Phosphorylation of E-cadherin/catenin complex and Rho family GTPase activities were also examined. Although CD148 introduction did not alter the expression levels and complex formation of E-cadherin, p120, and β-catenin, CD148 WT, but not CS, promoted cadherin contacts and strengthened cell-cell adhesion in A431D/E-cadherin WT cells. This effect was accompanied by an increase in Rac1, but not RhoA and Cdc42, activity and largely diminished by Rac1 inhibition. Further, we demonstrate that CD148 reduces the tyrosine phosphorylation of p120 and β-catenin; causes the dephosphorylation of Y529 suppressive tyrosine residue in Src, a well-known CD148 site, increasing Src activity and enhancing the phosphorylation of Y228 (a Src kinase site) in p120, in E-cadherin contacts. Consistent with these findings, CD148 dephosphorylated both p120 and β-catenin in vitro. The shRNA-mediated CD148 knockdown in A431 cells showed opposite effects. CD148 showed no effects in A431D and A431D/E-cadherin 764AAA cells. In aggregate, these findings provide the first evidence that CD148 promotes E-cadherin adhesion by regulating Rac1 activity concomitant with modulation of p120, β-catenin, and Src tyrosine phosphorylation. This effect requires E-cadherin and p120 association.CD148 is a transmembrane tyrosine phosphatase that is expressed at cell junctions. Recent studies have shown that CD148 associates with the cadherin/catenin complex and p120 catenin (p120) may serve as a substrate. However, the role of CD148 in cadherin cell-cell adhesion remains unknown. Therefore, here we addressed this issue using a series of stable cells and cell-based assays. Wild-type (WT) and catalytically inactive (CS) CD148 were introduced to A431D , A431D/E-cadherin WT (expressing wild-type E-cadherin), and A431D/E-cadherin 764AAA (expressing p120-uncoupled E-cadherin mutant) cells. The effects of CD148 in cadherin adhesion were assessed by Ca CD148  is a transmembrane PTP that is composed of an extracellular domain containing type III fibronectin repeats, a transmembrane segment, and a single intracellular catalytic domain CD148 was initially cloned from Hela cells as a PTP whose expression and activity is increased with cell density The cadherin/catenin complex is a major component of adherens junctions and plays a central role in cell-cell adhesion. The extracellular domain of cadherin mediates homophilic and calcium-dependent adhesion between adjacent cells and its cytoplasmic domain binds to the Armadillo-family proteins, p120 and β-catenin In this study, we investigated the effects of CD148 in E-cadherin mediated cell-cell adhesion by introducing CD148 and E-cadherin forms into the cadherin-deficient A431D cells and by functionally and biochemically assessing E-cadherin mediated cell adhesion events in these cells. The p120-uncoupled E-cadherin mutant was also introduced to determine the contribution of p120 to CD148 effects. Our data demonstrate that CD148 promotes E-cadherin cell adhesion by regulating Rac1 activity, concomitant with modulation of p120, β-catenin, and Src tyrosine phosphorylation, and that this effect requires E-cadherin and p120 association.The primary antibodies used for immunoblotting and immunoprecipitations: p120 (pp120), E-cadherin (34-E-cadherin), β-catenin, phospho-p120 (pY228), phosphotyrosine (pY20), Rac1, and Cdc42 were from BD Bioscience . β-actin (C-2), CD148 (143-41), and RhoA (119) were from Santa Cruz Biotechnology . Src was from Upstate Biotechnology . Phospho-Src (pY529) was from Invitrogen Corporation . Secondary antibodies for immunoblotting: HRP-conjugated anti-mouse or anti-rabbit IgG were from GE Healthcare Bio-Sciences . For immunoprecipitation of E-cadherin, anti-E-cadherin (HECD-1) from Takara Bio Company  was used. For flow cytometry, phycoerythrin (PE)-conjugated anti-CD148 (143-41) from R&D Systems  was used. Immunofluorescence staining was performed using anti-E-cadherin , anti-p120 , and the following secondary antibodies; FITC-conjugated anti-CD148 , Alexa Fluor 546-conjugated goat anti-mouse IgG2a , and Alexa Fluor 647-conjugated goat anti-mouse IgG1 (Invitrogen Corporation).A431D epidermoid cervical carcinoma cells and its stable cells expressing either wild-type (WT) or 764AAA E-cadherin Cells were plated onto glass coverslips  placed in a 12-well plate, cultured for 24 h, fixed in 100% methanol at −20°C for 7 min, and incubated with primary antibodies for 1 h at RT. The immunoreaction was visualized by the subsequent incubation with secondary antibodies (30 min at RT). Coverslips were mounted on glass slides with Fluorogel  and photographed using Zeiss LSM 510 confocal microscopy.2 was added for the indicated time. For immunofluorescence, cells were plated onto glass coverslips  placed in 12-well plate, cultured for 24 h, starved, and calcium-switch was carried out as described above. Then, the cells were fixed in 100% methanol at −20°C for 7 min, immunostained, and photographed as described above.Calcium-switch assay was performed as described previously 5 cells/ml in DMEM supplemented with 1% FBS. 2.5×104 cells (in 50 µl) were pipetted on the lid of a 24-well plate, inverted, allowed to aggregate for 12–14 h in a humid 5% CO2 incubator at 37°C. Cells were subjected to shear force by passing them through a standard 200-µl pipet tip 10 times and were photographed through a microscope  with 10×phase-contrast objective and Amscope digital camera . For Rac 1 inhibition, Rac 1 inhibitor NSC 23766  or Vehicle was added to the cell suspension at the concentration of 75 µM prior to hanging drop. For the assessment of Rho-family GTPase activity, cells were harvested at 0, 2, and 4 h after hanging drop, lysed, and the activities of Rac1, RhoA, and Cdc42 were measured as described below.Cell aggregation assay was performed as described previously 2, 0.5 mM NaCl, 2% NP-40, protease inhibitor cocktail ] and the same amount (300–400 µg) of cell lysates was incubated with either 50 µg of PBD-GST or RBD-GST protein beads to precipitate activated Rac1 and Cdc42 or RhoA. Active (GTP-bound) and total Rac1, Cdc42, and RhoA were assessed by immunoblotting with anti-Rac1 (BD Bioscience), anti-Cdc42 (BD Bioscience), and anti-RhoA (Santa Cruz) antibodies. Relative levels of active versus total Rac1, Cdc42, and RhoA were quantified by densitometric analysis with image J (NIH) software. Experiments were repeated at least 4–5 times and the data was expressed as means ± S.E.M. ANOVA was used for multiple-group comparisons and the unpaired Student’s t test was used for two-group comparisons. Differences were considered statistically significant when P<0.05.RhoA, Rac1, and Cdc42 activities were assessed using the Rac1 and RhoA pull-down activation assay kit  according to the manufacturer’s instruction. In brief, cells were lysed in lysis buffer . p120, β-catenin, and E-cadherin were immunoprecipitated from the lysates with specific antibodies. The immunoprecipitates were washed twice in wash buffer  and subsequently in succinate buffer . The beads were then suspended in 100 µl of succinate buffer with either GST or GST-CD148 proteins  and incubated for 30 min at 30°C. After washing with succinate buffer, the immunoprecipitates were subjected to immunoblotting. For the vanadate competition, 1 mM Na3VO4 was added to the reaction mixture prior to incubation.Figure S1). Shown in Figure S2).CD148 is abundantly expressed in epithelial cells of various tissues Figure S3), indicating that this effect is associated with E-cadherin cell adhesion. The CD148 WT induced increase of Rac1 activity was not observed with CD148 CS or in A431D/E-cadherin 764AAA cells. Second, we also addressed this issue using a hanging-drop cell aggregation assay as this assay was successfully applied to the assessment of E-cadherin function Figure S4) cells. Consistent with this finding, a Rac1 inhibitor (NSC23766) largely abolished the ability of CD148 WT to promote cell aggregation (panel C). These results were also supported by our cell density experiments showing that CD148 WT, but not CS, increases the Rac1 activity in a cell-density dependent manner in A431D/E-cadherin WT, but not 764AAA, cells (data not shown). Last, we asked if CD148 knockdown shows opposite effects on E-cadherin cell adhesion. For this experiment, we utilized A431 cells that endogenously express E-cadherin and CD148  in Src tyrosine kinase and increases its activity Figure S6). The β-catenin Y654 is also known to be phosphorylated by Src To determine the biochemical changes in E-cadherin/catenin complex, which are associated with the CD148 effects, we next investigated the tyrosine phosphorylation of E-cadherin/catenin complex in the setting of calcium-switch assay. E-cadherin/catenin complex was immunoprecipitated using an E-cadherin antibody and the tyrosine phosphorylation of p120 and β-catenin that are recruited to E-cadherin was assessed by immunoblotting. Shown in in vitro assays. First, we asked if the substrate trapping  form in vitro. Since previous studies have shown that E-cadherin is phosphorylated by tyrosine kinases including Src Figure S7), suggesting that Y228 may not serve as a dephosphorylation site.These results suggest that CD148 dephosphorylates p120 and β-catenin as well as Src. Therefore, we next addressed this issue using in vitro assays  CD148 causes the dephosphorylation of the suppressive tyrosine residue in Src in E-cadherin contacts, increasing Src activity and possibly increasing the phosphorylation of p120 Y228 and Vav2 Y172; 4) p120 is required for the CD148 and E-cadherin association.The present study demonstrates, for the first time, that: 1) CD148 promotes E-cadherin cell-cell adhesion concomitant with an increase in Rac1 activity; 2) CD148 reduces the tyrosine phosphorylation of p120 and β-catenin in E-cadherin contacts. Further, the The CD148 enhancement of E-cadherin cell adhesion is accompanied by an increase in Rac1 activity. A body of evidence has shown a critical role of Rac1 in the establishment of cadherin adhesion. Rac 1 was shown to be recruited to the sites of E-cadherin contacts in vitro assay as the phosphorylation of this residue is known to reduce the affinity of β-catenin for cadherin How does CD148 increase Rac1 activity in E-cadherin contacts? We hypothesize there may be three possibilities. First, it is well known that CD148 dephosphorylates the suppressive tyrosine residue in Src and increases its activity in vitro (data not shown). This finding suggests that p120 may mediate the association of CD148 with E-cadherin as well as serve as a substrate.Our data also demonstrate that p120 catenin is required for the association of CD148 with E-cadherin. Recent studies have shown that p120 prevents cadherin endocytosis and functions as a master regulator of cadherin stability and cell surface retention In conclusion, the present study provides for the first time the functional and biochemical evidence about CD148 regulation of cadherin cell adhesion. Further investigation of this pathway should give a new insight into PTP regulation of cadherin function.Figure S1Expression levels of the stably introduced CD148 are comparable to those in cultured human endothelial cells. CD148WT-introduced or CD148-negative A431D/E-cadherin WT cells and human renal microvascular endothelial cells  were stained with a PE-conjugated CD148 antibody (R&D System) and the expression levels of CD148 were assessed by flow cytometry  as described previously .(PDF)Click here for additional data file.Figure S2CD148 introduction promotes VE-cadherin contacts in HUVEC cells. The recombinant adenovirus encoding HA-tagged CD148 WT or β-galactosidase (LacZ) were infected to subconfluent human umbilical vein endothelial cells  at a multiplicity of infection of 100 as described previously . At 48 h post infection, the cells were washed with PBS and fixed with 100% methanol (for VE-cadherin) or 2% paraformaldehyde followed by permeabilization with 0.02% saponin (for HA). The cells were immunostained with VE-cadherin  or HA  antibodies followed by incubation with a secondary antibody . The nucleus (purple) was counterstained with TO-PRO-3 reagent . Cells were photographed with Zeiss LSM 510 confocal microscopy. CD148-overexpression expands VE-cadherin contacts, generating more continuous distribution, in HUVEC cells (upper panels). Anti-HA immunostaining indicates that HA-tagged CD148 is expressed in most of the cells (>90%) (lower panels).(PDF)Click here for additional data file.Figure S3E-cadherin blocking antibody abolishes the CD148 effects to increase Rac1 activity in a calcium switch assay. The CD148 effects increasing Rac1 activity were assessed by a calcium-switch assay in the presence (+) or absence (−) of an E-cadherin blocking antibody (1 µg/ml) .(PDF)Click here for additional data file.Figure S4Effects of CD148 in Rho-family GTPase activities in A431D cells. CD148 WT-introduced or CD148-negative A431D cells were subjected to a hanging-drop assay. Rac1, Cdc42, and RhoA activities were measured at the indicated time points. The data show means ± SEM of quadruplicate determinations. In contrast to A431D/E-cadherin WT cells Click here for additional data file.Figure S5Comparison of p120, β-catenin, and Src tyrosine phosphorylation between A431D/E-cadherin WT and A431D/E-cadherin 764 AAA cells. The tyrosine phosphorylation of p120, β-catenin and Src in E-cadherin contacts were compared between A431D/E-cadherin WT and A431D/E-cadherin 764 AAA cells (on the same gel) using a calcium-switch assay and immunoblot analysis. The membranes were reprobed with p120, β-catenin, and Src antibodies and a ratio of phosphorylated to total protein was quantified by densitometry.(PDF)Click here for additional data file.Figure S6CD148 WT increases the tyrosine phosphorylation (Y172) of the membrane-associated Vav2 in E-cadherin contacts. CD148WT-introduced or CD148-negative A431D/E-cadherin WT or A431D/E-cadherin 764AAA cells were subjected to a calcium switch assay. The cell membrane fraction was isolated using Qproteome Cell Compartment kit  according to the manufacturer’s instruction. Vav2 was immunoprecipitated with anti-Vav2  and the phosphorylation of Vav2 was assessed by immunoblotting with a phospho (pY172)-Vav2 antibody . The amounts of Vav2 were assessed by reprobing the membrane with anti-Vav2. Purity of the cell membrane fraction was assessed by anti-calnexin  and anti-α tubulin  immunoblotting. A ratio of phosphorylated to total Vav2 was quantified by densitometry (right panels). The data shows representative of four independent experiments. CD148 WT, but not CS, increases the phosphorylation of Vav2 in E-cadherin contacts in A431D/E-cadherin WT cells. This effect is not observed in A431D/E-cadherin 764 AAA cells.(PDF)Click here for additional data file.Figure S7CD148 dephosphorylation of p120 Y228 residue is limited in vitro. CD148 dephosphorylation of p120 Y228 was assessed in vitro, compared with the overall p120 tyrosine dephosphorylation. p120 was immunoprecipitated from the pervanadate (PV)-treated or untreated A431D/E-cadherin WT cells. The immunoprecipitates were incubated with GST or GST-CD148 proteins (1.0 µg) and its effects were assessed by immunoblotting using pY20 phosphotyrosine (pY) and p120 Y228 phospho-specific (Y228) antibodies. The amount of proteins was assessed by reprobing the membrane with a p120 antibody.(PDF)Click here for additional data file.


