Penicillium digitatum is the major postharvest pathogen of citrus fruit under Mediterranean climate conditions. Previous results have shown that proteases is the largest enzyme family induced by P. digitatum during fruit infection. In the present work, we addressed the study of the role of P. digitatum’s proteases in virulence following two complementary approaches. In the first approach, we undertook the functional characterization of the P. digitatumprtT gene, which codes for a putative transcription factor previously shown to regulate extracellular proteases in other filamentous fungi. Deletion of prtT caused a significant loss in secreted protease activity during in vitro growth assays. However, there was no effect on virulence. Gene expression of the two major secreted acid proteases was barely affected in the ΔprtT deletant during infection of citrus fruit. Hence, no conclusion could be drawn on the role of these secreted acidic proteases on the virulence of P. digitatum. In the second approach, we studied the effect of different protease inhibitors and chelators on virulence. Co-inoculation of citrus fruit with P. digitatum conidia and a cocktail of protease inhibitors resulted in almost a complete absence of disease development. Analysis of individual inhibitors revealed that the metalloprotease inhibitor, 1,10-phenanthroline, was responsible for the observed effect. The application of metal ions reverted the protective effect caused by the metallopeptidase inhibitor. These results may set the basis for the development of new alternative treatments to combat this important postharvest pathogen. Aspergillus or Penicillium, contain more than 200 and 100 annotated genes encoding for putative proteases in the MEROPS database (https://www.ebi.ac.uk/merops/), respectively. These enzymes play a major role in the physiology, morphogenesis, and metabolism of fungi. Their production is regulated in response to environmental signals, such as extracellular pH and carbon and nitrogen sources . T. TP. digprtT using the methodology previously described [P. digitatum. The first step of the gene deletion strategy was to construct the plasmid pDprtT using the USER-friendly cloning technique. Positive E. coli transformants were selected as kanamycin-resistant colonies and screened by PCR (data not shown). Afterward, the plasmid was introduced into electrocompetent A. tumefaciens AGL1 cells. The following step was the transformation of P. digitatum by co-cultivation with A. tumefaciens. Putative transformants were selected in the presence of hygromycin B.Targeted gene disruption of escribed  was perfprtT gene was verified by PCR using the primers 1F/HPHTer2 for the identification of the integration at the promoter region, and HPHPRO4/4R for the terminator region , whereas the ectopic mutant had the highest production .Conidia production of the parental strain Pd1, ectopic, and Δoduction A. No difprtT deletion mutant should be impaired in the production of extracellular proteases, a protein degradation assay was performed with the four strains in solid culture medium. We inoculated the strains onto filter discs on PDA plates containing 2 mg/mL of dichloran to restrict colony growth. After growth of the colonies during 4 days at 24 °C, the filters were removed and the plates were overlaid with a layer of agarose containing skim milk. Hydrolysis of milk proteins, mostly casein, at 37 °C during 3 days led to the appearance of a proteolytic halo. A clearing zone was observed only with strains expressing the prtT gene: The parental strain Pd1 and the ectopic mutant , but there was a statistical significance depending on the date of the experiment at the 95% confidence level .To test the role of PrtT in the pathogenicity of avelate’ A,B and ‘avelate’ C–F sweetavelate’ C,D was tP. digitatum [pep1), which codes for a putative aspartic endopeptidase Pep1. In the same study, other genes coding for putative proteases/peptidases were also detected: An aspergillopepsin (PDIP_06020), a tripeptidyl peptidase , a carboxypeptidase (PDIP_71590), and a serine peptidase (PDIP_67670). According to SignalIP prediction , four of them, except the serine peptidase, are extracellular proteases. As it was evident from the proteolytic assay that PrtT regulates the production of different extracellular proteases, we monitored the transcript levels of the prtT gene and the genes that encode the two major secreted proteases of P. digitatum during the infection process (PDIP_82060 and PDIP_12220). For this study, we collected spores and peel tissue of oranges infected with the parental strain Pd1, the ectopic eprtT3 mutant, and the knockout mutant ΔprtT70, at 12, 24, 48, and 72 hours post inoculation (hpi). A similar pattern of expression for prtT (PDIP_25240) was observed in both the parental strain Pd1 and the ectopic mutant , phosphoramidon , and the metal ion chelators EDTA, EGTA, ferrozine, and DETC  either alone or in the presence of 1,10-phenanthroline  Construction of prtT knockout mutants and characterization of the mutants during in vitro and in vivo growth, and (ii) the application of different protease inhibitors during the infection of P. digitatum in sweet oranges.This study aimed to characterize the role of secreted proteases in the virulence of ly clear . In our P. digitatum prtT gene, which encodes a putative transcription factor controlling the expression of multiple secreted proteases. The disruption of the prtT gene has been previously described in A. niger [A. fumigatus [P. oxalicum [P. digitatum  and the tripeptidyl peptidase aor1 encoding gene (PDIP_12220) [prtT gene, indicating that the regulation of these genes depends mostly on another factor(s). XprG is another transcription factor that regulates extracellular protease production in Aspergillus nidulans, a fungus that lacks a PrtT homolog. Deletion of both A. fumigatus xprG and prtT genes resulted in the generation of a mutant with almost no ability to degrade proteins; however, it retained wild-type virulence in murine systemic and pulmonary models of infection [P. digitatum, we identified a single XprG ortholog by amino-acid similarity to A. fumigatus XrpG (data not showed). The possibility that these two major proteinases are relevant for P. digitatum virulence in sweet oranges and the role of XrpG in the regulation of protease secretion should be further explored. In future experiments, we might generate P. digitatum deletion mutants in these two genes encoding major extracellular proteases and in the xrpG putative transcription factor gene.In order to determine the role of PrtT in virulence, the knockout Δ oranges . After 7l models ,16,31. Tte lysis ,16; howete lysis ,16. Our s fungus ,16. AnotP_12220) . The expP. digitatum. The presence of protease inhibitors has been described in plants and they are part of the pathogenesis-related proteins [Brassica oleracea by Lorito et al. [P. digitatum in oranges, and after 6 days post-inoculation, only 1,10-phenanthroline and the combinations containing this metalloprotease inhibitor were effective in controlling the development of P. digitatum in oranges. We tested other metalloprotease inhibitors, such as bestatin and phosphoramidon, and different metal ion chelators, such as EDTA, EGTA, ferrozine, and DETC  and antifungal  activities. Metal sequestration is also found in nature as a means to combat microbial infection. The process by which a host organism sequesters trace minerals in an effort to limit pathogenicity during infection has been designated ‘nutritional immunity’ [P. expansum could be involved in the formation of iron chelates, which could favor iron acquisition and pathogenicity [1,10-phenanthroline is a membrane permeable heterocyclic compound with the ability to sequester metal ions in biological systems, forming coordination compounds with them . It has gression . The utigression . Phenantmmunity’ ,41,42. Wmmunity’ ,41,42. Tmmunity’ ,44. Moregenicity .P. digitatum with some metal ions  in sweet oranges to further analyze the role of 1,10-phernatroline as a metalloproteinase inhibitor and as a chelator. We chose these four metal ions because 1,10-phenanthroline has a very high affinity for Fe2+, Zn2+, and Cu2+, but very low affinity for Mn2+. We hypothesized that the effect of these metal ions reverting the inhibition of 1,10-phenanthhroline would be related to their affinity to this chelator. The co-inoculation had no effect in the development of the pathogen, with an incidence of 100% of infected wounds after 7 dpi (Magnaporthe oryzae and Fusarium verticillioides [P. digitatum was partially reverted by application of CuSO4, and was completely reverted by the addition of ZnSO4 and FeSO4 (In the present work, we co-inoculated er 7 dpi B. Howevellioides . The effnd FeSO4 B, indicaP. digitatum during the infection of citrus fruits and that this gene is not involved in P. digitatum virulence. Furthermore, the good results in decay control obtained in the present study with 1,10-phenanthroline, a well-known metal chelator, warrants the exploration of a new possible target in fungal control: Metal chelation as a means to restrict micronutrient availability to pathogens.By way of conclusion, this study showed that PrtT, a putative transcription factor that regulates extracellular proteases, is not the major factor affecting the regulation of the two major extracellular proteases secreted by


Following the realization that eukaryotic RNA-binding proteomes are substantially larger than anticipated, we must now understand their detailed composition and dynamics. Methods such as RNA interactome capture (RIC) have begun to address this need. However, limitations of RIC have been reported. Here we describe enhanced RNA interactome capture (eRIC), a method based on the use of an LNA-modified capture probe, which yields numerous advantages including greater specificity and increased signal-to-noise ratios compared to existing methods. In Jurkat cells, eRIC reduces the rRNA and DNA contamination by >10-fold compared to RIC and increases the detection of RNA-binding proteins. Due to its low background, eRIC also empowers comparative analyses of changes of RNA-bound proteomes missed by RIC. For example, in cells treated with dimethyloxalylglycine, which inhibits RNA demethylases, eRIC identifies m6A-responsive RNA-binding proteins that escape RIC. eRIC will facilitate the unbiased characterization of RBP dynamics in response to biological and pharmacological cues. RNA interactome capture allows the detailed investigation of RNA-bound proteomes. Here the authors describe enhanced RNA-interactome capture using LNA-modified probes for increased sensitivity and specificity. RIC is based on the irradiation of living cells with ultraviolet (UV) light to generate covalent bonds between RNA and proteins that are in direct contact with each other. Subsequently, cells are lysed under denaturing conditions, and polyadenylated RNAs are isolated using deoxythymidine oligonucleotides (oligo(dT))-coupled beads. After extensive washes, the crosslinked proteins are eluted and identified by quantitative mass spectrometry2. RBP hits are defined as those proteins that are enriched in irradiated samples compared to controls that have not been irradiated but otherwise