Bunyaviridae, genus Orthobunyavirus, California serogroup. Studies indicate that INKV infection is mainly asymptomatic, but can cause mild encephalitis in humans. In northern Europe, the sero-prevalence against INKV is high, 41% in Sweden and 51% in Finland. Previously, INKV RNA has been detected in adult Aedes (Ae.) communis, Ae. hexodontus and Ae. punctor mosquitoes and Ae. communis larvae, but there are still gaps of knowledge regarding mosquito vectors and genetic diversity. Therefore, we aimed to determine the occurrence of INKV in its mosquito vector and characterize the isolates.Inkoo virus (INKV) is a less known mosquito-borne virus belonging to About 125,000 mosquitoes were collected during a mosquito-borne virus surveillance in northern Sweden during the summer period of 2015. Of these, 10,000 mosquitoes were processed for virus isolation and detection using cell culture and RT-PCR. Virus isolates were further characterized by whole genome sequencing. Genetic typing of mosquito species was conducted by cytochrome oxidase subunit I (COI) gene amplification and sequencing (genetic barcoding).Ae. communis mosquitoes were found positive for INKV RNA and two isolates were obtained. The first complete sequences of the small (S), medium (M), and large (L) segments of INKV in Sweden were obtained. Phylogenetic analysis showed that the INKV genome was most closely related to other INKV isolates from Sweden and Finland. Of the three INKV genome segments, the INKV M segment had the highest frequency of non-synonymous mutations. The overall G/C-content of INKV genes was low for the N/NSs genes (43.8–45.5%), polyprotein (Gn/Gc/NSm) gene (35.6%) and the RNA polymerase gene (33.8%) This may be due to the fact that INKV in most instances utilized A or T in the third codon position.Several Ae. communis is the key vector. The high mutation rate of the INKV M segment may have consequences on virulenceINKV is frequently circulating in northern Sweden and The online version of this article (doi:10.1186/s12985-017-0725-5) contains supplementary material, which is available to authorized users. Lepidus timidus) is speculated to be a reservoir host of INKV. Previous findings of INKV infections in human populations of northern Europe, and the possibility of the virus to be vertically transmitted by mosquitoes in endemic areas, highlights the need for further surveillance and characterization of virus strains to elucidate the impact of INKV infections. In this study, INKV was isolated from Ae. communis mosquitoes, and the full genome sequences of the three segments were obtained. In addition, further identification of mosquito vectors that may transmit INKV is necessary in the establishment of appropriate vector control, surveillance and infectious disease modelling.Inkoo virus (INKV) is endemic in northern Europe and may cause an influenza-like illness that may develop to a more severe form with encephalitic symptoms. Interestingly, the sero-prevalence is unexpectedly high among human populations in Finland and Sweden. In endemic areas, mammals have also been shown to have specific antibodies against INKV and the mountain hare  communis larvae in northern Sweden [Ae. communis is the most abundant species in the north during the first half of the summer [INKV belongs to family  Finland –9. Recenn Sweden , which sn Sweden , 12. TheThe clinical picture of INKV disease is not fully known. It is assumed that most infected persons are asymptomatic or present a mild febrile illness, which is self-limiting in most cases. However, in severe cases, it may cause a neuroinvasive disease that manifest in a form of encephalitis , 13. TheBunyaviridae family encode nonstructural NSs and NSm proteins from the S and M segments, respectively.INKV has a tri-segmented, negative-sense, single-stranded RNA genome that is approximately 12 kb in size. The S segment (1 kb) encodes the nucleocapsid protein (N), the M segment (4.5 kb) encodes for a polyprotein that is subsequently cleaved into two envelope glycoproteins Gn and Gc and the L segment (6.9 kb) encodes the RNA-dependent RNA polymerase , 17. In Orthobunyavirus genus, Bunyamwera sero-group can undergo genetic re-assortments that possibly occur during co-infection [Orthobunyavirus, but are classified in distinct serogroups; Bunyamwera and California respectively. The ability of La Crosse virus (a