching specific regions of each pMDC32-derived plasmids, and v) unknown sequences  or together with miR173 (library +miR173), respectively. To determine how expression of miR173 affects the patterns of sRNA populations, we classified sRNAs in five groups: i) matching to equences . Approxiectively . These pectively . As correctively , suggestectively .N. benthamiana [The size distribution profiles of the-miR173 and +miR173 sRNA libraries  confirmethamiana  and showN. benthamiana, Agrobacterium or unknown sRNAs and among sRNAs mapping to the common region of pMDC32- derived plasmids. In contrast, 34 and 22 sRNAs species derived from sequences downstream the miR173 target sites of syn-tasiR-CP and syn-tasiR-3NCR, respectively, met the threshold and increased when miR173 was expressed  and accumulated in larger amounts as compared to sRNAs originated in the absence of miR173 (at least 8 times more). A very low amount of species fulfilling these thresholds were detected among the xpressed .Seven PPV CP-specific sRNAs induced by miR173-expression were 21-nt in length and were in register with the expected miR173-guided cleavage site of the syn-tasiR-CP transcript . In agreThe results presented above clearly show that the sRNAs accumulation patterns observed upon expression of syn-tasiR-CP and syn-tasiR-3NCR constructs are significantly different. The sRNAs species observed from the processing of syn-tasiR-CP construct fulfill with the expected ones, that is, seven 21-nt phased sRNAs in register with the predicted miR173-guided cleavage site . HoweverN. benthamiana by agroinfiltration and the infiltrated leaves were challenged three days later by mechanical inoculation with purified virions of a recombinant PPV expressing GFP. Whereas an average of 2.4 infection foci per leaf were detected on the plants expressing only miR173, plants expressing syn-tasiR-3NCR or syn-tasiR-CP, either with or without miR173, showed high resistance to PPV: a single focus was detected on one of the 23 leaves treated with syn-tasiR-3NCR or syn-tasiR-CP plus miR173 and no focus was detected on any of the 24 leaves expressing syn-tasiR-3NCR or syn-tasiR-CP alone . The number of infection foci of plants expressing syn-tasiR-3NCR or syn-tasiR-CP, either plus miR173 or plus miR159 as a control, was drastically reduced, compared with plants agroinfiltrated with plasmids expressing miR173 or miR159, either alone or together with an empty vector (5 or 6 foci in 16 leaves in comparison with between 103 and 386 foci in 8 leaves) . ImmunodRNA silencing is a natural regulatory and defense mechanism that has been engineered as a useful biotechnological tool to downregulate plant gene expression and to provide antiviral resistance ,38. MIGSN. benthamiana. The two syn-tasiRNA constructs used in this study only differ in 126 nt which correspond to two different regions (CP coding sequence and 3’ NCR) of the PPV genome, placed downstream of a miR173 cleavage site. PTGS induction by these constructs was evidenced by accumulation of a large number of sRNAs spread through the complete T-DNA region, regardless coexpression of miR173. We also detected sRNAs specifically induced by miR173 expression which mapped almost exclusively downstream the miR173 target site, which is in agreement with previous reports showing that TAS1-like transcripts yield tasiRNA only from the 3’ RNA fragment generated by miR173-guided cleavage [TAS1c transcript [We have used the previously reported TAS1/miR173-related system ,32,43 tocleavage . Abundancleavage ,31,44. Aanscript , althouganscript , 44. Sinanscript , 46. In Montgomery et al.  showed tThe abundance of PPV-derived sRNAs in leaves agroinfiltrated with syn-tasiR-CP and syn-tasiR-3NCR in the absence of miR173 indicates that these constructs are inducing a strong sense PTGS reaction. In agreement with this fact, these agroinfiltrated leaves showed an