We go on to show that activation of Src42A is necessary to trigger a signaling cascade within the inflammatory cells involving the ITAM domain-containing protein Draper-I (a member of the CED-1 family of apoptotic cell clearance receptors) and a downstream kinase, Shark, that is required for migration to wounds. The Src42A-Draper-Shark-mediated signaling axis is homologous to the well-established SFK-ITAM-Syk-signaling pathway used in vertebrate adaptive immune responses. Consequently, our results suggest that adaptive immunoreceptor-signaling pathways important in distinguishing self from non-self appear to have evolved from a more-ancient damage response. Furthermore, this changes the role of H2O2 from an inflammatory chemoattractant to an activator signal that primes immune cells to respond to damage cues via the activation of damage receptors such as Draper.Tissue damage leads to a robust and rapid inflammatory response whereby leukocytes are actively drawn toward the wound. Hydrogen peroxide  and a Syk homolog (Shark) to regulate migration of macrophages to sites of damage in  The SFK most closely related to zebrafish Lyn in Drosophila is Src42A, and the critical redox-sensitive cysteine residue necessary for H2O2 detection in the fish (C466) is conserved in Src42A but is absent from the remaining SFKs and related non-receptor tyrosine kinases  [src42AE1 mutant embryos revealed that these inflammatory cells essentially ignored such wounds, exhibiting directionalities close to zero  [Because Htrophils , we bega btk29A) . Live im to zero . Immunos to zero A, and throphages C and S1Dg injury B′′ were ruitment C and S1DDN within macrophages did not alter overall cellular morphology, though Src42ADN macrophages exhibited slightly larger spread areas in vivo  domain is critical for these responses [shark also genetically interacts with src42A and draper [2O2 by Src42A in macrophages might trigger the same signaling pathway and be important for their migration to wounds. Live imaging macrophage responses to wounds in draper mutants revealed that these inflammatory migrations are severely impaired with fewer macrophages present at the wound 1 hr after wounding  . Draper- embryos A and 3B,2O2 and places the ITAM-containing Draper-I variant at the center of this damage-induced signaling cascade. However, draper encodes a homolog of the C. elegans apoptotic cell clearance receptor CED-1 [Drosophila macrophages and glia [Drosophila embryos [Drosophila embryonic macrophages actively prioritize apoptotic cells above the growth factor signals that guide their developmental migrations [draper mutant and draper RNAi-expressing macrophages appeared vacuolated, containing increased numbers of apoptotic corpses per cell compared to controls  [draper-null mutant background. Crucially, both Draper-I constructs can be expressed at comparable levels, including when expressed in macrophages in a draper mutant background , src42AE1 [src42AK10108 [src42Amyri [shark1 [shark-deleting deficiency Df(2L)BSC434 [draperΔ5 [UAS-draper RNAiHM501623 [UAS-src42ADN [UAS-draper-IWT [UAS-shark-2 [UAS-draper-IY949F line 24127-5 [UAS-draper-II line 03529-6 [CTG, TTG, or GAL4-independent fluorescent balancer chromosomes [For a full list of genotypes used in this study, see srp-GAL4 , crq-GALpxn-GAL4  were rec da-GAL4  was usedsrc42AE1 , src42AK2AK10108 , src42Am [shark1 , the shaL)BSC434 , draperΔHM501623 , UAS-srcaper-IWT , UAS-sha-shark-2 , UAS-draomosomes .2 of wound, normalized to the average wound response of the control). For a detailed description of image processing and analysis, see the For all live imaging experiments, stage 15 embryos were collected from overnight apple juice agar plates and mounted on slides in a minimal volume of 10S Voltalef oil (VWR), following dechorionation in bleach for 1 min and extensive washing in water. All imaging was carried out at room temperature. For dynamic imaging of wound responses, epithelial wounds were induced using a nitrogen-pumped Micropoint ablation laser tuned to 435 nm (Andor Technologies), as per Razzell et al. . EGFP orI.R.E., F.S.L.M.R., and W.W. conceived and designed the experiments; I.R.E., F.S.L.M.R., and E.L.A. performed the experiments; and I.R.E., F.S.L.M.R., E.L.A., and W.W. wrote the paper.


PIPO is expressed as a fusion protein with the N-terminal half of P3 (P3N-PIPO) via transcriptional slippage of viral RNA-dependent RNA polymerase (RdRp). We herein show that clover yellow vein virus (ClYVV) produces a previously unidentified factor, P3N-ALT, in the +1 reading frame via transcriptional slippage at a conserved G1–2A6–7 motif, as is the case for P3N-PIPO. The translation of P3N-ALT terminates soon, and it is considered to be a C-terminal truncated form of P3. In planta experiments indicate that P3N-ALT functions in cell-to-cell movement along with P3N-PIPO. Hence, all three reading frames are used to produce functional proteins. Deep sequencing of ClYVV RNA from infected plants endorses the slippage by viral RdRp. Our findings unveil a virus strategy that optimizes the coding capacity.RNA viruses use various strategies to condense their genetic information into small genomes. Potyviruses not only use the polyprotein strategy, but also embed an open reading frame,  Viruses that possess single-stranded, mRNA-sense genomes are called positive-strand RNA viruses. While most mRNAs in eukaryotic cells are monocistronic, positive-strand RNA viruses encode multiple proteins in single polycistronic genomes. Several strategies are adopted to translate viral internal genes, such as synthesis of subgenomic RNAs13479101114Potyviridae in the picornavirus-like supergroup of positive-strand RNA viruses, and comprise one of the largest genera of plant viruses. They cause diseases in many crops, resulting in severe economic losses. For example, plum pox virus has spread throughout Europe, Asia, North Africa, and South and North America. The estimated costs associated with sharka disease of stone fruits have exceeded 10 billion Euros in the last 30 yearspipo, was discovered within the P3 coding region in all members of the family Potyviridaepipo ORF exists in the –1 (or +2) reading frame relative to the polyprotein ORF. PIPO is expressed in planta as a fusion protein with the N-terminal half of P3  noninfectious to Nicotiana benthamiana22Arabidopsis thaliana was shown to interact with TuMV P3N-PIPO based on yeast two-hybrid analysisN. benthamiana homologue, NbDREPP, interact with the PIPO domain  to recruit P3N-PIPO to plasmodesmata in planta19Potyviruses belong to the family pipo ORF, there is a G1–2A6–7 motif that is highly conserved among potyviruses1–2A6–7 motif252628291–2A6–7 motif, Rodamilans et al.et al.At the 5′ end of the Clover yellow vein virus (ClYVV) belongs to the genus Potyvirus in the family Potyviridae. It causes severe systemic necrosis in legumes, including French bean, broad bean and pea3435via activation of salicylic acid-mediated defence signalling in susceptible pea accessions37via TS or FS, using an in vitro translation systemP3 cistron produces another alternative reading frame product, P3N-ALT, whose translation terminates immediately downstream of the G2A6 motif in the +1 reading frame. Although P3N-ALT is considered to be a C-terminal truncated form of P3, it acts cooperatively with P3N-PIPO in viral cell-to-cell movement. Amplicon sequencing analysis using RNAs obtained from ClYVV-infected broad bean plants indicates that genomes with nucleotide insertions or deletions at A6 in the G2A6 motif are produced during infection. This suggests that TS by the viral RdRp is responsible for the expression of P3N-PIPO and P3N-ALT.The objective of this study is to investigate the mechanistic details of how ClYVV establishes virulence. To characterize the expression mechanism of P3N-PIPO, the highly virulent ClYVV strain RBpipo ORF differed between the two isolates (P3 cistron derived from RB (RB-P3 mRNA) produced more P3N-PIPO than mRNA carrying that from Cl30 (Cl30-P3 mRNA) in an A. thaliana in vitro translation systemCl30-P3 and RB-P3 mRNAs  and RB-P3N-PIPOmk (about 25 kDa), respectively, were also synthesized in vitro. The RB-P3 RNA produced a detectable amount of P3N-PIPO, while Cl30-P3 RNA produced a trace amount of P3N-PIPO in the WGE  CS isolate–1) and P3(ALT:FLAG+1) express the FLAG-tagged P3N-PIPO proteins as –1 frame products and the FLAG-tagged P3N-ALT proteins as +1 frame products, respectively. P3N-PIPO:FLAGmk and P3N-ALT:FLAGmk were modified by mutations in the G2A6 motif to produce the FLAG-tagged proteins as zero-frame products, which were used as size markers  , were sualt ORF without affecting the amino acid sequences of P3 and P3N-PIPO; therefore, we designed a mutant of Cl30, termed Cl-P3ΔARFPs, in which alternative reading frame products (ARFPs) are not produced. In Cl-P3ΔARFPs, the G2A6 motif was mutated to G2AGA4, and a TGA stop codon was placed eight nucleotides downstream of the G2A6 motif in the pipo frame, such that it produces a full-length polyprotein from P1 to CP, but not P3N-PIPO or P3N-ALT (1920in trans. We used white clover mosaic virus (WClMV) vectorP3 cistron but expressed various reading frame products by mutating the G2A6 motif: WCl/P3N-PIPO–1, P3+1)–1, P3+1)  . This imIn another experiment in which broad bean plants were used, Cl-P3ΔARFPs co-inoculated with WCl/P3N-ALT and WCl/P3N-PIPO moved to systemic leaves, whereas Cl-P3ΔARFPs failed to infect the whole plant bodies when it was infected alone . This rein trans, although the percentage of multicellular