AbstractCrustacea, Peracarida) from the inner lagoon habitats from the Alacranes Reef National Park, the largest coral reef ecosystem in the Gulf of Mexico. The dataset contains information on records collected from 2009 to 2011. Data are available through Global Biodiversity Information Facility (GBIF).Alacranes Reef was declared as a National Marine Park in 1994. Since then, many efforts have been made to inventory its biodiversity. However, groups such as amphipods have been underestimated or not considered when benthic invertebrates were inventoried. Here we present a dataset that contributes to the knowledge of benthic amphipods . The biological material includes 588 data records with a total abundance of 6,551 organisms. The species inventory represents, until now, the richest fauna of benthic amphipods registered from any discrete coral reef ecosystem in Mexico. Alacranes Reef is a highly diverse protected coral reef ecosystem, located in the southern sector of the Gulf of México. It is considered as one of the largest coral reefs of the Gulf of Mexico . It was Alacranes Reef Biodiversity Expedition.Carlos Enrique Paz Ríos and Nuno Simões.The dataset was composed using information from benthic macrofauna samples randomly collected on different benthic habitats and artificial substrates between 2009 and 2011, mostly in the inner lagoon of Alacranes reef.Resources included funds to NS from the National Autonomous University of Mexico (UNAM) for supporting academic research (PAPIME-PE207210), funds from the Mexican Government for scientific research (SEMARNAT-CONACyT108285) and an international cooperation for marine scientific research between Mexico and USA (HRI-CONABIO-NE018) through the project “Actualización del conocimiento de la diversidad de especies de invertebrados marinos bentónicos de aguas someras (<50 m) del Sur del Golfo de México”. Funds to CEPR was provided by CONACyT scholarship.The dataset contains information on amphipods collected from three different expeditions: the first one in August 2009, the second one in April 2010 and the third one in October 2011. In each expedition a relatively high diversity of habitats was explored in order to increase the chance to capture the species diversity. In the 2010 and 2011 expeditions, special effort was made to collect information on navigation buoys and two wood docks nearby a sand island (isla Perez).Thalassiatestudinum .Amphipods were collected from 50 sampling sites: Forty-one randomly distributed on natural habitats from the inner lagoon, including different benthic habitats  and nine collected on artificial substrates . Diverse sampling devices were employed: PVC core for sandy substrates, shovel for patches of intertidal sand, suction device sampler for coral rubble, sand and shallow coral reef flats and, finally, a Riley push-net and trawl for shallow seagrass beds of Bemlos, Elasmopus, Idunella, Harpinia, Leucothoe, Maera, Psammogammarus); further specialised taxonomical effort will be needed before assigning a name. Species names were matched using the Taxon Match tool in the World Register of Marine Species (WoRMS) in order to corroborate, standardise and update if necessary. Sampling points in the reef, available in the Data resources (see below), were georeferenced and displayed into different maps using different software .Amphipod species were sorted and identified to the lowest possible taxonomical category, using specialised literature such as keys for identification, e.g. Once collected, the amphipods were first anaesthetised with magnesium chloride (4%) for 15 to 20 min. and then fixed with formaldehyde solution (10%) buffered with seawater before storage. In the laboratory, samples were washed through a 500 µm size mesh and preserved with 70% of ethanol solution. Finally, the organisms were stored in glass containers with a catalogue number according to the regional collection ‘Crustáceos de Yucatán’ (YUC-CC-255-11), from the ‘Universidad Nacional Autónoma de México, Unidad Sisal’ Mexico.2 , Colomastigidae (6 spp.), Lysianassidae (8 spp.), Leucothoidae (11 spp.), Maeridae (12 spp.) and Aoridae (13 spp.).The dataset contains information on 110 species , belonging to 71 genera, 33 families and three suborders. The suborder Data range: 2009-8-01 – 2011-10-17.OtherCreative Commons Attribution Non Commercial (CC-BY-NC) 4.0 LicenseBenthic amphipods from Alacranes Reef, Campeche Bank, Mexicohttp://ipt.iobis.org/caribbeanobis/resource?r=anfipodosbentonicos_bancodecampeche_mexicohttp://ipt.iobis.org/caribbeanobis/archive.do?r=anfipodosbentonicos_bancodecampeche_mexico1Benthic amphipods from Alacranes Reef, Campeche Bank, MexicoDarwin Core Archive (DwC-A)15UTF-81.0The dataset presents an occurrence data sheet with 15 columns including information for 588 records.


Further developments of PLA protocols will be helpful to dissect importin beta-1-orchestrated pathways during mitosis.Karyopherin beta-1/Importin beta-1 is a conserved nuclear transport receptor, acting in protein nuclear import in interphase and as a global regulator of mitosis. These pleiotropic functions reflect its ability to interact with, and regulate, different pathways during the cell cycle, operating as a major effector of the GTPase RAN. Importin beta-1 is overexpressed in cancers characterized by high genetic instability, an observation that highlights the importance of identifying its partners in mitosis. Here we present the first comprehensive profile of importin beta-1 interactors from human mitotic cells. By combining co-immunoprecipitation and proteome-wide mass spectrometry analysis of synchronized cell extracts, we identified expected  and novel mitotic interactors of importin beta-1, many with RNA-binding ability, that had not been previously associated with importin beta-1. These data complement interactomic studies of interphase transport pathways. We further developed automated proximity ligation assay (PLA) protocols to validate selected interactors. We succeeded in obtaining spatial and temporal resolution of genuine importin beta-1 interactions, which were visualized and localized  As such, it regulates key cellular functions7. Its interphase partners are both direct and indirect. Indirect partners include proteins carrying nuclear localization signals (NLS), recognized by an adaptor molecule belonging to the importin alpha subfamily. Using its C-terminal region, importin beta-1 interacts with importin alpha adaptor/NLS cargo, forming “classical” trimeric import complexes. In the nucleus, the GTPase RAN, loaded with GTP (RANGTP), binds importin beta-1 at its N-terminal region, thus destabilizing interactions at the C-terminus and triggering the cargo release from the import complex. Importin beta-1 also binds several proteins directly, including nucleoporins (NUPs) in their phenyl-glycine (FG)- and FxFG-rich regions, when crossing nuclear pores during nuclear import. Importin beta-1 also directly recognizes a variety of unrelated cargoes acting in different pathways8.Importin beta-1 has a well-characterized modular structure composed of HEAT repeats included in distinct functional domains10. In this mitotic mode, importin beta-1 generally inhibits factors with which it interacts2, and thus prevents their unscheduled activity and the premature onset of events during mitosis. Analogous to the mechanism operating in nuclear import, RANGTP binding to importin beta-1 releases mitotic “cargoes” in a free, biologically active form, thus enabling relevant mitotic event(s) to take place. When overexpressed in human cells, importin beta-1 overrides RANGTP regulation and keeps its mitotic partners in an inhibited state: this causes complex mitotic abnormalities, even under conditions under which nuclear import is not overtly affected14, indicating that mitosis is most sensitive to altered importin beta-1 levels. Regulated expression of importin beta-1 is therefore critical in control of cell division4. Importin beta-1 is overexpressed in many cancer types that generally display high genomic instability, e.g. cervical15 and gastric16 carcinoma, as well as diffuse large B-cell lymphoma17. Because many cancer types are dependent on importin beta-1 expression for their proliferation and survival20, importin beta-1 has been proposed as a novel therapeutic target21 and inhibitors are being developed, e.g. Importazole22 and INI-4323.After NE breakdown, importin beta-1 takes on global regulatory roles in mitotic spindle organization and function24, and, more recently, in a comparative study of twelve human importin family members to depict specificities in recognition of cargoes in distinct import pathways25. Studies of importin beta-1 mitotic interactors are less complete. In an early analysis of human importin beta-1 mitotic partners, we used MALDI-TOF mass spectrometry (MS) to examine importin beta-1 co-immunoprecipitates from mitotic-enriched cell cultures after thymidine arrest and release: we identified NUP358/ RAN-binding protein 2 (RANBP2), a large NUP with RAN-binding domains and E3 ligase activity for small ubiquitin-like modifier (SUMO) peptides, and RAN GTPase-activating protein 1 (RANGAP1) in the SUMO-conjugated form (RANGAP1-SUMO1)14. RANBP2 and RANGAP1-SUMO1 themselves form a stable complex throughout the cell cycle26. The complex  includes the SUMO E2 conjugating enzyme UBC9 and has enhanced SUMO ligase activity27. RANBP2 and RANGAP1-SUMO1 localize at the spindle microtubules (MTs) and accumulate to kinetochores (KTs) after MT attachment28. Importin beta-1 also localizes to the mitotic spindle MTs and poles12, and its interaction with RANBP2 prevents the RRSU premature localization at KTs prior to MT attachment29. Recent work has shown that this is essential to regulate the timing of SUMO conjugation of KT factors, and hence KT functions31. However, beyond individual studies of specific mitotic factors, a comprehensive view of importin beta-1 interactors in mitotic cells is currently lacking.Interactomic analyses of importin-1 beta have been developed in interphase cultures to get insight into mechanisms of transport, based on stable isotope labeling by amino acids in cell cultures (SILAC)In situ proximity ligation assays (is-PLA) is an informative detection technique to visualize protein interactions at their intracellular site32. We have developed an automated PLA protocol to validate selected importin beta-1 interactions: that has enabled us to visualize the interactions of interest during stages of mitotic progression. Coupling the interactome analysis with automated is-PLA imaging provides valuable information on processes through which importin beta-1 can operate in mitosis.Here we present a