efficient anti-PPV protection, which was not significantly enhanced by tasiRNA production induced by miR173 expression. Recently, Carbonell et al.  also shoTAS1c was used as a backbone to produce syn-tasiRNA as previously described ,29. The Primers for syn-tasiR-CP:5´GGGGACAAGTTTGTACAAAAAAGCAGGCTTTGTGATTTTTCTCTACAAGCGsyn-tasiR-CP-F: AATCAAAGGCATATCTGGCGAGG 3´5´GGGGACCACTTTGTACAAGAAAGCTGGGTTATGGCACATTTCAGTAACGT 3´syn-tasiR-3NCR-R: Primers for syn-tasiR-3NCR:5´GGGGACAAGTTTGTACAAAAAAGCAGGCTTTGTGATTTTTCTCTACAAGCGAAGAGAAAAGGATGCTAACAGGA 3´.syn-tasiR-3NCR-F: 5´GGGGACCACTTTGTACAAGAAAGCTGGGTATCGCAACATGCACAACCTCC 3´.syn-tasiR-3NCR-R: Arabidopsis thaliana MIR173 precursor (509nt) and MIR159 precursor (334nt) were amplified from Col-0 genomic DNA using the following primers, which included Gateway recombination sites:The 5´GGGGACAAGTTTGTACAAAAAAGCAGGCTATAATTAGCAAGTAATAAGG3´ MIR173-R: 5´GGGGACCACTTTGTACAAGAAAGCTGGGTATCTGTTATACAACCAAATCC3´MIR173-F: 5´GGGGACAAGTTTGTACAAAAAAGCAGGCTTTACAGTTTGCTTATGTCAGATCC3´MIR159-F: 5´GGGGACCACTTTGTACAAGAAAGCTGGGTTGACCCGGGATGTAGAGCTCCCTTCAATCC3´MIR159-R: All the purified PCR fragments were introduced between the CaMV 35S promoter and the NOS terminator sequences into the pMDC32 binary vector by Gateway recombination and termed syn-tasiR-3NCR, syn-tasiR-CP, MIR173 and MIR159.Nicotiana benthamiana plants were grown in a greenhouse maintained at 16 h light and 8 h dark photoperiod, temperature range 19–23°C. Plants with 5–6 true leaves were used for agroinfiltration as described [600 = 1 for each construct, when two constructs were agroinfilrated a mixed 1:1 (v/v) culture was prepared.escribed . CultureN. benthamiana leaves were dusted with carborundum and inoculated with 150 ng or 450 ng of purified GFP-tagged PPV [Two or three agroinfiltrated gged PPV . Virus agged PPV . PonceauN. benthamiana leaves were screened for GFP expression with a MZ FLII (Leica Microsystems) fluorescence stereomicroscope, using excitation and barrier filters of 480/40 nm and 510 nm, respectively. Images were collected with an Olympus DP70 digital camera with DP controller and DP manager software.Infected N. benthamiana leaves and subjected to Northern blot analysis as previously described [Small RNAs were purified from agroinfiltrated escribed .RNA samples from twelve leaves (2 leaves from each plant) agroinfiltrated with two independent clones of syn-tasiR-3NCR and two independent clones of syn-tasiR-CP, either in the presence or absence of miR173 precursor, were pooled. Small RNA libraries were prepared and subjected to deep sequencing by the Beijing Genomics Institute . Briefly, between 15–30 nucleotides (nt) RNAs were purified and ligated to the Illumina 5´ and 3´ adaptors, and further converted into single-stranded cDNA and then amplified by PCR. Purified PCR products were sequenced by an Illumina HiSeq 2000 Genome Analyzer.http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE69348 (accession number GSE69348).The high throughput sequencing data are freely available at GEO database http://srna-workbench.cmp.uea.ac.uk/tools/) [Clean reads were obtained after adapter trimming and removing low quality reads, adaptor null reads, insert null reads, rRNA and tRNA and reads with polyA tail using the software of The UEA small RNA Workbench (/tools/) . Clean r/tools/)  without S1 TableTotal small RNA species and 21nt, 22nt and 24nt species matching different sequences are given in columns. The overall number, the induced and its ratio are also detailed.(DOCX)Click here for additional data file.