foci in which Cl-P3ΔARFPs moved to two or more cells was much lower with P3N-ALT than with P3N-PIPO , GFP-G1A6(f2) and GFP-G1A6(f3) , GFP-G1A6(f2) and GFP-G1A6(f3) produced both –1 and +1 reading frame products with slightly different efficiencies (in vitro. Because FS efficiency is strongly dependent on the reading frames of slippery sequences1011in vitro experiments. This raises the possibility that P3N-PIPO and P3N-ALT are produced by TS of the ClYVV-encoded RdRp in planta.We investigated whether the expression of P3N-PIPO and P3N-ALT resulted from FS or TS using G1A6(f3) . Note th product . We analciencies . Thus, ri.e., NIb), the viral genomic region encompassing the G2A6 motif of ClYVV multiplied in planta was sequenced at a high coverage. To avoid possible carry-over of viral genomes containing insertions and/or deletions (indels), we used infectious cDNA clones as initial inocula instead of virions. The plasmids pCl30 and pRB carrying Cl30 or RB infectious cDNA, respectively, were biolistically inoculated into broad bean plants, which are suitable hosts for biolistic inoculations of leaves still attached to the plant. Viral RNA samples were prepared from systemically infected upper leaves. The 209 nucleotide (nt)-long DNA fragments were amplified using cDNA prepared from the total RNA. The first 49 nucleotides of the amplified fragments, containing the G2A6 motif, were sequenced using a HiSeq2000 instrument. As controls for indels potentially introduced during PCR amplification, library preparation and/or amplicon sequencing, the plasmid DNAs used for inoculations were also subjected to the same procedure.To examine the production of P3N-PIPO and P3N-ALT through TS by the viral RdRp  adenine (33A) occurred significantly more in both Cl30 and RB compared with their parental plasmids used for inoculation. Since it is unable to identify the exact position where indels had occurred within a homopolymer stretch such as A6 by sequencing, we interpreted these results to mean that indels had been introduced within the A6 sequence rather than only at 33A during viral RNA replication in planta, Further analysis using a negative binomial model fitting with indel counts observed in three, five, seven or nine base-windows  also supported the result obtained from Fisher’s exact test , because TS was reported to occur at G0A6+sequences in mammalian cells and Escherichia coli471–2A6–7 motif, but carries a G0A6 motif  revealed that out of 14,149 accessions, 5,133 carry a G1–2A6+ motif and 11,624 carry a G0A6+ motif Flaviviridae. Because these truncated proteins are accompanied by another alternative reading frame product as in the case of ClYVV, in which P3N-ALT is accompanied by P3N-PIPO 5152i.e., the truncated P3) is a functional protein during ClYVV infection  and pSP/Cl30-P3N-ALTmk (to express P3N-ALT size marker for Cl30). pSP/RB-P3 was used as a template to construct pSP/RB-P3N-PIPOmk (to express P3N-PIPO size marker for RB) and pSP/RB-P3N-ALTmk (to express P3N-ALT size marker for RB). The primers used for construction are shown in DpnI, PCR products were phosphorylated by T4 polynucleotide kinase and circularized using a Ligation-Convenience kit .For the l30 ref.  and RB38XhoI site, SpeI site, Gly/Ser linker, three copies of flag epitope tag coding sequenceXbaI site, by an overlap extension PCR. The PCR product was digested with XhoI and XbaI and inserted between the XhoI and SpeI sites of pTA7001 to generate pTA/XhSp-3FLAG.To construct plasmids for the agroinfiltration assay, we first generated vector pTA/XhSp-3FLAG, which is a modified version of pTA7001 ref. . We geneP3 cistron and 3′ end of pipo was amplified by PCR with primers 1009 and 1011 (+P3N-PIPO and P3N-PIPO–1) and pTA/RB-P3N-PIPO:FLAGmk, respectively. The cDNA product for pTA/Cl30-P3(PIPO:FLAG–1) was amplified by PCR using pCl30  was amplified by PCR using the infectious clone of BYMV-CSmk was amplified by two-step PCR using the BYMV infectious clone as a template and primers 1010, 1013, 3669 and 3668 , pTA/RB-P3(ALT:FLAG+1) and pTA/CS-P3(ALT:FLAG+1), respectively. To generate pTA/Cl30-P3N-ALT:FLAGmk, pTA/RB-P3N-ALT:FLAGmk and pTA/CS-P3N-ALT:FLAGmk, the cDNA fragments were amplified by PCR using primers 1009 and 1350 (+1) as the template, primers 1009 and 1349 (+1) as the template and primers 1010 and 1351 (+1) as the template, respectively. The amplified fragments were introduced into pTA/XhSp-3FLAG using XhoI and SpeI sites. The binary vector carrying a gene for yellow fluorescent protein (YFP) was prepared as follows. The cDNA of the YFP gene with the XhoI and SpeI sites was amplified by PCR using pGWB641  in this study.For the cell-to-cell movement assay, we modified the l30 ref. , as follEcoRI and extracted with phenol/chloroform (1:1 v/v), followed by ethanol precipitation. RNA was synthesized from the linearized plasmid DNA using the AmpliCap SP6 high-yield message maker kit  in the presence of a cap analogue, according to the manufacturer’s instructions. After the transcription reaction, the template DNA was digested with RNase-free DNase I and the mRNA was purified using an RNeasy Mini kit .The plasmid DNAs were linearized with 35S]methionine . The mixture was incubated at 25 °C for 90 min.Translation reaction cocktail (15 μl) for WGE (Promega) was prepared according to a previous reporthttp://rsbweb.nih.gov/ij)]. The % accumulation level for the –1 frame product A–1 was determined as follows:After incubation, the translation products were separated on a NuPAGE 4–12% Bis-Tris Gel  with MES running buffer (Invitrogen). The protein bands were visualized using a FLA-7000 image analyzer . For quantification, the acquired images were processed using the MultiGauge (Fuji Photo Film). The band intensities were measured using ImageJ , and the suspensions were adjusted to OD600 = 1.0. Acetosyringone was added to the suspensions , followed by incubation at room temperature for 2–4 h. The suspensions were infiltrated into N. benthamiana leaves using needleless syringes. Leaves were sprayed with 30 μM DEX solution containing 0.01% Tween-20 one day after agroinfiltrationAgrobacterium-mediated transient expression was conducted as described previouslyWestern blotting was conducted as described previouslyTo detect P3N-ALT, rabbit polyclonal antibodies were raised against the partial peptide of the N-terminal region of ClYVV P3, SLTGQVIQFDTKMLIS. After fractionation of the antibodies that possessed affinity to the immunized peptide, their specificity to the N-terminal region of P3 was confirmed . WesternDetached leaves of a susceptible pea line, PI 250438, were biolistically co-inoculated with tungsten particles coated with 800 ng of pCl-P3ΔARFPs and 200 ng of either of the pE2113 and WClMV vectors, as described previously38For GFP fluorescence and gel images, the acquired images were processed using Photoshop 12.0.4 or CS5 software . For gel images of Western blotting and autoradiograms, contrast was adjusted for the sake of visibility as appropriate.BglII and 2.5 units of NdeI  simultaneously, with adapter ligation using T4 DNA Ligase . Amplicon-adaptor complexes were purified using Agencourt AMPure XP  and were amplified by PCR with index and universal primers.ClYVV infectious plasmid clones, Cl30 and RBhttp://sourceforge.net/projects/bio-bwa/) to the 209 nt sequence of Cl30 or RB. We did not filter the raw reads before alignment but did remove reads with bases with low quality values around indels after the alignment. The quality values of inserted bases, and bases adjacent to inserted or deleted base(s) were analysed. The threshold of the quality value was set to 30. Base substitutions in the reads were not analysed in this study. Counts of reads with indels and total reads for each sample were obtained from the reads remaining after filtering. To analyse enrichment of particular sizes of indels, the counts were then fitted to a negative binomial model:The raw sequence reads were aligned using BWA 0.7.10 software , type of RNA sample , total number of reads of the sample, and residual, respectively. O is the log link function. Frequencies of indels per site were analysed by Fisher’s exact test using R software (3.1.2) and the glm.nb function in the MASS package (7.3–35).where http://www.ncbi.nlm.nih.gov/). The sequences were semi-automatically curated to exclude non-RNA viral genomes using a custom Perl script and by manual inspection. ORFs with annotations for noncanonical codon usage or in an unconventional translational manner, such as ribosomal frameshift were excluded. Next, using a custom Perl script, (i) sequences of ORFs with G1–2A6+ and G0A6+ motifs were parsed, (ii) translation products produced from RNA genomes with +1 or –1 base indels at each G1–2A6+ or G1A6+ motif were simulated, and (iii) the lengths of the simulated peptides were recorded. The length of a translation product of an ORF that contained no termination codons in the reading frame changed by a simulated indel was set to 0. In order to normalize difference of the number of entries of viral species in the database, the list of the motif site and the length of simulated peptides was then normalized per motif site by selecting only one entry (accession) with the longest predicted amino acid sequence following the simulated indel of a virus when multiple entries for the virus with the same length, and the same start and stop codon coordinates of the original ORF containing the motif exist.Full-length genomic sequences of RNA viruses were downloaded in the GenBank format from the NCBI website .