comprehensive identification of importin beta-1 interactors from homogeneously synchronized mitotic cells using co-immunoprecipitation (co-IP) experiments hyphenated to a high resolution MS analysis. We identify expected partners, e.g. members of the RAN and SUMO pathways, as well as interactors that had not been previously associated with importin beta-1. Most mitotic protein interactions are highly dynamic in time and in space, and innovative methodologies are being developed to unravel specificities and variations. To identify the interactions of importin beta-1 with protein partners during mitosis, we developed the workflow schematized in Fig. 33 analyses, we had to address two preliminary methodological issues, concerning the importin-beta-1 antibody bait and the purity of mitotic cell populations. Because importin beta-1 can engage various domains to interact with partners, different antibodies might be differentially hindered in co-IP experiments by importin beta-1 partners with which they may compete. We tested commercial antibodies against full-length or specific epitopes of importin beta-1  and released cell cultures collected at mitotic round-up using the “shake-off” procedure. After cross-linking to beads and incubation with equal extract amounts, the antibodies showed differential effectiveness in their yield of immunoprecipitated importin beta-1 34, which inhibits the kinesin Eg5 and arrests cells in late G2/early prometaphase with unseparated centrosomes. By immunofluorescence (IF), STLC-arrested and released cell cultures, though being more synchronous compared to thymidine release, included many mitoses still exhibiting monopolar spindles 1 hour after wash-out  was obtained, which includes known as well as novel importin beta-1 interactors. The full list of all 272 hits is shown in Supplementary Table 36; ii) importin alpha and importin-7, both of which cooperate with importin beta-1 in nuclear import of particular classes of cargoes in interphase3; and iii) components of the RRSU complex , as expected from previous studies37.Among well-established importin beta-1 interactors we pulled down various classes of factors: i) the GTPase RAN and RANBP1, both  with global roles in mitotic spindle organization and dynamics12, and NUSAP, a nucleolar protein in interphase that has tubulin-binding and MT-stabilizing activity in mitosis; through these activities NUSAP regulates the formation of asters and long MTs39. Interestingly, NUSAP is known to interact with both importin beta-1 and importin-7 (consistent with our detection of importin-7 in the importin beta-1 mitotic interactome); these two importins are reported to differentially inhibit NUSAP’s mitotic functions: importin beta-1 blocks aster assembly, while importin-7 enables the formation of small asters but prevents the assembly of long MTs38. Also of interest is the presence in the interactome of BUB3, a mitotic spindle checkpoint factor that promotes the stabilization of correct (“end-on”) MT attachments to KTs. BUB3 is required for BUB1 localization to KTs, and the BUB3/BUB1 complex cooperates with other spindle checkpoint factors to prevent premature anaphase onset. Importin-beta-1 was previously found to interact with BUB3, thus preserving it from the ubiquitin/proteasome system and regulating its stability40.The importin beta-1 co-IP also includes proteins with clear mitotic roles. Of note are tubulin beta  were associated with specific mitotic phenotypes  when their coding genes were silenced. Of the remaining annotated genes, 7 (3.6%) gave a predominantly nuclear phenotype , 10 (5%) affected cytokinesis, and 51 (26.15%) affected more general features . With all possible technical reservations inherent to the Mitocheck siRNA-based methodology, these observations support the conclusion that many importin beta-1 interactors identified in our study are indeed required, directly or indirectly, for some aspect of mitosis.Besides these well-characterized factors, we wished to analyse the profile of importin beta-1 mitotic interactors. As a first approach, we examined the list of all 272 identified hits , in order to get more insight into their nature and functions. Table 42, and also cooperates with dynein to maintain spindle orientation43. Heat shock proteins have roles in mitotic entry, progression and exit, often - though not exclusively - by regulating the stability of various mitotic proteins44. The HSP90B family members identified in our list were reported to interact with, and generally stabilize, G2/M checkpoint factors  and mitotic regulators . Some newly identified importin beta-1 interactors also exert global roles in mitosis, including the heterogeneous nuclear ribonucleoprotein C (HNRNPC)45, the translation elongation factor EEF246, and protein TFG (Trk-fused gene)47: all are involved in regulation of translation, which is essential in mitosis; consistent with this, all - when dysfunctional - yield abnormal mitoses or abrogation of mitosis.Some factors in the list have general roles in mitosis. Some of them, with signalling and chaperone activity, were known to bind importin beta-1. The 14–3–3 epsilon protein YWHAE regulates M entry by associating with and regulating the localization of cdc25B48. Prdx1 is inactivated by Cdk1-dependent phosphorylation in early mitosis, thus unshielding mitotic centrosomes from high H2O2 concentrations, and facilitating the inactivation of centrosomal phosphatases. Dephosphorylation and reactivation of Prdx1 in late mitosis then shields back the centrosome from H2O2, allowing the local reactivation of phosphatases. The abundance of importin beta-1 at spindle poles12, and its ability to interact with Prdx1 detected here, might provide an additional mechanism to regulate the timing of Prdx1 activity/inactivation at centrosomes. Interestingly, the importin beta-1 mitotic interactome also contains catalase  regulates H2O2 at the centrosome level and is crucial for centrosome function51. Interestingly, clathrin targets the MT-regulatory factor TACC-3 to the spindle MTs52 in an importin-beta-1-sensitive manner: importin beta-1 inhibits clathrin/TACC3 binding, while RANGTP restores it. Despite of this regulatory connection, a direct physical interaction between clathrin and importin beta-1 had not been shown before, suggesting the possibility that importin beta-1 regulates clathrin availability for TACC-3 by binding to it.Many identified interactors localize to the spindle, the main site of importin beta-1 localization in mitosis. Clathrin, a component of the endocytic pathway with RNA-binding capacity, and a recognized example of a moonlighting protein, localizes to MTs and regulates spindle functions, in part by localizing other proteins to the spindle54. Interestingly, HNRPU/SAF-A co-immunoprecipitates with Aurora-A and TPX2, which are required for HNRPU/SAF-A association with the spindle; in turn, HNRPU/SAF-A contributes to targeting of Aurora-A to the spindle MTs55. Importin beta-1 interacts with and negatively regulates TPX2 activity; the identification of HNRPU/SAF-A in the interactome suggests a possible interplay between importin beta-1, HNRPU/SAF-A, TPX2, and Aurora-A in steps of spindle assembly. Among newly identified importin beta-1 interactors localizing to the spindle, the RNA processing and transport protein EWSR1 is required for mitotic entry and, in cells that do enter mitosis, for proper spindle organization57. The PRP19 pre-mRNA processing factor affects the mitotic spindle MT density and chromosome alignment58. These newly identified interactors might hint at additional mechanisms through which importin beta-1 can regulate the mitotic spindle.In addition, both the ribosomal protein RPS3 and the ribonucleoprotein HNRPU/SAF-A localize at spindles in mitosis and their inactivation yields mitotic delay, spindle defects and chromosome misalignment59. Noteworthily, nucleolin associates with Hsp90, which contributes to stabilize it60, and co-immunoprecipitates with HNRPU/SAF-A55: the importin beta-1 interactome contains all these components. Several newly identified importin beta-1 interactors perform similar function. The spliceosome component ERH (Drosophila Enhancer of Rudimentary Homolog), which also interacts with the interactome member RPS3, regulates the levels of the CENP-E kinetochore-associated motor protein61, and hence KT interactions with MTs. The THO complex subunit 4 (ALYREF), involved in export of spliced mRNA, is also required for proper chromosome alignment62. The RNA helicases (DDX5 and DDX17) regulate the stability of Aurora-B mRNA, and hence the overall Aurora-B protein abundance63. The identification of diverse interactors that play roles in chromosome congression and MT/KT interactions thus expands the range of mechanisms that importin beta-1 might regulate.We also identified regulators of MT/KT interactions in the importin beta-1 interactome, the depletion of which generally results in chromosome misalignment and missegregation. A known example is nucleolin, the major nucleolar component, which interacts with the chromosomal periphery in mitosis, including around outer KTs, where it contributes to regulate MT interactions64. In addition, IQGAP recruits several proteins to the cleavage furrow in cytokinesis65;  importantly, it functions in recruitment of NUP98 and other NUPs (MAB414 antibody substrates) during NE reassembly at mitotic exit66, a process in which importin beta-1 plays crucial roles6. Together, the data identify a variety of factors and pathways potentially regulated by importin beta-1 at various steps during mitosis.Finally, the list of importin beta-1 interactors includes factors acting at mitotic exit. Here we identify KHDRBS1/SAM68, known to play roles in mitotic exitin situ proximity ligation assays (is-PLA) protocol to validate true importin beta-1 interactors in mitotic cells and depict where and when the interactions occur. Is-PLA uses ordinary primary antibodies to the proteins of interest, that are revealed by secondary antibodies conjugated to DNA oligonucleotides of complementary sequence: when the target proteins interact, or are in close proximity, the oligonucleotides can be ligated and amplified, and the amplification products are visualized at the site(s) and temporal window in which the interactions occur. We reasoned that the is-PLA method, if developed to be automated, could advance the validation of the interactomic results and add temporal and spatial information on the identified interactions.Experimental validation of protein partners after interactomic screening often relies on Western blotting assays of co-immunoprecipitating complexes, or other cell extract-based methods that fail to