Little is known about the molecular pathogenesis of ASD. One of the possible molecular pathogenesis is an imbalance of excitatory and inhibitory receptors linked with the NLGN-PSD-95-SHANK complex via postsynaptic density protein/Drosophila disc large tumor suppressor/zonula occludens-1 protein (PDZ) binding. In the present study, we focused on GPR85 as a candidate gene for ASD because the C-terminal amino acid sequence of GPR85 [Thr-Cys-Val-Ile (YCVI)] is classified as a type II PDZ-binding motif, and GPR85 is a risk factor for schizophrenia. GPR85 is an orphan receptor that regulates neural and synaptic plasticity and modulates diverse behaviors, including learning and memory. While searching for molecules that associate with GPR85, we found that GPR85 was associated with postsynaptic density protein (PSD)-95 linked with NLGN in the brain.Autism spectrum disorder (ASD) has a complex genetic etiology. Some symptoms and mutated genes, including GPR85 gene in patients with ASD. We used immunostaining to examine the intracellular localization of mutated GPR85 and its influence on the morphology of cells and neurons.We examined the proteins that associate with the C-terminal sequence of GPR85 by pull-down assay and immunoblot analysis and searched for a mutation of the GPR85: one had T1033C (M152T) and the other had G1239T (V221L). These mutations were located in a domain related to G protein interaction and signal transduction. In contrast to wild-type GPR85, mutated GPR85 was more preferentially accumulated, causing endoplasmic reticulum stress, and disturbed the dendrite formation of hippocampal neurons.The C-terminal sequence of GPR85 interacted with PSD-95 at PDZ1, while NLGN interacted with PSD-95 at PDZ3. Two male patients with ASD from independent Japanese families possessed inherited missense mutations at conserved sites in GPR85 associated with the PSD-95 linked with NLGN, which is related to ASD. GPR85 carrying the mutations detected in ASD patients disturbed dendrite formation that could be the candidate for molecular pathogenesis of ASD through the associated NLGN-PSD-95 receptor complex.The online version of this article (doi:10.1186/s13229-015-0012-5) contains supplementary material, which is available to authorized users. We receNRXN1, NRXN2, NLGN2, NLGN4, and SHANK3 genes related to ASD have been also found in schizophrenia patients [GPR85 located in the AUTS9 locus in the 7q31 region [GPR37 [Mutations in patients -13. Therpatients ,15. Pharpatients . Thus, t1 region  that harn [GPR37 .GPR85 is involved in predisposing patients to a high risk of schizophrenia; two GPR85 SNPs in moderate linkage disequilibrium have been associated with schizophrenia [GPR85 have not been found in patients with schizophrenia. GPR85 is an orphan receptor that is involved in determining brain size, regulating neural and synaptic plasticity, and modulating diverse behaviors (including learning and memory) [ophrenia , but mut memory) . The ami memory) ,20. GPR8 memory) . During GPR85 in ASD patients. Here, we show two independent missense mutations in the GPR85 gene of Japanese patients with ASD and describe the mutated GPR85-induced endoplasmic reticulum (ER) stress and deleterious effect on the dendrite formation of neurons.In the present study, we examined the interaction between GPR85 and PDZ proteins linked with NLGN and mutations of N = 622) samples, including male (N = 381) and female (N = 241) samples, were obtained from healthy Japanese adult volunteers after they provided written, informed consent. We also obtained the DNA of 200 Caucasian patients from the Autism Genetic Resource Exchange Consortium . AGRE samples included 172 males and 28 females; all were familial cases. Caucasian control DNA samples were obtained from the Coriell Institute .Lymphocytes were obtained from 72 unrelated Japanese ASD patients with autism or a pervasive developmental disorder not otherwise specified. Their conditions were diagnosed according to criteria outlined in the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV). Written informed consent was obtained from the parents of all patients. The patients included 57 males and 15 females, aged 3 to 23 years, with intellectual levels that varied from normal to