Kept together via an MS2 coat protein/RNA interface, it includes a fixed, polypeptidic transactivating domain and a variable RNA domain that recognizes the desired gene. Thanks to this device, we specifically upregulated five genes, in cell lines and primary cultures of murine pallial precursors. Gene upregulation was small, however sufficient to robustly inhibit neuronal differentiation. The transactivator interacted with target gene chromatin via its RNA cofactor. Its activity was restricted to cells in which the target gene is normally transcribed. Our device might be useful for specific applications. However for this purpose, it will require an improvement of its transactivation power as well as a better characterization of its target specificity and mechanism of action.To promote expression of endogenous genes  This goal has been achieved by short siRNA/miRNA-like molecules targeting gene promoters . Alternromoters –13 and Tromoters –16, DNA-romoters . Recentlromoters –23, alloromoters .To address these issues, we have conceived a novel, small and non-CRISPR-based device. This consists of a fully synthetic, ribonucleoprotein transcription factor, including a polypeptidic trans-activating domain as well as a non-coding RNA ‘bait’ domain. The former stimulates transcription. The latter specifically drives the whole ribonucleoprotein to the target gene. The two elements are kept together by two ancillary domains, a polypeptidic MS2 RNA-interacting domain , covalenFMR1, NAP22 and NRGN. Moreover, we employed this device to stimulate two transcription factor genes involved in early cortico-cerebral development, Emx2 and Foxg1. These genes control pallial field specification . Finally, performances of our transactivator could be optimized, by improving key features of its apofactor and cofactor components.Wild-type, CD1 strain mice used in this study were purchased from Harlan-Italy and housed at the SISSA mouse facility. Embryos were staged by timed breeding and vaginal plug inspection. Animals handling and subsequent procedures were in accordance with European laws  and with the National Institutes of Health guidelines. Embryos (E10.5 and E12.5) were harvested from pregnant dames killed by cervical dislocation and put in sterile ice-cold PBS supplemented with 0.6% glucose. Cerebral cortices (E12.5), mesencephalons (E10.5) and rhombo-spinal tracts (E10.5) were then dissected and collected in the same solution.Embryonic cortico-cerebral tissue was mechanically dissociated to single cells by gentle pipetting. Neural precursor cells were subsequently counted in a Burker chamber and plated in 24-multiwell plates , at the density of 1000 cells/μl, in a proliferative medium , 1X N2 (Invitrogen), 1 mg/ml BSA, 0.6% glucose, 2 μg/ml heparin (Stem Cell Technologies), 20 ng/ml bFGF (Invitrogen), 20 ng/ml EGF (Invitrogen), 1X Pen-Strept (Gibco), 10 pg/ml Fungizone (Gibco)). Neural precursors were acutely infected by recombinant lentiviruses and kept in culture up to 96 h.FMR1, NAP22 and NRGR experiments.HEK293T cells were cultured in Iscove's Modified Dulbecco's Medium 1X (Gibco) supplemented with 10% FBS (Sigma). They were used for lentiviral production and titration as well as for Nuclear-localization-signal, multimerized-MS2 peptide, Hemagglutinin-antigen, Virion-peptide-16) apo-transactivator is the following:The codon- and restriction enzyme-optimized coding sequence (cds) of NMHV , MS2_coat potein, HA_tag, AgeI restriction site, VP16_transactivating peptide (3x) and stop modules are highlighted by alternate styles, normal and underlined).ACCATGGTTTM Stbl 4TM (Invitrogen) cells and sequence-verified. LV_NMHV was used as an expression plasmid for FMR1, NAP22 and NRGN experiments in HEK293T cells. The same construct was utilized in producing the corresponding recombinant lentivirus, for Emx2 and Foxg1 assays in neural precursors.NMHV cds was synthesized, cloned into pUC57, grown in Xl1-blue cells and sequence-verified for us by GenScript. The cds was transferred as BamHI/SalI fragment into BamHI-SalI cut pCCLsin.PPT.hPGK.EGFP.Wpre . The resPlease note that pCCLsin.PPT.hPGK. EGFP.Wpre was used as the negative control for LV_NMHV and is referred to as ‘EGFP’, unless otherwise indicated.The NMHE expressing lentivector was obtained from LV_NMHV, replacing its AgeI/SalI fragment (encoding for the VP16 peptide) by the 141bp AgeI/SalI fragment ACCGGTTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAG CGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTC GGCATGGACGAGCTGTACAAGTAAAGCGGCCGCGTCGAC .The sequence of the RNA cofactor fragment MF6 was as follows (‘MS2 coat protein-binding stem-and-loop moieties’ are underlined):AAACATGAGGATCACCCATGTCG CCCGCTCCACCCAAACAACCCCCTAAACATGAGGATCACCCATGTCG AGGGCACCACCCAAACAAACAATGAAACATGAGGATCACCCATGTCG CATTCTCCAACCAACCAACCGGGGAAACATGAGGATCACCCATGTCG CCCCCTACACCCAAACAACCGCGCAAACATGAGGATCACCCATGTCG CGCGCATCACCCAAACAAACCAGGAAACATGAGGATCACCCATGTCG CCTGGGATCCACCGATATCMF6_AGATCTCGGGMF6 was cloned as BglII/EcoRV fragment into BamHI-EcoRV cut pcDNA3ΔPvuII, thus obtaining MF6-ø.FMR1-, NAP22- and NRGN-specific, wild-type ncRNA baits were amplified from genomic DNA by the oligos reported in Supplementary Table S1. NAP22.60L-derivative, mutant baits  were chemically synthesized as dsDNAs, flanked by sticky, BamHI/SalI compatible adaptors. All baits were then cloned as BamHI/XhoI or BamHI/SalI fragments into the BamHI/XhoI cut MF6-øplasmid, thus obtaining the corresponding MF6-bait constructs.Sequences of the RNA cofactor fragments MF1 and MF2 were as follows (here ‘MS2 coat protein-binding stem-and-loop moieties’ are underlined):AAACATGAGGATCACCCATGTCGCCCGCAGCGGATCCCCCGTCGACTTT TTTGGTACC;MF1_GATCGATATCCGGGAAACATGAGGATCACCCATGTCGCCCGCAGCGGATCGATATCCGGGAAACATGAGGATCACCCATGTCGCCCGCAGCGGAT CCCCCGTCGACTTTTTTGGTACC.MF2_GATCGATATCCGGGMF1 was cloned as BamHI-compatible/KpnI fragment into BamHI-KpnI cut pCCLsin.PPT.hPGK.EGFP.Wpre vector , thus obEmx2 and FoxG1 ncRNA baits were generally amplified from genomic DNA, by the oligos reported in Supplementary Table S2. The Emx2 30bp bait was obtained by annealing the two oligos reported in the same table. Baits were cloned as BglII/XhoI fragments into BamHI/SalI cut LV_MF1/2-øplasmids, thus obtaining the corresponding LV_MF1-bait and LV_MF2-bait constructs.The Plasmids driving constitutive expression of mCherry and EGFP referred to in Supplementary Figures S1 and S5, Pgkp1-mCherry and CMVp-EGFP, were obtained by replacing EGFP cds by mCherry cds in pCCLsin.PPT.hPGK.EGFP.Wpre and by cloning EGFP cds into pcDNA3ΔPvuII, respectively.6 cells were cotransfected by 0.75 μg of a ncRNA cofactor-expressing plasmid, 1.25 μg of an apofactor (or a control)-expressing plasmid and 0.25 μg of pcDNA3ΔPvuII-EGFP (see above), as internal control.Cells were generally cotransfected by LipoD reagent, according to Manufacturer's instructions. Where not otherwise specified, aliquots of 106 cells were cotransfected by 0.95 μg of ncRNA cofactor-expressing plasmid and 1.60 μg of apofactor (or control)-expressing plasmid.In specific cases (as highlighted in legends to figures), cells were cotransfected by Lipofectamine 3000 reagent, according to manufacturer's instructions. Here two rounds of cotransfection were performed, at a 12-hour interval. In each round, where not otherwise specified, aliquots of 10Recombinant third generation self-inactivating (SIN) lentiviruses were produced and titrated as previously described .http://blast.ncbi.nlm.nih.gov and run online with the following parameters: blastn, ‘for somewhat similar sequences’; expect threshold: 40; word size: 7; max matches in a query range : 0; match/mismatch scores, 2, -3; gap costs: existence 2, extension 2; filter low complexity regions: no; filter species-specific repeats: no). Homologous modules found by Blast were then prioritized and selected, based on their length (between 50 and 110 bp), gap density (<20%) and identity (>70%). Selected modules were subsequently mapped to the transcriptome, using the UCSC Genome browser  and the Ensemble-GENCODE track (accessible at http://www.ensembl.org). Lastly, they were filtered on the basis of their distance from transcriptional start-site (TSS) of potential off-target genes (from −0.5 kb to +0.5 kb). The results are summarized in Supplementary Tables S5 and S6.ncRNA bait sequences were blasted against human and murine genomes by Blast software  according to manufacturer's instructions. Agarose gel electrophoresis and spectrophotometric measurements (NanoDrop ND-1000) were employed to estimate its concentration, quality and purity.TM (Invitrogen) in the presence of random hexamers, according to the manufacturer's instructions. 1/100 of the resulting cDNA was used as substrate of any subsequent qPCR reaction. Limited to the intronless amplicons, prior to the retrotranscription, RNA preparations were treated by DNAseI (2U/μg of RNA) 1 h at 37°C, and processed by RNeasy Mini Kit (Qiagen). Next, negative control PCRs were run on RT− cDNA preparations. In general, PCR reactions were performed by the SsoAdvanced SYBR Green SupermixTM platform (Biorad), according to manufacturer's instructions. For each transcript under examination and each sample, cDNA was PCR-analyzed in technical triplicate, against absolute standards, and average results calculated. Averages were normalized against Gapdh and further normalized against controls. Experiments were performed at least in biological triplicate and analyzed by Student's t-test. Oligos were as in Supplementary Table S3.At least 0.5 μg of total RNA from each sample was retrotranscribed by SuperScriptIIINAP22) or neural cell cultures (case Emx2). Cells were transfected with constitutive expression plasmids (case NAP22) or acutely infected with bio-active and control lentiviruses (case Emx2), similar to transactivation assays. Then, they were kept in culture for 72 and 96 h, respectively. ChIP analysis was performed according to the MAGnify™ Chromatin Immunoprecipitation System protocol (Invitrogen), with minor modifications.The chromatin immunoprecipitation quantitative polymerase chain reaction assays (ChIP-qPCRs) were performed on chromatin extracted from HEK293T cells  into ∼600 bp fragments. Sonicated chromatin was immunoprecipitated for 2 h at 4°C, by 1.5 μg of an α-HA rat antibody , 2.5 μg of an α-RNApolII mouse antibody , or 2.5 μg of murine IgG (Invitrogen), in a final volume of 100 μl. Immunoprecipitated DNA was purified according to the manufacturer's instructions. Lastly, 1/30 of each immunoprecipitated (IP) DNA sample (case α-HA-IP) or 1/60 of it (case α-RNApolI-IP and IgG-IP controls) were amplified by qPCR. For each sample, qPCRs were performed in technical triplicate. Averages were normalized against input chromatin and further normalized against NMHV/MF6-øor NMHV/MF2-øcontrols, in case of NAP22 and Emx2 tests, respectively. Experiments were performed at least in biological triplicate and analyzed by Student's t-test. Oligos were as in Supplementary Table S4.For each ChIP assay, chromatin from 10HEK293T cells, naive or lipofected with LV_NMHV or LV_NMHV/pcDNA3ΔPvuII-MF6-NAP22, were grown on poly-L-lysine coated glass coverslips in 12 multiwell plates, fixed with 4% paraformaldehyde (PFA) for 20 min at 4°C and then washed three times in 1X PBS. Lentivirus-transduced, floating neural precursor aggregates were gently trypsinized to single cells and left to attach 1 h at 37°C to poly-L-lysine (200 μg/μl) coated SuperFrost Plus microscope slides (Menzel-Glaser). Here they were fixed by 4% PFA for 20 min at 4°C and washed three times in 1X PBS.In all cases, immunofluorescence was performed as previously described . The folTM. DAPI stained nuclei images were counted with ImageJ Cell Counter plug-in.HA, beta-actin and H3K9me3 immunofluorescences were photographed on a Nikon Eclipse TI microscope, equipped with a 40X objective and a Nikon C2 confocal system. For each sample, Z-stacks of 6, 0.5 μm-spaced optical sections, flattened and averaged, were shown. Tubb3 immunofluorescences were photographed on a Nikon Eclipse TS100 fluorescence microscope equipped with a DS-2MBWC digital microscope camera with a 20X objective. For each independent biological replicate, at least six fields, corresponding to at least 1500 cells, were analyzed by an operator blind of cells ‘genotype’. Images were processed by Adobe Photoshop CS2 softwareTM and Microsoft Excel 11 softwareTM.Western analysis was performed according to standard methods. Total cell lysates in CHAPS buffer were quantified by BCA protein assay kit (Fisher Scientific # 10678484) and denatured at 95°C for 5 min, prior to loading. Fifty microgram of proteins was loaded per each