provide spatial or temporal information on dynamic interactions. We decided to develop an Figure 22. Results are shown in Fig. Among importin beta-1 interactors to test in PLA assays, we selected RAN, RANBP1 and RANBP2 as established importin beta-1 interactors in all cell cycle stages, and NUSAP and BUB3 as mitotic factors. Two approaches were used to control the specificity of the PLA automated mode. First, as a negative control, native HeLa cells, not expressing GFP, were incubated with importin beta-1 and GFP-specific antibodies, followed by oligonucleotide-conjugated secondary antibodies: no PLA signal was detected, indicating that no aspecific ligation or amplification occurred Fig. . Second,The is-PLA protocol also visualized importin-beta-1 interaction products with the spindle-associated protein NUSAP and the spindle checkpoint component BUB3 Fig. : positiv30. Depicting the spatial map of importin beta-1 interactions with its partners can help to understand its modes of action in mitotic cells. To fully exploit the informative content of the PLA method, we carried out PLA assays in asynchronous HeLa cultures, where all stages of mitosis are present with physiological frequency, and examined the PLA interaction signals in higher magnification to characterize their localization and abundance with spatiotemporal resolution.During the unfolding of mitosis, importin beta-1 can use different mechanisms to repress its interactors. It inhibits certain MT-regulatory factors, for example TPX2, which it binds via an importin alpha molecule, by co-localizing with them and rendering them inactive in the interaction. In other cases, importin beta-1 displaces factors away from their site of action. For example, it prevents the unscheduled onset of KT functions by localizing certain regulatory factors  away from KTs67. RANGTP-specific conformational antibody68 also detected fractions at centrosomes69 and spindle MTs35. In PLA assays, RAN/importin beta-1 interactions were visualized around and within the spindle region ; its amount increased in cells treated with nocodazole (NOC), which prevented MT assembly and induced sustained activation of the spindle assembly checkpoint  in a study of transport pathways73. Elucidating the precise mechanism by which importin beta-1 acts on these factors, and their regulated pathways, will be important to uncover yet poorly investigated pathways of control of specific mitotic events by importin beta-1.We have searched several resources to obtain specific information on importin beta-1 mitotic interactors. Cross-comparison of our full interactome list  for 20 hours, then released in RO3306-free medium, monitored under an inverted microscope and collected at round-up by mechanical shake-off. For automated image acquisition, cells were grown on 4-chamber Culture Slides  (8 × 104 cells per 1.7 cm2 well), and either left to cycle asynchronously, or synchronized using RO3306 as above and fixed 60 minutes after RO3306 wash-out. Where indicated, cells were treated with 50 μM Importazole (Sigma) for the last 2–3 hours of culture before harvesting the cells.Human HeLa cervix carcinoma epithelial cells were cultured in DMEM supplemented with 10% fetal calf serum, 2% L-glutamine, and 2% penicillin/streptomycin in a humidified atmosphere at 37 °C in 5% COTo covalently bind antibodies to Protein G-conjugated sepharose beads , dimethylpimelimidate  was used as crosslinker. Importin beta-1 antibody Sigma I2534 , or mouse IgG (Santa Cruz Biotechnology sc-2025), were diluted in 1 ml of phosphate buffered saline (PBS), mixed with 15 μl of bead pellet, and incubated overnight at 4 °C on a rotating wheel. The mixture was then centrifuged , the pellet was washed with 10 volumes of 0.2 M sodium borate (pH 9.0) and suspended in 100 volumes of 0.2 M sodium borate (pH 9.0)/PBS, to which 20 mM DMP was added just before use. After rotation for 1 hour at room temperature (RT), the beads were washed in 0.2 M ethanolamine (pH 9.0). The remaining reactive amino groups were quenched by adding 0.2 M ethanolamine (pH 9.0) . The bead pellet was then centrifuged , washed with 0.1 M glycine (pH 2.5) to remove non-crosslinked antibodies, washed further 3 × in 10 volumes PBS, and stored in PBS at 4 °C.The importin beta-1 interactome was captured from mitotic HeLa cells using anti-Importin beta-1 antibody (Sigma-Aldrich I2534). Mouse IgG antibodies (Santa Cruz Biotechnology sc-2025) were used as a control for non-specific binding proteins. RO336-released mitotic cells were centrifuged at low speed, washed twice at 4 °C in cold PBS/0.5 mM PMSF and resuspended in lysis buffer (50 mM Tris HCl (pH 8.0), 150 mM NaCl, 1% NP40) containing protease and phosphatase inhibitors . After incubation on ice for 30 minutes, cells were gently homogenized using a Dounce homogenizer and the extent of mechanical disruption of the cells was checked by microscopic observation. After centrifugation , the supernatant was quantified by Bradford assay and 1 mg of lysate was immediately precleared with Protein G-Sepharose at 4 °C for 1 hour . Importin beta-1 interacting partners were co-immunoprecipitated overnight on a rotating wheel at 4 °C from the precleared mitotic supernatant with anti-importin-beta-1 antibody covalently coupled to Protein G-Sepharose . For control, the same amount of precleared mitotic extract was incubated with mouse non-specific IgG covalently coupled with Protein G-Sepharose. The mixture was then centrifuged at 3000 rpm, 4 °C for 15 minutes and the pellet was washed 5 × in lysis buffer and again centrifuged as above. After the last centrifugation step, protein complexes were eluted from the beads by a gentle 3 × washing cycle in 15 μl of 0.1 M glycine buffer (pH 2.5) at RT. After centrifugation , the pH of the eluted proteins was neutralized by adding 1.5 M Tris HCl pH 8.0.76, gel slices were processed as described77 and tryptic peptide mixtures extracted and desalted according to ref.78. LC-MS/MS analyses were performed by reverse phase chromatography , on an Ultimate3000 system . Peptides eluted by a two-step gradient of ACN containing 0.1% FA  at 300 nl/min flow rate, were directly injected into an LTQ-Orbitrap XL mass spectrometer (Thermo-Fisher Scientific). Data-dependent tandem MS were performed with full precursor ion scans (MS1) collected at 30,000 resolution, with an automatic gain control (AGC) of 1 × 106 ions, and maximal injection time of 1000 ms. The five most intense (>200 counts) ions with charge states ≥ +2 were selected for collision-induced dissociation (CID). Dynamic exclusion was active with 90 ms exclusion for ions selected twice within a 30 ms window. For MS/MS scanning, the minimum MS signal was set to 500, activation time to 30 ms, target value to 10,000 ions, and injection time of 100 ms. All MS/MS spectra were collected using a normalized collision energy of 35% and an isolation window of 2 Th. Spectra were searched against the Homo sapiens UniProtKB database  and common contaminant proteins using the software package MaxQuant . We set oxidation (methionine) and acetylation (protein N-terminus) as variable modifications, carbamidomethylation (cysteine) as fixed modification, mass tolerance of 20 ppm for the precursor ion (MS) and of 0.5 Da for the fragment ions (MSMS). High-confidence peptide-spectral matches were filtered at <1% false discovery rate. Due to the high sensitivity and resolving power of the mass spectrometric platform used here, together with the high power of the Andromeda search engine in assigning peptide sequences from tandem MS data, the MaxQuant platform yielded a large set of starting data. As a first step of our established proteomic pipeline in data analysis of the original outputs, we discarded identifications for all annotated potential laboratory contaminants , and for hits that the search engine identified in a reverse decoy database used as a false negative control. To extract meaningful information from this cleaned data, a higher confidence matrix was set up by filtering out protein groups recognized with a low confidence level. In particular, we removed proteins which the Andromeda algorithm identified based on an extremely limited number of assigned peptide sequences (i.e.: i) number of unique peptides ≤0, ii) number of peptides ≤1, and iii) only by a peptide carrying a post-translational modification, or iv) through less than 3 MS/MS spectra), or v) presenting an ambiguous association, e.g. associated with multiple genes; see Supplementary Table 79) . The resultant list of the 272 high confidence hits was analyzed by data mining against the interphase list24, the Mitocheck database version 2.1 , and the latest releases of UNiProt http://www.uniprot.org/, Biogrid https://thebiogrid.org/, MiCroKITS http://microkit.biocuckoo.org/ and Pubmed https://www.ncbi.nlm.nih.gov/pubmed.Aliquots of co-immunoprecipitates were diluted in Laemmli buffer and fractionated through SDS-PAGE on 4–20% Mini-PROTEAN TGX™ gel (Bio-Rad). After colloidal Coomassie stainingCells grown on coverlisps were fixed in 3.7% paraformaldehyde/30 mM sucrose (10 min), permeabilized in 0.1% Triton X-100, then blocked in 3% bovine serum albumin in PBS/0.05% Tween-20 . Antibodies used for image segmentation were: chicken anti-alpha-tubulin , mouse anti-phospho-Ser10-histoneH3 . Secondary antibodies were: anti-chicken FITC , anti-mouse FITC  and anti-rabbit Cy3 . DNA was stained with 0.1 μg/ml 4,6-diamidino-2-phenylindole . Slides were mounted in Vectashield (Vector Laboratories).