virus belonging to the same serogroup as INKV) to undergo reassortment in nature via vertical transmission was demonstrated by rearing mosquito eggs to adults and the virus RNA isolated from their abdomen. Analyses of the S, M and L of the La Crosse virus sequences, indicated that about a quarter of the infected mosquitoes contained reassorted genome segments [Tri-segmented mosquito-borne viruses from the nfection –20. For nfection . Geneticnfection . Ngari- segments . Sequencsegments .We performed characterization of the S, M and L segments of INKV isolated from a mosquito-borne surveillance exercise in northern Sweden, with an aim of investigating the genetic diversity of INKV in northern Sweden. Investigation of the codon usage of INKV and their vertebrate (human) and invertebrate (mosquito) hosts was performed. In the present study, we report the isolation and first whole genome analyses of the mosquito-borne INKV in northern Sweden. This information is vital in understanding the virusadaptation to vectors and hosts considering that arboviruses are utilizing both insect and mammalian cell machinery for replication.This study was conducted between June and September 2015, in selected areas of Västerbotten County, northern Sweden, see Fig. Two sites were selected in Lövånger due to a previous outbreak of Ockelbo disease that occurred in 2013 . The exaMosquitoes were collected every second week from June–September using propane mosquito magnets . The mosquitoes were gently removed from the nets and sorted into 50 mL tubes according to date, site and tube number before stored at −80 °C until processing.The mosquitoes were homogenized individually in 550 μL Dulbecco’s Modified Eagles Media , 2% HEPES . About 60 μL of ten individual mosquito homogenates (1×) were pooled to produce 10× pools (60 μL from 10 mosquito homogenates = 600 μL of 10× pool), and similarly, 60 μL from ten 10× mosquito homogenates were pooled together to make 100× pools (60 μL from ten 10× mosquito homogenates = 600 μL of 100× pool). The preparation of samples was performed at +4 °C, and subsequently stored at −80 °C.Extraction of viral nucleic acid was performed by Viral NA Extraction Kit using the magnetic bead based method . The procedure was performed according to the manual , with 250 μL of the homogenate and 10 μL of protein kinase K  in 1.5 mL sterile micro-centrifuge tubes. Afterwards, viral nucleic acid was extracted and collected in an elution volume of 50 μL.Orthobunyavirus genus-primers  see Additional file Orthobunyavirus positive samples, INKV specific PCR was also performed, using primers for S-, M- and L-segments (Eurofins Genomics) see Additional file RT-PCR was performed on the nucleic acid extracts with INKV positive RT-PCR-products were purified using QiaQuick purification kit  and ethanol precipitation. Purified samples were sent to Eurofins Genomics (Germany) for sequencing with INKV specific primers, see Additional file 2. Briefly, 50 μL of homogenate was inoculated on confluent 24-well plates seeded with Vero B4 and BHK-21 cells. The inoculated plates were incubated for one hour before adding 1 mL of pre-heated media and observed once per day for cytopathic effects. The media composition for Vero B4 cells included; DMEM supplemented with 2% fetal bovine serum (FBS) , 2% HEPES, 2% penicillin streptomycin (PEST)  and 2% L-glutamine, whereas that of BHK-21 contained; Glasgow MEM supplemented with 5% FBS, 1.3 g/L tryptose , 2% HEPES, 1 mM sodium pyruvate, 2% PEST and 2% L-glutamine.Samples of 100× homogenates, including all INKV RNA positive pools were added to BHK-21 and Vero B4 cells. Both cell lines were cultured in 24-well plates at 37 °C and 5% COwww.ncbi.nlm.nih.gov/blast) against GenBank databases. The sequences obtained from the study  segments were aligned to the respective segment sequences of the Finnish prototype strain KN3641 using Muscle. Amino acid substitution analysis of the INKV S, M and L-segment was performed using Muscle programme incorporated within MEGA6 [Virus sequences obtained in the study were identified by BLAST . The extracted DNA was amplified using qPCR as described before . Primershttp://www.endmemo.com/bio/gc.php. Codon usage was monitored using http://www.bioinformatics.org/sms2/codon_usage.html.In order to investigate the G/C content of the different segments of the Inkoo virus genome we