treated identically.RNA-binding proteins (RBPs) play essential roles in gene expression and other cellular functions. Thus their identification and the understanding of their mechanisms of action and regulation is key to unraveling physiology and disease. Recent approaches for the proteome-wide identification of RBPs, especially RNA interactome capture (RIC)Drosophila embryos, and plant seedlings7, successfully recovering many well-known RBPs. In addition, RIC has led to the identification of hundreds of new RBPs that were not previously related to RNA biology and that lack known RNA-binding domains (RBD)4. Nevertheless, it has been reported that RIC proteomes can be contaminated with DNA-binding and other proteins that could falsely be assigned as RBPs3.RIC has been applied to different eukaryotic systems, including mammalian cell lines, yeast, 9, experimental variability and technical noise limit its utility for such a challenging application.To unravel RBP function, we need to understand how RBPs respond to environmental and pharmacological cues. While RIC has successfully been employed toward this aimHere we describe enhanced RIC (eRIC), which combines the use of a locked nucleic acid (LNA)-modified capture probe and more stringent capture and washing conditions. The performance of the eRIC and RIC protocols was directly compared using Jurkat cells. eRIC markedly increases capture specificity, reduces material requirement, and improves signal-to-noise ratios compared to precedent techniques. eRIC is particularly suitable for the detection of unconventional RBPs and the identification of dynamic changes among the RNA-bound proteome.With suitable tools for de novo RBP discovery now in hand, we wanted to build on the principle of RIC and develop a method that is highly performant in comparative studies. To reach this goal, we aimed to reduce DNA and ribosomal RNA (rRNA) contaminations that could contribute inadvertently co-purifying proteins and increase background RBPs.7, followed by centrifugation to remove insoluble material . With this probe, all steps of the protocol, including capture and all washes, could be executed at 37–40 °C instead of 4 °C /poly(A) hybridization. This is followed by temperature-mediated elution at 50–55 °C Fig. . The inc28S rRNA is more drastic than of the 18S rRNA  RNA either by means of a sufficiently long poly(A) stretch that is bound by the capture probe, or by hybridization of the rRNA to complementary sequences within poly(A) RNA. In line with these considerations, efforts to further reduce 18S rRNA contamination were met by decreased poly(A) RNA yields. The qPCR results show that the higher temperature used in the eRIC protocol is not associated with RNA degradation  using intron-sensitive primers that amplify complementary DNA (cDNA) but not genomic DNA. Pull down of poly(A) RNA by eRIC is specific and capture probe-mediated, because no RNA is detected when uncoupled beads are used Fig. . CompareRNA Fig. . These dion Fig. .Fig. 2eRZNF80  RNA. Since the RNA analyses required heat rather than RNase elution of the eRIC samples for obvious reasons, the purity of eRIC samples may be even higher following the RNase-based elution of the original eRIC protocol.We then subjected the proteins eluted according to the two protocols to downstream analysis. Following eRIC, the eluates are vacuum-concentrated using a SpeedVac, instead of the Amicon filters that RIC employs and that are commonly associated with protein loss and size bias. To exclude technical bias, SpeedVac-mediated concentration was also applied to RIC samples (see below). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining of eluted proteins shows patterns that differ profoundly from the input samples and that are absent from the non-irradiated controls, indicating the enrichment of specific RBPs after UV crosslinking by both RIC and eRIC Fig. . The banSpecific enrichment for the known RBPs UnR, Nono, and HuR was confirmed in both the RIC and the eRIC samples by western blotting. Pull down of the RBPs by eRIC was at least equally efficient than for RIC Fig. .11. SP3 maximizes recovery of peptides for mass spectrometry and is compatible with the use of detergent throughout the procedure until the final wash. To facilitate the comparison between all other aspects of the RIC and eRIC protocols, and as described for the concentration step, SP3 was applied to both eRIC and RIC samples. Consequently, we used equal cell numbers for both methods, focusing on the differences in ribonucleoprotein (RNP) capture per se.To minimize pre-analytical sample loss, we introduced the highly sensitive Single-Pot Solid-Phase-enhanced Sample Preparation (SP3) protocolt test) and fold change (FC) > 2) were considered as “hits.” Applying this criterion, we identified 683 and 588 RBPs in eRIC and RIC samples, respectively  and subjected to liquid chromatography–tandem mass spectrometry (LC-MS/MS) Fig. . Proteinely Fig. . When weely Fig. . Thus eR12) were similarly identified by the two approaches  and FC > 2). Unsupervised clustering and posterior Gene Ontology (GO) analysis of these proteins revealed a high enrichment for terms associated with mRNA, such as “mRNA-processing,” “mRNA splicing,” and “mRNA transport” among the proteins preferentially recovered by eRIC  of DMOG for only 6 h. After crosslinking and lysis, we compared eRIC and RIC using two complete sets of biological replicates  as a test case, because α-ketoglutarate is required as a co-factor by RNA demethylasestes Fig. . eRIC letes Fig. , confirmt test) and a consistent FC of at least 10% in each replicate. eRIC recovered a specific group of 20 RBPs with increased and of 41 RBPs with decreased RNA-binding after DMOG treatment  RNA purified from Jurkat cells treated with 0.5 mM DMOG (or DMSO) for 6 h. This analysis indicated that DMOG incubation indeed increased the m6A modification of poly(A) RNA . Of the 61 DMOG-regulated RBPs that we identified by eRIC, at least 18 (30%) were previously shown to be affected by m6A21  RNA-bound proteome concordant with the increase in steady-state m6A levels that are missed by RIC, further supporting the superior performance of eRIC in comparative studies.22. We describe a method developed on the basis of existing approaches, which fills a critical void in the available repertoire: a robust, highly reproducible, and sensitive method to detect biologically relevant changes in the RNA binding of RBPs in response to biological cues.RBPs are central to every aspect of gene regulation and many other processes in cell biology5. Especially methods applied to living cells and organisms based on covalent RBP–RNA crosslinking have received much attention24. Taken together, these methods have revealed that the biological repertoire of RBPs is at least twice as large as previously anticipated, posing challenges and yielding opportunities for future discovery.During the past few years, different methods have been developed to uncover and study the proteome-wide repertoire of RBPs25. These strategies involve the in vivo incorporation of nucleotide analogs (such as 4-thiouridine or 5-ethynyluridine) into cellular RNA. However, metabolic labeling of RNA can inhibit rRNA synthesis and cause nucleolar stress26, which is expected to cause significant secondary effects. Furthermore, the preferential labeling of newly synthesized RNAs, especially for those with high turnover rates, can bias RBP-binding analyses. Most recently, the discovery that silica-based matrices not only retain nucleic acids but also proteins that are covalently bound to these has added a simple and powerful approach to the methodological repertoire for the discovery and isolation of RBPs, which is applicable to all classes of RNA27. However, this simple new method may offer limited selectivity especially against DNA-binding proteins, since silica matrices can bind both classes of nucleic acid28.RIC derived its popularity from the stringency of purification enabled by the poly(A) tail–oligo(dT) hybridization. However, this method is by definition limited to RBPs bound to polyadenylated RNAs. To address this, new approaches that also capture non-polyadenylated RNAs have been developed25. Poly(A) RNA represents only a small fraction of the total cellular RNA, and the absolute levels of many regulatory RBPs bound to this minority fraction are also low. By contrast, RBPs bound to non-poly(A) RNA such as ribosomal proteins, rRNA processing factors, and ribosome-associated proteins represent a large fraction of MS/MS peptides and can mask the detection of poly(A) RNA-associated RBPs. For this reason, methods based on global RNA purification are expected to underperform in comparative studies aimed to identify responses of RBPs bound to poly(A) RNAs such as mRNAs and long non-coding RNAs.In addition to the above considerations, the advantages of global RNA purification may be offset by disadvantages in the detection of poly(A) RNA-bound RBPs3. This problem is particularly evident with cells with a relatively small volume   were identified by RIC Fig. . The 22 31. The identity of the RBPs that are involved is still largely unknown. Two recent studies discovered RBPs with altered binding to m6A compared to unmodified RNA in vitro21. To the best of our knowledge, no equivalent proteome-wide studies have been performed on living cells. Remarkably, among the RBPs that eRIC identified to respond to DMOG treatment, we found a significant enrichment for proteins previously linked to m6A   display the responses that would be expected based on prior knowledge of their modes of interaction with m6A  is one of the most abundant posttranscriptional modifications on mRNAs. The m6A modification of mRNAs responds to environmental cues and appears to be relevant in, e.g., stem cell differentiation, T cell function, and tumorigenesisPs) Fig. 20,21. Mom6A Fig. . Since sHaving used DMOG treatment as a proof of concept for the use of eRIC for comparative analyses, this work provides a first in vivo analysis of the changes of the RNA-bound proteome in response to the inhibition of RNA demethylases and constitutes the first attempt to characterize the repertoire of m6A-regulated RBPs in vivo. Our datasets also implicate mTOR signaling in DMOG-induced effects on translation initiation factors and translation Fig. . Thus, w2 flasks  in RPMI 1640 medium  supplemented with 10% heat-inactivated Fetal Bovine Serum  and penicillin/streptomycin  in a humidified incubator at 37 °C and 5% CO2.Jurkat cells  were maintained as a suspension culture in 175 cm10. The probe was resuspended in nuclease-free water (Ambion) to a final concentration of 100 μM and coupled in DNA low binding tubes (Eppendorf) to carboxylated magnetic beads  through the 5’ amine as follows or kept at −20 °C until coupling. Bead slurry (50 mg/mL) was washed 3 times with 5 volumes of 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer pH 6. A 20 mg/mL solution of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride  in MES buffer was freshly prepared. Five volumes were combined with 1 volume of 100 μM probe solution and this was added to pelleted washed beads originated from 1 volume of bead slurry (for one capture: 1.5 mL EDC solution + 300 μL probe solution + 