severely disabled. Four probands had siblings with ASD, and others were sporadic. Control .GPR85 regions. The primers are listed in Additional file Genomic DNA was extracted from peripheral blood lymphocytes or lymphoblasts using a standard method described by the manufacturer. We used polymerase chain reaction (PCR) primers to amplify each exon and its boundaries in the CGGCATTTCCCCCGGTTTT-3′; antisense primer: 5′-AAAACCGGGGGAAATGCCGTGGCCACAGACAGAG-3′; for mutation of V221L, sense primer: 5′-GAAGCCAGTCCAGTTTTTAGCAGCAGTCAGCCA-3′; antisense primer: 5′-TGGCTGACTGCTGCTAAAAACTGGACTGGCTTC-3′. Underlined nucleotides were mutated sites. Mutations were confirmed by DNA sequencing.The full length of GPR85 cDNA was isolated from human cDNA library . To generate GPR85 mutants, site-directed mutagenesis were performed by using QuikChange II Site-Directed Mutagenesis kit (Stratagene) with the following primers: for mutation of M152T, sense primer: 5′-CTCTGTCTGTGGCCAKpn1 sites of the pcDNA4/TO/myc-His expression vector, and GPR85-C and GPR85-deltaC fragments were subcloned into BamH1 sites pGEX4T-3 vector . FLAG-GPR85 wild type and mutants in pGEM-T easy vector were subcloned into EcoR1 sites pcDEF3-FLAG vector.The enzyme-digested PCR DNA fragments corresponding to full-length GPR85 and full-length GPR85-deltaC for the pcDNA4/TO/myc-His expression vector  or pcDEF3-FLAG vector [The experiments were approved by the Genetic Modification Safety Committee of Jichi University  and were carried out under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology.We followed the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology. All protocols for animal handling and treatment were reviewed and approved by the Animal Care and Use Committee of Jichi University  and by the International University of Health and Welfare .in vitro (DIV) 3 to 5). Briefly, hippocampalis were dissected in Hepes-buffered saline solution and dissociated by adding 10% trypsin and 1% DNase and incubating for 15 min. Neurons were plated at a density of 2 × 104 cells/cm2 on 0.05% polyethylenimine-treated, 1.2 mm-diameter cover slips  and incubated in Neurobasal™ medium with B-27 supplement (Invitrogen) and 2% fetal bovine serum. Cultures were maintained at 37°C in 5% CO2. Half of the growth medium was exchanged with fresh medium once a week. Neurons with developed dendritic arbors were used for the experiments. COS and C2C5 [2.Primary culture of hippocampal neurons was prepared from mouse wild-type embryos at embryonic day (E) 18 as previously described . Neuroneand C2C5  cells weGPR85 (M152T or V221L)-myc-his, pcDEF plasmid containing FLAG-wild-type, mutated GPR85 (M152T or V221L), or wild-type, mutated GPR85 (M152T or V221L)-deltaC and PSD-95-GFP [Neurons (cultured for 7 days (DIV 7)) and C2C5 cells were transfected with pcDNA4 plasmid containing wild-type or mutated D-95-GFP  construct-test .C2C5 cells were lysed in buffer  and protease inhibitor or buffer A in ProteoExtract® Native Membrane Protein Extraction Kit according to the manufacturer’s protocol . Separated proteins were transferred to nitrocellulose transfer membrane by electroblotting. Nonspecific binding was blocked with 0.5% nonfat dry milk with 0.1% Triton X-100 in PBS. After centrifugation, 40 μg of total protein was subjected to SDS-PAGE (7.5% to 12%). Then, blots were incubated overnight at 4 °C with mouse anti-His ; mouse anti-CHOP  levels were assessed by adding alkaline phosphatase-conjugated and goat anti-rabbit or anti-mouse immunoglobulins (Promega) and detecting the signals generated in reactions with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-1-phosphate. Data from three experiments were scanned and analyzed for quantification with Image J software . Results compared with wild type were analyzed using Student’s Cells were fixed in 4% paraformaldehyde (PFA) or methanol in PBS. After they were incubated