Echinococcus. In this work, we have characterised the ex-RNA profile secreted by in vitro grown metacestodes of Echinococcus multilocularis, the casuative agent of alveolar echinococcosis.Extracellular RNAs (ex-RNAs) are secreted by cells through different means that may involve association with proteins, lipoproteins or extracellular vesicles (EV). In the context of parasitism, ex-RNAs represent new and exciting communication intermediaries with promising potential as novel biomarkers. In the last years, it was shown that helminth parasites secrete ex-RNAs, however, most work mainly focused on RNA secretion mediated by EV. Ex-RNA study is of special interest in those helminth infections that still lack biomarkers for early and/or follow-up diagnosis, such as echinococcosis, a neglected zoonotic disease caused by cestodes of the genus Echinococcus miRNAs in patient samples by RT-qPCR yielded negative results suggesting the tested miRNAs may not be good biomarkers for this disease.We have used high throughput RNA-sequencing together with RT-qPCR to characterise the ex-RNA profile secreted towards the extra- and intra-parasite milieus in EV-enriched and EV-depleted fractions. We show that a polarized secretion of small RNAs takes place, with microRNAs mainly secreted to the extra-parasite milieu and rRNA- and tRNA-derived sequences mostly secreted to the intra-parasite milieu. In addition, we show by nanoparticle tracking analyses that viable metacestodes secrete EV mainly into the metacestode inner vesicular fluid (MVF); however, the number of nanoparticles in culture medium and MVF increases > 10-fold when metacestodes show signs of tegument impairment. Interestingly, we confirm the presence of host miRNAs in the intra-parasite milieu, implying their internalization and transport through the tegument towards the MVF. Finally, our assessment of the detection of A comprehensive study of the secretion mechanisms throughout the life cycle of these parasites will help to understand parasite interaction with the host and also, improve current diagnostic tools. Echinococcus multilocularis.Extracellular RNAs (ex-RNAs) are secreted by cells through association with proteins or extracellular vesicles (EV). In the context of parasitism, ex-RNAs represent novel communication intermediaries with promising potential as biomarkers. In order to better understand the role ex-RNAs may play in the context of the zoonotic disease echinococcosis, we have characterised the RNA profile secreted by the larval stage (metacestode) of Echinococcus miRNAs in patient samples yielded negative results suggesting the tested miRNAs may not be good biomarkers for this disease.By analysing the products secreted towards the extra- and intra-parasite milieus, we demonstrate that the metacestode displays a polarized secretion of different classes of small non-coding RNAs (sRNAs). In addition, we show that EV secretion occurs mainly towards the inner fluid of the metacestodes. Interestingly, we confirm the presence of host sRNAs in the intra-parasite milieu, implying their internalization and transport through the tegument. Finally, the detection of E. multilocularis metacestode together with information on the distribution of the detected RNA classes in different extracellular compartments. This information is of importance to better understand host-parasite interaction and also, to improve current diagnostic tools.In summary, our results provide a detailed description of the ex-RNA landscape of the  SequencNAs from ,40 and mNAs from . The valE. multilocularis and M. musculus were identified (≤ 1 SNP), the origin of the miRNA was established by taking into consideration: i) the pre-miRNA sequence to which the highest number of reads could be mapped and ii) the absence of mismatches to the pre-miRNA in the most frequent read sequence. When the terminal 3´nucleotide corresponded to a non-templated U it was not considered as a mismatch but as a non-templated added nucleotide [When miRNAs with high identity between cleotide .Identification of other sRNAs: High quality reads were also aligned to the E. multilocularis (v4) and M. musculus (GRCm38) genomes using Bowtie  E. multilocularis: miRNA and pre-miRNA sequences from [http://rnacentral.org/) and ii) M. musculus: miRNA and pre-miRNA sequences from miRBase, tRNAs from GtRNAdb (http://gtrnadb.ucsc.edu/genomes/eukaryota/Mmusc10/Mmusc10-summary.html) and those non-coding RNA sequences classified as “Other” in RNA central database excluding miRNAs and tRNAs. tRNA sequence annotation was performed with tRNAscan-SE [https://github.com/MCucher/Emul_exRNA_scripts_and_data.git. The final step was to parse the tabular blastn-short tables to excel and only reads that fulfilled the following criteria were retained: gap = 0, hit start 1 or 2, mismatch = 0, read coverage ≥ 95 and read count number ≥ 350. Those sequences that passed these filters and were present in E. multilocularis and M. musculus datasets from the same library were individually analysed to determine their origin. This was established by i) determining the percentage of coverage over sequence length and ii) identifying the number of mapping reads per locus. The final selection was made on highest coverage and highest number of mapping reads to each genome. When the origin of the sequence could not be confidently determined by these criteria, sequences were termed Ambiguous and were not used for further analyses. Otherwise, they were termed Unambiguous and used to classify the different types of RNAs detected in each sample.reads 8, ) to obtareads 8, ). These ces from ,40 and nAscan-SE . In-housFor generating an abundance ranking specific for each sRNA-type and in order to compare sRNA proportions among samples, every sRNA sequence was normalized with respect to the total number of counts associated to the corresponding sRNA biotype  in a given sample ,40. The All bioinformatics analyses were performed in a local server at IMPaM, which is part of Sistema Nacional de Computación de Alto Desempeño (SNCAD), ID 924 Ministerio de Ciencia, Tecnología e Innovación Productiva (MINCyT), Argentina.Echinococcus multilocularis samples: Samples from patients with presence of liver lesion detected by ultrasonography were defined positive if immunodiagnosis results were positive for E. multilocularis. Negative samples corresponded to patients with negative immunodiagnosis.E. granulosus s.l. (Fumouze)) and ELISA . Samples were kept at -20°C until RNA isolation.Serum (250 μl) from 3 alveolar echinococcosis patients taken before and after treatment  and from 3 negative patients were obtained from the Serology Department of the Institute of Hygiene and Microbiology, University of Würzburg, Germany. Diagnosis was performed by HAT  collected in BD Vacutainer EDTA tubes were obtained from Policlinico San Matteo Hospital Foundation, Pavia, Italy. Cysts were classified according to the WHO-IWGE classification and patients were tested for routine diagnostic purposes in the laboratory of Parasitology of San Matteo Hospital Foundation, using ELISA . An equal number of samples from each of the following groups were analysed: active (CE1-2), transitional (CE3b), inactive  and control group (negative patients with no parasitic cysts). All the CE enrolled patients have one single cyst localized in the liver. Samples were kept at -80°C with Trizol LS until RNA isolation.Culture medium samples: Individual culture medium or pool of samples were used, and RNA isolation was performed as described in section “Small RNA library construction and sequencing”. Transitional cultures contained 15–30% of phenol red-stained metacestodes. Only the < 200-nt RNA was purified. After precipitation and drying, RNA was resuspended in 20 μl of RNAse-free water.Glu) or poly-A  RT. Stem-loop (SL) RT was performed as follows: 5 μl of RNA were added to 2 μl of RNAse-free H2O and incubated 5 min at 94°C, followed by ≥ 1 min in ice. Then, the following reagents were added to reach the indicated final concentrations: reaction buffer 1X, DTT 5 mM, dNTPs 0.5 mM, SL primers 0.0125 μM each, RNAse OUT 0.4 U/μl, SuperScript 4 10U/μl, RNAse-free H2O to final volume 20 μl. Reactions were incubated 30 min at 16°C, 30 min at 50°C, 10 min at 80°C. Poly-A reaction was performed in a final volume of 10 μl with reaction buffer 1X, ATP 1 mM, E-PAP  0.5 U/μl. Incubation was performed 1 h at 37°C followed by 10 min at 65°C. RT was carried out in 13 μl of reaction with 5 μl of poly-A RNA, poly-T adaptor 2.8 μM and 0.77 mM dNTPs. This was incubated 5 min at 65°C, followed by ≥ 1 min in ice. Then, reaction buffer 1X, DTT 5 mM, RNAse OUT 0.4 U/μl, SuperScript 4 10U/μl, RNAse-free H2O to final volume 20 μl were added and incubated 30 min at 50°C, followed by 10 min at 80°C. Stem loop primers and Poly T adaptor sequences are described in cDNA synthesis was performed by stem-loop  (Solis Biodyne) and 0.2 μM of each primer. Real time PCR was performed in a StepOne Plus cycler (Applied Biosystems). The cycling conditions were: 95°C 15 min, followed by 40 cycles of 95°C 15 sec, annealing temperature  with 5´FAM and 3´end MBG modifications  was used as described above.Plasma/sera samples: RNA isolation, cDNA synthesis and qPCR were performed as described above. After precipitation and drying, RNA was resuspended in 20 μl of RNAse-free water. Five μl of RNA were used as input for the SL RT reaction. As positive amplification control, endogenous hsa-miR-423-5p was used  from culture media and MVF from active cultures  and to a lesser extent clearly mapped to the M. musculus genome (0.1% - 8.4%). The size distribution of E. multilocularis mapping reads showed that P100 samples displayed three peaks: 19 bp, 32 bp and 40 bp for culture medium and 22 bp, 33 bp and 40 bp for MVF  and tRNA-halves (~ 32–33 bp) [GlyGCC, tRNALysCUU and tRNAGluCUC in P100    RNA was observed in S100 from culture medium. SRP is a universally conserved ribonucleoprotein complex involved in targeting transmembrane and secretory proteins to the endoplasmic reticulum . Here, wthe gene .M. musculus genome, we only focused on those vertebrate sRNAs detected in the MVF. Echinococcus multilocularis metacestodes multiply asexually in vitro in co-culture with hepatocytes, which are removed when axenic parasites are needed [With respect to reads mapping to the e needed . HoweverE. multilocularis metacestode eventually develops as a heterogeneous multivesicular mass with regions of necrotic