300 μL bead slurry). Coupling was performed for 5 h at 50 °C and 800 rpm, with occasional pelleting. Beads were then washed twice with phosphate-buffered saline (PBS) and then incubated with 200 mM ethanolamine pH 8.5 for 1 h at 37 °C 800 rpm to inactivate any residual carboxyl residue. Coupled beads were finally washed three times with 1 M NaCl and stored in 0.1% PBS–Tween at 4 °C.The capture probe  is composed of a primary amine at the 5’ end, a flexible C6 linker, and 20 thymidine nucleotides in which every other nucleotide is a LNA: /5AmMC6/+TT+TT+TT+TT+TT+TT+TT+TT+TT+TT Coupled beads can be reused several times. To do so, the poly(A) stretches interacting with the LNA probe, expected to be resistant to the RNA digestion, have to be eluted by other mean. Also, any trace of RNases from the elution has to be eliminated. Beads were reused a few times in this work and at least eight times in other works, with optimal results.Coupled beads employed for a capture (300 μL) were resuspended in 400 μL of nuclease-free water (Ambion), transferred to a 1.5 mL tube, and incubated for 5–10 min at 95 °C 800 rpm. Immediately afterwards, before bead slurry cool down, beads were collected by magnetic force, and the supernatant discarded. Beads were then washed 3 times with 5 volumes of water and 3 times with 5 volumes of lysis buffer and stored in 0.1% PBS–Tween at 4 °C until use.8 proliferating Jurkat cells at a density of about 1–1.5 × 106 cells/mL were employed per sample. Where stated, 0.5 mM DMOG  or an equivalent volume of DMSO  was added to the culture medium for 6 h prior to processing. DMSO concentration in the medium was 0.023% v/v. Cells were collected by centrifugation at 400 × g for 5 min at 4 °C, resuspended in 40 mL of cold PBS, and split into two 145 × 20 mm2 petri dishes , which were deposited on a metal plate pre-cooled on ice and irradiated with 150 mJ/cm2 at 254 nm UV light in a Spectrolinker XL-1500 (Spectronics Corporation). Irradiation was omitted in −UV controls. While constantly maintaining 4 °C, cells were transferred to 50 mL conical centrifuge tubes, pelleted at 400 × g for 5 min, and lysed in 7.5–10 mL of ice-cold lysis buffer (see composition below) supplemented with cOmplete Protease Inhibitor Cocktail . To enhance homogenization, samples were passed 3–5 and 6–10 times through syringes with 22-Gauge (0.7 mm diameter) and 27-Gauge needles (0.4 mm diameter), respectively, snap frozen in liquid nitrogen, and kept at −80 °C for several days until further processing.In all, 1.0–1.3 × 10Cell lysates were thawed in a 37 °C water bath, incubated for 15 min at 60 °C, quickly cooled down on ice, and clarified 5 min at max speed and 4 °C. Dithiothreitol (DTT) extra (5 mM) was added to the samples. LNA2.T-coupled beads were equilibrated in lysis buffer (3 buffer exchanges with 3 volumes of lysis buffer each). After saving 100 μL as input, lysates were incubated with 300 μL of equilibrated LNA2.T-coupled beads for 1 h at 37–40 °C (inside an incubator) with gentle rotation to capture RNA–protein complexes. Beads were collected with a magnet, and the supernatant was transferred to a fresh tube for a second round of capture. Beads were subjected to successive rounds of washes, each of them performed for 5 min with gentle rotation at 37–40 °C (inside an incubator) with 10 mL of the corresponding buffer pre-warmed to 37–40 °C. We performed 1 wash with lysis buffer and 2 successive washes with each of the buffers 1, 2 and 3 (see composition below). Pre-elution was performed in 220 μL of nuclease-free water (Ambion) for 5 min at 40 °C and 800 rpm. Afterwards, the bead suspension was divided into two aliquots, one of 200 μL for the RNase-mediated elution for protein analysis and one of 20 μL that was heat-eluted for RNA/DNA analyses. For the RNase-mediated elution, beads were resuspended in 150 μL of 1× RNase buffer (see composition below), 5 mM DTT, 0.01% NP40, ∼200 U RNase T1 , and ∼200 U RNase A  and incubated at 37 °C 800 rpm for 30–60 min. Beads were then collected with a magnet, and the supernatant transferred to a fresh tube, which was placed again on a magnet (to fully remove any trace of beads) before saving the supernatant. Eluates were maintained on ice until finishing the second round of capture. Then combined eluates were supplemented with 2 μL 10% SDS and concentrated using a SpeedVac until reaching a volume of <100 μL (30–45 min at 37 °C), snap frozen, and stored at −80 °C. Heat elution was performed on the beads reserved ad hoc with 15 μL of elution buffer (see composition below) at 95 °C 800 rpm for 5 min. Beads were immediately collected, and the supernatant quickly recovered (before temperature drops). Any trace of beads was eliminated by a second round of collection as explained before.Lysis buffer: 20 mM Tris-HCl (pH 7.5), 500 mM LiCl, 1 mM EDTA, 5 mM DTT, 0.5% (w/v) LiDS.Buffer 1: 20 mM Tris-HCl (pH 7.5), 500 mM LiCl, 1 mM EDTA, 5 mM DTT, 0.1% (w/v) LiDS.Buffer 2: 20 mM Tris-HCl (pH 7.5), 500 mM LiCl, 1 mM EDTA, 5 mM DTT, 0.02% (v/v) NP40.Buffer 3: 20 mM Tris-HCl (pH 7.5), 200 mM LiCl, 1 mM EDTA, 5 mM DTT, 0.02% (v/v) NP40.10× RNase buffer: 100 mM Tris-HCl(pH 7.5), 1.5 mM NaClElution buffer: 20 mM Tris-HCl (pH 7.5), 1 mM EDTA.25 magnetic beads (NEB) at 4 °C for 1 h with gentle rotation. Beads were washed with 10 mL of ice-cold buffers. RNP complexes were eluted in 165 μL of elution buffer for 5 min at 55 °C and 800 rpm. An aliquot of 15 μL was taken and used for RNA/DNA analyses. The remaining 150 μL were combined with 10× RNase buffer, 1 M DTT, and 1% NP40  and ∼200 U RNase T1 and RNase A (Sigma-Aldrich). RNA was digested for 60 min at 37 °C. Two rounds of capture were performed and combined eluates were concentrated and stored as described for eRIC. Final volume of RIC eluates was adjusted to the volumes of the corresponding eRIC eluates.Lysates were thawed in a 37 °C water bath, and after taking 100 μL as input, they were incubated with 300 μL of equilibrated oligo(dT)11. Proteins were digested by trypsin (Promega) at 37 °C overnight using an enzyme-to-protein ratio of 1:50. Peptides were labeled with TMT10plex Isobaric Label Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. For further sample clean up, an OASIS HLB µElution Plate (Waters) was used. Offline high pH reverse phase fractionation was carried out on an Agilent 1200 Infinity high-performance liquid chromatography system, equipped with a Gemini C18 column .Captured proteins were reduced in 10 mM DTT in 50 mM HEPES pH 8.5 at 56 °C for 30 min and alkylated with 20 mM 2-chloroacetamide in 50 mM HEPES pH 8.5 for 30 min at room temperature in the dark. Samples were prepared using the SP3 protocolAn UltiMate 3000 RSLC nano LC system (Dionex) fitted with a trapping cartridge  and an analytical column  was employed. Trapping was carried out with a constant flow of solvent A (0.1% formic acid in water) at 30 µL/min onto the trapping column for 6 min. Subsequently, peptides were eluted via the analytical column with a constant flow of 0.3 µL/min with increasing percentage of solvent B (0.1% formic acid in acetonitrile) from 2 to 4% in 4 min, from 4 to 8% in 2 min, from 8 to 28% in 96 min, and finally from 28 to 40% in 10 min. The outlet of the analytical column was coupled directly to a QExactive plus mass spectrometer (Thermo Fisher Scientific) using the proxeon nanoflow source in positive ion mode.m/z in profile mode in the FT with resolution of 70,000. The filling time was set at maximum of 100 ms with a limitation of 3 × 106 ions. Data-dependent acquisition was performed with the resolution of the Orbitrap set to 35,000, with a fill time of 120 ms and a limitation of 2 × 105 ions. A normalized collision energy of 32 was applied. The instrument was set to alternate between MS and data-dependent MS/MS-based acquisition with up to a maximum of 10 MS/MS events per cycle. A minimum AGC trigger of 2e2 and a dynamic exclusion time of 30 s were used. The peptide match algorithm was set to “preferred” and charge exclusion to “unassigned,” charge states +1 and +5 to +8 were excluded. MS/MS data was acquired in profile mode.The peptides were introduced into the QExactive plus via a Pico-Tip Emitter 360 µm OD × 20 µm ID; 10 µm tip (New Objective) applying a spray voltage of 2.3 kV. The capillary temperature was set at 320 °C. Full mass scan was acquired with mass range 350–1400 32 and Mascot (v2.2.07) were used to process the acquired data, which was searched against the Uniprot Homo sapiens proteome database UP000005640, which contains common contaminants and reversed sequences. The following modifications were included into the search parameters: Carbamidomethyl (C) and TMT10 (K) (fixed modifications), Acetyl (N-term), Oxidation (M), and TMT10 (N-term) (variable modifications). A mass error tolerance of 10 ppm and 0.02 Da was set for the full scan (MS1) and the MS/MS spectra, respectively. A maximum of two missed cleavages were allowed and the minimal peptide length was set to seven amino acids. At least two unique peptides were required for protein identification. The FDR on peptide and protein level was set to 0.01. The R programming language (ISBN 3–900051–07–0) was used to analyze the raw output data of IsobarQuant. Potential batch effects were removed using the limma package33. A variance stabilization normalization was applied to the raw data using the vsn package34. Individual normalization coefficients were estimated for crosslinked and non-crosslinked conditions. During the DMOG versus DMSO comparison, an additional blocking factor for the protocol (RIC or eRIC) was chosen. Normalized data were tested for differential expression using the limma package. The replicate factor was included into the linear model. For comparisons between crosslinked versus non-crosslinked, hits were defined as those proteins with an FDR <5 % and an FC > 2. In eRIC/RIC comparative experiments, proteins were first tested for their enrichment over −UV controls, and the intensity of the proteins enriched in at least one condition were compared in the corresponding +UV samples. The R package fdrtool35 was employed to calculate FDRs using the t values from the limma output. Proteins with an FDR < 5% and a consistent FC of at least 10% in each replicate were defined as hits. The ggplot2 R package36 was used to generate the graphical representations.IsobarQuant5) and that lack known RNA-binding functions . Overlaps with other RIC data sets were displayed in UpSet plots37.In the “single point” eRIC/RIC experiments, enzymes were defined as those proteins listed in the six enzyme commission groups (EC 1–6) in the IntEnz database (release May 2017). Metabolic enzymes were defined as those enzymes that map to Metabolism in Reactome, plus the subunits of the ATP synthase and the respiratory chain complexes. enigmRBPs are defined as those eRIC/RIC hits that do not have a known RNA-binding domain , http://bioinfogp.cnb.csic.es/tools/venny/index.html). Analysis were restricted to those proteins detected by the eRIC/RIC comparative experiments. Fisher’s exact tests were used to calculate enrichment of m6A-responding proteins among eRIC and RIC samples.Comparison of DMOG-responsive hits identified by eRIC and RIC with previously reported m6A-regulated RBPsHomo sapiens ; annotation data