used the Endmemo software Orthobunyavirus RNA see Table Orthobunyavirus RNA positive pools and later, when using INKV specific primers, demonstrated to be INKV. To confirm the viability of INKV positive 100× pools, inoculation of the corresponding samples on BHK-21 and Vero B4 cells was done. Two of the 100× pools showed a clear cytopathic effect in culture and were further verified as INKV by RT-PCR and sequencing.Approximately 125,000 mosquitoes were sampled from the indicated sites. Of these, a representative selection of 10,000 mosquitoes that were distributed equally between sampling sites and dates was selected. Four of the one hundred (100×) pools were positive for Ae. communis. However, the source of the isolates from the 100× pools may not be known due to the pooling method used.The COI gene of the three INKV positive individual mosquito homogenates was amplified for species identification. The findings indicated that all positive INKV individual mosquito homogenates were identified as Ae. communis, indicated both synonymous and non-synonymous mutations in all three RNA segments). The S segment showed two non-synonymous and 14 synonymous mutations in the coding sequence of the nucleocapsid gene and non-structural (NSs) gene. Interestingly, the M segment presented 46 non-synonymous mutations and 234 synonymous mutations in contrast to the much longer L segment that had only 18 non-synonymous mutations among the 381 nucleotide changes. Thus, the M-segment had the highest frequency of non-synonymous mutations, 10/1000 bases, compared to 2/1000 and 3/1000 bases for the S- and L-segment, respectively. A phylogenetic analysis indicated that the S segment (KX554935) was closely related to previous INKV isolates as well as Jamestown Canyon virus (JCV) , M (KX554936) and L (KX554937) segments of the present INKV isolate, in comparison with the Finnish prototype strain KN3641 strain isolated from 5, M , Bunyaviridae (e.g. Tahyna virus and INKV) and Flaviviridae (e.g. West Nile virus) are present in Europe [The European continent, in comparison to other continents such as Africa and Asia, has so far experienced a lower activity of mosquito-borne viruses, possible due to its temperate climate conditions. This may also be linked to the limited diversity and abundance of virus competent mosquito species. However, viruses belonging to n Europe , 3. Nonen Europe . It is pPhylogenetic analyses of the S, M and L segments of the obtained INKV isolate showed that it was most closely related to the other three INKV strains presently available in genetic databases. This finding implied that the INKV strains, including the study isolate, might have a similar genetic background and evolution history. However, more genetic data on INKV is needed to support this hypothesis.Ae. communis in Finland, which points toward the existence of diverse INKV strains. The high genetic variation of in total nearly 50 amino acids could in the long run have impact on virus infectivity, host range and adaptation to environmental conditions.The present INKV sequence included numerous non-synonymous mutations, especially in the M-segment of the study isolate, in comparison to prototype KN3641 strain, isolated from When comparing the S, M and L segments of this study, the M segment showed a 3–5 times higher frequency of non-synonymous mutations, in comparison to the S- and L segments. This may be attributed to the presence of conserved sequence motifs located close to the transcription termination site of the S-segment , 30 and Flaviviruses have been shown to have lower G/C content compared to other Flaviviruses transmitted by mosquitoes and vertebrate hosts [The actual drivers of codon usage bias among RNA viruses are not known. However, nucleotide base composition (G/C content) and codon usage are shown to vary amongst different organisms, for example G/C content in prokaryotes ranges between 0.25 to 0.75 whereas that of vertebrates is between 0.3 to 0.65 , 33. Thite hosts . In the te hosts . HoweverAe. communis as the species associated with all three INKV positive mosquito individuals confirmed its role as a key vector for INKV [Ae. communis is the most common and widely distributed mosquito species in northern Sweden [The identification of for INKV . Ae. comn Sweden , and is n Sweden , 36, 37.n Sweden .Aedes communis as the principal vector of