parasite tissue [8 (1.11 x 108–7.65 x 108) nanoparticles/ml which represents an increase of ~ 20-fold with respect to P100 from the active cultures described above (p = 0.05) (9 (3.60 x 109–9.02 x 109) nanoparticles/ml, i.e. a significant increase of ~12-fold nanoparticles with repect to MVF from active metacestodes (p = 0.05) (8 (8.74 x 108–13.49 x 108) nanoparticles/ml secreted into the MVF  . The det = 0.05) . Viable  the MVF . Nanopar the MVF . EV pres the MVF .In contrast to active cultures, RNA could be readily detected by electrophoresis in all the extracellular compartments, including the P100 fraction from culture medium where the RNA size profile showed high similarity to that of MVF Figs . As descGeneral sequencing results are shown in In comparison to samples from active metacestode culture, P100 from transitional culture medium contained more miRNAs (16 vs 5) . Among tGlu RNAs, from one or two isoacceptors, in P100 samples  in culture medium and stained MVF, and 5´end-tRNAPro(UGG) and 3´end-tRNALys(CUU) in culture medium  could be detected by RT-qPCR both plicates . Notablyplicates . Since mplicates . In CE splicates . In all Finally, the presence of host miR-122-5p could be detected in MVF .in vitro by E. multilocularis metacestodes. Since this larval stage is a fluid-filled vesicle, the germinal layer has an apical surface that secretes molecules towards the extra-parasite milieu (culture medium) and a basal surface whose secretions accumulate in the intra-parasite milieu (MVF). One such type of molecules are nucleic acids which have been described in other organisms to be secreted in soluble ribonucleoprotein complexes or associated to EV or lipoproteins. Previously, we have determined by transmission electron microscopy that EV could be hardly detected in the culture medium of E. multilocularis metacestodes in contrast to media conditioned by metacestodes from T. crassiceps and M. corti [In this work, we have characterised the profile of ex-RNA secreted M. corti . In thatEchinococcus spp. EV can alter the cellular and functional phenotypes of immune cells in vitro [E. multilocularis EV release in vivo may represent a strategy to divert the immune system while allowing parasite survival through stem cell metastasis [In this way, we observed in active cultures that EV are mainy secreted towards the intra-parasite milieu since a significant higher number of nanoparticles were present in the MVF. However, in transitional cultures there was an increase of more than 10-fold in the number of nanoparticles detected in the extra-parasite milieu and the MVF of senescent metacestodes. As the tegument is responsible for synthesizing the laminated layer , we hypoin vitro ,28, E. mtastasis . In addiEchinococcus spp. metacestodes release proteins to the extra-parasite medium in vivo due to the presence of host circulating antibodies generated against parasite antigens [Trichinella spiralis muscle stage larvae in vitro secrete unprotected sRNAs suggesting the existence of an alternative RNA secretion mechanism than those described to date [In line with these observations, ex-RNAs in active cultures were enriched in the S100 (EV-depleted) fraction of culture medium in comparison to the P100 (EV-enriched) fraction, while in transitional cultures ex-RNAs could be abundantly detected in the P100 fraction. This implies that in undisrupted metacestodes ex-RNAs may be secreted in soluble ribonucleoprotein complexes or lipoproteins. It is known that antigens ; however to date  that mayE. multilocularis metacestodes mainly secrete sRNAs (< 200 nt) in agreement with previous reports on ex-RNAs present in EV-enriched and EV-depleted samples from medium conditioned by helminth parasites [4- and 3´OH- present in miRNAs and tRNAs [In this work, we demonstrated that arasites ,21,24,54nd tRNAs , since nHaemonchus contortus but not in S100 nor P100 from adults [H. polygyrus shows an inverse proportion, with 75% reads mapping to miRNAs in P100 and only 10% in S100 [The ex-RNA profile from the S100 samples of culture media showed a predominant proportion of miRNAs in contrast to observations made on ex-RNAs from mammalian cell lines ,56,57. Tm adults . Another in S100 . AlthougE. multilocularis metacestodes from experimental infections accounting for 8.7% (± 0.27) of total miRNA expression [in vivo [in vitro generated metacestodes, these results suggest that not only the most abundant miRNAs expressed in parasite tissues are secreted. miR-71-5p and miR-4989-3p have been detected in cestodes EV [H. polygyrus could not be detected in sera from infected mice while miRNAs from the circulating filarial nematode Litomosoides sigmodontis could [H. contortus could be detected in serum from infected sheep [E. multilocularis miRNAs in sera from experimentally infected mice [Previously, we reported that miR-71-5p has a moderate expression level in pression . Here, i[in vivo . Even thtodes EV ,30,58 anis could . In addied sheep  althoughed sheep . Altogetted mice ; howeverde novo [E. multilocularis infiltrates and destroys the host liver parenchyma, liver fibrosis is a common pathological feature of AE which is also associated with abundant vascular structures proximal to the parasite [Several parasite miRNAs enriched in the extra-parasite S100 fraction share the seed region with their homologous host miRNAs, suggesting that the parasite could mimic those miRNAs to regulate the same pathways. Particularly, miR-96-5p was reported to regulate cholesterol uptake and biosynthesis ,62. Givede novo , it woulde novo . HIF1 isparasite . In thisE. multilocularis metacestodes in vitro is carried out in co-culture with rat hepatocytes in the presence of fetal bovine serum [Interestingly, we confirmed the presence of host miRNAs in the intra-parasite milieu, implying their transport through the tegument towards the MVF. In particular, the most abundant miRNA was miR-122-5p. This miRNA is not encoded in invertebrate genomes, it has liver-enriched expression and it is known to be secreted and present in bovine serum . The asene serum . Due to Glu, tRNAGly and tRNAAla. We further confirmed by RT-qPCR that these sRNAs were detected in S100 from culture medium, in agreement with findings in human cell lines, where tRNA-derived sRNAs were predominantly detected in EV-depleted fractions and mostly corresponded to tRNAGlu and tRNAGly [Schistosoma mansoni though their relative abundance in relation to other RNA biotypes was not assessed [With respect to other ex-sRNAs, tRNA-derived sequences were found. These sequences mapped mainly to the 5´end of certain tRNAs, such as tRNA tRNAGly ,56. In p tRNAGly ,22, wherassessed . tRNA-deassessed . Our resin vitro and in the absence of host stimuli, a polarized secretion of sRNAs takes place in the non-vesicular fraction (S100) of E. multilocularis metacestodes, with miRNAs mainly secreted to the extra-parasite milieu and rRNA- and tRNA-derived sequences secreted to the intra-parasite milieu. Further studies are necessary to determine if E. multilocularis ex-RNAs play a role in targeting neighbouring host cells and if they can efficiently reach the host bloodstream, for instance, in early metacestodes with a still non consolidated laminated layer.Here we have shown that We believe that a comprehensive study of the secretion mechanisms throughout the life cycle of these parasites will help to improve current diagnostic tools by targeting those molecules with higher chances to be detected in host body fluids according to parasite state of development.S1 FigA) Classification of Active and Transitional metacestodes according to phenol red staining  and eosin staining . B) Collapsed metacestodes with loss of turgency were excluded from cultures. White arrows show collapsed metacestodes. Scale bars indicate 1 cm.(TIF)Click here for additional data file.S2 FigE. multilocularis metacestodes. M: marker. FU: fluorescence units. Size distribution of reads mapping unambiguously to the E. multilocularis genome detected in the P100 and S100 fractions of culture medium  and metacestodes vesicular fluid  of metacestodes.Analysis of the long RNA (> 200 nt) content present in the P100 and S100 fractions of culture medium  and metacestode vesicular fluid  of active (TIF)Click here for additional data file.S3 FigM. musculus homologous miRNAs and level of enrichment are shown.(A) Linear regression of miRNA abundance in fraction S100 of active culture medium and in metacestode tissue. Each dot represents a miRNA. Red dots indicate miRNAs enriched in S100 fraction of active culture medium; hollow dots indicate miRNAs enriched in metacestode tissue; black dots indicate miRNAs with equal abundance. (B) miRNAs enriched in the S100 fraction of active culture medium. The sequence, (TIF)Click here for additional data file.S4 FigA) Length distribution of reads detected in culture medium of active metacestodes. Sequences corresponding to reads mapping to one specific region (±1 nt) and account for ≥ 50% of total read counts for this gene are shown. Only the 3´-end of the gene is displayed. B) RT-qPCR detection of SRP-derived sequence in P100 and S100 from active and transitional cultures. N = 3 each. Lines indicate the median values.(TIF)Click here for additional data file.S5 FigE. multilocularis active (A) and transitional  cultures. Host microRNAs detected in the S100 fraction from metacestode vesicular fluid of active (D) and transitional  cultures.Analysis of the P100 and S100 fractions of metacestode vesicular fluid of (TIF)Click here for additional data file.S6 FigE. multilocularis genome detected in culture medium  and MVF (Q-T).Analysis of the small RNA content (< 200 nt) present in the P100 and S100 fractions of metacestodes vesicular fluid (MVF) from viable (non-stained)  and senescent (stained)  metacestodes. Size distribution of the three main RNA biotypes detected in MVF from viable  and senescent  metacestodes. Analysis of the large RNA content (> 200 nt) present in the P100 and S100 fractions of culture medium  and MVF (K-N). M: marker. FU: fluorescence units. General size distribution of reads mapping unambiguously to the (TIF)Click here for additional data file.S7 FigRNA biotypes identified in the P100 and S100 fractions of MVF from viable (non-stained) (A) and senescent (stained) (B) metacestodes. Most abundantly detected miRNAs  and tRNA-derived sequences  in non-stained and stained MVF.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.