lane and was run on a 12% acrylamide −0.1% SDS gel. NAP22 was detected by a primary rabbit anti-BASP1 polyclonal antibody (Sigma #SAB2107478), used at 1:2500, and a secondary HRP-conjugated anti-rabbit antibody (LifeTech # 32260), used at 1:2000. As previously reported , under tEach ‘biological replicate’ included cells pooled from at least two independent wells/petri dishes. Numbers of biological replicates analyzed in each experiment are shown under the corresponding graphs. Each biological replicate was scored at least in technical triplicate. Data were normalized as reported in figure legends, averaged and statistically evaluated by Student's t-test . Variability was graphically shown by standard error of mean bars.To overactivate our GOIs by a programmable device, we first assembled a chimeric cDNA encoding for a novel polypeptidic apo-transactivator, NMHV Figure . This inThen, for each GOI we built the DNA copy of a dedicated non-coding RNA cofactor Figure . This inFMR1, NAP22 and NRGN, robustly expressed by these cells (http://webserver.mbi.ufl.edu/∼shaw/293.html). We built the cDNAs encoding for the NMHV apo-transactivator and the MF6-containing cofactors specific for these genes , 83±16% for NAP22 (P < 0.004) and 25±4% for NRGN (P < 0.014)  to HEK293T cells, in different combinations. After three days, the transfected cells were profiled for the three GOIs by qRTPCR Figure –F. Each ) Figure –F. Conve) Figure –F and thRemarkably, gene transactivation was specific. In fact, each NMHV/ncRNA pair, while stimulating the corresponding GOI, did not affect the other two genes . Moreover, selected genes sharing extensive homologies with the three GOIs in the surroundings of their TSSs (Supplementary Table S5) were not affected . Transactivation did not spread along the chromosome far from the intended bait target . Conversely, it was restricted to transcription units having their TSSs located in the 5′ and 3′ surroundings of such target ]. We found that, upon delivery of NMHV and MF6-NAP22, the qRTPCR signal corresponding to distinct regions of NAP22-pre-mRNA was differentially affected. This signal was unchanged across the splice donor site, while it was upregulated by +41±4%  closer to the splice acceptor site   Figure . With th2 Figure , a potenNAP22-TSS. In turn, this seems to promote the progression of RNApolII from the region immediately downstream of the TSS toward the 3′ end of the gene and results in increased transcription.All this suggests that, in the presence of the MF6-NAP22 cofactor, NMHV specifically binds to the surroundings of in vitro model of this process. We stimulated the embryonic patterning gene Emx2 in high-density, primary cultures of murine, cortico-cerebral precursors. Due to poor transfectability of these cells, we moved to lentiviral vectors. As the hexameric MF6 moiety resulted unstable in our lentiviral backbone . Neither NMHV nor MF1-Emx2 alone elicited any significant effect .Encouraged by these results, we decided to test the portability of this strategy to a gene mastering cortico-cerebral histogenesis, in an established backbone , we replpressing , murine pressing  and SuppR Figure . Comparet Figure . MoreoveEmx2 in pallial precursors slows down their neuronal differentiation  within engineered neural cultures , meaning that gene upregulation obtained by our strategy, albeit small, can yield robust biological effects.It has been reported that upregulation of ntiation ,30,33. TEmx2 upregulation elicited could be due to suboptimal interaction between the apo-transactivator and its ncRNA cofactor. Therefore, we tried to improve performances of our device, addressing this issue in two different ways. First, it has been reported that RNAs containing increasing numbers of MS2-binding stem-and-loop fingers interact with the MS2 coat protein in a progressively stronger way , while MF2-Emx2 alone did not elicit any significant effect   Figure .Emx2, we monitored the expression levels of six other genes , randomly chosen among those active in pallial precursors . Four additional genes , sharing extensive homologies with Emx2 in the surroundings of their TSSs (Supplementary Table S6), were neither affected . Notably, the replacement of the original ncRNA bait with its reverse complementary counterpart abolished Emx2 transactivation .To assess if MF2-Emx2/NMHV-dependent stimulation was restricted to ecursors , that weEmx2-bait by qPCR . No enrichment was conversely observed for the chromatin of three potential LV_MF1-Emx2 off-targets, Ptpn12, CachD1 and Dvl3   .To generalize these results, we built a ncRNA cofactor specific for another key gene involved in brain patterning, 1 Figure . Coexpre) Figure . This le) Figure  and D. TEmx2- and Foxg1-promoting protocols to neural precursors taken from two regions of the embryonic neural tube which do not express these two genes, rhombo-spinal tract , and assayed the corresponding MF6-containing cofactors with the NMHV apofactor, in HEK293T cells. All four baits were able to support transactivation of the intended target genes Figure  and B.Emx2-activating device, in cortico-cerebral precursors. Here, reducing the ncRNA bait from 179 to 60 bases (Supplementary Table S1) did not jeopardize device activity, which was slightly increased  was detected in a cell population infected at moi's of 15 and 5 .Notably, the results referred to above were obtained upon cotransfection of about only one-third of the total HEK293T cell population . When tHowever, incomplete cell population transduction could not be the only cause of limited gene expression gain and additional issues likely contributed to it. Among them there is the poor stability of our apoenzyme/coenzyme complex. In fact, better performances were elicited, as the number of MF fingers included into the RNA cofactor increased Figures ,E and 4BEmx2 and Foxg1 upregulation obtained in this study was <2-fold, it was sufficient to robustly change the behavior of neural precursors . The RNA bait could be shortened up to 60 bases, in the absence of adverse effects Figure –C. MutagThird, the bait-dependent transactivating effect exerted by NMHV was tightly restricted to the surroundings of the bait target and depended on bait orientation. TSSs located >1 kb far from the bait target were not affected . Conversely, transcription units having their 5′ ends <0.5 kb far from the bait target displayed a more articulated behaviour. Upon NMHV overexpression, they were generally stimulated by sense-oriented baits (Figures To summarize, as length, orientation and distance from TSS of the RNA bait were properly tuned, our device appeared to work specifically and reliably. As many as six primary baits out of six tested, 115–179 bases long, sense-oriented and directed against TSS-proximal targets Figures –C.During the execution of this study, several groups reported the successful creation of a novel RNA-programmable transactivator type –23, origIn theory, the best performing RNA-baits used in this study, being co-oriented with the upregulated mRNAs Figures  and 3G. NAP22, FMR1 and Emx2 loci, the most responsive to NMHV/RNA-cofactor stimulation (Figures NRGN and Foxg1 loci, less responsive to NMHV/RNA-cofactor, because of their lower abundance (Figures We do not know how our transactivator recognizes its target and promotes transcription. Concerning gene recognition, should the RNA cofactor directly interact with its cognate DNA, this would hardly occur via a triple helix structure, as triple helix formation requires long homopurinic-homopyrimidinic traits, absent in our baits . Convers Figures ,B,E,F. S Figures ,B. InterNAP22 intronic regions, but not for NAP22-TSS surroundings (Figure Regardless of the mechanism of chromatin recognition, RNA-mediated tethering of NMHV to 5′ TSS surroundings might promote transcription by a variety of VP16-dependent mechanisms –63. VP16s Figure . This isIn conclusion, here we described a novel programmable transactivator, including a polypeptidic effector and an RNA bait in charge of recognizing the intended target gene. We provided a basic characterization of its functional properties. We preliminarily addressed molecular mechanisms mediating its action. This device might be useful for some specific applications. Presently, however, it is a simple prototype, which still needs a substantial improvement of its transactivation power as well as a better characterization of its target specificity and mechanism of action. These issues will be hopefully subject of a dedicated follow-up study.


Bacteroides fragilis are rare in the United States. We isolated a multidrug-resistant anaerobe from the bloodstream and intraabdominal abscesses of a patient who had traveled to India. Whole-genome sequencing identified the organism as a novel Bacteroides genomospecies. Physicians should be aware of the possibility for concomitant carbapenem- and metronidazole-resistant Bacteroides infections.Metronidazole- and carbapenem-resistant  Bacteroides fragilis  mass spectrometry and 16S rRNA sequencing. New rim-enhancing fluid collections in the abdomen and pelvis were noted on computed tomographic scan images, and percutaneous drainage fluid from these collections grew 3+ (moderate) quantities of B. fragilis. Isolates from blood culture and abscess fluid were resistant to multiple classes of antimicrobial drugs, including metronidazole and imipenem . Annealed Y adaptors (5′-(PO4-) GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG-3′ and 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′) were ligated by using T4 DNA Ligase in Rapid Ligation Buffer . The library was PCR-amplified with KAPA HiFi HotStart ReadyMix  by using primer 1 (5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGC-3′) and primer 2 (5′-CAAGCAGAAGACGGCATACGAGATCAAGGTCACGGTCTCGGCATTCCTGCTGAACCG-3′). For sequencing, 250-bp paired-end reads were used with a custom index primer (5′-AGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCG-3′); sequencing was performed to an estimated coverage of 61× per base. Oligonucleotides were synthesized by Integrated DNA Technologies . De novo genome assembly was performed by using the ABySS v1.3.5 assembler . In brief, DNA was digested by using NEBNext dsDNA Fragmentase and then end-repaired and A-tailed by using B. fragilis reference genomes  showed a high degree of sequence divergence (Bacteroides species and observed similar results (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (ANIb) analysis  sequence database that contains published bacterial sequences; confirmation of the relationship indicated that the organism most closely resembles B. fragilis 638R and is not better classified as an alternative species already present in GenBank.Initial comparison of the clinical isolate with the 3 completed vergence . We expa results . An averanalysis  Table 1.B. fragilis reference strains, and 43.81% GC content. The shotgun sequence encodes 5,053 predicted genes, including 1,479 hypothetical proteins without inferred function. This number is significantly (1-tailed z-score = 1) larger than the average predicted gene content of B. fragilis reference genomes , although we cannot rule out minor contributions from plasmid DNA. Of 1,696 B. fragilis core genes , 1,508 (88.9%) were present in the clinical isolate.The clinical isolate contained an estimated 5.50 Mbp of DNA, ≈20% more than sequenced B. fragilis has been shown to result from up-regulation of the cfiA metallo-β-lactamase . Although the exact mechanism of metronidazole resistance is unknown, it has been attributed to chromosomally or plasmid-encoded nitroimidazole resistance (nim) genes encoding nitroimidazole reductase  and tetQ genes (tetracycline resistance). The isolate had a substitution in gyrA (Ser82 to Phe) known to confer resistance to fluoroquinolones  incorrectly identified this organism as B. fragilis (B. fragilis might instead represent this novel genomospecies, we compared ANIb values for other isolates with those of the clinical isolate in our study. ANIb values indicate that 2 of the multidrug-resistant B. fragilis isolates sequenced in an earlier report (cfiA–mediated carbapenem resistance and overexpression of efflux genes, respectively, have been also described and may contribute to the high levels of resistance observed with this isolate.Genomic sequencing revealed the isolate in this study to be a genomospecies related to, but distinct from, r report  Table 3.Enterobacteriaceae worldwide poses a major public health concern, often requiring use of more toxic “last-line agents,” such as colistin and polymyxin B. Although rare thus far, multidrug-resistant Bacteroides infections present a similar challenge to the treating physician and have the potential to become increasingly common in the future. Thus, providers should be aware of the possibility for concomitant carbapenem and metronidazole resistance in these organisms. Future studies will be required to determine what proportion of multidrug-resistant isolates can be ascribed to the novel genomospecies of Bacteroides reported here and whether this particular genomospecies offers any opportunities for targeted therapies.The increasing prevalence of carbapenem-resistant Bacteroides species and antibiotic resistance genes in clinical isolate.Values for the clinical isolate and other