®In Situ Detection Reagents Red DUO92008 (Sigma-Aldrich). Cells were fixed, blocked, and incubated with primary antibodies as for conventional IF; we used combination of mouse and rabbit primary antibodies for each protein pair , DUO92002, and Duolink®In Situ PLA® Probe Anti-Mouse MINUS, Affinity purified Donkey anti-Mouse IgG (H + L) DUO92004, respectively, Sigma-Aldrich) were then added  and incubated in a pre-heated humidity chamber . Subsequent ligation  and amplification  steps were performed following the Olink Bioscience protocol. To localize PLA signals, cells were co-stained using DAPI and chicken anti-alpha tubulin antibody, followed by FITC-conjugated anti-chicken IgG.Cells grown on 4-chamber Culture Slides  were processed for PLA in four steps: i) incubation of fixed cell slides with primary specific antibodies for the proteins under examination; ii) incubation with secondary antibodies conjugated with complementary oligonucleotide tails ; iii) ligase addition; when the target proteins interact or are very close, the ligation step will produce a DNA circle; iv) rolling circle amplification. All steps were performed using the DuolinkSegmentation is used to partition a digital image to identify objects or extract relevant information. After a background subtraction step, image segmentation on Maximum Intensity Projections was performed using Nis-Elements HC 4.2 (Nikon). We used the Threshold function of the Nis-Elements HC 4.2 software to define regions in the images where we intended to perform the analysis , based on signal intensity, size and circularity. Segmentation masks for chromosomes and spindles were saved and automatically reloaded for each new image set. All steps were performed on a single.nd2 file for the whole experiment acquisition. To evaluate the effectiveness of segmentation, manual analysis of the same images was performed in parallel. For the automated analysis of PLA signals, the “Automated measurements results” function of the software was used: Sum Intensity values for PLA signals within the selected masks were obtained and exported to Excel. Data were statistically analyzed using Graph Pad Prism 6.ad-hoc macro was then applied for the automated generation of 2D projection (Maximum Intensity Projection) of all acquired images within a single.nd2 file at the end of the acquisition process.Images from cell samples processed for PLA in 4-chamber culture slides were acquired under a Nikon Eclipse Ti microscope equipped with the Perfect Focus System, a Nikon DS-Qi1 Cooled Digital Monochrome Camera and a 40 × objective , using the Nis-Elements AR 3.2 software. A grid (15 fields per well) was generated, saved and re-loaded for all acquisitions, with a focus adjustment step performed for each new slide. In order to collect the whole spindle-associated signal from rounded-up mitoses, images were acquired over an 8 μm range along the z-dimension (z-step: 0.8 μm). An Fixed samples on microscope slides were analyzed under a Nikon Eclipse 90i microscope equipped with a Qicam Fast CCD camera (Qimaging). Single-cell images were taken using an immersion oil 100 × objective (NA 1.3). Images (.nd2 format) were acquired using NIS-Elements AR 3.2 software (Nikon) along the z-axis ; out of focus planes were uniformly removed before processing. After a background subtraction step, images were deconvolved using the ‘AutoQuant’ deconvolution module of NIS-Element HC . Image projections were created using the Extended Depth of Focus (EDF) function of NIS-Element HC 5.02. Focused images were uniformly processed using the LUT (Look Up Table) tool of NIS-Element HC 5.02, and exported in TIFF format for Figure composition using Photoshop CS6.The data that support the findings of this study are available from the authors upon reasonable request.Supplementary material


The split G-quadruplex DNAzyme has emerged as a valuable tool for visual DNA detection. Here, we successfully integrated colorimetric split G-quadruplex DNAzyme assay with nucleic acid sequence-based amplification to generate a novel detection approach, allowing visual and rapid detection for the RNA of Shimen and HCLV strains of Classic Swine Fever Virus (CSFV). CSFV is a RNA virus that causes a highly contagious disease in domestic pigs and wild boar. With this method, we were able to detect as little as 10 copies/ml of CSF viral RNA within 3 h in serum samples taken from the field. No interference was encountered in the amplification and detection of Classic Swine Fever Virus in the presence of non-target RNA or DNA. Moreover, Shimen and HCLV strains of Classic Swine Fever Virus could be easily differentiated using the NASBA-DNAzyme system. These findings indicate the NASBA-DNAzyme system is a rapid and practical technique for detecting and discriminating CSFV strains and may be applied to the detection of other RNA viruses. Sensitive method for nucleic acid detection is very crucial for clinical diagnosis and virus identification. However, many of these strategies are either time-consuming or require sophisticated instruments that may not be available in laboratories with fewer resources. More sensitive, simple and rapid strategy for nucleic acid detection will always be in demand for the laboratory and point-of-care diagnostic applications. Recently, visual nucleic acid detection has attracted a lot of attention because of its satisfactory sensitivity, simplicity and portability1232−) by H2O2 to produce a colorimetric signal that can be visualized by the naked eye5678411Mycobacterium tuberculosisStaphylococcus aureusThe G-quadruplex/hemin complex, acting as an activated horseradish peroxidase (HRP)-mimicking DNAzyme, can catalyze the oxidation of 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTSClassic swine fever virus (CSFV) is the etiologic agent of classic swine fever (CSF), which causes a highly contagious disease in domestic pigs and wild boar. Outbreaks of CSF usually lead to significant economic losses in many countries worldwide19Due to the big loss CSF may cause, many CSFV detection methods has been set up in pig herds. The two most-used assays are the virus neutralization test and ELISA. Although the virus neutralization test has been regarded as the “gold standard”, it is work-intensive and time-consuming, and thus not suitable for detection of large numbers of samplesIn this study, we utilized the colorimetric split G-quadruplex DNAzyme system combined with NASBA technique to generate a novel CSFV detection approach. This approach adopted a split probe targeting system, designed with G-rich sequences, which reassembles in the presence of target RNA amplified by NASBA, and producing G-quadruplexes with a catalytic activity. With this approach, we could detect Shimen strain (the most widespread and highly virulent wide type CSFV strain in China) in serum samples and distinguish them from the HCLV strain (the most used vaccine strain in China) within 3 hours through a simple colorimetric change.2O2 -mediated oxidization of ABTS2−, which results in the formation of the oxidation product ABTS and a discernible green color. In our study, viral RNA (CSFV RNA) was first extracted from the cell or serum samples. Then single-stranded target RNA was amplified by NASBA  in Shimen strain to “5′ UGCGACU 3′” (3431–3437nt) in HCLV. Therefore, the 39 nt sequence of the CSF viral genome containing this sequence difference was used as the “detection sequences” to design our detection probes. We designed two groups of probes to detect the RNA of Shimen strain or the RNA of HCLV, each group contains three probes . The 5′ end of probe A has the same sequence as the 5′ end of the “detection sequences” both in Shimen´s and HCLV´s genome, and could hybridize with the 3′ end of the minus strand of the detection target DNA/RNA. The 3′ end of probe B-S or B-H could hybridize with 5′ end of the minus strand of detection target DNA/RNA of Shimen strain or HCLV. The 3′ end of probe A and the 5′ end of probe B-S or B-H could form a G-quadruplex structure when probe A and B were pulled together through hybridize with the adjacent sequence in target viral DNA/RNA. Probe C-H and C-S were designed as the competitive probes which are complementary with the minus strand of the detection target DNA/RNA of HCLV or Shimen, these two probes would compete with probes B-S and B-H in binding to the target DNA/RNA and thus may reduce false positive results  into minus- and single-stranded RNA copies. Then probes, substrates, and reagents were add to the purified RNA templates and incubated for 30 sec before a subtle color change could be observed. When using group 1 probes for Shimen RNA detection, a discernible green color change was observed with the sample of Shimen-infected PK15 cells, while the samples of the mock-infected PK15 cells and HCLV-infected PK15 cells were almost colorless . Besides10 copies/ml of Shimen-RNA and 2.5 × 1010 copies/ml of HCLV-RNA. As shown in 8 dilutions for Shimen strain 411The ability of the method to detect and differentiate CSF viral RNA in serum samples suggested that it is applicable for field investigation of CSFV infection. The whole detection procedure could be completed within 3 hour without the use of complex instruments. Only a thermostatic water bath is needed. The results are visual, with the positive results indicated by a green color that is clearly observable by the naked eye. These advantages make this method an ideal option for field assays. Further works are still needed to simplify the purification procedure after NASBA and to apply the assay for other RNA virus detections.CGUGACA 3′” in wild-type strains to “5′ UGCGACU 3′” in HCLV . Therefore, we designed the detection probes according to a consensus sequence located in the conserved region of E2 gene, where a distinctive difference was found between 9 wild-type strains in China and HCLV, converting from “5′  in HCLV . The NASin China . This inIn conclusion, this work has strongly confirmed the effectiveness of the NASBA-DNAzyme method in the detection and differentiation of CSFV RNA in cell and serum samples. This approach offers several advantages over other molecular techniques, including high sensitivity and specificity, rapid visual readout, ease-of-use, and inexpensiveness. In future, we will try to simplify the detection steps further and accommodate this method to the detection of other RNA viruses.2O2 (Sinopharm), Tris-HCl (Sinopharm), KCl (Sinopharm), NaCl (Sinopharm), T7 RNA Polymerase (Promega), AMV Reverse transcriptase (Promega), RNase H (Thermo Scientific), BAS (TaKaRa), M-MLV (Invitrogen). For the hemin solution, hemin was dissolved in DMSO to obtain a final concentration of 10 mM. For the ABTS solution, ABTS was dissolved in DMSO to obtain a final concentration of 250 mM.Hemin (Acrose), HEPES (Amresco), dimethyl sulfoxide , ABTS , HThe absorption spectrum of ABTS was measured at the wavelength of 414 nm using a SpectraMax i3 multiscan Spectrum . A Canon digital camera was used to take all the photographs in the experiment. A water bath  was used for the NASBA procedure.The cells infected with 2 CSFV strains (Shimen and HCLV), 1 BVDV strain (NADL), 1 PRRSV strain (Hubei), 1 PRV strain (HB), 1 PPV strain (HN99), 1 PCVII strain (HBZX) were provided by China Institute of Veterinary Drug Control.RNAsimple Total RNA kit  was used to extract total RNA from cell and serum samples, according to the manufacturer’s protocol. Ten-fold serial dilutions of RNA from virus isolates were prepared.The NASBA procedure used in this study has been previously described by KievitsProbes were prepared at 4 °C as 20 μM stock solutions and then stored for several months at −20 °C. The probe mixtures for group detection were prepared as 5 μM stock solutions at 4 °C and stored for several months at −20 °C. The sequences of probes and primers are listed in 4OH, 20 mM KCl, 200 mM NaCl, 400 nM probe A, 