Digital multiplex gene expression profiling is overcoming the limitations of many tissue-processing and RNA extraction techniques for the reproducible and quantitative molecular classification of disease. We assessed the effect of different skin biopsy collection/storage conditions on mRNA quality and quantity and the NanoString nCounter™ System’s ability to reproducibly quantify the expression of 730 immune genes from skin biopsies.n = 6) obtained from skin sections from four patients undergoing routine abdominoplasty were subject to one of several collection/storage protocols, including: i) snap freezing in liquid nitrogen and transportation on dry ice; ii) RNAlater (ThermoFisher) for 24 h at room temperature then stored at − 80 °C; iii) formalin fixation with further processing for FFPE blocks; iv) DNA/RNA shield (Zymo) stored and shipped at room temperature; v) placed in TRIzol then stored at − 80 °C; vi) saline without RNAse for 24 h at room temperature then stored at − 80 °C. RNA yield and integrity was assessed following extraction via NanoDrop, QuantiFluor with RNA specific dye and a Bioanalyser . Immune gene expression was analysed using the NanoString Pancancer Immune Profiling Panel containing 730 genes.Healthy human skin punch biopsies , with integrity lowest from the saline stored sample (RQS < 2). There was a high degree of reproducibility in the expression of immune genes between all samples with the exception of saline, with the number of detected genes at counts < 100, between 100 and 1000 and > 10,000 similar across extraction protocols.A variety of processing methods can be used for digital immune gene expression profiling in mRNA extracted from skin that are comparable to snap frozen skin specimens, providing skin cancer clinicians greater opportunity to supply skin specimens to tissue banks. NanoString nCounter technology can determine gene expression in skin biopsy specimens with a high degree of sensitivity despite lower RNA yields and processing methods that may generate poorer quality RNA. The increased sensitivity of digital gene expression profiling continues to expand molecular pathology profiling of disease. Molecular profiling of tissue for insight into mechanisms of disease, stratification of individuals for disease risk and to monitor therapeutic responses is rapidly increasing due to advances in technology. Driven by high-throughput molecular technology, such as digital sequencing, there is a growing body of molecular biomarker data across cancer phenotypes that aim to allow for personalised medical approaches that minimise unnecessary treatment.A key consideration in molecular biomarker analysis is the need to extract high quality RNA from tissue samples . The croVarious methods are also available for RNA protection, such as with TRIzol  or RNAlaThis was a comparison of immune gene expression from six skin tissue biopsy RNA extraction methods collected from three healthy patients undergoing abdominoplasty, with biopsies 3 and 4 collected from the same patient. All six methods were performed on abdominoplasty tissue collected from each person. Following excision of tissue, six 4 mm biopsies were collected with standard techniques. The study was conducted under approval from the Griffith University Human Research Ethics Committee and the United HealthCare Human Research Ethics Committee (HMR/05/15/HREC).Following collection of the six skin biopsies from tissue from each patient the following storage and transport procedures were used: i) snap freezing in liquid nitrogen and transportation on dry ice; ii) RNAlater  for 24 h at room temperature then stored at − 80 °C; iii) formalin fixation and storage of FFPE blocks at room temperature; iv) DNA/RNA Shield  stored and shipped at room temperature; v) placed in TRIzol  then stored at − 80 °C; vi) 0.15 ml saline without RNAse for 24 h at room temperature then stored at − 80 °C. First homogenization of skin biopsies using ZR BashingBead Lysis Tubes (Zymo) and Tissue Lyser II (Qiagen) was unsuccessful, therefore it was re-done using gentleMACS octo and M tubes (Miltenyi Biotec). For the samples processed with liquid nitrogen, saline and RNAlater, RNA was extracted using the Maxwell® RSC simplyRNA Tissue Kit . For the FFPE samples the RNeasy® mini kit  and ReliaPrep™ FFPE Total RNA Miniprep System (data is not shown) were used for RNA extraction. From samples in TRIzol RNA was extracted using the Direct-Zol™ RNA kit  while the Quick RNA™ Miniprep Kit  was used for extraction of RNA from DNA/RNA shield  stored biopsies. After isolation RNA samples were aliquoted and stored at − 80 °C until further analysis.260 / A280 ratio was measured with the NanoDrop 1000 UV-Vis spectrophotometer  with an A260 / A280 ratio > 1.9 considered an indicator of pure RNA. RNA quality score (RQS) was calculated by a LabChip 24 bioanalyzer (PerkinElmer). Based on data using RNA Pico Sensitivity Reagent Kit, all RNA samples except LN1, LN2, RL1 and RL2 were concentrated using the Zymo RNA Concentrator kit (Zymo). After concentration, RNA was assessed using Quantifluor direct RNA dye (Promega) and LabChip 24 RNA Pico Sensitivity Reagent Kit (PerkinElmer).RNA extraction was performed in an RNAse-free environment following the manufacturer’s protocol for each kit. The concentration of extracted RNA (ng/μL) was assessed using three different methods: i) UV-spectrophotometry ; ii) LabChip24 with Standard and Pico sensitivity RNA reagents (PerkinElmer); iii) Quantifluor direct RNA dye (Promega). An = 730 genes of key inflammatory pathways and n = 40 reference/housekeeping genes. The manufacturer’s protocol was followed with small modification in that 300 ng of total RNA extracted from skin biopsies was hybridised with probes at 65 °C for 24 h. Samples were processed on the NanoString Prep Station and the target-probe complex was immobilised onto the analysis cartridge. Cartridges were scanned by the nCounter Digital Analyser for digital counting of molecular barcodes corresponding to each target at 280 fields of view.Immune gene expression analysis was undertaken using the NanoString nCounter analysis system  using the commercially available nCounter PanCancer Immune Profiling panel kit. The PanCancer Immune profiling panel contains http://mev.tm4.org). The Advanced Analysis Module enables quality control (QC), normalisation, cluster analysis, differential gene expression (DGE), Pathview Plots and immune cell profiling. Raw data was normalised by subtracting the mean plus one standard deviation of eight negative controls while technical variation was normalised through internal positive controls. Data was corrected for input volume via internal housekeeping genes using the geNorm algorithm. Immune cell scores were determined using cell specific gene expression from The Cancer Genome Atlas (TCGA) as detailed in [p < 0.001.Gene expression data was analysed using the Advanced Analysis Module in the nSolver™ Analysis Software version 4.0 from NanoString Technologies  and TIGR Multi-Experiment Viewer .Counts for genes above background threshold, below 100, between 101 and 1000 and above 1000 by sample are shown in Table The role of molecular profiling in pathology to classify disease was recognised in 2014 through the formalisation of an informatics subdivision within the Association for Molecular Pathology given the growing use of high throughput quantitative data to deliver health care . A recogVariation in RNA quality results in inaccurate and misleading changes in molecular profiling, underpinning the need for reliable and reproducible protocols for the processing of tissue and extraction of RNA . NumerouThe NanoString nCounter Analysis system has been one of the latest advances in genomic technology for molecular profiling. As a hybridisation-based system, the technology eliminates the need for amplification bias common to PCR for direct counting of molecular transcripts. Research has demonstrated that the NanoString System is able to quantify transcripts from total RNA of lower quality and quantity, potentially providing researchers with additional options for the collection of tissue for molecular profiling. We utilised the PanCancer Immune Profiling kit to undertake broad-based molecular profiling of mRNA extracted from tissue using the various tissue processing techniques. The technology had high sensitivity of target detection across the sample set even at lower quality scores and yields, which is consistent with previous research , 12. AbsIn conclusion, our study shows that several tissue processing and extraction techniques successfully isolate RNA for analysis using high throughput digital counting. We observed substantial variation in the quality and yield of these techniques, with tissue stored in FFPE blocks providing the most consistent yield and quality scores in all participants. We note a number of limitations, in particular the small number of samples per protocol, that each processing method utilised a different RNA extraction method, that the results relate to skin samples only and that these samples were fresh tissue not older samples so caution should be taken in extrapolating these results. Many of these limitations are consistent with clinical research and increases the ecological validity of the results for research and pathology purposes. Despite the variation and quality of mRNA, the NanoString nCounter analysis system was able to quantify 730 genes across protocols with a high degree of similarity, highlighting the benefits of hybridisation-based technology for molecular profiling.


C. elegans RyR gene unc-68. We show that specific isoform subsets are created via alternative promoters and via alternative splicing in unc-68 Divergent Region 2 (DR2), which actually corresponds to a region of high sequence variability across vertebrate isoforms. The expression of specific unc-68 alternative exons is enriched in different tissues, such as in body wall muscle, neurons and pharyngeal muscle. In order to infer the function of specific alternative promoters and alternative exons of unc-68, we selectively deleted them by CRISPR/Cas9 genome editing. We evaluated pharyngeal function, as well as locomotor function in swimming and crawling with high-content computer-assisted postural and behavioral analysis. Our data provide a comprehensive map of the pleiotropic impact of isoform-specific mutations and highlight that tissue-specific unc-68 isoforms fulfill distinct functions. As a whole, our work clarifies how the C. elegans single RyR gene unc-68 can fulfill multiple tasks through tissue-specific isoforms, and provide a solid foundation to further develop C. elegans as a model to study RyR channel functions and malfunctions.Ryanodine receptors (RyR) are essential regulators of cellular calcium homeostasis and signaling. Vertebrate genomes contain multiple RyR gene isoforms, expressed in different tissues and executing different functions. In contrast, invertebrate genomes contain a single RyR-encoding gene and it has long been proposed that different transcripts generated by alternative splicing may diversify their functions. Here, we analyze the expression and function of alternative exons in the  in vivo using classical genetic approaches is challenging, notably because different isoforms may work in a partially redundant manner for many of their functions. Logically, research has progressed more rapidly in discovering the function of whole genes than that of specific isoforms. Using C. elegans model, we take advantage here of the recent development of efficient genome editing techniques to create alternative exon-specific molecular lesions and infer their specific roles in vivo by using a large set of phenotypic measures. We applied this approach to elucidate the role of alternative exons in the Ryanodine receptor (RyR) gene unc-68, which encodes the largest ion channel reported so far. Our results have implications for the understanding of the functional diversity of RyR channels across phyla and