INKV. The sequence data obtained from this study will aid in building up of the scanty genetic data available in the sequence archives. These findings clearly highlight the importance of surveillance for early detection and response to new potential threats and when designing appropriate prevention and control strategies. The study was limited to northern Sweden and therefore the findings may not be a true reflection of the actual scenario in other parts of the country. Though the pooling approach employed in the study saved the cost incurred in purchasing reagents and supplies, it was not sensitive enough to allow detection of lower copies of the virus since it created a dilution effect. Also, it was difficult to design appropriate broad-range primers targeting all the viruses within the California serogroup.In conclusion, the study provides additional evidence on Additional file 1: Table S1.Orthobunyavirus genus specific primers used for RT-PCR. (PDF 586 kb)Sequences of Additional file 2: Table S2.Sequences of INKV specific primers used for RT-PCR and sequencing. (PDF 187 kb)


Malassezia is the dominant fungus in the human skin mycobiome and is associated with common skin disorders including atopic eczema (AE)/dermatitis. Recently, it was found that Malassezia sympodialis secretes nanosized exosome-like vesicles, designated MalaEx, that carry allergens and can induce inflammatory cytokine responses. Extracellular vesicles from different cell-types including fungi have been found to deliver functional RNAs to recipient cells. In this study we assessed the presence of small RNAs in MalaEx and addressed if the levels of these RNAs differ when M. sympodialis is cultured at normal human skin pH versus the elevated pH present on the skin of patients with AE. The total number and the protein concentration of the released MalaEx harvested after 48 h culture did not differ significantly between the two pH conditions nor did the size of the vesicles. From small RNA sequence data, we identified a set of reads with well-defined start and stop positions, in a length range of 16 to 22 nucleotides consistently present in the MalaEx. The levels of small RNAs were not significantly differentially expressed between the two different pH conditions indicating that they are not influenced by the elevated pH level observed on the AE skin. Extracellular vesicles (EV) are released not only from different mammalian cell-types but also from microorganisms and parasites and have the capacity to transfer complex biological information12341119Magnaporthe oryzaeSclerotinia sclerotiorumBotrytis cinereaPhytophthora sojaeNeurospora crassaS. sclerotiorum, a plant pathogenic fungi, is an example of the former where it has been proposed that two milRNAs are involved in vegetative developmentB. cinerea, an aggressive fungal pathogen that is able to infect more than 200 plant species, uses small RNAs to interfere with the host RNA interference (RNAi) machinery and selectively silences host immunity genes to achieve infectionPseudomonas aeruginosa is able to reduce the host immune response by releasing EVs containing small RNA that inhibit the IL-8 secretion of airway epithelial cellsmiRNAs are small non-coding RNAs with a length between 20 and 22 nucleotides (nt)Malassezia is a commensal yeast that colonizes the human skin right after birth and predominates the human fungal skin microfloraMalassezia sympodialis, which is associated with several common skin disorders such as atopic eczema (AE)/dermatitisM. sympodialis in terms of specific IgE-and T-cell reactivity and/or positive atopy patch test (APT) reactions, indicating a link between AE and M. sympodialisM. sympodialis allergens have been sequenced so farM. sympodialis cultured at pH 6.1, which reflects the elevated skin pH of AE-patients, secrets more allergens compared to cultured at pH 5.5, which represents the normal skin pHM. sympodialis secretes nanosized exosome-like vesicles30M. sympodialis host-microbe interactions we here aimed to assess whether small RNAs are present in MalaEx and, if so, address whether the levels of these RNAs differ in MalaEx isolated from M. sympodialis cultured at normal skin pH compared to the higher pH on the skin of AE patients.To elucidate M. sympodialis cells was similar between the two different pH conditions after 48 h culture  we also investigated the