set: GO biological process complete or GO cellular component complete, as indicated; test type: Fisher’s exact with FDR multiple test correction. Overrepresented GO terms were manually curated, and only selected terms were included in the main figures due to space constrains. The full lists of GO-enriched terms are provided in Supplementary Data 36 was used to generate the graphical representations.GO-term enrichment analysis were performed with AmiGO 2 (powered by PANTHER), using the following parameters: analysis type: PANTHER overrepresentation test; reference list: The concentration of captured RNA (heat-eluted) was estimated using a NanoDrop spectrophotometer (Thermo Fisher Scientific). To determine the profile of captured RNA, 1 µL of each sample was diluted to 5–10 ng/µL and analyzed in an Agilent 2100 Bioanalyzer System using the RNA 6000 Pico Kit, following the manufacturer’s indications. Where stated, total RNA from whole-cell lysates was purified using the Quick-RNA MicroPrep Kit (Zymo) and analyzed similarly.2: β-actin , GAPDH  and 18 S rRNA . From ref. 3: L1.3 .In all, 2–5 µL of the undiluted captured RNA were reverse transcribed into cDNA using SuperScript II (Life Technologies) and random hexamers (Life Technologies), according to the manufacturer’s instructions. Real-time qPCR was performed using SYBR Green PCR Master Mix  in a QuantStudio 6 Flex system (Life Technologies) with the following primers : 28 S rRNA , RPS6 , ZNF80 . From ref. Proteins co-purified by eRIC or RIC or present in whole-cell lysates (inputs) were separated by SDS-PAGE and subjected to silver staining following standard procedures or transferred onto a nitrocellulose membrane and analyzed by western blotting. Primary antibodies against the following proteins at the indicated dilutions were used: Cold shock domain-containing protein E1 (CSDE1)/UNR , Non-POU domain-containing octamer-binding protein (NonO) , and ELAV-like protein 1 (ELAVL1)/Hu-antigen R (HuR) . As a secondary antibody, a Goat anti-rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP)  was employed. Uncropped scans are provided in Supplementary Fig. 6 cells/mL were incubated with 0.5 mM DMOG  or an equivalent volume of DMSO  for 6 h. Subsequently, cells were pelleted (400 × g 5 min 4 °C), washed with ice-cold PBS, and lysed in ice-cold RIPA buffer  supplemented with proteinase inhibitors  and phosphatase inhibitors . After clarification, proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. Primary antibodies to the following proteins at the indicated dilutions were used: anti-GAPDH , all the rest from Cell Signaling: phospho-4EBP1 (Ser65) , 4EBP1 , phospho-p70 S6 Kinase (Thr389) , p70 S6 Kinase , phospho-ULK1 (Ser757) , and ULK1 , phospho-mTOR(Ser2448) . A Goat anti-rabbit IgG HRP  was used as secondary antibody. Uncropped scans are provided in Supplementary Fig. Proliferating Jurkat cells at a density of about 1 × 102 at 254 nm in a Spectrolinker XL-1500 (Spectronics Corporation). The membrane was washed with 0.05% PBS–Tween (PBS-T) and blocked for 1 h with 5% skimmed milk in PBS-T. Primary antibodies were incubated in blocking solution overnight at 4 °C, followed by 3 rinses with PBS-T, secondary antibody incubation in blocking solution for 1 h at room temperature, 3 washes with PBS-T, and development using ECL . Antibodies used were: anti-m6A  and Goat anti-Rabbit IgG-HRP .Aliquots of the heat-eluted RNA of the same eRIC samples analyzed by MS (−UV controls) were used to estimate m6A levels. RNA was incubated for 10 min at 65 °C and immediately placed on ice. Concentration was estimated with a NanoDrop spectrophotometer (Thermo Fisher Scientific) and serial dilutions were prepared in order to obtain 100, 50, 25, and 12.5 ng/µL. One microliter of each dilution was directly pipetted onto a Zeta Probe membrane (Bio-Rad), air dried for ~10 min, and crosslinked twice with 120 mJ/cmSupplementary InformationPeer Review FileSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Description of Additional Supplementary Files


Physcomitrella patens DYW-type PPR proteins alone can perform efficient C-to-U editing in Escherichia coli reproducing the moss mitochondrial editing. Single amino acid exchanges in the DYW domain abolish RNA editing, confirming it as the functional cytidine deaminase. The modification of RNA targets and the identification of numerous off-targets in the E. coli transcriptome reveal nucleotide identities critical for RNA recognition and cytidine conversion. The straightforward amenability of the new E. coli setup will accelerate future studies on RNA target recognition through PPRs, on the C-to-U editing deamination machinery and towards future establishment of transcript editing in other genetic systems.RNA editing converting cytidines into uridines is a hallmark of gene expression in land plant chloroplasts and mitochondria. Pentatricopeptide repeat (PPR) proteins have a key role in target recognition, but the functional editosome in the plant organelles has remained elusive. Here we show that individual  E.coli system is an easy to manipulate platform for future studies on RNA target recognition and C-to-U RNA editing.Bastian Oldenkott et al. show that single moss pentatricopeptide repeat proteins with a DYW domain are sufficient to drive efficient C-to-U RNA editing in Escherichia coli. They demonstrate that the  Core to the recognition of specific cytidines for conversion into uridines are plant-specific RNA-binding pentatricopeptide repeat (PPR) proteins4. PPR arrays in these editing factors are of the PLS type with long  and short  PPR variants alternating with the canonical P-type PPRs of 35 amino acids. The three carboxy-terminal PPRs of PLS-type PPR proteins generally differ in their amino acid conservation and are labeled P2, L2, and S2. Many additional trans-acting proteins contribute to complex editosomes in flowering plants7. Early land plant lineages, however, seem to lack such additional components. All of the few C-to-U RNA editing sites in the moss Physcomitrella patens have been assigned to specific editing factors addressing one or maximally two edits, respectively10. All these editing factors are PPR proteins with a terminal DYW domain, which had been proposed to be the cytidine deaminase needed for the biochemical conversion of cytidine into uridine11. Further circumstantial evidence supported that concept16, but other studies questioned the idea of the DYW domain as a cytidine deaminase18.C-to-U RNA editing of organellar messenger RNAs (mRNAs) is nearly omnipresent in land plants and can affect up to thousands of sites in chloroplast or mitochondrial transcriptomes, respectivelyStudying plant RNA editing in vivo is time-consuming and labor-intensive and hindered by occasionally strong mutant phenotypes, lack of a transformation protocol for plant mitochondria, and the background of interacting proteins in the native system.Escherichia coli. Single amino acid mutations in the DYW domain clearly demonstrate its role as the cytidine deaminase acting on polyribonucleotides. Mutations in the cognate RNA targets and within the PLS-type PPR arrays essentially confirm the current PPR-RNA recognition code, but also reveal that some conceptually improved PPR-RNA matches may decrease rather than improve RNA editing and that at least some L-type PPRs evidently play an important role in the process. We demonstrate that changing key amino acid positions in a PPR will allow for a corresponding nucleotide change in the RNA, resulting in a designed target switch. The identification of numerous off-targets in the E. coli background transcriptome will accelerate our understanding of RNA target recognition by PLS-type PPRs.Here we demonstrate that single DYW-type plant editing factors can faithfully edit their corresponding targets in P. patens mitochondria , allowing insertion of target sequences behind the editing factor coding sequences on the same transcript -induced E. coli cultures edited the target cytidine very efficiently from 70 up to 100%  in each PPR to complement the findings for target mutations. Improving the fit of P-6TD to the A by converting it to P-6TN yields perfect editing Fig. . The nat2+ ion coordination . A single amino acid conversion into alanine at any of the important residues led to a complete loss of RNA editing. Position 4 of this signature is alternatively lysine (K) or arginine (R) in other editing factors. Converting the native arginine of PPR65 into lysine, however, reduced editing efficiency to ca. 47%. Similarly, the tyrosine (Y) of the name-giving tripeptide motif at the end of the DYW domain is occasionally replaced by phenylalanine (F), like in PPR65. The substitution of the one aromatic amino acid for the other did not result in a detectable reduction of editing efficiency  transcriptome analyses after expression of the two editing factors. This revealed only seven off-targets for PPR65, most with editing efficiencies below 10%, but 79 sites of C-to-U editing for editing factor PPR56, with one site in a 5′-untranslated region even edited in 87% of the transcripts  RNA structures to be correlated with reduced or increased editing efficiencies  was modified to obtain pET41Kmod by creating a multiple cloning site  in the former XhoI restriction site downstream of the gateway cassette. Protein coding sequences lacking most of the N-terminal targeting sequences were amplified with Phusion High-Fidelity DNA Polymerase  and introduced into the Gateway pDONR/Zeo Entry vector (Thermo Fisher Scientific). Plasmid clones were used as template for mutagenesis with appropriate primers in rolling-circle PCRs32. Protein coding regions were transferred into pET41Kmod by Gateway cloning  to create MBP fusion proteins .RNA editing target sequences (including at least 33 bp upstream and 5 bp downstream of the editing site) flanked by appropriate restriction sites were generated by primer annealing. Twenty microliters of a mixture of 50 mM forward and reverse primer each was heated to 95 °C for 5 min and left to cool down to 40 °C. The double-stranded product was phosphorylated  and inserted into the dephosphorylated  pET41Kmod vector or the respective plasmid clones of editing factors. Constructs containing editing factors and downstream targets (Supplementary Data E. coli starter cultures (Luria Broth with 50 µM kanamycin and 17 µM chloramphenicol) were grown overnight. 