send an encouraging signal for the future application of genome editing to elucidate the functions of alternative exons in C. elegans.The use of alternative exons allows a vast diversification of the isoform sequences encoded by single genes. The functional study of gene product isoforms  Red signal was detected only in pharyngeal muscle ::CeBFP protein. Blue signal was detected only in neurons, suggesting exon 13 exclusion.In the first approach , we creal muscle , suggest[Ex13Scar/BFP] reporter in which exon 13 inclusion creates a red UNC-68(ex12/ex13/ex14)::mScarlet protein, whereas exon 13 exclusion leads to a frame shift over the mScarlet sequence (nevertheless devoid of stop codons) and the production of a blue UNC-68(ex12/ex14)::out-of-frame mScarlet::CeBFP reporter protein. We observed red signal in the pharyngeal muscle  null mutants, and mutant lines that we engineered to lack specific alternative exons and/or promoters  and a slightly less pronounced reduction in Δex1.1 animals . Pharyngeal pumping was normal in Δex1.2 animals, but significantly reduced in Δprom2:ex1.2 animals. Collectively, these data suggest (i) that exon 1, promoter 1 and promoter 2 are all important for unc-68-mediated pharyngeal pumping control, (ii) that exon 1.2 is dispensable, but (iii) that exon 1.2 can partially compensate for the lack of exon 1.1 if promoters 1 and 2 are intact.Pharyngeal pumping in s system . This prfindings , 22, we unc-68, we expanded the phenotypic characterization of unc-68 mutants using high-content computer-assisted crawling behavior analysis , DAG634 domEx634 [unc-68p2::mNeonGreen::unc-54 3’UTR], DAG635 domEx635[unc-68p2::mNeonGreen::unc-54 3’UTR], DAG636 domEx636[unc-68p1::mNeonGreen::unc-54 3’UTR], DAG637 domEx637[unc-68p1::mNeonGreen::unc-54 3’UTR], PHX214 unc-68(syb214), PHX215 unc-68(syb215), PHX216 unc-68(syb216), PHX217 unc-68(syb217), PHX218 unc-68(syb218), PHX219 unc-68(syb219);PHX220 unc-68(syb220), PHX471 unc-68(syb471), PHX729 unc-68(syb729), DAG862 domEx862, DAG865 domEx865, DAG866 domEx866, DAG938 domEx938, DAG939 domEx939, DAG940 domEx940, DAG949 domEx949, DAG1150-1152 domEx1150-1152.The strains used in this study were the following: N2, TR2171 unc-68(syb218) and PHX220 unc-68(syb220) were outcrossed four times with wild type .unc-68 promoter 1 or promoter 2. To that end, we amplified N2 genomic DNA by PCR using the following primers flanked with attB4 and attB1r recombination sites:The three-fragment MultiSiteGateway system (Invitrogen) was used. We first created slot 1 Entry plasmids containing either attB4unc-68p1_F: ggggacaactttgtatagaaaagttgATCGTTGGTTAATAATTGTTGGCTAACCGTattB1runc-68p1_R: ggggactgcttttttgtacaaacttgTCTGTAAAACAAAAAAACTAGAGGTGCTGGattB4unc-68p2_F: ggggacaactttgtatagaaaagttgATACAAAGTTCAAGTTGACAATTAGTTCTattB1runc-68p2_R: ggggactgcttttttgtacaaacttgTTTCTTGGAACTAACTAATCATATCACTG[slot1 Entry unc-68p1] and dg603 [slot1 Entry unc-68p2].PCR products were then cloned into pDONR-P4-P1R vector (Invitrogen) by BP recombination to create: dg604 mNeonGreen slot2 Entry plasmid (dg398)  and dg576 [unc-68p2::mNeonGreen::unc-54 3’UTR]. dg735 [unc-68prom1::NLS::mScarlet::unc-54UTR] was created via a LR reaction combining dg604, dg651 [slot2 Entry NLS::mScarlet], pMH473 and pDEST R4R3.These Entry plasmids were then each recombined with a  (dg398) , an unc-The different minigenes were obtained by gene synthesis , an unc-54 3’UTR slot3 Entry plasmid (pMH473) and a pDEST R4R3 destination vector. Resulting expression plasmids were named dg648 [unc-68p1::QF::unc-54 3’UTR] and dg649[unc-68p2::QF::unc-54 3’UTR].The QUAS promoter was used in the two-component Q-system, where it can be activated by the QF transcription factor, expressed under the control of a second promoter from a separate construct . To geneunc-68p1::QF construct, unc-68p2::QF construct, or both of them at the same time. Our initial goal was to express these reporters in all UNC-68-expressing tissues using both promoter 1 and 2 to drive the transgene expression. However, despites numerous attempts, we never detected signal in body wall muscle, but only in neurons and pharyngeal muscle. Of note, we easily obtained transgenics when using only a promoter 2 construct, obtained no transgenic line when using only a promoter 1 construct, and obtained transgenic lines at a dramatically reduced frequency when using a mix of the two constructs. In the latter case, expression was absent in body wall muscles. A possible explanation is that our minigenes display some toxicity when expressed in body wall muscles, strongly counter-selecting transgenic animals with detectable expression in this tissue. The exon 13 tissue-specific analysis presented in the Result section is therefore limited to pharyngeal muscle and neurons.When generating transgenic lines, we combined each QUAS-minigene construct with either TM HP Plasmid miniprep kit (Sigma) and was microinjected at a concentration of 20 ng/μl according to a standard protocol [[unc-122p::GFP] or [unc-122p::RFP] were used as co-injection markers.DNA was prepared with a GenEluteprotocol . Either For fluorescent reporter imaging, we used either a Zeiss Axio Plan 2 fluorescence microscope  or a Leica TCS SPE-II confocal microscope , equipped with a 488 nm wavelength diode laser and an ET525/50m emission filter. Z-stack images were acquired across whole animal thickness and maximal intensity projections are depicted.unc-68 transcript isoforms, respectively, using the following primers , PCR products (~4.7 kb in size) were cloned into the pCR4-TOPO TA vector (Invitrogen), and resulting clones sequenced from the 3’ extremity to determine the specific exon combination in the DR2 region of each clone.unc-68 transcript. Based on genomic coordinates, we could differentiate between exon 1.1-containing and exon 1.2-containing transcripts. Raw data are available in For the analysis reported in Videos of grinder movements of adult animals on food were recorded at a 160X magnification thanks to a stereomicroscope (Leica M2015FA) equipped with a camera (Leica DFC345FX), as previously described . GrinderFirst day adult animals were transferred to 24-well plates with M9 buffer and videos were recorded using a camera (DMK33UX250).Motility of worms was assessed as the number of waves of body bending per min in M9 buffer. Body bending was scored using a computer assisted analysis in Image J as previously described .E. coli seeded NGM petri dishes were video-recorded and analyzed with the Tierpsy tracker [T-tests, using Holm-Bonferroni correction for multiple comparisons (P < .01).First day adult animals crawling on OP50  tracker , 30. At S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.


The pharmacokinetics of a drug is dependent upon the coordinate work of influx transporters, enzymes and efflux transporters . The transporter–enzyme interplay may occur in liver, kidney and intestine. The influx transporters involving drug transport are organic anion transporting polypeptides (OATPs), peptide transporters (PepTs), organic anion transporters (OATs), monocarboxylate transporters (MCTs) and organic cation transporters (OCTs). The efflux transporters are P-glycoprotein (P-gp), multidrug/toxin extrusions (MATEs), multidrug resistance-associated proteins (MRPs) and breast cancer resistance protein (BCRP). The enzymes related to drug metabolism are mainly cytochrome P450 enzymes (CYP450s) and UDP-glucuronosyltransferases (UGTs). Accumulating evidence has demonstrated that diabetes alters the expression and functions of CYP450s and transporters in a different manner, disordering the transporter–enzyme interplay, in turn affecting the pharmacokinetics of some drugs. We aimed to focus on (1) the imbalance of transporter-CYP450 interplay in the liver, intestine and kidney due to altered expressions of influx transporters , efflux transporters  and CYP450s  under diabetic status; (2) the net contributions of these alterations in the expression and functions of transporters and CYP450s to drug disposition, therapeutic efficacy and drug toxicity; (3) application of a physiologically-based pharmacokinetic model in transporter–enzyme interplay. The pharmacokinetics of a drug is determined by absorption, distribution, excretion and metabolism. Drug absorption, distribution and excretion are mainly controlled by drug transporters, while drug metabolism is mediated by metabolic enzymes. These drug transporters are classified into the ATP binding cassette (ABC) family and the solute carrier (SLC) family. The main SLC transporters involved in drug transport are multidrug/toxin extrusions (MATEs), organic anion transporting polypeptides (OATPs), monocarboxylate transporters (MCTs), organic anion transporters (OATs), peptide transporters (PepTs), and organic cation transporters (OCTs). Most SLC transporters belong to influx transporters, except for MATEs. The identified ABC transporters related to drug efflux include P-glycoprotein (P-gp), multidrug resistance-associated proteins (MRPs) and breast cancer resistance protein (BCRP). These transporters are widely expressed in the intestine, liver and kidney. They affect drug therapeutic effects/toxicity via regulating drug uptake or secretion. Enzymes involved in drug metabolism mainly include cytochrome P450 enzymes (CYP450s) and UDP-glucuronosyltransferases (UGTs). These enzymes are also widely distributed in the liver, intestine and kidney. Drug disposition in tissues is highly dependent on the coordinate work of these influx transporters, enzymes and efflux transporters, termed as the “interplay of transporters and enzymes” ,2,3. MorDrugs are eliminated in the liver mainly via metabolism and biliary excretion. The liver highly expresses various drug transporters  A and druOATPs, expressed at the basolateral membrane of hepatocytes, mediates hepatocyte uptake of many anions from the blood, which becomes a rate-limited process of hepatic clearances for some drugs . It was P-gp, expressed at the canalicular membrane of hepatocytes, mediates biliary excretion of its substrates. Diabetes was reported to upregulate the expression of hepatic Mdr1b (ABCB1b) mRNA and P-gp protein in 8-day diabetic Wistar rats (type 1 diabetes) induced by STZ, which was associated with the activation of protein kinase C alpha (PKCα) and nuclear factor kappa-B (NF-κB) . A similBCRP, expressed at the canalicular membrane of hepatocytes, is also involved in the biliary excretion of its substrates. Altered expressions of hepatic BCRP under diabetic status are dependent on the type of diabetes, and diabetic progression. For example, expressions of hepatic BCRP mRNA in 5-week and 8-week diabetic rats induced by STZ were significantly downregulated, but expression of BCRP protein only showed a trend to decrease . SimilarMRP2 is co-expressed with P-gp and BCRP at the canalicular membrane of hepatocytes. Alterations in the expression of MRP2 by diabetes are also dependent on species. STZ-induced diabetic Donryu rats showed significantly lower expression of hepatic MRP2 mRNA, which was in line with lower biliary excretion of pravastatin . A similGrowing evidence has demonstrated that diabetes increases the expression of some CYP450s  in rats ,28,29,30Hyperketonemia (such as acetone) is considered as a reason for diabetes inducing CYP2E1. Data from primary cultured rat hepatocytes demonstrated that insulin decreased the expression of CYP2E1 and CYP2B protein and mRNA, inferring that a deficiency of insulin is also a reason that diabetes induces the expression of CYP2El and CYP2B . FurtherO-demethylase and 7-ethoxyresorufin-O-deethylase were decreased. Expressions of other CYP450s, such as CYP1B1, CYP2B1, CYP2C11 and CYP2E1, were unaltered, inferring that diabetes regulates the expression of hepatic CYP450s in an isoform-specific manner  estrone sulfate in renal cortical slice. These decreases were in line with the activation of PKCα and NF-κB pathways, increased nuclear factor erythroid 2-related factor 2 (Nrf2) and oxidative stress [The basolateral membrane of renal