R)-2-Hydroxy-4-phenylbutyric acid [(R)-HPBA] is a key precursor for the production of angiotensin-converting enzyme inhibitors. However, the product yield and concentration of reported (R)-HPBA synthetic processes remain unsatisfactory.(d-lactate dehydrogenase (d-nLDH) in Lactobacillus bulgaricus ATCC 11842 was found to have high bio-reduction activity toward 2-oxo-4-phenylbutyric acid (OPBA). The mutant d-nLDHY52L/F299Y was then coexpressed with formate dehydrogenase in Escherichia coli BL21 (DE3) to construct a novel biocatalyst E. coli DF. Thus, a novel bio-reduction process utilizing whole cells of E. coli DF as the biocatalyst and formate as the co-substrate for cofactor regeneration was developed for the production of (R)-HPBA from OPBA. The biocatalysis conditions were then optimized.The Y52L/F299Y mutant of NAD-dependent R)-HPBA in 90 min. Given its high product enantiomeric excess (>99%) and productivity (47.9 mM h−1), the constructed coupling biocatalysis system is a promising alternative for (R)-HPBA production.Under the optimum conditions, 73.4 mM OPBA was reduced to 71.8 mM ( R)-2-hydroxy-4-phenylbutyric acid [(R)-HPBA] and ethyl (R)-2-hydroxy-4-phenylbutyrate [(R)-HPBE] can be used as the key precursors for the production of angiotensin-converting enzyme (ACE) inhibitors R)-HPBA or (R)-HPBE. In recent years, great success has been achieved in asymmetric synthesis of (R)-HPBE catalyzed by recombinant reductases Escherichia coli strain harboring CgKR2 and glucose dehydrogenase (GDH) were applied in preparing (R)-HPBE with high concentration, desirable enantiomeric excess (ee) (>99%) and yield R)-HPBE, the product yield and concentration of the reported (R)-HPBA synthesis processes remained unsatisfactory (R)-HPBA. Compared with enzymatic resolution catalyzed by hydrolases, especially lipases R)-HPBA from OPBA through bio-reduction, highly efficient reductases and cofactor regeneration systems are needed.In previous studies, enzymatic resolution and asymmetric reduction were used in the biological production of (R)-HPBE preparation processes, which often utilize a specific carbonyl reductase, the production of (R)-HPBA from OPBA is catalyzed by 2-ketoacid reductases, especially NAD-dependent d-lactate dehydrogenase (d-nLDH) d-nLDH, OPBA could not be efficiently catalyzed by the biocatalyst because of its large aromatic group at C-4.On the other hand, cofactor regeneration systems that utilize glucose as a co-substrate in (R)-HPBE production may not be the proper choice in the (R)-HPBA production. The addition of glucose to the reaction system may result in the production of organic acids (such as gluconic acid and lactic acid) as byproducts and increase the complexity of the (R)-HPBA separation process d-nLDH was used to transform OPBA to (R)-HPBA. The cofactor NADH was regenerated by formate dehydrogenase (FDH) present in whole cells of Candida boidinii ATCC 32195. Although this NADH regeneration system produced CO2 as the only byproduct, which facilitated the isolation of (R)-HPBA, the whole cells of C. boidinii should be pre-treated with toluene to make them permeable In contrast to the  were assayed. The most active reconstructed d-nLDH was co-expressed with FDH from C. boidinii NCYC 1513 in E. coli BL21 (DE3). Then, a novel process utilizing whole cells of recombinant E. coli was developed for efficient production of (R)-HPBA from OPBA , phenylmethanesulfonyl fluoride (PMSF), and (R)-HPBA were purchased from Sigma-Aldrich. (S)-HPBA was purchased from J&K Chemical. All other chemicals in this study were of reagent grade.OPBA was purchased from Gracia Chemical Technology Co., Ltd. Chengdu (China). Isopropyl-β-E. coli DH5α and BL21 (DE3) were used for general cloning and expression procedures, respectively. E. coli WD, E. coli D1, and E. coli D2 were used to express wild d-nLDH, d-nLDHF299Y, and d-nLDHY52L/F299Y, respectively E. coli PD containing the vector pETDuet-1 was used as a control. The plasmid pETDuet-ldhDY52L/F299Y-fdh was constructed as follows: the ldhDY52L/F299Y gene was amplified using primers D.f and D.r with plasmid pETDuet-ldhDY52L/F299Y as a template. The fdh gene was amplified using primers F.f and F.r with genomic DNA of C. boidinii NCYC 1513 as a template. The resulting PCR products ldhDY52L/F299Y and fdh were digested with NcoI-BamHI and NdeI-XhoI, respectively, and cloned into MCS1 and MCS2 of pETDuet-1 successively to construct pETDuet-ldhDY52L/F299Y-fdh. The plasmid pETDuet-ldhDY52L/F299Y-fdh was then transformed into E. coli BL21 (DE3) to construct E. coli DF. All of the E. coli strains were grown in Luria-Bertani (LB) medium, and ampicillin was added at a concentration of 100 µg m1−1 if necessary.The bacterial strains, plasmids, and oligonucleotide primers used in this study are listed in E. coli PD, E. coli WD, E. coli D1, E. coli D2, and E. coli DF were all cultured in LB medium (100 µg ml−1 ampicillin) at 37°C to an optical density of 0.6 at 600 nm. IPTG (1 mM) was then added to induce protein expression, and cultures were grown at 16°C for a further 12 h. Cells were harvested by centrifugation at 6,000 rpm for 10 min, washed twice with 67 mM phosphate buffer solution (pH 7.4), and then subjected to successive biotransformation.The recombinant strains of −1. Samples (0.2 ml) were collected periodically and centrifuged at 12,000 rpm. The concentrations of OPBA and (R)-HPBA in the supernatant were analyzed by a high-performance liquid chromatography (HPLC) system .To optimize the biotransformation conditions, 5-ml reaction mixtures were incubated at 37°C and 120 rpm in a 25-ml flask. The pH was adjusted from 5.5 to 8.5. The concentrations of OPBA and formate were 25–175 mM. The concentration of the whole cells was 1–8 g dry cell weight (DCW) lE. coli PD, E. coli WD, E. coli D1, E. coli D2, and E. coli DF were harvested, suspended in 67 mM phosphate buffer solution (pH 7.4) containing 1 mM PMSF, and then disrupted by sonication  for 5 min in an ice bath. Thereafter, intact cells and cell debris were removed by centrifugation, and the resultant crude extracts were subjected to successive d-nLDH activity assays. The reduction activities of d-nLDH wild-type and mutants toward OPBA were assayed at 37°C in 1 ml of 50 mM Tris-HCl buffer (pH 7.5) containing 0.2 mM NADH, 10 mM OPBA, and the crude extracts of E. coli PD, E. coli WD, E. coli D1, E. coli D2, and E. coli DF. The rate of NADH decrease was determined by measuring the absorbance change at 340 nm d-nLDH activity was defined as the amount that catalyzed the oxidation of 1 µmol of NADH per minute. The protein concentration was determined by the Lowry procedure by using bovine serum albumin as the standard Cells of R)-HPBA were measured by HPLC (Agilent 1100 series) equipped with an Agilent Zorbax SB-C18 column  and a variable-wavelength detector at 210 nm. The mobile phase consisted of 1 mM H2SO4 and acetonitrile with a ratio of 85∶15 (v/v) at a flow rate of 0.7 ml min−1 at 30°C. Stereoselective assays for (R)-HPBA and (S)-HPBA were performed by HPLC analysis by using a chiral column  and a tunable UV detector at 254 nm. The mobile phase was 2 mM CuSO4 and acetonitrile with a ratio of 85∶15 (v/v) at a flow rate of 0.5 ml min−1 and a temperature of 25°C. The ee of (R)-HPBA was defined as [((R)-HPBA−(S)-HPBA)/((R)-HPBA+(S)-HPBA)]×100%.OPBA and (R)-HPBA by d-nLDH, the wild type d-nLDH from L. bulgaricus ATCC 11842 and its mutants were overexpressed in E. coli BL21 (DE3). Crude extracts of E. coli PD, E. coli WD, and E. coli D1 exhibited rather low OPBA reduction activity -HPBA from OPBA.To evaluate the possibility of transforming OPBA into -HPBA. OPBA at 50 mM was used as the substrate. Whole cells of E. coli PD, E. coli WD, E. coli D1, and E. coli D2 at a concentration of 8 g DCW l−1 were added to the reaction broth. The reaction was conducted at 37°C for 2 h. Here, NADH was regenerated through the direct addition of 50 mM glucose in the reaction system. Whole cells of E. coli D2 exhibited higher (R)-HPBA producing capability than did cells of E. coli PD, E. coli WD, and E. coli D1(R)-HPBA productivity (3.7 mM h−1) was still rather low because of the low efficiency of the NADH regeneration system. Additionally, organic acids, including pyruvic acid, lactic acid, and acetic acid, accumulated in the reaction broth (Asymmetric reduction of OPBA by whole cells of . coli D1. Howeveron broth .d-nLDHY52L/F299Y in E. coli DF and the (R)-HPBA production capability of the novel biocatalyst was investigated. Formate (50 mM) was added to the reaction broth for the regeneration of NADH. Although the activity of d-nLDHY52L/F299Y in the crude extract of E. coli DF was lower than in the extract of E. coli D2, whole cells of E. coli DF exhibited much higher (R)-HPBA producing capability than other biocatalysts (R)-HPBA at 49.0 mM was obtained from 50 mM OPBA. The productivity of (R)-HPBA was 24.5 mM h−1. Thus, whole cells of E. coli DF were selected as biocatalysts for (R)-HPBA production in the subsequent experiments.FDH is a good choice for NADH regeneration in a biocatalysis system because its substrate, formate, has a low cost and its product, carbon dioxide, is easily separated atalysts . (R)-HPBR)-HPBA production from OPBA by using whole cells of E. coli DF were optimized. The influence of the reaction pH was determined in reaction mixtures containing 13 g DCW l−1 whole cells of E. coli DF, 50 mM OPBA, 50 mM sodium formate, and 200 mM phosphate buffer (pH ranging from 5.5 to 8.5). After bioconversion at 37°C for 15 min, the highest (R)-HPBA production was detected at pH 6.5 -HPBA production was detected when 75 mM OPBA was used . When the reactions were conducted to approximate 80% theoretical yield, the highest specific productivity was observed at a biocatalyst concentration of 6 g DCW l−1  were conducted at pH 6.5 and 37°C for 30 min. The highest (was used . The eff DCW l−1 .R)-HPBA from OPBA was developed. Biotransformation was conducted at 37°C in 200 mM phosphate buffer (pH 6.5) with 6 g DCW l−1 whole cells of E. coli DF as the biocatalyst. As shown in R)-HPBA with a high enantiomeric purity -HPBA was produced with a yield of 60.9% after 360 min -HPBE and (R)-HPBA through bio-reduction R)-HPBE production processes, the product concentrations of the reported (R)-HPBA synthesis processes were rather low (d-LDH from Staphylococcus epidermidis and FDH from Candida boidinii were applied for (R)-HPBA production. (R)-HPBA at a concentration of 182 mM was produced, which is the highest reported yield of (R)-HPBA to date d-nLDH and FDH were co-expressed in E. coli DF and used for (R)-HPBA production from OPBA. The productivity (47.9 mM h−1) and ee (>99%) of the product were rather high for (R)-HPBA production. Additionally, given the simple composition of the biocatalytic system, separation of (R)-HPBA from the biocatalytic system would be relatively inexpensive. Therefore, the novel process established in this study could also be used as a promising route for the production of highly optically pure (R)-HPBA.Many biocatalysts have been used in the enantioselective production of -HPBA production from OPBA. After optimization of the biotransformation conditions, 73.4 mM OPBA was reduced to 71.8 mM (R)-HPBA with a high productivity of 47.9 mM h−1 and an excellent ee (>99%). The constructed coupled biocatalysis system developed in this work may be a promising alternative for the production of the key medical intermediate (R)-HPBA.In summary, whole cells of Figure S1HPLC analysis of the product of the catalytic reaction by using whole cells of E. coli D2 (A) as the biocatalyst and glucose as the substrate for NADH regeneration or whole cells of E. coli DF (B) as the biocatalyst and sodium formate as the substrate for NADH regeneration.(TIF)Click here for additional data file.Figure S2HPLC analysis of the product of the catalytic reaction utilizing the whole cell biocatalyst. (A) HPLC analysis of (R)-HPBA and (S)-HPBA. (B) Product of the catalytic reaction.(TIF)Click here for additional data file.


Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are widely used to treat HIV-1-infected individuals; indeed most first-line antiretroviral therapies typically include one NNRTI in combination with two nucleoside analogs. In 2008, the next-generation NNRTI etravirine was approved for the treatment of HIV-infected antiretroviral therapy-experienced individuals, including those with prior NNRTI exposure. NNRTIs are also increasingly being included in strategies to prevent HIV-1 infection. For example: (1) nevirapine is used to prevent mother-to-child transmission; (2) the ASPIRE (MTN 020) study will test whether a vaginal ring containing dapivirine can prevent HIV-1 infection in women; (3) a microbicide gel formulation containing the urea-PETT derivative MIV-150 is in a phase I study to evaluate safety, pharmacokinetics, pharmacodynamics and acceptability; and (4) a long acting rilpivirine formulation is under-development for pre-exposure prophylaxis. Given their widespread use, particularly in resource-limited settings, as well as their low genetic barriers to resistance, there are concerns about overlapping resistance between the different NNRTIs. Consequently, a better understanding of the resistance and cross-resistance profiles among the NNRTI class is important for predicting response to treatment, and surveillance of transmitted drug-resistance. Lys3 molecule that is hybridized to the primer binding site (PBS) of the viral RNA genome. This nascent DNA strand is elongated by the RNA-dependent DNA polymerase activity (RDDP) of RT until the 5' end of the HIV-1 RNA is reached. RT then uses its RNase H activity to hydrolyze the RNA strand of the RNA/DNA duplex which allows the DNA to hybridize with a repeat sequence at the 3' end of the HIV-1 RNA. Following this strand transfer, the viral DNA strand is elongated by the RDDP activity of RT until the entire RNA template has been copied. The RNase H activity of RT also hydrolyzes the HIV-1 RNA during synthesis of (−) strand DNA, except for a purine rich sequence, termed the polypurine tract (PPT), which serves as a primer for the initiation of (+) strand DNA synthesis. The DNA-dependent DNA polymerase (DDDP) activity of RT elongates the PPT primer. Removal of the PPT and tRNA primers by RT RNase H activity then allows a second strand transfer to take place by interaction of complementary PBS sequences. The HIV-1 RT DDDP activity including strand-displacement activity completes the synthesis of the double stranded proviral DNA precursor.Reverse transcription of the single-stranded (+) RNA genome into double-stranded DNA is an essential step in the HIV-1 replication life-cycle [Due to its essential role in HIV-1 replication, RT is a major target for antiviral drug development and two classes of inhibitors, (1) the nucleoside and nucleotide RT inhibitors (NRTIs) and 2) the nonnucleoside RT inhibitors (NNRTIs), have been approved by the FDA for the treatment of HIV-1 infection. The NRTIs are analogs of naturally occurring dNTPs that lack a 3'-hydroxyl group on the ribose sugar/pseudosugar [ the nonnet al. reported that NNRTI binding distorts the precise geometry of the DNA polymerase catalytic site, especially the highly conserved tyrosine-methionine-aspartic acid-aspartic acid (YMDD) motif and proposed that this class of drugs inhibits DNA polymerization by locking the polymerase active site in an inactive conformation [et al. observed that NNRTI binding deformed the structural elements that comprise the “primer grip”, a region in RT that is involved in the precise positioning of the primer DNA strand in the polymerase active site [et al. proposed that the NNRTI-BP may normally function as a hinge between the palm and thumb domains [NNRTIs interact with HIV-1, but not HIV-2, RT by binding to a single site on the p66 subunit of the HIV-1 RT p66/p51 heterodimer termed the NNRTI binding pocket (NNRTI-BP) that is situated approximately 10 Å from the RT DNA polymerase active site . Crystal domains . Since tNNRTIs are widely used to treat HIV-1-infected individuals . Indeed,NVP is a dipyridodiazepinone inhibitor , and wasp < 0.001) [vs. 69.3%; lower limit of one-sided 97.5% CI–10.9%). In addition, more patients discontinued in the maraviroc compared with the EFV arm due to lack of efficacy (11.9% vs. 4.2%). The 004 study compared the efficacy and tolerability of raltegravir vs. EFV, both combined with tenofovir and lamivudine [vs. 81%; p = 0.003) [EFV, a benzoxazinone inhibitor , is the < 0.001) . The ran< 0.001) . At 48 wmivudine . At 96 wmivudine ,25,26, bmivudine . Of note= 0.003) . Howeverin vitro activity against a broad range of HIV-1 groups and subtypes, including strains that exhibit resistance to EFV and NVP [in vitro include E138K, Y181C/I, V179F, G190E and M230L [vs. 39% in DUET-1, p < 0.01 and 62% vs. 44% in DUET-2, p < 0.001, respectively). A comprehensive analysis of study-entry resistance data from the DUET studies identified 17 ETR resistance-associated mutations, namely: V90I, A98G, L100I, K101E/H/P, V106I, E138A, V179D/F/T, Y181C/I/V, G190A/S, and M230L [et al. reported that in a protease inhibitor-naïve population, with baseline NRTI and NNRTI resistance and NRTI recycling, TMC125 was not as effective as first use of a protease inhibitor due to baseline NRTI and NNRTI resistance [ETR is a potent diarylpyrimidine (DAPY) analog which shows  and NVP . Comparend M230L . ETR effnd M230L . At entrnd M230L . Importand M230L . In the sistance .50 values range from 0.07 to 8.45 nM) [in vitro experiments, RPV shows a resistance profile and a genetic barrier to the development of resistance comparable to those of ETR [vs Efavirenz) and ECHO (Early Capture HIV Cohort Study) studies [vs. EFV in combination with two NRTIs. In both studies, RPV was found to be non-inferior to EFV in treatment-naïve participants. However in both trails, RPV-treated participants had higher rates of virologic failure than EFV-treated participants (10% vs. 6%), particularly in individuals with pre-treatment HIV-1 viral loads >100,000 copies/mL (17% virologic failure in the RPV arm vs. 7% in the EFV arm). Resistance analyses demonstrated that the most common NNRTI-resistant mutation that emerged when participants failed RPV was E138K, typically in combination with M184I or M184V/I mixtures. Viral fitness studies have suggested that E138K may compensate for the fitness deficits of both M184I and M184V and restore the replicative capacity of viruses containing M184I/V [p < 0.01) in four independent genotype databases [Like ETR, RPV is a DAPY analog that shows potent activity against wild-type HIV-1 group M isolates (0.07 to 1.01 nM) and group O isolates (EC8.45 nM) . In in ve of ETR . NNRTI r studies ,37. Thes M184I/V . However M184I/V . Other matabases . Importaatabases . As suchA parentral, long-acting form of RPV (RPV-LA) has been developed with the goal of improving treatment adherence and for potential use as a PrEP agent . A RPV-LIn vitro, DAP resistance correlates with the following mutations: V90I, L100I, K101E, V106I, V108I, E138K, E138G, Y181C, and Y188L [DAP is also a DAPY analog that demonstrates potent, dose-dependent inhibitory effects against a broad panel of HIV-1 isolates from different clades . DAP wasnd Y188L .50 of <1 nM). MIV-150 has been extensively evaluated in challenge models of HIV and the herpes simplex virus (HSV) when combined with carrageenan , and zinc acetate [MIV-150 is a PETT-urea analog with potent antiviral activity Nearly all of the NNRTI resistance mutations are within or adjacent to the NNRTI-binding pocket. Indeed there is no evidence that any one mutation only confers resistance to a single agent: most NNRTI-resistance mutations reduce susceptibility to two or more NNRTIs .(ii)i.e., Y181I/V) a single mutation may be sufficient.The genetic barrier to NNRTI resistance is low. Typically, EFV, NVP and RPV require only a single mutation to reduce clinical efficacy. ETR requires two mutations, but in certain circumstances  which share over-lapping resistance pathways. Consequently, a better understanding of the resistance and cross-resistance profiles among the NNRTI class is important for predicting response to treatment, and surveillance of transmitted drug-resistance.