400 nM probe B, and 400 nM probe C for CSFV RNA detection. The final volume of mixtures were 97 μL adjusted by ultrapure water. The NASBA products were denatured at 65 °C for 3 min and then hybridized with probes at 40 °C for 3 min. Then 1.6 μL of 100 μM hemin was added to the system to ensure that the final concentration was 1.6 μM and kept at room temperature for 5 min. Then 2 μL of 50 mM ABTS and 0.15 μL of 1 M H2O2 were added to the system and incubated at room temperature for 2 min before the readout of the absorbance of the oxygenation product ABTS at 414 nm in a 100 μL reaction in a 96-well plate (Corning). Photograghs were immediately taken after colorimetric reaction. All reaction had been performed in triplicate to ensure reproducibility.NASBA products were added directly into the probe mixture containing 25 mM HEPES-NHThe CSF viral RNAs from virus isolates (cell lysate) were also detected by RT-PCR (Reverse Transcription PCR) method. The reaction premix consisted of 5 μL of RNA free water, 5 μL of 10 μM forward primer, 5 μL of RNA template. The premix was incubated at 65 °C for 5 min and then on ice for 2 min. Then 2 μL of 10 mM dNTP, 100 U of M-MLV, 5 μL of M-MLV 5 × buffer and 5 μL of RNA free water were added to the premix, followed by incubation at 42 °C for 60 min and 75 °C for 10 min. 2 μL of the RT products were used as template for later PCR procedure. The PCR conditions were initial incubation at 94 °C for 2 min, followed by 30 cycles of denaturation at 94 °C for 30 sec, primer annealing at 60 °C for 30 s and extension at 72 °C for 30 sec. An addition step was conducted at 72 °C for 5 min.How to cite this article: Lu, X. et al. Visual detection and differentiation of Classic Swine Fever Virus strains using nucleic acid sequence-based amplification (NASBA) and G-quadruplex DNAzyme assay. Sci. Rep.7, 44211; doi: 10.1038/srep44211 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Penicillium italicum, but the possible action mechanism remains undetermined. In present study, α-terpineol was evaluated for antibacterial activity against Penicillium digitatum along with the mode of their antibacterial action.Plant essential oils could act effectively against postharvest diseases, α-terpineol, a typical terpenoid of plant essential oils, exhibited strong antifungal activity in against P. digitatum was strongly inhibited by α-terpineol, with the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of 2.00 and 8.00 µl/ml, respectively. Scanning electron microscopy observation revealed that α-terpineol obviously altered the morphology of P. digitatum hyphae by causing the loss of cytoplasm and distortion of mycelia. A rapid increase in the membrane permeability of P. digitatum was observed after treated with MIC or MFC of α-terpineol, evidenced by the release of cell constituents, the extracellular conductivity, and the extracellular pH. In addition, α-terpineol apparently induced a decrease in total lipid contents of P. digitatum cells, indicating the destruction of cell membrane structures after treatment.The results showed that mycelial growth of P. digitatum.Based on our study, α-terpineol might affect the cell wall synthesis and lead to the disruption of cell wall. The cell wall disruption affected fungal morphogenesis, the integrity of membrane and leakage of intracellular components, these results suggested that α-terpineol treatment inhibited the growth of  Penicillium digitatum is the most devastating pathogen and causing about 90 % of production losses during postharvest handling of the fruit  at 25 ± 2 °C. The spores’ concentration was adjusted to 5 × 105 cfu/ml using a hamacytometer.The fungal pathogen α-Terpineol (90 %) was obtained from Sigma-Aldrich . Cholesterol (95 %) and phosphovanillin (98 %) were purchased from TCI Shanghai .P. digitatum were evaluated by the poisoned food technique  were used for all SEM observations  were initially collected by centrifugation at 4000g for 20 min, washed three times with sterilized double distilled water, and resuspended in 100 ml phosphate buffered saline (pH 7.0). After that, the suspensions were treated with α-terpineol at various concentrations  for 0, 30, 60 and 120 min. Then, 1 ml of supernatant was used to measure the absorbance at 260 nm with a UV-2450 UV/Vis spectrophotometer .The release of cell constituents was measured according to the method described previously  and a Delta-320 pH-meter  following the instructions, respectively. The P. digitatum cells treated by MIC or MFC of α-terpineol were selected at 0, 30, 60 and 120 min of treatment. Control flasks without α-terpineol were also tested.The measurement of extracellular conductivity and extracellular pH of P. digitatum cells with α-terpineol at various concentrations  was determined using phosphovanillin method  in a clean dry test tube with vigorous shaking for 30 min. The tubes were centrifuged at 4000g for 10 min. The lower phase containing lipids was thoroughly mixed with 0.2 ml saline solution and centrifuged at 4000g for 10 min. Then, an aliquot of 0.2 ml chloroform and lipid mixture was transferred to a novel tube and 0.5 ml H2SO4 was added, heated for 10 min in a boiling water bath. After that, 3 ml phosphovanillin was added and shake vigorously, and then incubated at room temperature for 10 min. The absorbance at 520 nm was utilized to calculate total lipid content from the standard calibration curve using cholesterol as a standard.Total lipid content of P < 0.05 and P < 0.01.All experiments were performed as three biological replicates and statistical analysis (ANOVA) was performed using Statistical Package for the Social Science (SPSS) statistical software package release 16.0 . Statistical significance was set at P. digitatum was affected by α-terpineol in a dose-dependent manner (P < 0.01). High α-terpineol concentration (≥2.00 μl/ml) completely inhibited the mycelial growth of P. digitatum, whereas α-terpineol at a concentration lower than 1.00 μl/ml only showed moderate antifungal activity against P. digitatum after 2 days of incubation. After 4 days of incubation, the mycelial growth of P. digitatum was still totally inhibited by the application of 8.00 μl/ml α-terpineol. Thus, the MIC and MFC of α-terpineol for P. digitatum were measured to be 2.00 and 8.00 μl/ml, respectively.As shown in Table P. digitatum, and the inhibitory effect was positively correlated with the α-terpineol concentration. The MIC of only showed partially antifungal activity against P. digitatum after 4 days of incubation, but the MFC of α-terpineol totally inhibited the growth of P. digitatum  when P. digitatum was treated with early exposure of MIC or MFC  than that of the control (0.02) but significantly lower (P < 0.05) than that with MFC of α-terpineol (0.52). The OD260 value of P. digitatum suspensions treated with MIC of α-terpineol maintained a smooth ascending trend after 30 min of exposure, whereas that with MFC continuously increased after 30 min of exposure, and reached to the highest absorbence of 0.70 at 120 min of exposure.The release of cell constituents significantly increased  than that in control (70.8 μs/cm).Although α-terpineol treatment did not affected the extracellular conductivity at initial exposure time, α-terpineol caused a significant increase after 30 min incubation Fig. b. MoreovP. digitatum suspensions treated by α-terpineol is presented in Fig. P. digitatum suspensions with MIC or MFC of α-terpineol rapidly increased in the first 30 min of exposure, and followed by a moderate ascending trend in the subsequent stage. The extracellular pH values in P. digitatum suspensions after incubation with MFC and MIC of α-terpineol for 120 min were 5.83 and 4.90, respectively, which were significantly higher than that of the control (4.56) (P < 0.05).The extracellular pH in the control maintained a stable level during the entire incubation. The extracellular pH in P. digitatum was poorly understood. It is generally accepted that the lipophilicity of terpenoids enabled them to preferentially partition from an aqueous phase into membrane structures of the fungi, resulted in membrane expansion, increased membrane fluidity and permeability, and induced the leakage of ions and other cellular contents  than that of the control (323.31 mg/g dry weight).The total lipid of ent Fig. . After 1P. digitatum (Fig. P. digitatum. Thus, this result confirmed that α-terpineol could damage the cell membrane integrity of P. digitatum, affected the cell membrane structure after the disruption of cell wall and inhibited the growth of P. digitatum.Lipids were the main components of biological membranes (Watkins et al. tum Fig. , and we tum Fig.  and decrP. digitatum, leading to the disruption of cell wall, and subsequently resulting in irreversible deleterious morphological alterations, disturbance of membrane structure, increase in membrane fluidity, leakage of ions and other cell contents. The present data indicated that α-terpineol could become a possible alternative to synthetic fungicides in the fight against P. digitatum.Based on the present study, it could be concluded that α-terpineol possess fungitoxic activities inhibiting the growth of