250 µL of the pre-culture were used to inoculate 25 mL of the same media supplemented with 0.4 mM ZnSO4 in 100 ml Erlenmeyer flasks with baffles. Cultures were grown at 37 °C until an OD600 of 0.4–0.6 was reached. Cultures were cooled on ice for a minimum of 5 min before adding 0.4 mM IPTG for induction of construct expression. Cells were incubated at 16  °C and 180 rpm for 20 h before harvesting 1.5 mL samples. Shorter incubation times after induction of expression were applied test wise in selected experiments  with the recommended option of a pre-incubation with lysozyme . RNA was treated with DNaseI (Thermo Fisher Scientific) and complementary DNA (cDNA) was synthesized using random hexanucleotide primers . A reverse primer upstream of the T7 terminator stem-loop sequence and a forward primer binding in the PPR protein coding region were used for reverse transcription-PCR amplification. PCR amplification assays contained 1 µL template of cDNA, 0.4 µM of each primer, 1× recommended PCR buffer, 0.8 mM dNTPs, 1U GoTaq polymerase , and double-distilled water in a final volume of 25 µL. Amplification assays included 5 min initial denaturation at 94 °C followed by 35 cycles each with 30 s denaturation at 94 °C, 30 s annealing at 52 °C, 2.30 min synthesis at 72 °C, and a final step of synthesis for 5 min at 72 °C. Oligonucleotides are listed in Supplementary Data To check for RNA editing in https://dna.macrogen.com). Sequencing chromatograms were analyzed with MEGA 7 and Bioedit 7.134  with the default settings and the temperature of 16 °C.PCR products were gel-purified (Macherey and Nagel) and sequenced directly  and Illumina sequencing (150 bp paired-end) was done by Novogene (https://en.novogene.com/).To identify off-targets in the E. coli BL21 genome (CP010816.1) in parallel with genomic DNA reads (SRX326827: SRR941832) using GSNAP37 v. 2017-11-15 with proposed settings38. RNA − DNA differences to the E. coli reference genome were called using JACUSA39. In parallel, the RNA-seq data were mapped against the pET41Kmod vector sequence with the respective PPR protein and the editing target included. For a strict identification of RNA editing sites, U vs. C or A vs. G differences in RNA vs. DNA, reads were considered only when: not filter-flagged  in the JACUSA output, detected in the RNA-seq data of both technical replicates, not present in RNA-seq reads of wild-type BL21 DE3 included as a control (SRX4183661: SRR7280082) or of the respective other PPR clone, and at sites showing clean DNA background (C or G > 98%) and RNA reads (T + C or G + A > 98%). The level of RNA editing was defined as the ratio of altered to total RNA reads subtracted by the same quotient for the corresponding DNA reads  at each site. The original transcript mapping data are available as separate Excel sheets as Supplementary Data To identify candidate sites of C-to-U editing, transcriptome reads were aligned against the Further information on experimental design is available in the Reporting SummarySupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8


In Sweden, human tissue samples obtained from diagnostic and surgical procedures have for decades been routinely stored in a formalin-fixed, paraffin-embedded, form. Through linkage with nationwide registers, these samples are available for molecular studies to identify biomarkers predicting mortality even in slow-progressing prostate cancer. However, tissue fixation causes modifications of nucleic acids, making it challenging to extract high-quality nucleic acids from formalin fixated tissues.In this study, the efficiency of five commercial nucleic acid extraction kits was compared on 30 prostate biopsies with normal histology, and the quantity and quality of the products were compared using spectrophotometry and Agilent’s BioAnalyzer. Student’s t-test’s and Bland-Altman analyses were performed in order to investigate differences in nucleic acid quantity and quality between the five kits. The best performing extraction kits were subsequently tested on an additional 84 prostate tumor tissues. A Spearman’s correlation test and linear regression analyses were performed in order to investigate the impact of tissue age and amount of tissue on nucleic acid quantity and quality.Nucleic acids extracted with RNeasy® FFPE and QIAamp® DNA FFPE Tissue kit had the highest quantity and quality, and was used for extraction from 84 tumor tissues. Nucleic acids were successfully extracted from all biopsies, and the amount of tumor (in millimeter) was found to have the strongest association with quantity and quality of nucleic acids.To conclude, this study shows that the choice of nucleic acid extraction kit affects the quantity and quality of extracted products. Furthermore, we show that extraction of nucleic acids from archival formalin-fixed prostate biopsies is possible, allowing molecular studies to be performed on this valuable sample collection. Prostate cancer (PCa) is the most common male cancer in Europe, with almost 400,000 new diagnoses each year . The natFinding prognostic biomarkers for PCa in prospective studies requires large study populations with many years of follow-up to ensure appropriate end-point data. By conducting nested case-control studies in large repositories of archived prostatic tissue, the efficiency and cost-effectiveness of prognostic studies can be strongly improved, while preserving the validity of a prospective cohort study. In Sweden, virtually all human tissue samples obtained from diagnostic and surgical procedures have for decades been routinely stored in formalin-fixed, paraffin-embedded, (FFPE) form . ThroughNucleic acids of high quality are vital for downstream molecular applications, however fixation of tissues causes modifications of biomolecules such as cross-linkage of nucleic acids with proteins, covalent modifications of both DNA and RNA, and fragmentation of RNA, making it challenging to extract nucleic acids of high quality from FFPE tissues –5. The qIn order to assess the ability of five commercially available kits to obtain high-quality DNA and RNA from FFPE tissues, we carried out four comparative trials on archival FFPE prostate needle biopsies. Furthermore, the best performing kits were used in order to assess the quantity and quality of nucleic acids extracted. The results from this study will subsequently be implemented in the Program for prediction of Mortality in prostate cancer (PROMORT) study, investigating prognostic biomarkers in prostate biopsy tissues .In this study, we included FFPE prostatic needle biopsy tissues from men who underwent prostate biopsies for suspicion of PCa at the Department of Urology, University hospital in Örebro, Sweden between 1992 and 2002. In the first part of the study, in order to assess the performance of five commercial kits for extraction of nucleic acids from FFPE biopsy tissues, we included 30 biopsies with normal prostatic histology. In the second part of the study, in order to assess the quantity and quality of the nucleic acids extracted from the FFPE biopsies, we included 84 biopsies with cancer. The study was approved by the Ethical committee in Stockholm, Sweden .In the comparison between a) the High Pure FFPE RNA Micro kit and the RNeasy® FFPE kit and b) the High Pure FFPET DNA Isolation kit to the QIAamp® DNA FFPE Tissue kit, three serial sections of 10 μm were cut from ten biopsies with normal histology. In each case, the first section was discarded to exclude negative effects from exposure to air. The two remaining sections were placed on separate slides; tissues were scraped and put in separate Eppendorf tubes, and were subsequently used for RNA/DNA extraction using either one of the two different extraction kits being compared. The extraction procedure followed the manufacturers’ instructions. RNA was eluted with 20 μl elution buffer while DNA was eluted with 50 μl.For comparison between the best performing RNA and DNA kits and the AllPrep® DNA/RNA FFPE kit, four serial sections of 10 μm were cut from the third set of ten biopsies with normal histology. In each case, the first section was discarded to exclude negative effects from exposure to air. Each subsequent section was placed on separate slides; tissues were scraped, put in separate Eppendorf tubes and were subsequently used for extraction of nucleic acids using the three different extraction kits. The extraction procedure followed the kit instructions and RNA/DNA was eluted using 20 μl and 50 μl of elution buffer, respectively.The study material, consisting of 30 FFPE prostate biopsies with normal histology, was randomly divided into three different groups; 1) ten biopsies for comparison between the High Pure FFPE RNA Micro Kit  and the RNeasy® FFPE kit , 2) ten biopsies for comparison between the High Pure DNA FFPET Isolation Kit  and the QIAamp® DNA FFPE Tissue kit , and 3) ten biopsies for comparison of the best performing DNA and RNA kits from the two previous steps to the AllPrep® DNA/RNA FFPE kit . In order to minimize the risk of variation due to different operators, the same operator performed all extractions with one kit .The study pathologists (F.G and M.F) assessed the prostatic biopsies for millimeter of tumor (tumor length), percentage of tumor tissue and Gleason score. They also circled tumor areas on the hematoxylin and eosin (H&E) slides corresponding to the tissue blocks. Three slices of 10 μm were subsequently sectioned from each biopsy tissue block; the first section was discarded while the remaining two sections from each case were used for macro-dissection of the tumor area using an RNase-free scalpel for each case. The macro-dissected tumor area from one tissue section was used for RNA isolation using the RNeasy® FFPE kit  and the tumor area from the second section was used for DNA isolation using the QIAamp® DNA FFPE Tissue kit , following the manufacturers’ instructions.The quantity and purity (A260/A280) of the extracted DNA and RNA was measured using the NanoDrop ND-2000 Spectrophotometer . When using a spectrophotometer, an OD260/280 greater than 1.8 or 2.0 is generally considered to be an indicator of good RNA and DNA quality, respectively. Lower OD ratios could indicate the presence of contaminants, or very low concentrations (< 10 ng/μl) of nucleic acids in the sample. The RNA integrity was assessed using the Agilent 2100 BioAnalyzer and the Agilent RNA 6000 Pico kit . The RNA Integrity Number (RIN) is a score of the RNA degradation ranging on a scale from 1 to 10, where 1 represents the most degraded RNA and 10 represents a completely intact RNA .T-values of < 38 were considered positive.The endogenous control gene 18S rRNA is typically employed to allow quantitation of relative gene expression in DNA/RNA samples. In order to determine the presence of amplifiable 18S rRNA in the extracted samples, a qPCR was performed. Ten nanograms of total RNA were converted to cDNA using the SuperScript VILO master mix . The resulting cDNA or 10 ng of DNA from each sample were used in a qPCR reaction using a TaqMan probe for RNA18S5 (Hs03928985_g1) and TaqMan Fast Universal PCR Master Mix  and run in a 40-cycle reaction on the 7900 HT system . The threshold value was automatically calculated using the RQ Manager Version 1.2.1 software and CT-values between the kits. A Bland-Altman analysis was performed in order to investigate the level of agreement of the two extraction kits. A fixed difference indicates that one method yields higher/lower values compared to the other method by a constant amount, while a proportional difference indicates if one method yields higher/lower values than those from the other method, which is proportional to the level of the measured variable [For assessment of the quality and quantity of the nucleic acids extracted with the five commercial extraction kits, both a paired Student’s t-test and a Wilcoxon matched-pairs signed rank test was performed in order to evaluate differences in yields, purity (A260/A280), RIN- and Cvariable .In order to test if the percentage of tumor cells, length of the tumor tissue, or year of biopsy were associated with the quantity and