Objective: The selectins are cell adhesion molecules that mediate the interactions among leukocytes, activated platelets, and endothelial cells. We aimed to investigate whether P-selectin polymorphisms are associated with thrombosis in patients with antiphospholipid syndrome (APS). Materials and Methods: The diagnosis and classification of APS were based on the report of an international workshop. Genomic DNA was extracted from citrated blood samples of all subjects. Three single nucleotide polymorphisms associated with the P-selectin coding region  were assessed. Results: There were 26 APS (65%) patients with thrombosis. The number of patients without thrombosis was 14 (35%). The frequency of the N562D-DN genotype was significantly higher in patients with APS than in healthy controls (p=0.003). The frequency of this genotype was significantly higher in patients with APS with thrombosis compared with patients with no thrombosis (p=0.03). The N562D-NN genotype was found at a higher frequency in patients with APS than in healthy controls (p=0.004). Conclusion: Our results suggest that the N562D polymorphism of the DN genotype of P-selectin is associated with an increased risk of thrombosis in patients with APS. Antiphospholipid syndrome (APS) is an autoimmune disease characterized by pregnancy morbidity and arterial-venous thrombosis in the presence of antiphospholipid antibodies (aPLAs) . AlthougPatients and ControlsForty adult patients with APS and 40 healthy subjects with no history of thrombosis or autoimmune disease were included in the study. The history of disease, physical examination, and screening of lupus anticoagulant (LA) and serum anticardiolipin IgG and IgM levels were assessed for all patients. The diagnosis and classification of APS were based on an international consensus statement . The subLA was diagnosed using activated partial thromboplastin time, kaolin clotting time, and Russell’s viper venom test according to published criteria . IgG andThe study protocol was approved by the local ethics committee, and written and signed informed consent was obtained from all participants.GenotypingGenomic DNA was extracted from citrated blood samples. Three single nucleotide polymorphisms associated with the P-selectin coding region  were assessed. Polymerase chain reaction (PCR) was done in a total volume of 25 µL containing 2 U of Taq DNA polymerase (Fermentas), 2 mmol/L MgCl2, 0.2 mmol/L of each dNTP, 2.5 µL of 10X PCR buffer, and 50 ng of genomic DNA. Allele specific primers were used in the following concentrations: 15 pmol of 290N-R (5’-TAAATGAATTCAGTCCATGGTTCCTACAT-3’), 5 pmol of 290S-R (5’-CACAGTCCATGGTTCCTTGAC-3’), 11 pmol of 290common (5’-TGTGTGGCTTTTCTCCTTTC-3’), 2 pmol of 562D-R (5’-ATTGCCCTACCAGCTTAAAGCCG TAGTC-3’), 7 pmol of 562N-R (5’-CTCCAGCTTAAAGCCGTTCTT-3’), 10.5 pmol of 562common (5’-TGAATATATAAGTGA ATGAACTTTGTG-3’), 3.5 pmol of 715P-R (5’-CCT GCT TGATAG GTT GCC ACG GAA GG-3’), 8 pmol of 715T-R (5’-GCAGGT TGG CAC GGT TGT-3’), and 9 pmol of 715common (5’-CTGTGA AAT GCT CAG AAC TAC ATG-3’). PCR amplification was carried out in a GeneAmp PCR System 9700 Thermo Cycler  using 36 cycles of 94 °C for 25 s, 57 °C for 25 s, and 72 °C for 25 s. PCR products were separated on agarose gels and stained with ethidium bromide. The PCR products were 115, 205, and 182 bp long for S290N, N562D, and T715P, respectively.Data are expressed as mean ± SD, number (%), or median (range). Test statistics were computed using the Mann-Whitney U test and the Kruskal-Wallis test. The chi-square test and odds ratio were used to calculate the 95% confidence intervals. Correlation coefficients and significance were calculated by Spearman’s test to assess the differences between groups. For all tests, a 2-tailed p-value of <0.05 was considered statistically significant. Statistical analyses were performed using the software package SPSS 15 running on Windows NT.There were 26 APS (65%) patients with thrombosis, 12 (46%) of which cases involved veins, 10 38%) arteries, and 4 (15%) veins and arteries together. The number of APS patients without thrombosis was 14 (35%). The mean age of patients  was 39.4±9.5 years. The characteristics of patients are seen in Table 1. The frequency of the N562D-DN genotype was significantly higher in patients with APS than healthy controls (p=0.003). The frequency of this genotype was significantly higher in patients with APS with thrombosis compared to patients without thrombosis (p=0.03). The N562D-NN genotype was found at a higher frequency in patients with APS than in healthy controls (p=0.004). The frequency of the N562D-NN genotype was not different between patients without thrombosis and control subjects (p=0.21). On the other hand, S290N and T715P polymorphisms were not different between patient and control groups  and 3. T8% arteriP-selectin is expressed on activated platelets and endothelial cells. P-selectin glycoprotein ligand-1 (PSGL-1) is found in neutrophils and monocytes, and these are from microparticles. Connecting P-selectin/PSGL-1 activated leukocytes for endothelium rolling, and providing the release of tissue factor initiates thrombosis . PreviouOn the other hand, the relationship between polymorphism of PSGL-1 and ischemic cerebrovascular disease was shown previously . Roldan In conclusion, our results suggest that the N562D polymorphism DN genotype of P-selectin is associated with an increased risk of thrombosis in patients with APS. The NN genotype of the same polymorphism might be protective against thrombosis in those patients. The effect of N562D polymorphism on sP-selectin levels will be studied in future work.Conflict of Interest StatementThe authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.


The mechanisms contributing to transcription-associated genomic instability are both complex and incompletely understood. Although R-loops are normal transcriptional intermediates, they are also associated with genomic instability. Here, we show that BRCA1 is recruited to R-loops that form normally over a subset of transcription termination regions. There it mediates the recruitment of a specific, physiological binding partner, senataxin (SETX). Disruption of this complex led to R-loop-driven DNA damage at those loci as reflected by adjacent γ-H2AX accumulation and ssDNA breaks within the untranscribed strand of relevant R-loop structures. Genome-wide analysis revealed widespread BRCA1 binding enrichment at R-loop-rich termination regions (TRs) of actively transcribed genes. Strikingly, within some of these genes in BRCA1 null breast tumors, there are specific insertion/deletion mutations located close to R-loop-mediated BRCA1 binding sites within TRs. Thus, BRCA1/SETX complexes support a DNA repair mechanism that addresses R-loop-based DNA damage at transcriptional pause sites.  •Endogenous BRCA1 and senataxin (SETX) interact in a BRCA1-driven process•BRCA1/SETX complexes are recruited to R-loop-associated termination regions (TRs)•BRCA1/SETX complexes suppress transcriptional DNA damage arising at nearby R-loops•BRCA1 breast cancers reveal indel mutations near BRCA1 TR binding regions Transcriptional R-loops represent a potential threat to genome integrity. Hatchi et al. show that BRCA1, in partnership with SETX, is engaged in a DNA repair mechanism that deals with R-loop-associated genomic instability at transcriptional termination pause sites. BRCA1) significantly increase the risk of developing breast and ovarian cancers  across the genome (Our laboratory has identified senataxin (SETX) as a putative BRCA1 binding partner in a yeast two hybrid and several, independent TAP-MS-based genome-wide BRCA1/protein interaction screens  binding assays  Figure D. The reIn light of the existence of BRCA1/SETX binding, we tested for BRCA1 co-recruitment at transcription termination sites associated with R-loops by chromatin immunoprecipitation (ChIP) performed on the endogenous human β-actin gene D Skourt. Cross-lSpecifically, we found that in mock siRNA-transfected cells BRCA1 binding was significantly enriched (3- to 6-fold) at the transcription termination site (5′ pause and pause site probes) when compared to the signals obtained following BRCA1 depletion F. NotablTo search for a connection between the formation of R-loops and the recruitment of BRCA1/SETX at the β-actin termination site, we first asked whether RNA:DNA hybrids forming within this gene are affected by BRCA1 and SETX depletion. We measured R-loop formation over the β-actin gene, employing DNA:RNA hybrid immunoprecipitation (DIP) of uncross-linked DNA using a monoclonal Ab specific for RNA:DNA hybrids (S9.6) . As showWe then tested BRCA1 and SETX chromatin occupancy over the β-actin gene following R-loop suppression. ChIP experiments were performed in HeLa cells before and after overexpression of GFP-tagged RNaseH1 A, which Thus, our results suggest that BRCA1/SETX complexes bind at termination region (TR) sites in response to local R-loop formation and that they also suppress R-loop abundance, in part, by participating in the regulation of transcription and of transcription termination.Since R-loop accumulation can increase the risk of genomic instability by promoting DNA damage , we testSince several studies suggest that the ssDNA of an R-loop is prone to transcription-associated mutations or DNA breaks , we adapIn the absence of BRCA1 or SETX, significant signals were observed when using 5′ pause and pause site primers, thereby reflecting the existence of one or more ssDNA breaks located near the termination region C and S3BWe then asked whether RNaseH1 overexpression suppressed the appearance of ssDNA breaks in the β-actin gene in BRCA1- or SETX-depleted cells. The background signals in control region C remained barely detectable, while RNaseH1-expressing BRCA1- or SETX-depleted cells revealed a significant signal decrease within the 5′ pause and pause regions C and S3BENSA, Gemin7) in which transcription termination is also dependent on R-loops  mechanism  , we furtSince the DNA damage that developed over R-loop-associated TRs upon BRCA1/SETX depletion is proposed to be R-loop and, therefore, transcription dependent, we analyzed the expression levels of these BRCA1 TR genes. RNA-seq data were used to analyze HeLa cell transcriptomes available from ENCODE. The expression profiles of genes whose filtered TRs did or did not overlap BRCA1 ChIP-seq peaks were compared. The data showed that BRCA1 TR genes clearly displayed higher mRNA levels (p = 1.1E-7), when compared to genes whose termination sites are not associated with a BRCA1 binding site  . Here weThese results suggest that in mammalian cells BRCA1 binds to TR (associated with paused RNAPII) in a substantial subset of highly transcribed genes, whose pause sites reflect a strong tendency to form R-loops. Taken together, our findings indicate that BRCA1 and SETX participate in the prevention/repair of ssDNA damage occurring at specific regions (TRs) in response to the transcription-associated formation of R-loops in physiological conditions. Conceivably, these genes require BRCA1/SETX-dependent DNA damage surveillance to ensure regulation of transcription .We next asked whether a defect in the newly detected BRCA1/SETX function could participate in the pathogenesis of BRCA1-deficient breast cancer tumors. Recent large-scale DNA sequencing screens performed in various human cancers have shed light on the nature and location of associated somatic mutations . We perfWe divided the 21 patient datasets into 3 breast cancer types: 12 sporadic , 5 BRCA1, and 4 BRCA2 mutant tumors. In parallel, we also performed a global mutation analysis among the “R-loop free” CoTC-regulated genes (“negative” genes)  where BRTo further investigate the mutational profile at the 3′ ends of the BRCA1 TR genes, we focused on a narrower region defined as: TTS ± 4 kb. Strikingly, the only significant enrichment observed was for indels in BRCA1 mutant tumors  B. PrecisOverall, these results show that BRCA1-deficient tumors are significantly enriched for indels in BRCA1 TR genes compared to BRCA2 and sporadic breast cancers. A significant fraction of the BRCA1 tumor-associated indels lie in the vicinity of BRCA1 binding TR loci, supporting the view that BRCA1 plays a critical role in preventing/repairing R-loop-mediated damage in the vicinity of R-loop-associated TR.−/− breast cancer genomes.In this study, a combination of biochemical, molecular, and genetic data provides evidence that a newly identified BRCA1/SETX complex is required to restrain the development of R-loop-mediated DNA damage at specific genomic regions. In particular, we have delineated a sequence of events that is predicted to occur over a substantial subset of R-loop-associated transcriptional pause sites and, when impaired, results in the BRCA1 TR-associated indel mutations observed in BRCA1β-actin, Gemin7, and ENSA  and in the processing of R-loops in partnership with RNA processing factors suggest that these structures are a source of cancer-related instability .BRCA1- and BRCA2-mutant cancers exhibit differences in histopathology, gene expression profiles, and clinical course, even though they share similarities in their marked levels of genomic instability  were prepared as previously described , except ChIP experiments on BRCA1, SETX, and γH2AX were performed using a modified version of N.J.P. laboratory’s protocol , as detaDetailed description of the computational and statistical analyses is available in The integrative Genomics Viewer (IGV) genome browser was used to visualize the indels and the BRCA1 and RNAPIISer2P ChIP-seq profiles across the termination regions of the BRCA1 TR genes .S.J.H. and D.M.L. originally suggested and first conceived the rationale for this project. E.H. designed the overall project with help from K.S.-S., K.M.M., D.M.L., and N.J.P. E.H. performed the experiments and analyzed the experimental data unless otherwise stated. E.H. received advice, reagents, and help for the ChIP experiments and the co-immunoprecipitation from K.S.-S., K.K.M., S.P., and S.D. K.S.-S. performed and analyzed the BRCA1 and SETX DIP and part of BRCA1 and SETX ChIP experiments. A.Y., M.L.E., and M.K. performed the genomic analysis of BRCA1 ChIP-seq data and K.K.-G. the meta-analyses correlating RNAPII and DRIP datasets. S.V., L.P., and G.P. performed the global mutational analyses of the breast tumors. E.H. and D.M.L. wrote the manuscript. All authors contributed to the editing of the manuscript.