Artificially synthesized RNA molecules provide important ways for creating a variety of novel functional molecules. State-of-the-art RNA inverse folding algorithms can design simple and short RNA sequences of specific GC content, that fold into the target RNA structure. However, their performance is not satisfactory in complicated cases.We present a new inverse folding algorithm called MCTS-RNA, which uses Monte Carlo tree search (MCTS), a technique that has shown exceptional performance in Computer Go recently, to represent and discover the essential part of the sequence space. To obtain high accuracy, initial sequences generated by MCTS are further improved by a series of local updates. Our algorithm has an ability to control the GC content precisely and can deal with pseudoknot structures. Using common benchmark datasets for evaluation, MCTS-RNA showed a lot of promise as a standard method of RNA inverse folding.https://github.com/tsudalab/MCTS-RNA.MCTS-RNA is available at The online version of this article (doi:10.1186/s12859-017-1882-7) contains supplementary material, which is available to authorized users. RNA inverse folding .The target structure Fig.  determinsingle sites. They are not constrained and can be assigned with any base . The event that a pair site  is assigned with a base pair XY is described as {XYi,j}. For a single site, it is described as {Xi}. Random assignment of a site is defined as follows. If it is a paired site, a base pair is chosen from  with equal probabilities. If it is a single site, a base is chosen from  with equal probabilities.The remaining free positions are called ℓ, the maximum depth of the tree is ℓ. A path from the root to a leaf represents a partially determined sequence. In the first round of MCTS-RNA, only the root node exists in the search tree. From ℓ sites, a site is chosen randomly. If it corresponds to a single site, four child nodes containing bases  are created under the root node. Otherwise, six nodes with base pairs  are created. Each node i contains three variables: the visit count vi represents the number of visits in the search process, zi denotes the immediate merit of node i evaluated by sequence generation, and the cumulative value wi is defined as the sum of zj for all descendant nodes including itself. The UCB score  randomly. (ii) If two non-essential positions are paired in the predicted structure and not paired in the target structure, do the following: If they are AU or UA, replace them with AA or UU randomly.If they are GC or CG, replace them with CC or GG randomly.If they are GU or UG, replace them with one of  randomly.In the first step of the local update, we obtain the predicted structure of the current sequence, then apply rewriting rules as many times as possible. There are two rewriting rules: (i) If two non-essential positions are paired in the target structure, but not in the predicted structure, replace them with one of [AU3,11} and {AU4,10} are updated to {GC3,11} and {AU4,10}, respectively. {GC2,13} is updated to {CC2,13} due to the second rule.The first rule is expected to form a base pair, while the second one breaks the pair. The three options in the second rule are designed to avoid changing the number of GCs in the sequence. Figure RGC was fixed to 1 and δ was set to 0.01 for nested structures and 0.02 for pseudoknot structures. As shown later, this setting resulted in relatively strict control of the GC content in comparison with competing methods. If more efficiency is required, one can decrease RGC or increase δ to relax the control. The number of local updates k was set to 50. In all competing methods, we employed their default parameters unless otherwise stated. Experiments were done on a CentOS 6.7 PC with 2.6 GHz CPU and 256 GB memory.Following , we usedsuccess, if it could generate, within 10 min, at least one compliant sequence whose secondary structure matches perfectly with the target structure. If there is at least one success for a target structure, the structure is regarded as solved.Given a target structure, the performance of an inverse folding method is measured as follows. For a nested structure, an inverse folding method is applied 50 times to the same structure with different random seeds. For a pseudoknot structure, the number of applications is reduced to 10 times due to heavy computational cost. Each run is considered as a β and trade-off parameter C, we applied MCTS-RNA to five small datasets with different values of β∈{1,2,3} and C∈{0.01,0.05,0.1,0.3,0.4,0.5,0.6,0.7,0.9,1.0}. Each dataset consists of four nested Rfam structures and four Pseudobase++ structures, which were randomly selected. For each dataset, MCTS-RNA was performed ten times per each structure with seven different GC content values. This resulted in total 560 MCTS-RNA runs for each of five datasets. The average number of successes over the five datasets was used to measure the performance of each parameter setting. As shown in Fig. C=0.5 and β=1 turned out to be the best setting. These values will be used in all remaining experiments.To identify the best values of expansion threshold In this experiment, MCTS-RNA is compared with existing tools with GC content control: AntaRNA and MODENA. RNAifold and IncaRNAtion are omitted, as Kleinkauf et al.  showed tTable To compare MCTS-RNA with ERD, we also performed experiments without GC content control. Table We applied MCTS-RNA, antaRNA and MODENA to 249 pseudoknot structures. Figure α∈{0.2,0.5,1.0,2.0,4.0}, β∈{0.2,0.5,1.0,2.0,4.0},ρ∈{0.05,0.1,0.2}, As shown in Additional file α=0.2,β=0.2,ρ=0.05 turned out to be the best. Additional file In most literature about RNA inverse folding, software tools are evaluated with their default parameters , becausThe accuracy of MCTS-RNA may be positively biased for the structures used in parameter optimization. In Additional file MCTS-RNA consists of MCTS and local search. In this section, we investigate how much these two parts contribute to accurate inverse folding and how they interact. For easy problems, local search from random initial sequences may suffice, but the addition of MCTS would seem necessary in difficult cases. In the following experiments, we used the 29 nested structures.Figure To measure the effect of MCTS, we compared MCTS-RNA with a simpler method of applying the local search to randomly designed initial sequences Fig. . DetaileIn this research work, we introduced MCTS-RNA based on Monte Carlo Tree Search to solve RNA inverse folding problem. A characteristic of this approach is that the sequence space is represented as a tree of assignment events. MCTS-RNA outperformed existing tools based on evolutionary algorithms and provided an efficient way to search in the GC-content-specific sequence space. Evolutionary algorithms keep a population of intermediate solutions and update them simultaneously. The update is designed such that a certain level of diversity is maintained to avoid falling into local minima. MCTS offers a more specific way to perform trial-and-error by setting up a search tree and allowing backtracking when the current branch turns out to be non-promising according to the UCB score.playout algorithm that randomly creates the remaining moves according to the rules of the game [We believe that it is easy to deploy MCTS to other real-life optimization problems, thanks to its clear separation between the problem-dependent part of the algorithm and the general search. In MCTS-RNA, the local search is the problem-dependent part, while in computer Go, it corresponds to the the game . By contthe game . In fututhe game  and discthe game .


Highly pathogenic avian influenza viruses with H5 and H7 hemagglutinin (HA) subtypes evolve from low-pathogenic precursors through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been observed to occur naturally only in these HA subtypes, little is known about the genetic basis for the acquisition of the polybasic HA cleavage site. Here we show that consecutive adenine residues and a stem-loop structure, which are frequently found in the viral RNA region encoding amino acids around the cleavage site of low-pathogenic H5 and H7 viruses isolated from waterfowl reservoirs, are important for nucleotide insertions into this RNA region. A reporter assay to detect nontemplated nucleotide insertions and deep-sequencing analysis of viral RNAs revealed that an increased number of adenine residues and enlarged stem-loop structure in the RNA region accelerated the multiple adenine and/or guanine insertions required to create codons for basic amino acids. Interestingly, nucleotide insertions associated with the HA cleavage site motif were not observed principally in the viral RNA of other subtypes tested . Our findings suggest that the RNA editing-like activity is the key mechanism for nucleotide insertions, providing a clue as to why the acquisition of the polybasic HA cleavage site is restricted to the particular HA subtypes. Influenza A viruses are divided into subtypes based on the antigenicity of the viral surface glycoproteins hemagglutinin (HA) and neuraminidase. Of the 16 HA subtypes (H1 to -16) maintained in waterfowl reservoirs of influenza A viruses, H5 and H7 viruses often become highly pathogenic through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been known since the 1980s, the genetic basis for nucleotide insertions has remained unclear. This study shows the potential role of the viral RNA secondary structure for nucleotide insertions and demonstrates a key mechanism explaining why the acquisition of the polybasic HA cleavage site is restricted to particular HA subtypes in nature. Our findings will contribute to better understanding of the ecology of influenza A viruses and will also be useful for the development of genetically modified vaccines against H5 and H7 influenza A viruses with increased stability. Influenza A viruses (IAVs) are widely distributed in avian and mammalian species, including humans. IAVs have eight-segment negative-sense RNA genomes and are divided into subtypes on the basis of combination of two viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). IAVs with H1 to -16 HA and N1 to -9 NA subtypes have been isolated from wild aquatic birds, especially migratory ducks, which serve as a natural reservoir of IAVs in nature 13. Upon thttp://www.who.int/en/]). Some reassortant H5 viruses with different NA subtypes , originating from the same ancestral H5N1 virus, have recently emerged in China and spread to other countries in Eurasia and North America (9Highly pathogenic avian influenza (HPAI) viruses with the H5 and H7 HA subtypes produce high mortality in poultry and have caused devastating losses in poultry production worldwide. H5N1 HPAI viruses, first reported in Hong Kong in 1997, have been circulating in poultry for almost two decades . Since tAmerica 914.It is known that HPAI viruses evolve from low-pathogenic H5 and H7 viruses maintained in the natural reservoirs and that the key determinant for the different pathogenicities is the proteolytic cleavage of HA, which is required for infectivity of IAVs –19. Low-–20–24–27–Most outbreaks of HPAI are caused only by IAVs with the H5 and H7 subtypes. However, it was reported that an H6 virus with an artificially introduced polybasic HA cleavage site acquired high HA cleavability without trypsin and had a typical HPAI phenotype in experimentally infected chickens . An H9 vIn this study, we focused on the A/whistling swan/Shimane/499/83 (H5N3) (ShimH5) strain, which was originally isolated as a low-pathogenic strain and shown to become highly pathogenic after passaging through experimentally infected chickens . During We established a reporter assay to detect nucleotide insertions into the RNA sequence encoding amino acids across the HA cleavage site  of the strain ShimH5  to C. InTo evaluate this system, we first tested the plasmids containing the linkers , all of which had the sequence derived from the parent ShimH5 strain . QT6 celWe then hypothesized that a stretch of adenine residues in the RNAseqHAclv region might affect the frequency of nucleotide insertions, as has been suggested with the RNA editing mechanisms of some RNA viruses –39. Thus–We