quality of the extracted nucleic acids, a Spearman’s correlation test was performed. A multivariate linear regression models was performed in order to investigate associations between tumor length (mm), age of FFPE tissue, Gleason score and measurements of RNA/DNA quantity and quality. Tumor length and percentage of tumor cells was categorized and a Mann-Whitney U-test or a Kruskal-Wallis one-way analysis of variance was performed in order to test for differences in quantity and quality of nucleic acids between the categories. All statistical analyses were performed in SPSS Statistics version 22.p < 0.001). Furthermore, both the A260/A280 ratios and RIN-values in samples extracted with the RNeasy® FFPE kit were higher  compared to samples extracted with the High Pure kit  . To evaluate the quality and efficiency of the extracted RNA, the expression levels of 18S5 rRNA was measured by qPCR. All samples were amplifiable as indicated by a CT-value < 38. Samples extracted with the RNeasy® FFPE kit yielded lower CT-values compared to samples extracted with the High Pure kit (mean CT 25.4 versus 26.4), however this difference was not statistically significant  ranging from 8.1 ng/μl to 20.8 ng/μl compared to the samples extracted with High Pure FFPE kit (mean yield 5.6 ng/μl) ranging from 2.2 ng/μl to 11.2 ng/μl (p = 0.48) or purity , although a proportional difference was evident for RIN-values .Figure T-values (27.4 versus 28.0) between these two kits, although higher RIN-values were seen for samples extracted with the RNeasy® FFPE kit (Table p = 0.604), purity , or RIN-values  , A260/A280 ratios (1.47 versus 1.50) or Cp = 0.002). To evaluate the quality and efficiency of the extracted DNA, a qPCR measuring the expression levels of 18S5 rRNA was performed, and all samples were amplifiable as indicated by a CT-value < 38. A difference in CT –values could be seen for samples extracted with the two kits , with samples extracted with the QIAamp® kit yielding higher CT-values compared to samples extracted with the High Pure DNA kit (Table p = 0.162) or purity  . However, samples extracted with the QIAamp® FFPE kit had higher A260/A280 ratios compared to samples extracted with the High Pure DNA kit . Furthermore, samples extracted with the AllPrep® kit yielded lower CT –values  (Table p = 0.856) or A260/A280   for samples extracted with the two kits, however higher A260/A280 ratios were seen in samples extracted with the QIAamp® kit  and RIN-values   . A weak correlation between age of the FFPE tissue and DNA yield  was observed, but not RNA yield . Furthermore, there was no significant correlation between percentage of tumor cells or Gleason scores with DNA/RNA yield, A260/A280 ratios, RIN- or CT-values (data not shown). A multivariate regression analyses confirmed that tumor length was the main determinant for both RNA and DNA yield, for each additional millimeter tumor the quantity of RNA/DNA increased with 0.53 ng/μl (95% CI: 0.18–0.88) and 0.24 ng/μl (95% CI: 0.11–0.37), respectively. However, an association between tumor length and RIN-values was not found . Age of the FFPE tissue was associated with DNA yield , and Gleason score .There was evidence of correlation between tumor length (in mm) and RNA yield (1) Table . When diWe compared the quality and quantity of nucleic acid extraction in five commercially available extraction kits, using prostate needle biopsies with normal histology, assuming that normal tissue is less heterogeneous than malignant tissue, thus reducing the risk of biased comparison. We found that RNA extraction with RNeasy® had higher yields of RNA, purity (A260/A280) and RIN-values. Furthermore, the agreement in RNA yield and quality  between the two compared kits was low, with consistently lower yields and purities in the High Pure kit. RNA of high quality is essential when performing molecular studies, and thus even small differences in purity and RIN-values are important to consider when choosing extraction kit. Based on the results in this trial, the RNeasy® kit would be preferred for extraction of high quality RNA from FFPE prostate biopsies.In a second trial, the RNeasy® FFPE kit was compared to the AllPrep® DNA/RNA FFPE kit. The two kits produced similar yields and purity of the extracted RNA, however, higher RIN-values were found in samples extracted with the RNeasy® kit. Even though it would typically be preferable to extract both DNA and RNA from the same tissue section, the consistently lower quality of RNA produced with the AllPrep® kit suggests that using two separate extraction kits for DNA and RNA would be preferable if high quality RNA is desired.A possible explanation for the kit-to-kit variations in RNA quantity and quality could be the conditions recommended for the proteinase K digestion. The use of proteinase K digestion is necessary to release RNA from the meshwork of cross-linked proteins and nucleic acids, and it has also been shown to play an important role in the extraction of longer stretches of nucleic acids >200 bp) , 19, 20.0 bp , 19In a third trial, we compared the two DNA extraction kits QIAamp® DNA FFPE Tissue kit and High Pure FFPET DNA Isolation kit, and found similar DNA yields, although with higher purity (A260/A280) in the QIAamp® kit. One sample extracted with the QIAamp® kit had a high A260/A280 ratio (2.66), which is probably due to a RNA contamination within this sample. Based on these data, we chose the QIAamp® kit for comparison with the AllPrep® DNA/RNA FFPE kit. In this final trial, similar DNA yields in samples extracted with the two kits were found, although once again with higher purity (A260/A280) in the QIAamp®. Thus, our analyses suggest the QIAamp® kit as the best performing DNA extraction kit from FFPE prostate biopsies.Performing molecular studies on archival FFPE needle biopsy material have potential limitations. Prostate needle biopsies are often performed using an 18-gauge needle, yielding a biopsy which is roughly 1 mm in diameter and 12–15 mm long. Sectioning such a biopsy would in theory yield 100 sections of 10 μm. However, the biopsy has typically been sectioned previously as a part of clinical evaluation, and thus only a limited amount of tissue with representative disease histology is available for research studies. Furthermore, according to Swedish laws one must make sure that there is always representative cancer tissue left after sections are taken for research studies. Thus, in practice, it might be possible to take four to five 10 μm sections for research studies. The aim of this second part of the study was thus to investigate if it is possible to extract nucleic acids from archival FFPE prostate biopsies using one 10 μm section of tissue. We also wanted to investigate the quantity and quality of the nucleic acids extracted from the biopsies in order to investigate which type of molecular studies could be performed., who observed a significant association between DNA yield and quality and storage time [T < 38) in 83 of 84 (98.8%) RNA samples and 81 of 84 (96.4%) DNA samples. The RNA sample that was not amplified had the lowest purity of all samples (A260/A280 = 0.7), however for the DNA samples there was no apparent reason to why the amplification failed. These results indicate that archival FFPE prostate biopsy tissues are suitable for PCR amplification even though the small quantity and poor quality of the isolated nucleic acids.Based on the results from the assessment of nucleic acid extraction kits, nucleic acids were extracted using the QIAamp® DNA FFPE Tissue kit and RNeasy® FFPE kit. DNA was successfully extracted from all 84 biopsies with a mean yield of 3.7 ng/μl and a purity of 1.7. One DNA extract had a high A260/A280 ratio (5.3), indicating a RNA contamination of this sample. RNA was also successfully extracted from all 84 samples, with a mean yield of 10.6 ng/μl. The purity of the extracted RNA had a mean value of 1.55 and the RIN-value a mean of 2.3. The expression of 18S5 RNA showed high variability between samples, indicating that this is not an optimal endogenous control gene for prostate tissues. The amount of tumor (in millimeters) had the strongest association with both quantity and quality of the nucleic acids extracted from the biopsies within this study, which is consistent with a previous study showing an association between tumor amount, measured by millimeter tumor of a renal biopsy, and purity of RNA (A260/A280) . Our resage time . Nucleicage time –27. In tIn order to gain new insights to pathological processes, quantitative measures of mRNA levels and DNA analyses investigating for example mutations, polymorphisms and epigenetic changes have become essential. It is recommended that only high-quality DNA and RNA are used for these analyses. In some instances, however, such as in studies of biomarkers of prostate cancer mortality, high quality samples may not be available. Typically using low-quality samples introduces bias toward the null, because the degradation of samples entails random variability that is similar across comparison groups. Therefore, precision and power is impaired, and the risk of false negative associations, but not false positive, increases.T-values and limited concentrations of the extracts [Many studies have investigated the effect of poor RNA quality  and DNA quality, using techniques such as qPCR, microarrays and NGS , 28–35. extracts , 48, howextracts . Targeteextracts , and theextracts , 52.n = 10). Further studies on larger tissue materials are needed in order to validate the results found within the present study. 3) Even though the tissue sections used for comparisons of extraction kits were cut immediately adjacent to one another, there could still have been variations in the tissue area that influenced the DNA/RNA yield. 4) Due to the limited amount of tissue available in a prostate biopsy, extractions could not be performed in duplicates from the same biopsy. Furthermore, two different operators performed the extraction of nucleic acids with the kits to be compared, which could have affected the outcome due to inter-operator variability [An advantage with our study is that the extraction kits have been compared using serial sections of the same biopsies, which decreases the risk of differences between the kits due to variations in the tissue histology. Furthermore, the extraction kits were tested on biopsies with normal histology, which further decreases tissue heterogeneity. The best performing extraction kits for DNA and RNA was subsequently used for extraction of nucleic acids on 84 biopsies with malignant histology, yielding similar quantities and quality of the nucleic acids as in the normal biopsies. The study has a number of limitations. 1) The limited number of nucleic acid extraction kits tested. Many other kits for extraction of nucleic acids from FFPE tissues exist on the market, with the potential to perform better than the kits used in this study. 2) The small number of comparisons performed for each extraction kit (iability .To conclude, our results indicate that it is possible to utilize archival FFPE prostate biopsies in biomarker studies for PCa, and that the choice of extraction kit could be of importance to obtain high quality DNA and RNA from FFPE tissues.