proximal tubule cells highly expresses OAT1 and OAT3, mediating the excretion of their substrates including ACE inhibitors, angiotensin II receptor blockers, diuretics, β-Lactam antibiotics, antiviral agents and endogenous compounds [e stress ,116. Alte stress . Clinicae stress .Diuretics show their diuretic effect via affecting proximal tubular epithelial cells. Some diuretics (such as torasemid and furosemide) are also substrates of OATs . Several1-methylnicotinamide [N1-methylnicotinamide  in both mid (504 ± 293 mL/min) and late pregnancy (557 ± 305 mL/min) were reported to be higher than in postpartum (240 ± 106 mL/min). The renal secretion of N1-methylnicotinamide was 3.5-fold higher in mid pregnancy and 4.5-fold higher in late pregnancy compared with postpartum [Renal OCT1/2, mainly expressed at the basolateral membrane of tubule cells, transport a variety of organic cations, such as metformin, cisplatin, cephalexin and acyclovir. In STZ/HFD-induced diabetic rats, it was found that OCT2 protein expression was only 50% of control rats . Similartinamide ,127. Thetinamide ,127, inftinamide . The dectinamide . A clinitinamide  showed ttinamide . Renal cstpartum . In linestpartum , and lowstpartum ,132 compstpartum ,134,135.stpartum ,137. Howstpartum .Diabetes induced an approximately two-fold increase in renal PepT1 protein and slightly induced mRNA of PepT1 and PepT2 . ReportsFor MRP2, increased expression of renal MRP2 protein was found in STZ/HFD-diabetic rats  and in SThe identified renal CYP450s include CYP2C, CYP2J, CYP2E1, CYP4As and CYP4Fs. They mainly mediate local biotransformation of endogenous compounds such as arachidonic acid. For example, arachidonic acid is metabolized by CYP4As and CYP4Fs to 20-hydroxyeicosatetraenoic acid (vasoconstrictor) and by CYP2C and CYP2J to epoxyeicosatrienoic acid (vasodilator), synergistically regulating renal vasoactivity. Several reports have shown that diabetes also affect the expressions of renal CYP450s. In mice, it was found that HFD feeding significantly decreased activities of CYP3A (1′-hydroxymidazolam), CYP2E1 (hydroxychlorzoxazone), CYP2J (ebastine hydroxylation) and CYP2B6 (hydroxybupropion) and CYP4A (12-hydroxydecanoic acid), but increased the activities of CYP2C (hydroxytolbutamide) and CYP2D  . DiabeteTransporter interplay occurs in the kidney. For example, methotrexate is eliminated mainly via renal active excretion, which is involved in OAT1/3 at basolateral membrane of tubule and BCRP and MRP2 at brush-border membrane of tubule, these transporters in series work to regulate renal active excretion of methotrexate. Both higher plasma concentration of methotrexate and higher toxicity of methotrexate in STZ-diabetic rats  may partThe above statements demonstrated that diabetes may simultaneously affect functions and expressions of transporters and enzymes in the intestine, liver and kidney. Their contributions to the disposition of drugs are often opposite. Moreover, some physiological parameters such as blood flow rates, intestinal transit and the free fraction of drug in plasma are often altered. Thus, the effects of diabetes on pharmacokinetics of drugs should be the integrated effects of these alterations, which may be accomplished using PBPK.Atorvastatin is a substrate of P-gp, OATPs, BCRP and CYP3As. Diabetes reduced expressions of intestinal P-gp, CYP3A and OATP1A5, while induced expressions of liver OATP1B2 and CYP3A. Intestinal BCRP was highly dependent upon diabetes course, which was decreased at an early phase of diabetes (10-day) but induced at a late phase (22-day). A pharmacokinetic study showed that oral plasma exposure was increased in 10-day diabetic rats, but decreased in 22-day diabetic rats. PBPK simulation demonstrated that contributions of these targeted protein to altered oral plasma exposure of atorvastatin were intestinal BCRP > hepatic OATP1B2 > hepatic CYP3A > intestinal CYP3A > intestinal P-gp > intestinal OATP1A5. The increased oral plasma atorvastatin exposure in 10-day diabetic rats and the decreased oral plasma atorvastatin exposure in 22-day diabetic rats were attributed to the altered intestinal BCRP .Verapamil is substrate of CYP3As and P-gp. Rat experiments showed that diabetes increased plasma exposure of verapamil following an oral dose but decreased plasma exposure of verapamil following an intravenous dose . A semi-liver) of furosemide was reported to be 2.20 mL/min/kg [int,liver was 0.75 mL/min/250g. Diabetes increased the expression of CYP2E1 and CYP3As by 3-fold [int,liver and CLliver were estimated to be 0.75 mL/min/250g and 2.20 mL/min/kg. The estimated CLliver of furosemide was near to observed data (2.98) in diabetic rats [Rat experiments demonstrated that diabetes decreased plasma furosemide exposure following oral dose but increased plasma furosemide exposure following intravenous dose . IntestiL/min/kg  and the y 3-fold  and the y 3-fold . Thus, ftic rats . Diabetetic rats . PBPK siA whole-body PBPK model was also used to predict the pharmacokinetics of metformin in diabetic patients . The resGrowing evidence has demonstrated that diabetes often simultaneously affects transporters and enzymes in the intestine, liver and kidney. Moreover, these alterations are dependent on tissue and diabetic progression. For example, diabetes induced the expression of hepatic CYP3A, but downregulated the expression of intestinal CYP3A. In STZ/HFD-induced diabetic rats, 10-day diabetes decreased the expression of both intestinal and hepatic BCRP, but 22-day diabetes induced the expression of intestinal BCRP not hepatic BCRP protein . Moreove


C9orf72 hexanucleotide repeat expansion. However, the mechanism(s) remain unclear. Karyopherins, including importin β and its cargo adaptors, have been shown to co-precipitate with the C9orf72 arginine-containing dipeptide repeat proteins (R-DPRs), poly-glycine arginine (GR) and poly-proline arginine (PR), and are protective in genetic modifier screens. Here, we show that R-DPRs interact with importin β, disrupt its cargo loading, and inhibit nuclear import of importin β, importin α/β, and transportin cargoes in permeabilized mouse neurons and HeLa cells, in a manner that can be rescued by RNA. Although R-DPRs induce widespread protein aggregation in this in vitro system, transport disruption is not due to nucleocytoplasmic transport protein sequestration, nor blockade of the phenylalanine-glycine (FG)-rich nuclear pore complex. Our results support a model in which R-DPRs interfere with cargo loading on karyopherins.Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases, including ALS caused by a  C9orf72 is the most common known cause of amyotrophic lateral sclerosis (ALS) and is also a major cause of frontotemporal dementia (FTD) and the ALS/FTD overlap syndrome (C9orf72 HRE is thought to cause disease by a toxic gain of function involving expanded repeat RNA and dipeptide repeat proteins (DPRs) produced by repeat-associated (non-AUG) translation, although a modest reduction in C9ORF72 protein is also seen  and antisense  directions have been identified in postmortem tissue (A GGGGCC hexanucleotide repeat expansion (HRE) in syndrome . The C9om tissue , and ovem tissue  and animm tissue .Growing evidence suggests that disruption of nucleocytoplasmic transport (NCT), the regulated trafficking of proteins and ribonucleoprotein complexes between the nucleus and cytoplasm, is a major pathophysiologic mechanism in neurodegenerative diseases  and they operate in parallel to transport a repertoire of distinct cargoes defined by their nuclear localization signals (NLS)   , which c cargo’) . Since c cargo’) , we hypo cargo’) . However cargo’) . We obseTo test the functional consequence of R-DPR-importin β interactions for nuclear import, we performed the permeabilized cell assay , in whicUsing this method, we performed live imaging of nuclear import of Rango, a direct importin β cargo whose Ran-, importin β-, and energy-dependent nuclear translocation is conferred by the IBB domain . We veriThe mechanisms of cargo recognition for importin β differ even from its structurally closest relative transportin, whose cargos are marked by the PY-NLS motif . HoweverTo further validate the direct interaction between R-DPRs and importin β, we performed the bead halo assay. This equilibrium-based binding assay is capable of identifying both low- and high-affinity interactions between ‘bait’ proteins immobilized to beads, and fluorescent ‘prey’ in the surrounding buffer, which forms a fluorescent halo on the bead surface . First, To test if the disruption of nuclear import resulted from changes in the passive exclusion limit of NPCs, we tested the effects of R-DPRs on the passive influx of small cargoes. Passive diffusion of GFP and small fluorescent dextrans into the nuclei of permeabilized HeLa cells was imaged at 10 s intervals for 5 min, and nuclear fluorescence quantified over time. All experiments were done in the context of energy and cell lysate, identical to the active transport conditions, so as not to miss putative effects that may depend on simultaneous active transport (i.e. recruitment of importins and DPRs to the NPC). Under these conditions, we observed the expected differences in the rates of passive influx of 10-, 40-, and 70-kD dextrans, and verified that addition of energy and lysate did not affect the baseline rate of passive influx of GFP  . When weC9orf72 DPRs and FG-domains of yeast homologs of Nup62 (Nsp1) and Nup98 (Nup100 and Nup116) in the bead halo assay . For PR10, FG-binding could be augmented (to Nup100 and Nsp1 fragments) by adding unlabeled importin β to the assay, suggesting that recruitment of PR to FG-domains at the NPC could be mediated in part by an indirect interaction through importin β. Overall, these results support modest direct and indirect binding of R-DPRs to FG domains, which based on our passive transport data, do not decrease NPC permeability and even increase passive influx, by a mechanism that remains to be elucidated.We again observed non-selective binding by the R-DPRs, including to beads coated with a Nup116 F->A mutant construct. However, quantification of the halo intensities showed modest selective binding of GR10 and PR10 to GLFG-domains of Nup100, but not Nsp1 or Nup116, in a pattern that appeared primarily dependent on the length of the FG-domains tested , were then analyzed by immunoblotting. In this manner, we assessed the the partitioning of NCT proteins, Nups, and LCD-containing proteins between the soluble and aggregate fraction of the extracts . We saw These data confirm that R-DPR aggregates can recruit NCT constituents in addition to a host of nucleic acid-binding proteins. However, NCT proteins were not substantially depleted from the supernatant even in the presence of 100 µM GR10 and PR10, suggesting that sequestration of critical NCT factors in these insoluble protein assemblies is unlikely to fully explain the failure of nuclear import in the transport assays.Cytoplasmic aggregate formation, a pathological hallmark of neurodegenerative disease, has been proposed as a general mechanism for impairment of NCT by sequestration of critical NPC and NCT proteins . HoweverNext, we tested the effect of increasing the concentration of RNA, based on the growing evidence that RNA mediates the solubility of intrinsically disordered proteins, including FUS, TDP-43, and RNA-dependent DEAD-box ATPases , and attSince the above approaches for mitigating aggregate formation were either ineffective or incompatible with the transport assay, we modified the assay to clarify the role of R-DPR induced aggregates in the disruption of nuclear import. Before adding the R-DPR-treated cell lysates to permeabilized cells, we separated the soluble phase of the extracts (supernatants) from the aggregates (pellets) by centrifugation , such K/R-rich NLSs include the ‘classic’ mono- and bipartite NLS for importin α, the R-rich NLS of ribosomal proteins that directly bind importin β, importin 5, and importin 