then compared the predicted secondary structures of RNAs corresponding to the Linker29, Linker29-24a, Linker29-24a2b, and Linker29-12A sequences  and 3E. 10.1128/mBio.02298-16.3FIG S1 FIG S1, PDF file, 0.2 MB.RNAfold-predicted RNA structures of the linker sequences. The predicted RNA (positive-sense) secondary structures of Linker29 (A), Linker29-24a (B), Linker29-24a2b (C), Linker29-12A (D), and Linker29-12A-NL (E) are shown. The structures are colored by base-pairing probabilities indicated with color bars. For unpaired regions, the color denotes the probability of being unpaired. Download Copyright © 2017 Nao et al.2017Nao et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02298-16.4FIG S2 FIG S2, PDF file, 0.5 MB.Predicted RNA structures of longer sequences, including the linker sequences. The RNA (positive-sense) secondary structures were generated by the Quickfold (A) and RNAfold (B) programs for Linker29, Linker29-24a, Linker29-24a2b, Linker29-12A, and Linker29-12A-NL. The structures are colored by base-pairing probabilities indicated with color bars (B). For unpaired regions, the color denotes the probability of being unpaired. Download Copyright © 2017 Nao et al.2017Nao et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To verify the presence of vRNA containing the expected nucleotide insertions, we analyzed the ShimH5 HA vRNA incorporated into purified virions using a deep-sequencing approach. Reassortant viruses between A/Puerto Rico/8/1934 (H1N1) (PR8) and ShimH5 strains (rgPR8/ShimH5-HA [PR8 whose HA gene was replaced with that of ShimH5] and rgPR8/ShimH5 24a2b-HA [PR8 whose HA gene was replaced with that of ShimH5 24a2b]) were generated from cloned plasmids to standardize the viral polymerase activity and minimize the background genetic heterogeneity in the original population of the ShimH5 strains. As expected, we found single-nucleotide insertions of adenine residues into the 3 consecutive adenines at nucleotide positions 1047 to 1049 in rgPR8/ShimH5-HA  at a com10.1128/mBio.02298-16.5FIG S3 FIG S3, PDF file, 0.2 MB.Comparison of RNAfold-predicted secondary structures of the RNAseqHAclv regions of ShimH1, HokH2, HKH3, HokH4, and ShimH5. The predicted RNA (positive-sense) secondary structures of the RNAseqHAcly region of ShimH1 (A), HokH2 (B), HKH3 (C), HokH4 (D), and ShimH5 (E) are shown. The structures are colored by base-pairing probabilities indicated with color bars. For unpaired regions, the color denotes the probability of being unpaired. Download Copyright © 2017 Nao et al.2017Nao et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Finally, we compared the predicted secondary structures of the RNAseqHAclv region among HA subtypes using database sequences of low-pathogenic IAVs isolated from ducks . Download Copyright © 2017 Nao et al.2017Nao et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02298-16.2DATA SET S2 DATA SET S2, XLSX file, 0.03 MB.Comparison of RNA secondary structures predicted by Quickfold and RNAfold. The secondary structures of the RNAseqHAclv region of low-pathogenic IAVs isolated from waterfowl  of low-pathogenic IAVs isolated from waterfowl (441 strains). All structures are shown as positive-sense RNA sequences. Download Copyright © 2017 Nao et al.2017Nao et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Escherichia coli RNA polymerase during RNA elongation at runs of 10 or more adenines or thymines results in the addition of nontemplated uracil or adenine residues, leading to the restoration of the normal reading frame from out-of-frame lacZ constructs  to create the polybasic HA cleavage sites of IAVs, which has only been discussed hypothetically , 43. TakAlthough we detected nucleotide insertions during the vRNA synthesis of the parent ShimH5 strain, the frequency did not seem to be high compared to the RNA editing observed in other viruses , 36, 39.2 incubator.IAV strain ShimH5 was kindly provided by T. Ito (Tottori University), and ShimH1, HokH2, HKH3, and HokH4 were kindly provided by H. Kida (Hokkaido University). These viruses were propagated in the allantoic cavities of 10-day-old embryonated chicken eggs at 35°C for 48 h and stored at −80°C until use. MDCK cells  were groRenilla luciferase transfection control reporter plasmid (Promega), and a mixture of PB2-, PB1-, PA-, and NP-expressing pCAGGS in quantities of 150, 150, 150, and 300 ng, respectively, using FuGENE HD (Promega) according to the manufacturer’s protocol. At 24 h posttransfection, luciferase (firefly and Renilla luciferase) activities of cell lysates were measured with a GloMax96 Microplate luminometer (Promega) using the dual-luciferase assay system (Promega) according to the manufacturer’s protocol. Firefly luciferase activities were standardized to the transfection control Renilla luciferase activities .We used the modified pHW72 plasmid , pHW72-LThe vRNAs of ShimH1, HokH2, HKH3, HokH4, and ShimH5 were extracted from infectious allantoic fluids using a QIAamp viral RNA minikit (Qiagen) and reverse transcribed with Moloney murine leukemia virus reverse transcriptase (Invitrogen) using the uni12 primer (5′-AGCAAAAGCAGG) . For theCultured MDCK cells maintained in Eagle’s minimal essential medium (MEM) containing bovine serum albumin (0.3%), penicillin (100 U/ml), and streptomycin (0.1 mg/ml) were infected with rgPR8/ShimH1-HA, rgPR8/HokH2-HA, rgPR8/HKH3-HA, rgPR8/HokH4-HA, rgPR8/ShimH5-HA, or rgPR8/ShimH5-24a2b-HA at a multiplicity of infection of 0.001 and incubated at 37°C for 48 h. Then the infectious supernatant was collected. Virus particles were concentrated and purified by high-speed centrifugation  of the supernatant through a 10 to 50% sucrose density gradient. vRNAs were extracted from purified virus particles using TRIzol LS reagent (Sigma) according to the manufacturer’s protocol.cDNA libraries were prepared from vRNA without any amplification procedures to minimize the potential errors during the sequencing reaction, and the high depth of coverage of sequencing  with a high-quality score (no less than 30) enabled us to analyze the vRNA quasispecies, including infrequent nucleotide insertions that might not be detectable by Sanger sequencing. Briefly, extracted vRNA (10 μg) was used for the synthesis of double-stranded cDNA of the partial HA gene containing the sequence encoding the HA cleavage site with a PrimeScript double-strand cDNA synthesis kit (TaKaRa) using the HA gene-specific primers H1-996F (5′-GGAGAATGCCCTAAATATGTTAAAAGC), H2-1000F (5′-GCCCCAAATATGTAAAATCGGAGAG), H3-1001F (5′-GCCCCAAGTATGTTAAGCAAAACAC), H4-985F (5′-GCCCCAAATATGTTAAACAGGGCTC), and H5-963F (5′-GTATGCCTTTCCACAATATTCATCC). The synthesized double-stranded cDNAs (approximately 750 bp) were tagged with sequencing adapters having indexes by using a TruSeq DNA PCR-free sample prep kit (Illumina). The cDNA libraries were verified on a high-sensitivity DNA chip on a Bioanalyzer (Agilent Technologies) and quantified with real-time PCR using an Illumina compatible kit and standards (KAPA) before loading on the sequencing chip. Then the indexed libraries were sequenced using a MiSeq v3 600-cycle kit (Illumina) to perform 300-bp paired-end sequencing on a MiSeq instrument (Illumina), according to the manufacturer’s instructions. After the sequencing run, reads with the same index sequences were grouped.The sequencing reads were aligned with the reference HA sequences  determined by Sanger sequencing using Bowtie 2  with defhttp://unafold.rna.albany.edu/?q=DINAMelt/Quickfold) (http://rna.tbi.univie.ac.at/) and the ViennaRNA package (The Quickfold program (ickfold)  and RNAf package  were use


A report on the Second Aegean International Conference on the Long and the Short of Non-Coding RNAs, held in Heraklion, Greece, 9–14 June 2017. Investigations into gene regulation and disease pathogenesis have been protein-centric for decades. However, in recent years there has been a profound expansion in our knowledge of the variety and complexity of eukaryotic RNA species, particularly the non-coding RNA families. Vast amounts of RNA sequencing data generated from various library preparation methods have revealed these non-coding RNA species to be unequivocally more abundant than canonical mRNA species. Furthermore, insight into the diverse mechanisms and functional roles of these RNA transcripts is emerging, pointing to their roles in maintaining cellular homeostasis and gene regulation and/or directing various pathologies.Drosophila, plants and mammals, even presenting the therapeutic potential of these molecules in various diseases. Plenary talks were further complemented by active poster sessions, where participants engaged with more than 85 poster presenters during conference breaks.The Second Aegean International Conference on the Long and the Short of Non-Coding RNAs focused on the key contemporary findings in this field. Organised by a scientific committee comprising Judy Lieberman , Zissimos Mourelatos  and Andrei Thomas-Tikhonenko , the conference was held at the Aldemar Knossos Royal Village Conference Center in Heraklion, Crete (Greece), 9–14 June 2017, and was attended by 125 delegates from numerous countries. There were no defined keynote talks, but 25 invited talks and 15 short contributed talks, covering a breadth of small and long non-coding RNA species from yeast, In this report, we briefly recount the conference content by providing condensed, headline-style summaries of the research described in the talks and some of the posters. Within the scope of this brief report we have highlighted three key research themes: non-coding RNAs in disease; circular RNAs and their formation and function; and RNA stability. However, we cannot possibly do justice to the quality and quantity of all work presented. We would like to stress that omissions from this report are not based on quality, but simply a personal judgement as to which material could be most coherently presented within limited space.Perhaps the most pivotal discovery regarding non-coding RNAs are that they are not simply transcriptional by-products, or splicing artefacts, but comprise a diverse population of actively synthesised and regulated RNA transcripts. These transcripts can—and do—function within the contexts of cellular homeostasis and human pathogenesis. Technologies based on next-generation sequencing have revealed many novel classes of non-coding RNA transcripts, but biochemical characterisation to ascribe the mechanisms behind their functions, especially in vivo, provides an essential roadmap to guide the research field. Two overriding questions remain to be answered: 1) how is long non-coding RNA (lncRNA) targeted to the genome, and 2) are lncRNAs functionally important?Khps1 