with blocking reagent  for 1 h at room temperature, they were then subjected to the immunostaining using mouse anti-His, rabbit anti-His , mouse anti-FLAG, rabbit anti-FLAG (Sigma), rabbit anti-eIF2α , mouse anti-CHOP (Santa Cruz Biotechnology), mouse anti-MAP2 (Sigma), mouse anti-PSD-95 , and rabbit caspase-3 . Alexa-Ft-tests .The percentages of C2C5 cells that showed apoptotic morphology and CHOP, P-eIF2, and caspase-3 positivity were determined from a total of 200 cells that expressed wild type or 200 cells that expressed mutant GPR85 at 28 to 48 h after transfection. The percentage neurons with long dendrites were determined from a total of 100 neurons that expressed wild type or 100 neurons that expressed mutant GPR85 at 28 to 48 h after transfection. The experiments were repeated three times. The values represent averages of the percentages of the number of cells obtained in three experiments. Results were analyzed using Student’s in vitro pull-down assays, glutathione S-transferase  and the GST fusion proteins GST-GPR85-C including amino acids (SRLPREPYCVI)  and GST-GPR85-deltaC   were purified by Glutathione Sepharose 4B  from the extract of Escherichia coli containing each plasmid. GST fusion proteins bound to Sepharose beads were incubated with lysates from the brain or COS cells transfected with GFP-PSD-95 and GFP-SAP102 for 2 h at 4°C. The beads were washed three times with lysis buffer and then eluted with 50 mM Tris-HCl (pH 8.0) containing 20 mM reduced glutathione. The bound proteins were then separated by SDS-PAGE and detected by immunoblotting using the following primary antibodies: rabbit anti-SAP102 , rabbit anti-PSD-95 (Millipore), mouse anti-Mupp1 , mouse anti-Neuroligin (Synaptic Systems), mouse anti-GFP , and rabbit anti-GST (Santa Cruz Biotechnology).For The C-terminal amino acid sequence of human and mouse GPR85 (YCVI) is classified as a type II PDZ-binding motif . We emplNext, we examined the binding domain in detail during the interaction between GPR85 and PSD-95 Figure . GPR85-CGPR85 that caused amino acid substitutions: one was T1033C (M152T) and the other G1239T (V221L)  domains. These four GPR85 RNA isoforms are registered in GenBank as NM_001146265.1, NM_018970.6, NM_001146266.1, and NM_001146267.1. The two mutations found in this study, M152T and V221L, were located in exon 3, which is present in all four RNA isoforms. One mutation was located in the fourth TM domain and the other in the cytoplasmic region adjacent to the fifth TM domain  with PSD-95 in C2C5 cells. Wild-type GPR85, but not GPR85-deltaC, co-localized with PSD-95 , short dendrites , and no dendrites (<100 μm)  punctate stainings were detected on the dendrites, and some of them did not co-localize with PSD-95  knock-in mice.However, impaired dendrite formation or synaptic formation has not been observed in the brain of ent mice , suggestNRXN1, NRXN2, NLGN2, NLGN4, SHANK3, and SynGAP, have been also found in the schizophrenia patients [de novo mutations (R1117X and R536W) were identified in the SHANK3 gene of two families of schizophrenia [Impaired synaptic functions are shared with other psychiatric disorders including schizophrenia. Some of the mutations of the genes related to ASD, including patients -13; for Gpr85 (M152T) knock-in mice.Thus, the putative ligand of GPR85 and its signaling pathways may be a common molecular target for ASD and schizophrenia as described in -15. We wGPR85 interacted with PSD-95, associating with NLGN related to ASD. Two independent GPR85 mutations, T1033C (M152T) and G1239T (V221L), were found in male autism patients from Japanese families. Mutated GPR85 preferentially accumulated, causing ER stress and disturbing the dendrite formation of hippocampal neurons. Thus, mutated GPR85 could be associated with the molecular pathogenesis of ASD, and GPR85 may be a common molecular target for ASD and schizophrenia.