7 , the basPreviously published interactomes  and mechhttps://predictprotein.org/; Throughout this study, we observed marked differences in the behavior of GR and PR. Although both readily displaced importin β from the Rango FRET sensor, PR showed greater potency for inhibiting nuclear import in the permeabilized cell assay and higher relative selectivity for importin β in the bead halo assays. Moreover, while both PR and GR readily induced aggregate formation in the transport lysates, PR showed greater tendency to dissociate from the aggregates . Based oThus, although in a purified system GR and PR show a similar propensity to bind importin β, in the more complex environment of the cell, our findings predict PR to be the more potent disruptor. Micromolar concentrations of R-DPRs were required to observe functional import blockade in the permeabilized cell assay; however, 20mers were on average 3.3-fold more potent than 10mers across all cargoes, suggesting that longer DPRs, as are likely present in patients, may be significantly more potent. However, the size of polyGR and polyPR peptides in patients is unknown. GGGGCC repeat lengths in the 1000s have been reported in postmortem brain , althougBased on growing evidence that RNA is integral to the solubility of disordered protein assemblies , and polC9orf72-ALS were based on genetic modifier screens of expanded repeat RNA and DPRs in Drosophila and yeast. These studies implicated numerous karyopherins, Nups, and Ran cycle proteins as potential direct and indirect mediators of C9orf72-ALS pathogenesis  and cysteine  were synthesized by Genscript  and 21st Century Biochemicals  and verified by mass spectrometry to be free of trifluoroacetic acid adducts. Lyophilized powder was diluted in 0.1x XB’ buffer  and frozen in single use 10 mM aliquots at −80°C after snap freezing in liquid nitrogen.S. cerevisiae Nsp1(497-608) FxFG domain was obtained from M. Rexach (Restriction cloning was used to insert the ORF from pQE-ZZ-RanQ69L  between . Rexach .E. coli BL21(DE3) cells  that were cultured in 1L batches of LB media contained in 2.8L baffle-free Fehrnbach flasks. Protein expression was induced with 0.3 mM IPTG. Centrifugation was used to collect the cells and wash them in the ice-cold protein-specific buffer, as indicated below. Unless otherwise specified, all buffers were pH 7.4. The washed cell pellets were flash-frozen in liquid nitrogen and stored at −80°C, and lysed in ice-cold conditions and in the presence of protease inhibitors, using French pressure cell or microfluidizers. After dialysis in the protein-specific buffer, protein concentration was measured with the Bradford assay , and single-use aliquots of all proteins were stored at −80°C after flash-freezing in liquid nitrogen.Unless otherwise specified, recombinant proteins were expressed in 600nm = 0.1–0.3. The cultures were cooled to room temperature (22–25°C), and protein expression was induced at 22–25°C for 12–14 hr.For expression of proteins containing GFP variants, including Rango (pK44 and pK188), YFP-M9-CFP, GST-GFP-NLS, and GFP-Avitag, the cells were first outgrown at 37°C until reaching OD2, 1 mM MgCl2, pH 7.7).Cells expressing 6His-tagged fluorescent proteins  were washed and lysed in 10 mM imidazole/PBS and purified with either Ni-NTA agarose  or HIS-Select HF Nickel Affinity Gel . The lysates were clarified  and incubated with Ni resin . The resin was placed into small chromatography columns, washed with ice-cold 10 mM imidazole/PBS, and the proteins eluted with increasing concentration of imidazole/PBS (25–300 mM). SDS-PAGE was used to select and pool batches with the highest purity, prior to dialysis in PBS or XB buffer , the lysate clarified, and the protein affinity-purified on glutathione sepharose . After washes with TBSE, the proteins were eluted with TBSE containing increasing concentrations of glutathione (2.5–10 mM). Proteins eluted with 2.5 and 5 mM glutathione were pooled and dialyzed in PBS before storage.nm = 0.6, cooled to 22°C and the expression was induced with 0.3 mM IPTG. After 12 hr induction, cells were harvested with the CARR continuous flow centrifuge and lysed in PBS with 10 mM imidazole and 5 mM TCEP with a 110EH Microfluidizer  using 2 passes at 10,000 PSI under chilled conditions. The lysates were flash-frozen in liquid nitrogen and stored at −80°C. After thawing, the lysates were clarified and incubated with HIS-Select HF Nickel Affinity beads. The beads were washed with 10 mM imidazole/PBS and the protein eluted with 200 mM imidazole/PBS before dialysis in XB. The purified importin β was combined with a freshly thawed aliquot of Rango2-α1 (pK188) at 2.5:1 molar ratio ratio  and supplemented with 3% glycerol. Measurement of Rango fluorescence emission in a spectrometer (see below) was used to verify the FRET-off state of the Rango/importin β mixture before freezing.Full-length human importin β was expressed from pET30a-WT importin β pKW485;  at the P600nm = 0.4, followed by incubation at 22°C for 4 hr. The cells were washed with PBS, 10 mM Imidazole, 1 mM MgCl2, 5 mM TCEP, 0.2 mM AEBSF, pH 8.0, and lysed by ice-cold microfluidizer. The clarified lysates were used to isolate the zz-RCC1 proteins on HIS-Select HF Nickel Affinity beads, as described above. Proteins eluted with 0.2M imidazole/PBS were dialyzed in PBS before storage. The zz-RanQ69L  was expressed from BL21DE3 cells at PEL in an 80L bioreactor, using conditions described for importin β above, except that expression was induced with 0.3 mM IPTG at 37°C for 3 hr, and lysis was performed in PBS with 10 mM Imidazole, 5 mM TCEP, 2 mM MgCl2, and 1 mM GTP. The lysates were clarified and bound to HIS-Select HF Nickel Affinity beads (Millipore Sigma). The Ni resin was washed with ice-cold 10 mM imidazole/PBS and the protein eluted with 0.2M imidazole/PBS, followed by dialysis in XB. After measuring the concentration, 60 µM zzRCC1 and 2.4 µM zzRCC1 were combined in XB containing 2 mM GTP. Before aliquoting and storage, the measurement of Rango fluorescence emission in a spectrometer (see below) was used to verify that the zzRanQ69L-GTP-containing mix robustly induced Rango dissociation from importin β.The expression of zz-RCC1 was induced at OD600nm + 0.4–0.6, the cultures were cooled to room temperature, supplemented with 100 µM D-biotin, and the expression was induced with 0.3 mM IPTG at room temperature for 8–11 hr (pKW762). Proteins were purified on Ni-NTA resin as described for the non-biotinylated importin β fragments.To prepare biotinylated WT importin β-Avitag , BL21DE3 cells  were co-transfected with the respective plasmids together with pAC-biotin ligase (Avidity), followed by plating and growth in LB media containing ampicillin and chloramphenicol. After the 37°C cultures reached OD(497-609) in BL21(DE3) cells grown in LB media was induced at OD600nm = 0.4–0.6, followed by incubation at 37°C for 3–5 hr. The GST-tagged S. cerevisiae pGEX-Nup100(1-307), Nup100(1-610), Nup116(348-458) and Nup116(348-458)F > A were expressed in T7 Shuffle cells (NEB) that were grown in Dynamite media  diluted to 10 µM in XB was combined with 10-molar excess of Alexa Fluor 647 NHS ester (ThermoFisher), using freshly-prepared 10 mM dye in anhydrous DMSO. After incubation on ice for 2 hr, the sample was dialyzed in PBS and concentrated on 30kD MWCO filter (Millipore Sigma) before storage. A similar protocol was used to label BSA with 10-molar excess Sulfo-NHS-LC-biotin (ThermoFisher), followed by dialysis in PBS.Just before labeling, Alexa Fluor 488 C5-maleimide (ThermoFisher) was diluted to 20 mM in anhydrous DMSO and further diluted to 1.6 mM in XB’ buffer. Freshly thawed 10 mM aliquots of DPRs were diluted to 2 mM with 0.1x XB’ and combined with an equal volume of 1.6 mM Alexa Fluor 488 C5-maleimide and kept overnight at 4°C. The unreacted maleimide was quenched by 1:50 (v/v) 100 mM DTT before aliquoting and storage at −80°C. To verify labeling, DPRs were separated by SDS-PAGE followed by fluorescence detection.FRET assays for DPR-induced dissociation of the importin β-Rango complex were performed using a mix of 5 µM Rango-2/α1 (pK188) and 12.5 µM importin β (pKW485) prepared as described above. After thawing on ice, the mix was diluted to 20 nM Rango and 50 nM importin β in TBS, pH 7.4, 0.01% Tween-20 (TTBS), supplemented with increasing DPR concentrations, and mixed by brief vortexing at low speed. The positive control reactions for RanGTP-induced Rango-importin β dissociation were prepared by adding increasing concentrations of ZZ-RanQ69L-GTP to the samples, using a freshly thawed aliquot of 60 µM ZZ-RanQ69L, 2.4 µM ZZ-RCC1, 2 mM GTP in XB. The assay buffer alone was used as a blank. A Fluoromax-2 spectrometer  was used to detect the Rango emission spectra  while exciting the samples at 435 nm. The excitation and emission bandpass were set to 5 nm and integration time to 0.05 s. Peak emissions were recorded at 480 nm (donor) and 535 nm (acceptor) in all samples, and background emission subtracted at the same wavelengths in the blank. The FRET signal was calculated as the ratio of background-subtracted acceptor/donor emissions. The signal detected in the untreated sample , was then subtracted from the resulting values. Prism v6  was used to calculate the non-linear fit with one site-specific binding model while using the D’Agostino and Pearson K2 test to verify the normality of residuals and the Runs test to assure non-significant deviation from the model.An aliquot of 5 µM Rango-2/α1 + 12.5 µM importin β mix was diluted to 20 nM Rango and 50 nM importin β in TTBS, supplemented with increasing concentrations of GR10 or PR10, mixed by vortexing, and incubated for 30 min at room temperature. GFP-Trap magnetic beads  were washed and resuspended in TTBS. At the end of incubation, 8 µl bead suspension was added to each sample and mixed by rotation for 15 min. The supernatant was removed and beads washed 3 times with TTBS before boiling in 20 µl SDS-PAGE sample buffer with 2% β-mercaptoethanol. Samples were separated by SDS-PAGE and anti-GFP western blot performed as detailed below to detect Rango. After detecting the ECL signal, membranes were stained with Coomassie Brilliant R250 to detect importin β. Background-subtracted signals were determined by Image Lab 6.01 (BioRad) and the Rango ECL signal normalized to the importin β signal within each lane.Aliquots of total HEK RNA (3 µg) were mixed with either 4 µl 50 µM DPR-AF488 in 0.1x XB’ or with 1 µl 0.2% SYBR Gold nucleic acid stain (ThermoFisher) diluted in water. After 5 min incubation at room temperature, the samples were supplemented with Fast Digest loading buffer  and separated by electrophoresis on native 1% agarose gel in TBE, alongside with lanes containing HEK RNA (3 µg) or RNA ladder mixed with SYBR Gold. Immediately after electrophoresis, the gels were photographed with Bio-Rad ChemiDoc XRS+ using UV transillumination to simultaneously visualize the AF488-labeled R-DPRs and SYBR Gold-labeled RNA signals (where added).2, 1 mM DTT, 0.1% Tween-20). Immediately prior to the assay, fluorescent bait proteins and beads were combined with 4x assay buffer  to a total of 40 µL per well, in optical glass-bottom 96-well plates . Reactions were allowed to equilibrate at room temperature for a minimum of 30 min prior to imaging at 100x on an LSM800 confocal microscope . Intensity profiles comparing the maximum rim intensity to the background were plotted in ImageJ (NIH) by an investigator blinded to experimental conditions.The bead halo assay was carried out as described with minor modifications , using 62, 5 mM NaOAc, 0.5 mM EGTA, 250 mM sucrose, pH 7.3, with protease inhibitor cocktail). All rinse and assay buffers were supplemented with 50 mg/mL BSA. The optimal hypotonic buffer and BSA concentration varied by batch, and was optimized prior to each set of assays for ability to permeabilize the majority of plasma membranes while maintaining nuclear exclusion of a 70 kD fluorescent dextran (ThermoFisher).All animal procedures