activates expression of its cognate mRNA, SPHK1 (sphingosine kinase 1). This is achieved through forming a DNA–RNA triplex that recruits histone acetyltransferase p300/CBP and other associated effector proteins to the gene promoter. By developing a biochemical purification method for DNA–RNA triplexes as an alternative to ChiRP-seq (chromatin isolation by RNA purification), the Grummt group proposed a hotly debated model that these triplexes might be a general characteristic of target gene recognition by chromatin-interacting lncRNAs. While genome-wide binding sites for MEG3 lncRNA supported this new model, Jun-An Chen’s group  found the same lncRNA to be highly expressed in motor neurons, following systematic profiling studies on embryonic motor neuron-enriched lncRNAs. Loss of Meg3 directs motor neuron subtype identity by regulating the epigenetic landscape through PRC2, resulting in altered expression of homeotic genes and homeotic transformation in knockout mice. Martin Crespi  reported another homeotic transformation effect for a lncRNA in Arabidopsis, whereby broad modulation of alternative splicing resulted in altered root branching. Together, these reports endorse lncRNAs as having a function in homeotic transformation.Ingrid Grummt  elegantly demonstrated that an E2F1-regulated antisense lncRNA named Iannis Aifantis  performed a genome-wide lncRNA reverse-genetics screen with an enhanced Cas9 repressor protein to investigate regulation of key targets downstream of Myc. He identified a conserved lncRNA that coordinated chromosome conformation  in T-cell acute lymphoblastic leukaemia, with knockdown studies reducing tumours in vivo.These systematic analyses were also employed by other researchers at the meeting, including Andrea Ventura , whose talk “Cancer Modeling in the CRISPR Age” reaffirmed this approach as the gold-standard for genetic screens in cell lines and model organisms alike. Joshua Mendell  reported on a Cas9 genome-wide loss-of-function screen to identify new regulators of the microRNA (miRNA) pathway. This screen revealed a novel phosphorylation and dephosphorylation cycle for Argonaute 2 (AGO2), which is stimulated by regulated interactions between miRNA and targets to maintain the global efficiency of miRNA-mediated silencing. Collaborative distribution of the outcomes of these CRISPR screens might greatly accelerate research outputs, and was strongly encouraged by meeting delegates.SAMMSON. They demonstrated that the “addiction of melanoma” to this lncRNA is mediated through its interaction with distinct proteins, with contrasting functions, to regulate melanoma progression. This finding prompts an interesting hypothesis that lncRNA might function as a molecular switch to rewire RNA binding proteins in cancer.In concatenated talks, Jean-Christophe Marine and Eleonora Leucci  narrated an elegant study on a nearly ubiquitous, melanoma-specific oncogene (MITF)-related lncRNA, Multiple high-profile presenters, including David Bartel  and David Corey , thematically questioned whether ncRNA copy numbers were sufficiently abundant to co-regulate target molecules  in a physiological context. Corey further encouraged all attendees to assess absolute abundance of focal ncRNA and their target(s) and also their subcellular localisation—an approach that contributed greatly to his own study on factors involved in Argonaute-mediated nuclear RNAi. Quantifying the stoichiometric relationships between ncRNAs and their targets will likely be necessary in future research reports.As “new kids on the block” in the non-coding RNA family, the circular RNAs (circRNAs) are a large group of uniquely alternatively spliced RNA molecules, which are ubiquitous among eukaryotes and are proving functional in a plethora of contexts. Historically much maligned because of their relatively low abundance and high diversity, it may be surprising that five individuals’ presentations focused predominantly or exclusively on discrete aspects of circRNA biogenesis, recognition and regulation. Some presented on circRNA functions hitherto unheard-of for any ncRNA.Mariangela Morlando  showed that, while not widespread, certain circRNAs are not strictly non-coding RNAs at all. An example being circ-ZNF609, these circRNAs retain endogenous internal ribosome entry sites from the cognate mRNA, which enables their translation into protein. She also identified a new circRNA biogenesis factor, FUS, whose pathogenic mutations in amyotrophic lateral sclerosis and autism are linked to altered circRNA formation. Gregory Goodall  reported on the role of Quaking, an RNA binding protein that regulates circRNA biogenesis in epithelial–mesenchymal transition, a model of cancer metastasis. Nikolaus Rajewsky  described how CRISPR-Cas9-based genome editing could be used to remove the expression of one brain-specific circRNA, CDR1as. This is a ‘sponge’ for human microRNA-7 (hsamiR-7), which yields mice with a normal phenotype but mildly altered neurological responses.Arabidopsis and its cognate DNA locus via complementary base-pairing to produce an RNA:DNA hybrid, or R-loop, is sufficient to promote alternative splicing of its cognate mRNA, altering floral morphology. The capacity of circRNAs to bind DNA was supported by Ingrid Grummt, who reported that circRNAs, as well as other ncRNAs and linear RNAs, associate with DNA without complementary base-pairing in DNA–RNA triplexes. Yet, the hyperstability of circRNAs means they are potent mediators in any landscape, particularly in dynamic chromatin.Simon Conn  reported another seminal finding by demonstrating that circRNAs are stoichiometrically favoured to bind certain targets in the cell. Specifically, interactions between a circRNA from It has long been recognised that single-stranded RNA viruses, recognised as foreign entities, can drive host immune responses. However, the fact that circRNAs are structurally identical led opening speaker Howard Chang  to reason that circRNAs might do the same. As shown by ChiRP–MS (comprehensive identification of RNA binding proteins by mass spectrometry), recognition of self versus non-self circRNAs depends on their biogenesis and marking by endogenous protein co-factors. Importantly, the use of circRNAs seems as effective as current metal ion adjuvants in promoting immune responses to vaccination and to block viral transduction.Taken together, we have learned much about circRNAs since their systematic identification by NGS in 2013. It is increasingly clear that they are not accidental by-products of splicing. The talks summarized above provide insights into the ways in which circRNAs are involved in neurodevelopment and degeneration, plant development, and cancer. Studies of how circRNA is generated and its functions at the organismal level will surely proceed rapidly in the coming years.The functional consequence of miRNA binding to mRNA was once considered a one-way street, whereby miRNAs induce mRNA target instability. However, the reverse situation, called target-induced miRNA degradation (TIMD), is now emerging as a physiologically relevant phenomenon. Although miRNA biogenesis is well characterised, its regulation and the mechanism underlying miRNA turnover are not yet entirely understood.Both Paulina Pawlica from Joan Steitz’s group  and Francesco Nicassio  presented data on TIMD, including a proposed general mechanism for miRNA-degradation elements: the miRNA-binding region may not only be complementary to the 3′ end of miRNA, but with conformational flexibility can efficiently mediate miRNA decay. Nikolaus Rajewsky demonstrated that loss of a circRNA that acts as an miRNA ‘sponge’ also destabilises its miRNA and deregulates expression of its target mRNAs. Lynne Maquat  also revealed a new pathway for miRNA decay: functional miRNAs are degraded in human cells by the endonuclease Tudor-SN, and this is functionally critical for cell cycle progression. David Bartel found that RNA secondary structures are more prevalent within extended 3′-end regions of poly(A) signals than upstream regions. Experiments have demonstrated that this folding both enhances processing and increases mRNA metabolic stability, adding another possible layer of complexity to the model that miRNAs can target non-contiguous, flanking sequences of these structured regions.The myriad functions of miRNA have been well illustrated over the past decade, yet we are in a renaissance age of studying miRNA functions in animal models. Several miRNA knockout studies and salient phenotypes were revealed at this meeting. Robert Blelloch  posed the question as to why some evolutionarily conserved miRNAs have the same seed sequences and similar expression patterns, yet manifest distinct phenotypes. His findings suggest that the same-seed miRNA family share redundant and non-redundant roles. In agreement, Lin He  showed that knockouts of other compound mutants of the same-seed miRNA cluster family showed redundancy, until complete knockout was achieved. Taken together, these knockout analyses of miRNAs and lncRNAs indicate that ncRNAs are certainly important in embryonic development and disease progression. As miRNA and lncRNA knockout mice become increasingly available, perhaps we will learn that ncRNAs function in organ development and immunity, and link them to human diseases.Although many more interesting topics were discussed at the Crete meeting, including covalent RNA modifications, epitranscriptomics  and regulated patterns of RNA cleavage/polyadenylation , these could not be covered here.Lin He: novel methodology for efficient, cost effective CRISPR-targeted mice, called CRISPR-EZ.Zissimos Mourelatos: co-translational degradation of engaged mRNAs (ribothripsis).Drosophila embryo gene expression profile.Nikolaus Rajewksy: droplet-seq to digitally reconstruct, cell-by-cell, the The first long and short ncRNA meeting took place in Crete two years ago, bringing together ncRNA biologists from a narrow range of disciplines. Since then, our understanding of the molecular basis of the key processes underlying ncRNA regulation and their implications for disease and development has considerably increased. We now understand more about the molecular cues that direct the production of circRNAs, and their functions are emerging. Complementing these advances is our progress in understanding the mechanisms regulating long and short ncRNAs. The first and second meetings on ncRNAs in Crete will accelerate developments in the field of ncRNA biology by bringing together a more interdisciplinary field of scientists and, consequently, more systematic analyses. We enthusiastically await and can only imagine the advances to be presented at the third meeting in 2019.