While cognitive dissonance is an influential concept of social psychology, its relations with consciousness and episodic memory remain strongly debated. We recently used the free-choice paradigm (FCP) to demonstrate the crucial role of conscious memory of previous choices on choice-induced preference change (CIPC). After choosing between two similarly rated items, subjects reevaluated chosen items as more attractive, and rejected items as less attractive. However such a CIPC was present exclusively for items that were correctly remembered as chosen or rejected during the choice stage, both in healthy controls and in amnesic patients. In the present work, we show that CIPC can be modulated by suggestive quotes promoting self-coherence or self-incoherence. In addition to the crucial role of memory of previous choices, we discovered that memory of the suggestive quotes was correlated to the modulation of CIPC. Taken together these results suggest that CIPC reflects a dynamic homeostatic regulation of self-coherence. While it is very intuitive that our current preferences and values influence our future choices, the reverse causality is much less easy to predict and explain. However several empirical observations have confirmed that our previous choices can indeed influence our values and preferences. Such a reverse causality has been coined “cognitive dissonance” resolution, and is one of the most influential concept of social psychology since the seminal work of Festinger in the 50s . For insTaking these artefacts into account, we recently solved this debate about CIPC and memory, by reproducibly showing that CIPC occurs exclusively for items that were correctly remembered as chosen or rejected during the choice stage , 4. MoreInspired by this demonstration that CIPC depends on conscious episodic memory, and therefore that it is not an automatic and irrepressible process, we decided to address the following question: could CIPC be modulated by suggestive quotes promoting self-coherence of self-incoherence?To our knowledge, very few studies explored this issue in other paradigms traditionally associated with cognitive dissonance, such as the classical “counter-attitudinal statement” paradigm, in which subjects asked to write a counter-attitudinal essay tend to change their mind accordingly . Bator aIf CIPC could be modulated by suggestion, this would further demonstrate its non-automatic nature. Such a result obtained by manipulating self-coherence would also further confirm the importance of frontal lobe mediated executive control mechanisms at work in CIPC , 14–16.In the present study we provide such a demonstration, and reveal that episodic memory of the suggestive quotes is correlated to the modulation of CIPC in the FCP.This experiment has been approved by the Pitié-Salpêtrière ethical committee. All the 73 subjects gave their written informed consents, and were paid 10 Euros to participate in the experiment. All investigations were conducted according to the principles expressed in the Declaration of Helsinki.3,4, and to other studies from other groups.27 healthy participants were included in the Control group , 24 healthy participants were included in the Coherence group , and 22 healthy participants were included in the Incoherence group  leading to a total of 73 participants. They reported normal, or corrected-to-normal, visual acuity. This sample size was estimated according to our two previous studies using the very same free-choice paradigm Stimuli consisted of 160 (80 for each part: A & B) equalized images of holiday destination , with the name of the destination written underneath it, on the center of the screen (font size = 30). In 'Rating' blocks, one image appeared at the center of the screen in each trial, whereas in 'Choice' blocks, two targets were presented 4.8° off-center, to the left and to the right, in each trial.We used fictional quotes quotes that we attributed to Socrates, Buddha and Einstein in order to manipulate subjects’ attitude toward self-coherence according to 3 conditions , which included 80 trials. Each trial began with a fixation point presented during 1.5 second. Then one vacation destination was centrally presented for 3 seconds, followed by a blank screen lasting until subjects responded manually. They were requested to report how much they would like to spend their vacation in this destination using an eight-point scale . Subjects responded using the 1–8 number pad buttons of a regular keyboard. This first block was followed by a choice (Choice 1-A for the RCR condition), which included 20 trials (40 destinations were coupled). Participants were presented with pairs of destinations they had rated equally (difference of R1 scores≤ 1) and had to indicate with a button press at which one they would rather take their vacation. Note that all individual medians of R1 scores differences were null.In this choice block, every trial started with a fixation point presented during 1.5 second. Then, two destinations were presented side-by-side for 5 seconds. Subjects had to report manually their choice using the left/right arrows keyboard keys. Importantly, destinations were coupled according to Rating 1-A block. Each couple of destinations was composed of destinations rated similarly. Then, subjects had to perform another block of rating (Rating 2-A) on the same images as in the Rating 1-A phase. This Rating 2-A was followed by a new choice (Choice 2-A for the RRC condition). This time, subjects had to perform a choice between the other 40 destinations that had not showed during Choice 1-A. In the same way, those destinations were coupled according to Rating 1-A. This block was the last one of the part A.Part B of the experiment started with a rating block (Rating 1-B). This block followed the very same description that Rating 1-A block except that 80 new destinations were concerned. Following Rating 1-B stage, subjects were engaged in the Choice 1-B block which was structured similarly to the Choice 1-A block except that destinations were coupled according to Rating 1-B block. After this Choice 1-B, the manipulation period started. A confederate then entered in the experimental room requesting the examiner to come with him to solve an urgent problem. The examiner accepted to accompany the confederate but asked her to pause the ongoing experiment. Unbeknownst to the subject, the confederate set up a desktop background with one of the 3 quote categories (Coherence/Incoherence/Control) that would then be presented on the screen for 3 minutes , and both the examiner and the confederate left the room. This design allowed a clear double blind experiment whereby neither subjects nor experimenter knew which category the subjects were in. Upon the return of the examiner, subjects had to perform another block of rating (Rating 2-B). They had to rate the same destinations that has been rated during Rating 1-B. This Rating 2-B was followed by a new choice (Choice 2-B). This time, subjects had to perform a choice between the other 40 destinations that had not been shown during Choice 1-B. In the same way, those destinations were coupled according to Rating 1-B.The last block of the experiment was a memory block. Participants were asked, for each destination whether they had chosen or rejected it. Then, they were asked to report their confidence in their response. For both questions, subjects had to answer manually using the left/right keyboard arrows. As we were interested in testing whether subjects remembered the episode of the choice they made, we considered items as remembered only if subjects correctly reported whether they had chosen or rejected each of the two coupled items.At the end of the experiment subjects filled out a debriefing form in which they were asked to recall and write down the three quotes they were exposed to, and to report whether they interpreted the experiment interruption as intentionally planned by the experimenters. We then calculated a quote memory score for each subject using the following criteria: 1 point if the quote was remembered; 0.5 point if some crucial words were remembered; 0.25 point if only the name of the alleged author of the quote was remembered; 0 point if nothing relevant was remembered. Note that spreading of alternatives is computed as (second rating for a chosen item–first rating for a chosen item)–(second rating for a rejected item–first rating for a rejected item).We used linear mixed-effects models that offer the possibility to handle the heteroscedasticity related to the unbalanced number of items in each condition . SignifiWe first replicated our previous finding about the impact of memory on choice induced preference change in the ‘Neutral’ group , 4, by o−5 and for the ‘Coherence’ group: F = 15.6; p <10−4). Importantly, the impact of quotes on CIPC was not affected by participants’ interpretation of experiment interruption: we performed an ANOVA on RCR spread including this new factor (i.e. categorizing participants as considering the interruption of the experiment as planned (N = 11) or unplanned (N = 62)). The triple interaction between this factor and Group and Choice memory(2) factors was not significant  = 1.86; p = 0.16).We then confirmed the predicted impact of quotes on choice induced preference change, as a significant interaction between group , and choice memory (remembered vs forgotten choices) on RCR trials  = 3.88, p = 0.02; see We then probed the potential impact of episodic memory of quotes on quote-induced modulation of preference change. We split each experimental group in two subgroups according to subjects’ individual score on the quotes memory task, relative to the median value of this score calculated across the whole group. As for the previous analysis we could first control data quality by computing the 3-factors ANOVA on the ‘Neutral’ group only. As expected, the behavioral signature of memory dependent choice-induced preference change was not affected by the memory for neutral quotes  = 0.037; p = 0.84; see This result could be explained by a process of coherence exerted on rating 1 scores rather than on choice performance only. Post-hoc tests strengthened this interpretation by revealing that memory of citations impacted half-spread (R2-R1 for a given item) of chosen items in the predicted direction: the more subjects remembered the quotes, the closer were R2 and R1 . It is noticeable that this impact was not observed on rejected items (p = 0.34). This asymmetry was confirmed on a dedicated ANOVA calculated on half-spreads, as a significant interaction between chosen/rejected and low versus high memory of citations  = 7.86; p = 0.005). Note that this strategy,—observed in the ‘Coherence’ group -, of privileging coherence on rating 1 score rather than on choice stage performance, would not lead to similar result if applied in the ‘Incoherence’ group.In order to better understand the impact of quotes, we then analyzed the RRC trials. As expected, no significant interaction between group and choice memory was observed  = 0.88, p = 0.41), and we then focused on the impact of quotes. A significant interaction was found between group and quotes memory factors  = 3.41, p = 0.04). Post-hoc analyses revealed that while quotes memory did not modulate RRC spreads in the neutral  = 0.46,p = 0.50) and incoherence  = 0.87, p = 0.36) groups, a significant effect was present for the coherence group exclusively  = 8.42,p = 0.008). A smaller spread was observed for well memorized quotes. This result strengthens our proposed interpretation for RCR trials: when participants memorize the quotes, they tend to exert their coherence on the R2/R1 matching rather than on R2/choice matching.−5). This forgetting effect was most probably due to the long time interval (>30 minutes) separating RCR choices of ‘Part A’ from the memory task, and by the existence of memory interference related to the 4 distinct choice phases (RRC and RCR in both parts).). In spite of this forgetting effect, we confirmed the absence of difference of CIPC across the three groups in Part A: there was no significant triple interaction between Group, Condition and Memory factors on spread values  = 0.58; p = 0.56).Our experimental design also aimed at collecting an additional control condition for each subject by using the ‘Part A’ data see  and M&M.Finally, too few choices were confidently remembered in the present study, prevailing any analysis of confidence. This was probably due to the high-load of items, choices, experimental stages and experiment duration, as compared to our previous reports in which both objective responses and subjectively confident measures of episodic memory were very close and led to similar results .Taken together, our results demonstrate that CIPC can be manipulated by suggestive instructions, and that this manipulation is dependent of explicit memory.We discovered a significant impact of quotes promoting incoherence as a general cognitive posture, on CIPC, as compared to neutral quotes and to quotes promoting coherence. This suggestibility effect was even stronger for those individuals who consciously remembered the quotes the best. In agreement with our previous finding on the necessary role of episodic memory of previous choices on CIPC , 4, our The absence of difference between neutral quotes and coherence promoting quotes may be explained by two non-mutually exclusive hypotheses. First, this absence of difference may reflect a floor effect due to a general bias toward search of coherence: if individuals are spontaneously looking for coherence,—as reflected by the spontaneous CIPC effect -, we may have missed an increase of this trend. Additional experiments using more efficient quotes or longer exposure to quotes may reveal a possible increase of CIPC. An alternative hypothesis of this absence of difference between neutral and coherence promoting quotes may be related to the impact of quotes: as suggested by our additional analyses, quotes may have emphasized both a coherence effect related to previous choices (and leading to an increase CIPC), but also a coherence effect related to Rating 1 (and leading to a null CIPC). Therefore, we may still increase CIPC with coherence-promoting quotes, provided that they are more directive and more clearly addressed to previous choices.We conclude by raising the puzzling issue of consciousness and agentivity during CIPC: if CIPC only occurs for consciously remembered items, and if CIPC is more subject to suggestion when we remember better the instructions, one may wonder up to which point CIPC is a voluntarily/involuntarily and conscious/unconscious process. Is CIPC a non-reportable non-conscious process that requires an information (previous choice or current instruction) to be consciously accessed first? Or is CIPC accessible to conscious introspection, and even subjectively considered as an intentional act within the experimental context? Future psychological studies should better explore these two questions, now that the impact of episodic memory and of suggestion have been reliably shown on CIPC.S1 Experimental Dataset(ZIP)Click here for additional data file.S1 Quotes(DOCX)Click here for additional data file.