were approved by the Johns Hopkins Animal Care and Use Committee. Timed pregnant C57BL/6J females  were sacrificed by cervical dislocation at E16, cortex dissociated, and cells plated at 50,000/well on poly-D-lysine/laminin-coated, optical glass-bottom 96-well plates. Growth medium consisted of Neurobasal supplemented with B27, Glutamax, and penicillin/streptomycin (Gibco/ThermoFisher). At 5–7 days in vitro, neurons were rinsed in prewarmed PBS and permeabilized for 4 min. at 37° in a hypotonic solution containing 0–40 µM Tris-HCl pH 7.5 (to cause osmotic swelling) and 50–150 mg/ml BSA . Following permeabilization, cells were placed on ice and rinsed 2 × 5 min in transport buffer 2, 5 mM NaOAc, 0.5 mM EGTA, 250 mM sucrose, pH 7.3, with protease inhibitor cocktail). The optimal digitonin concentration varied by cell density and passage number, and was optimized prior to each set of assays for the ability to permeabilize the majority of plasma membranes while maintaining nuclear exclusion of a 70 kD fluorescent dextran (ThermoFisher).A single cell-derived clone of HeLa cells  were maintained in OptiMEM (Gibco/ThermoFisher) with 4% FBS and plated on uncoated optical glass-bottom 96 well plates, at appropriate densities to reach 70–90% confluence on the day of the transport assay. To permeabilize, cells were rinsed for 2 min in ice-cold PBS, and permeabilized on ice for 10 min in 15–30 µg/mL digitonin  in permeabilization buffer 2, 0.5 mM EGTA, 250 mM sucrose, pH 7.5, with protease inhibitor cocktail). Following permeabilization, cells were placed on ice and rinsed 3 × 5 min in transport buffer Nuclear import was carried out essentially as described  with modReaction mixes consisting of 2.5 mg/ml lysate, ER, fluorescent cargo , Hoechst, DPRs, RNA, or inhibitors ; 0.8 mg/mL wheat germ agglutinin  were assembled on ice during cell permeabilization. DPRs or inhibitors were allowed to equilibrate in cell lysate for at least 30 min prior to initiation of transport. Every plate included: (1) Cargo alone: fluorescent cargo, but no ER or lysate, (2) Untreated controls: fluorescent cargo, ER, and lysate, and (3) Inhibitor: fluorescent cargo, ER, lysate, and IPZ (Rango and GST-GFP-NLS reactions) or WGA (YFP-M9-CFP). Preassembled transport reactions were then transferred onto permeabilized cells via multichannel pipette, and allowed to proceed at room temperature for 2 hr  or 4 hr (GST-GFP-NLS). Cells were fixed in 4% paraformaldehyde/PBS, rinsed 2x with PBS, and transferred to 50% glycerol/PBS for immediate imaging.For a subset of neuron transport assays, transport was monitored live via time lapse imaging every 5 min for 30 min. In a subset of HeLa assays, transport reactions were centrifuged before use at 14,000g × 15 min to separate soluble and insoluble fractions. In another variation, the transport lysate + DPR and ER mix was allowed to preincubate on the permeabilized HeLa cells for 1 hour prior to initiation of transport, rinsed 1x with TRB, and transport initiated with fresh lysate, cargo, and ER.Multiple non-overlapping fields per well , were captured at 40x on an ImageXpress Micro XLS high-content microscope , and the ratio of nuclear to cytoplasmic fluorescence intensity was calculated using the MetaXpress automated translocation-enhanced module. Raw data were filtered to exclude autofluorescence and the mean N/C ratio from wells without ER or cell lysate was subtracted from all values. Resulting N/C ratios were expressed as % untreated, to permit comparisons across biological replicates.HeLa cells were permeabilized as above, rinsed 3x with TRB, and reaction mix containing 2.5 mg/ml HEK lysate, ER (to mimic the active import conditions), Hoechst, and/or DPRs were added and allowed to preincubate directly on the permeabilized cells for at least 30 min. 0.8 mg/ml WGA was used as a positive control. Cells were mounted on a Zeiss LSM800 confocal microscope, reaction mix withdrawn, and immediately replaced with fluorescent dextran (ThermoFisher) or recombinant GFP (pK803) in fresh lysate/ER mix to initiate the passive import reaction. A single 40x frame (containing 20–30 cells/well) was imaged per well, with images collected every 10 s for 5 min. The ratio of nuclear fluorescence intensity to local background at each timepoint was analyzed using Imaris , and values for each cell were expressed as a ratio of time 0 .2- and CaCl2- free DPBS (ThermoFisher), then flash-frozen in liquid nitrogen and stored at −80°C before further processing and analysis by the Johns Hopkins Mass Spectrometry and Proteomics core facility. Pellets were reduced/alkylated with DTT/IAA, reconstituted in TEAB/acetonitrile, and sonicated for 15 min prior to overnight digestion with Trypsin/LysC  at 37°C. Some precipitate remained; supernatants were desalted and analyzed by LC/MS/MS on a QExactive_Plus mass spectrometer (ThermoFisher). MS/MS spectra were searched via Proteome Discoverer 2.2 (ThermoFisher) with Mascot 2.6.2  against the RefSeq2017_83_ human species database (NCBI). Protein probabilities were assigned by the Protein Prophet algorithm  via the PRIDE partner repository (50 µM GR10 or 25 µM PR10 (in duplicate) were added to 5 mg/ml HEK whole cell lysate (in TRB with ER), incubated for 60 min at 37°C and aggregates were pelleted by centrifugation at 16,000 g for 10 min. Supernatants were removed and pellets washed 2x and resuspended in MgCllgorithm . Proteinpository  with the202 for 20 min  were prepared as in the nuclear transport assays, by adding 100 µM 10mers to 2.5 mg/ml HEK lysate in 100 µL TRB. Supernatants were boiled in Laemmli (BioRad) for 5 min. Pellets were boiled for 15 min followed by sonication in order to fully disperse aggregates for SDS-PAGE. Equal volumes of supernatant and pellet fractions were run on 4–12% Bolt Bis-Tris Plus gels (ThermoFisher), transferred to nitrocellulose membrane using an iBlot2 dry blotting system (ThermoFisher). Protein loading was analyzed by BLOT-Faststain , according to the manufacterers’ instructions. For immunodetection, membranes were blocked with 5% non-fat milk in TBST and probed by sequential incubation with the primary antibodies as detailed in the table below. Detection was by HRP-conjugated secondary antibodies/chemiluminescence using an ImageQuant LAS 4000 system . To permit sequential probing of membranes without stripping, signals were quenched by incubation with prewarmed 30% Hr 20 min . Band inData analysis, graphing, and statistical analyses were carried out using Prism v6-v8 (Graphpad), according to methods detailed under each experimental approach above and in the figure legends. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:C9orf72 affect nuclear import and will be of interest to a broad audience.We recognize that you and your colleagues have taken the earlier advice from our reviewers seriously. We believe that the revised manuscript represents a sophisticated set of approaches to identify how the two arginine containing polydipetide repeat proteins encoded by the ALS-causing repeat expansion in Decision letter after peer review:C9orf72 arginine-rich dipeptide repeat proteins disrupt importin β-mediated nuclear import" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen Huda Zoghbi as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The reviewers agree that your work represents a comprehensive, sophisticated, well-designed set of experimental efforts that test how the arginine containing polydipeptide repeat (R-DPRs) proteins produced in an inherited form of ALS affect nucleocytoplasmic transport. All reviewers agreed that the FRET and biochemical assays, including in situ permeabilized cell reconstitutions, do establish a direct interaction with importin β which disrupts its cargo loading, and correspondingly, inhibiting nuclear import in a manner that can be diminished by increasing RNA concentration (with the RNA presumably competing with importin β for R-DPR binding). Additionally, your efforts support that transport inhibition is not through sequestration of nuclear import components or blockage of the nuclear pore complex. One reviewer reported that the text and figures have been beautifully prepared, producing a compelling manuscript that was a pleasure to read.That said, there are areas where we would like further clarification.Essential revisions:C9ALS. However, subsequent experiments argue against this mechanism, and indicate that the action of GR differs from that of PR. By the end, we felt as if the original hypothesis had been disproved to some degree, but an alternative hypothesis had not been presented or adequately explored. The result was some confusion over what, precisely, R-DPRs are doing and how their actions result in NCT impairment.1) Your effort begins by probing a link between R-DPRs and importin β, and the first few figures appear to confirm this link as being important for interruption of NCT in 2) The experiments raise several questions that are not addressed in the text or in subsequent investigations. These include:a) The modest effect of PA and GA upon NCT ;b) The inhibition of Trp1-mediated NCT  which argues against importin β as a mechanism of action; the inconsistent effects of R-DPR length, particularly with regards to the Rango reporter ;c) The troublesome nonspecific binding of GR/PR in Figures 2 and 3;d) The increase in PR binding with addition of lysate ;e) The apparent increase in passive NCT flux with PR/GR ; andf) The isolation of more proteins by GR than by PR  despite the more pronounced effect of PR in most assays.3) The DPRs used in this study are considerably shorter than those observed in patients. Indeed, the purified DPRs used in this study are 10-mers and 20-mers, well within the normal range for non-ALS individuals, and used at μM amounts. Even though there is evidence of toxicity with these repeat sizes in various cellular systems, exploration of key aspects of this mechanism in the context of repeat sizes more reflective of those observed in patients should be considered.4) The majority of assays are executed in HeLa cells. Given data that there may be important differences in NCT in different cell types, the main claims should be explored in neurons; if not at least discussed. Essential revisions:1) Your effort begins by probing a link between R-DPRs and importin β, and the first few figures appear to confirm this link as being important for interruption of NCT in C9ALS. However, subsequent experiments argue against this mechanism, and indicate that the action of GR differs from that of PR. By the end, we felt as if the original hypothesis had been disproved to some degree, but an alternative hypothesis had not been presented or adequately explored. The result was some confusion over what, precisely, R-DPRs are doing and how their actions result in NCT impairment.Thank you for this feedback. It seems that in trying to be very clear about the model, we oversimplified things, both in terms of the expected differences between GR and PR and the narrow focus on importin β. Indeed, the action of GR clearly differs from PR, throughout our current study and in other recent publications. Moreover, while we focused on the possibility that importin β could be targeted by R-DPRs that mimic its cargoes, we assume that in parallel, R-DPRs could disrupt other nuclear transport receptors (karyopherins), as supported by our transportin (M9) data. All of this would be predicted based on the divergent molecular properties of GR and PR and the role of basic residues in nuclear import complex formation. We have therefore changed the title from “importin β” to “karyopherin” and made major edits throughout the manuscript reflecting both of the above topics.Based on their structure and published studies, the differing behavior of GR and PR is not surprising. On one hand, both are positively charged , with a predicted disordered state and polynucleotide-binding activity . However, because glycine is conformationally flexible and its side chain is comprised of a single hydrogen, while the integrated proline cyclic side-chain induces a rigid bend in the peptide backbone, poly-GR is predicted to accommodate many different macromolecular ligands, while poly-PR may be more selective. As recently reviewed by Freibaum and Taylor, 2017, GR and PR have differing half-lives, differing intracellular localization, and differing properties with regard to stress granule dynamics and mitochondrial function. Moreover, in yeast the modifiers of GR and PR toxicity showed little overlap .Accordingly, although both GR and PR showed low nanomolar displacement of importin β from the Rango (IBB) FRET sensor , in the presence of cell lysate, PR had greater potency for inhibiting importin- and transportin-mediated nuclear import , higher relative selectivity for importin β , and differing behavior with respect to the soluble phase of nuclear import . Although not quantitative in this regard, our mass spectrometry results showed 21% of hits uniquely bound GR10, and 11% PR10. This was even higher at 35% (GR50) and 25% (PR50) unique hits in the data set from Lee and colleagues (2016). We have edited the manuscript to discuss the differences between GR and PR in our assays and how that relates to the conclusions. Namely, that although in a purified system both of the R-DPRs show a similar propensity to bind importin β , in the more complex environment of the cell, our findings predict PR to be the more potent disruptor.vis-à-vis importin β, although we find that PR acts as a more selective inhibitor relative to GR, and there are broader implications of our findings for other karyopherins.In summary, our results do support our initial hypothesis 2) The experiments raise several questions that are not addressed in the text or in subsequent investigations. These include:a) The modest effect of PA and GA upon NCT ;In permeabilized primary neurons exposed to 100 µM GA10 and PA10, we saw a mild but statistically significant decrease in nuclear enrichment of the Rango import cargo at steady state . This was a late effect (>30 min), as we did not see any separation from controls within the first 30 minutes of nuclear import . The potential mechanism and physiologic relevance are not clear, given the high concentrations used and the lack of a similar effect in HeLa cells . In both cases the reaction mixture  is equivalent, therefore it is possible that high concentrations of GA and PA may somehow react differently with either the neuronal nuclear pore complexes or nuclear contents required for the transport reaction. In more complex in vitroand in vivomodel systems, GA has been proposed to sequester nuclear transport components , although we did not observe that in the sedimentation assay when compared to R-DPRs at equivalent concentrations . We have added additional comments to the Discussion.b) The inhibition of Trp1-mediated NCT  which argues against importin-ß as a mechanism of action; the inconsistent effects of R-DPR length, particularly with regards to the Rango reporter ;The nuclear import and export pathways, including those that are mediated by transportin and importin β, do not operate in series, but rather simultaneously in parallel, and for a wide repertoire of distinct cargoes. In the revised manuscript, we describe how the known features of importin β/α and transportin NLSs, and the properties of R-DPRs, predict that R-DPRs could employ distinct mechanisms to disrupt cargo loading on both transportin and importin β, in parallel.In case of transportin cargos, the positive charge of arginines in R-DPRs could promote their electrostatic interactions with the aromatic ring of the side chain of tyrosine (cation-pi interactions) which is the essential part of the PY-NLS of transportin. Since the PY-NLS also contains an N-terminal basic and R-containing region, R-DPRs could at the same bind transportin via cargo mimicry. Both mechanisms may be contributing. Although we focused on mechanistic implications for importin β, we did not want to overlook what we anticipated would be parallel biology affecting the transportin pathway. We have elaborated further on this issue, and have substituted “karyopherin” for “importin β” in the title, to reflect the importance of this observation.c) The troublesome nonspecific binding of GR/PR in Figures 2 and 3;The GR and PR peptides indeed showed a high propensity for nonspecific binding, as would be expected for these highly-charged peptides. To mitigate this, the bead halo assays were done at neutral pH with EDTA (to reduce cation-mediated interactions), NaCl (to disrupt weak ionic interactions), and Tween (to disrupt weak hydrophobic interactions). 10 mg/ml (150 µM) BSA was also included as an additional blocking agent. Nevertheless, such measures did not eliminate the nonspecific binding, either in this paradigm or in our attempts to optimize conventional immunoprecipitation assays with these peptides (not shown).Within the techniques available to us, we found the bead halo assay to be the most reproducible and quantitative approach to account for this nonspecific binding. We have included well-defined positive and negative controls for expected specific binding, and bare beads as well as sham-coated beads , to quantify the level of nonspecific binding for valid comparison. Based on minor comment 13, we also added Figure 2—figure supplement 2, confirming the ability of free importin β to compete for R-DPR binding in a dose-dependent manner. This further supports the specificity of the binding.Moreover, the effect of adding cell lysate in Figure 2 , precisely predicts the results of Figure 5 in the permeabilized cell assay. That is, in the context of cell lysate, PR retains its specific binding for importin β, whereas GR is consumed by binding to other targets (even the nonspecific binding goes away).Additional comments about this have been added to the description of the bead halo results.d) The increase in PR binding with addition of lysate ;This may be due to recruitment of additional importin β- and PR-binding partners from the cell lysate onto the beads, providing additional sites for AF488-PR to bind. We added this comment to the Results section describing these data.e) The apparent increase in passive NCT flux with PR/GR ; andThe acceleration of passive transport that we observed for PR and GR , was indeed surprising. There are several possible scenarios to explain this, outlined below and discussed in the revised manuscript. Please see also further comments under minor comment #14.Studies in yeast suggest that to pass through the NPC, cargos must overcome collisions with highly mobile, disordered FG domains, particularly the Nup98 homologues Nup100 and Nup116 . If R-DPRs target these FG domains, as indicated by the findings of Shi and colleagues (2017) and by our bead halo studies , this could drastically increase their positive charge or reduce their mobility, thus reducing their gate-keeping function. The passive cargo exclusion properties of the NPC have also been shown to depend on importin β. By superresolution microscopy, Ma and colleagues (2012) showed that when extra importin β accumulates within the NPCs, concentrating inside the peripherally-localized active transport zone, the central passive transport channel widens, thus allowing faster passive cargo flux. Alternatively, when Kapinos et al., 2017, monitored the passive cargo influx rate in permeabilized cells, they showed that when importin•cargo complexes were depleted from the NPC, the pore became functionally leaky. Although our permeabilized cell experiments indicated the R-DPR-induced accumulation of AF647-labeled importin β at the nuclear rim , concomitant with the transport blockade, these assays did not apply sufficient resolution to conclude if importin β was accumulated in or depleted from NPC channels. Superresolution microscopy studies would be needed to show the precise site of importin β accumulation , to test which of the above effects may be contributing. Single molecule tracking of passive transport through NPCs would also aid in definitively establishing the precise effect of R-DPRs on putative opening of the NPC permeability barrier.Nevertheless, our passive transport data  do not support the notion that R-DPRs induce block to nuclear transport by occluding the NPC transport channels. Such a conclusion is also strongly supported by the results of the “nuclei preincubation” experiment , where the NPCs were exposed to high R-DPR concentration for 1h before the start of the nuclear import assay, and no deceleration of nuclear import was observed.f) The isolation of more proteins by GR than by PR  despite the more pronounced effect of PR in most assays.This is explained by the structural differences in these two R-DPRs as discussed above in #1. Poly-GR is predicted to accommodate many different macromolecular ligands, while poly-PR is expected to be more selective, due to the marked difference in the flexibility of the peptide backbone. Our assays consistently demonstrate that PR has a higher selectivity for importin β in conditions mimicking the complex cellular milieu, consistent with the more pronounced inhibition of nuclear import.3) The DPRs used in this study are considerably shorter than those observed in patients. Indeed, the purified DPRs used in this study are 10-mers and 20-mers, well within the normal range for non-ALS individuals, and used at μM amounts. Even though there is evidence of toxicity with these repeat sizes in various cellular systems, exploration of key aspects of this mechanism in the context of repeat sizes more reflective of those observed in patients should be considered.C9orf72 repeat sequences occurs in neurologically healthy individuals, nor has deposition of GR and PR been reported in individuals with normal C9orf72 repeat length. Although all individuals possess short C9 RNA repeats, there are no data at this time to support the idea that non-ALS individuals express short DPRs.The length of GR and PR in affected individuals remains unknown due to technical challenges in determining protein size. This issue aside, there is no evidence, to our knowledge, that RAN translation of <30 E. coli was met with significant difficulty. These remain very technically difficult experiments to do, due to the nature of these proteins.The reviewers’ suggestion to try longer repeats is nevertheless very reasonable, and was why we compared 10mers to 20mers in the transport assays in Figure 1. The marked increase in transport inhibition that we observed for 20mers compared to 10mers does suggest that we would see even more potent effects at longer repeat lengths. However for both of the commercial entities we contracted with to make the peptides for this study, even producing 20mers by solid phase synthesis was technically challenging. Our own attempts at purifying recombinant DPRs in 4) The majority of assays are executed in HeLa cells. Given data that there may be important differences in NCT in different cell types, the main claims should be explored in neurons; if not at least discussed.Certainly, we agree with the critical need to verify the major claims of this paper in neurons. That was the basis for our extensive efforts  to optimize a method for selective permeabilization of neuronal nuclei. When we first encountered difficulty with the fragility of neurons compared to HeLa, we searched the literature and found no convincing reports of this assay in neurons. We then devised a novel method that allowed us to selectively permeabilize neurons without also rupturing the nuclear membrane. To our knowledge, this is the first convincing and reproducible use of the nuclear transport assay in permeabilized neurons.Upon doing so, we observed that the effects of GR and PR on Rango nuclear import (the direct importin β cargo) were very reminiscent of that seen in HeLa. Having verified this, we did not see a scientific rationale for continuing to duplicate all assays in neurons, which is significantly more time- and resource-intensive.