Myiopsitta monachus), identified by metagenomic next-generation sequencing during an investigation into a disease outbreak in a captive parrot breeding facility. Based on divergence from known strains, this sequence represents a new subgroup of avian metapneumovirus.Here, we report the coding-complete genome sequence of an avian metapneumovirus from a monk parakeet ( Myiopsitta monachus), identified by metagenomic next-generation sequencing during an investigation into a disease outbreak in a captive parrot breeding facility. Based on divergence from known strains, this sequence represents a new subgroup of avian metapneumovirus.Here, we report the coding-complete genome sequence of an avian metapneumovirus from a monk parakeet ( Metapneumovirus, family Pneumoviridae) cause disease in birds and mammals, including humans. Avian strains are important pathogens of commercial poultry, causing acute upper respiratory illness that is often complicated by secondary bacterial infections in chickens and turkeys . RNA was extracted using a Direct-zol RNA MiniPrep Plus kit (Zymo Research) with DNase treatment (Qiagen) and quantified by Nanodrop. Sequencing libraries were prepared from 100 ng of extracted RNA with 25 pg of spike-in control RNA from the External RNA Controls Consortium (ERCC) collection (Thermo Fisher Scientific), using an NEBNext Ultra II Directional RNA library prep kit for Illumina (New England Biolabs). A water sample was processed in parallel. Paired-end 150-nucleotide (nt) sequencing on an Illumina HiSeq 4000 instrument yielded 35,053,607 raw read pairs.At necropsy of an affected monk parakeet chick, the lungs, liver, and spleen were sampled and stored at −80°C. For RNA extraction, ∼50 mg of combined tissues was homogenized in 2 ml of DNA/RNA Shield (Zymo Research) using 2.8-mm ceramic beads (Omni) on a TissueLyser II instrument (Qiagen) with 5 cycles of 30 Hz for 30 sec followed by 1 min on ice. Samples were centrifuged at 16,000 × Psittacidae, parrots) available in the National Center for Biotechnology Information (NCBI) database as of 7 December 2018. Host subtraction and quality control were performed as described previously  9224 (eviously . The remThese metapneumovirus reads were used as seeds for Paired-End Iterative Contig Extension (PRICE) v1.2 (with the settings “–fpp <R1> <R2 > 350 99 –mol 30 –target 80 8 2 2 –nc 10 –lenf 500 8”) to assemble the full-length genome . The finOrthopneumovirus and 61 to 66% identity to representative members of the genus Metapneumovirus, indicating that this sequence represents a new subgroup of metapneumoviruses (The most similar sequences in the NCBI nt/nr reference databases identified by BLAST search were metapneumoviruses . Phylogeoviruses  8). Ana. AnaOrthWe have identified the first member of a new subgroup of metapneumoviruses, distinct from avian metapneumoviruses A, B, C, and D. Despite similarities between this outbreak and outbreaks of avian metapneumovirus in commercial poultry, it remains unknown whether the virus identified here directly caused the symptoms observed in this individual and/or flock.MK491499.The avian metapneumovirus sequence described here has been deposited at GenBank under the accession number


Drug transporters mediate the absorption, tissue distribution, and excretion of drugs. The cDNAs of P-glycoprotein, multidrug resistance proteins (MRPs/ABCC), breast cancer resistance protein (BCRP/ABCG2), peptide transporters (PEPTs/SLC15), proton-coupled folate transporters (PCFT/SLC46A1), organic anion transporting polypeptides (OATPs/SLCO), organic anion transporters (OATs/SLC22), organic cation transporters (OCTs/SLC22), and multidrug and toxin extrusions (MATEs/SLC47) have been isolated, and their functions have been elucidated. Enantioselectivity has been demonstrated in the pharmacokinetics and efficacy of drugs, and is important for elucidating the relationship with recognition of drugs by drug transporters from a chiral aspect. Enantioselectivity in the transport of drugs by drug transporters and the inhibitory effects of drugs on drug transporters has been summarized in this review.  S)-enantiomers exhibit stronger inhibitory potencies against cyclooxygenases [S)-enantiomer of naproxen is commercially available. We examined the enantioselective effects of flurbiprofen on the disposition of lithium in rats, and demonstrated that the (S)-enantiomer, but not the (R)-enantiomer, decreased the renal clearance of lithium with the impairment of renal function [S)-Warfarin has greater anticoagulant potency than the (R)-enantiomer [S)-Warfarin is metabolized by cytochrome P450 (CYP) 2C9, and the metabolism of the (R)-enantiomer is mediated by CYP1A2 and CYP3A4 [S)-enantiomer of omeprazole, which is the racemate. Both enantiomers exert identical effects on H+, K+-ATPase [R)-enantiomer [R)-enantiomer is mainly mediated by CYP2C19, which exhibits a genetic polymorphism, and the contribution of CYP2C19 to the metabolism of esomeprazole is negligible [Many drugs are chiral, and each enantiomer may exhibit specific therapeutic efficacy. For example, several nonsteroidal antiinflammatory drugs (NSAIDs) have an asymmetric carbon in their chemical structures, and the (ygenases . The (S)function . The antantiomer . (S)-Warantiomer . Furthergligible .In 1988, CYP2D6 was the first cytochrome P450 enzyme whose cDNA was identified . The cDNRepresentative drug transporters functioning in the intestinal absorption, biliary excretion, and renal tubular secretion of drugs have been summarized in this review. The interactions between chiral drugs and drug transporters have also been discussed. +-electrochemical gradient [Drugs are absorbed by the small intestine after their oral administration. gradient . PEPT1 rgradient . PEPT1 igradient . The proton-coupled folate transporter PCFT (SLC46A1) is localized to the brush-border membrane of enterocytes, and mediates the absorption of folate . PCFT reThe organic anion transporting polypeptide OATP2B1 (SLCO2B1), which is expressed in the brush-border membrane of intestinal epithelial cells, is crucially involved in the uptake of fexofenadine, celiprolol, and montelukast from the lumen ,15. P-glycoprotein  and the breast cancer resistance protein BCRP (ABCG2) are ATP-binding cassette (ABC) transporter proteins that are expressed in the brush-border membrane of enterocytes. These transporters are efflux pumps. P-gp transports various types of drugs, including anticancer agents, antihypertensive agents, antiarrhythmics, antidepressants, antimicrobial agents, anti-human immunodeficiency virus agents, anticonvulsants, antiemetics, immunosuppressants, neuroleptics, and opioids, and there is a broad range of overlapping substrate specificities for CYP3A4 and P-gp . BCRP haIn the basolateral membrane, the organic cation transporter OCT1 SLC22A1) and the multidrug resistance protein MRP3 (ABCC3) mediate drug transport . In OCT1A1 and thDrug transporters in the plasma membrane of hepatocytes are shown in The renal tubular secretion of drugs is mediated by drug transporters, as shown in +/organic cation antiport, which involves electroneutral transport [The organic cation transport system consists of the uptake type of OCT2 (SLC22A2) and the efflux modes of MATE1 and MATE2-K (SLC47A2) ,32,41. DP-gp functions in the renal tubular secretion of the cardiac glycoside, digoxin . AlthougS)-enantiomers were found to have greater affinity to PCFT than each (R)-enantiomer. Menter et al. performed a pharmacokinetic study on aminopterin in dogs and patients with psoriasis, and the findings obtained revealed a correlation with enantioselective absorption and in vitro findings [Previous studies reported enantioselectivity in drug transport by drug transporters, with PCFT as a representative. The antifolates methotrexate and aminopterin have an asymmetric carbon in their structures , and thefindings .1-receptor antagonist, has non-sedative properties that have been attributed to the restriction of its brain penetration by P-gp [R)-fexofenadine were higher than those of the (S)-enantiomer after the single oral administration of racemic fexofenadine to healthy volunteers [R)-fexofenadine in Xenopus oocytes injected with OATP2B1 cRNA than that of the (S)-enantiomer [S)-fexofenadine was higher than that of the (R)-enantiomer [Fexofenadine, a histamine H by P-gp . Enantiolunteers . Sakugawlunteers . To my klunteers ,50,51,52lunteers . OAT3 pllunteers ,55. At lantiomer . They alantiomer . Accordiantiomer ,56, wherantiomer . In addiantiomer . UnidentEnantioselective drug transport was demonstrated with the combination of pantoprazole and BCRP. BCRP affects the absorption, distribution, and excretion of drugs, and BCRP actively secretes xenobiotics, including drugs and carcinogens, and riboflavin into milk ,59,60. WR)-disopyramide inhibited the uptake of tetraethylammonium by HeLa cells expressing OCT1 more strongly than the (S)-enantiomer [S)-enantiomer is stronger than for the (R)-enantiomer [50 values of each enantiomer were described in Enantioselectivity was shown in the inhibitory effect of disopyramide and propranolol on OCT1. The -Verapamil, (S)-atenolol, and (S)-propranolol showed the higher affinities than each enantiomer. In d values are summarized.Moaddel et al. studied the binding of drugs to OCT1 with a liquid chromatography stationary phase containing immobilized membranes obtained from a cell line that expresses OCT1, and estimated their binding affinities using frontal displacement chromatography with [pranolol ,65. (R)-Xenopus oocyte expression system in order to examine enantioselectivity in the inhibition of OAT1 and OAT3 by NSAIDs [S)-enantiomers of flurbiprofen, ibuprofen, and naproxen on the transport of p-aminohippurate and methotrexate by OAT1 than that of each (R)-enantiomer. The inhibitory mechanisms of flurbiprofen were investigated, and both enantiomers were found to competitively inhibit OAT1. Enantioselective differences were not observed in the inhibition of OAT3. The inhibition of renal organic anion transporters leads to the delayed elimination of their substrates from the circulation. NSAIDs interfere with the renal excretion of methotrexate, and this combination is a representative among drug interactions via OAT1 and OAT3. The interaction is fatal when high-dose methotrexate therapy is given to a patient ,67. Prevy NSAIDs . The finS)-lansoprazole on the transport of estrone sulfate, methotrexate, and pemetrexed by OAT3 were stronger than those of the (R)-enantiomer. Furthermore, enantioselectivity was not recognized against OAT1. Enantioselectivity has been demonstrated in the pharmacokinetics of lansoprazole. The slower elimination of the (R)-enantiomer from plasma has been reported in healthy subjects [S)-lansoprazole by human liver microsomes was also shown [Proton pump inhibitors also interact with methotrexate ,75; OAT1subjects . The fasso shown . This inS)-enantiomers of flurbiprofen, ibuprofen, and naproxen than by each (R)-enantiomer [R)-enantiomers exerted strong inhibitory effects. The enantioselective inhibitory effects of NSAIDs on MRP2 and MRP4 have been examined. Kawase et al. performed uptake experiments on methotrexate using MRP2- and MRP4-expressing inside-out vesicles, and showed the stronger inhibition of MRP2 by the  and pharmacophore approaches have become more widely applied to assess interactions between drugs and drug transporters and predictions for substrates and inhibitors for several transporters were described . BecauseIn the future, research on drug transporters from the chiral aspect of drugs will provide important insights into pharmacokinetics, pharmacodynamics, and drug toxicity.