fraction of reads that mapped to annotated RNA) and un-annotated (non-coding) regions. We found that ~55% of reads mapped to the non-coding regions  only contains annotation for coding regions. We therefore investigated the mapped read set by considering whether any of the non-coding small RNA reads had any significant homology to identified small RNA classes in other similar genomes. To this end, we first considered Ustilago maydis33Malassezia than other model ascomycete fungi such as S. cerevisiae. For comparative purposes, we also considered the reference genomes used by Peres et al.M. sympodalis genome  against this reference database. We found significant hits to segments of several tRNA entries, as well as a single hit to a snRNA in S. cerevisiae (db_xref = SGD:S000006478), indicating that these features were present in the M. sympodialis genome. However, none of these features overlapped with the small RNA features we identified in the MalaEx NGS data.The annotated Although there were no highly expressed features that were predicted to be differentially expressed between the two pH conditions, many of the features were consistently expressed across almost all samples (i.e. at least 9 of the 10 pairwise cultured samples) and for both pH conditions . As a fuBased on this, we next investigated these features to determine whether we could establish if they were of functional significance, rather than a consequence of random events (such as degradation products). MalaEx:500:15:30) based on the stability of predicted hairpin loops generated from each small RNA plus flanking sequence and compared this to the corresponding distributions for (i) randomly selected sequences from the M. sympodialis genome sequence , (ii) human miRNAs, and (iii) human cytomegalovirus (HCMV) miRNAs  in miRBase release 20MalaEx small RNAs and random sequence were most similar and notably less stable than the highly similar human and HCMV MFE distribution  distribution of the complete small RNA set  set that mapped to the genome, but after filtering for a minimum average count >1000, only features msy-10193 and msy-4613 were above the threshold , an exonuclease called QDE-2-interacting protein (QIP), an Argonaute-protein (QDE-2) and Dicer proteinsMalassezia species have recently been reported to lack homologs of the canonical RNAi pathway, including Dicer, Argonaute, and RNA-dependent RNA polymeraseWe then assessed the presence of small RNAs in MalaEx and addressed if the levels of these RNAs differ between the two pH conditions. Different mechanisms have been proposed for the generation of milRNAs in fungi. In Malassezia. Here we specifically examined RNAs smaller than 30 bp that were predicted to be highly enriched in mature miRNA or siRNA. While we also identified a candidate feature set, examination of the mean free energy suggested they were unlikely to form hairpin loops, providing evidence that miRNAs are not carried in M. sympodialis extracellular vesicles. It remains to be tested whether this is a biological difference or simply a result of differences in experimental and analytical approach.A previous study of fungal extracellular vesicles examined both mRNAs and smaller size fractions (less than 200 bp) using Solid sequencingN. crassa. Dicer-independent small interfering RNAs (disiRNAs) originate from overlapping sense and antisense transcripts in N. crassa, but do not require any of the known RNA machineryM. sympodialis may represent a second example of disiRNA production, although our preliminary examination revealed no evidence for correlation of these small RNA features and base modifications  was cultured on Dixon agar plates8 cells were added to 300 ml mDixon broth supplemented with 50 mM MES (2-(N-Morpholino) ethanesulfonic acid) . The broth had been ultracentrifuged over night at 100.000× g and filtered through a 0.22 μm filter  to remove possible nanovesicle contaminants. M. sympodialis was cultured pairwise at different pH values (pH 6.1 and 5.5) for 48 h at 32 °C and at 200 rpm. pH was measured at the start and end of culture using a pH-meter . At each culture step blood and Sabourad agar plates were inoculated in parallel to exclude bacterial and Candida contaminations, respectively.M. sympodialis cells were separated by centrifugation at 1200× g for 5 min and the culture supernatant was used for the isolation of MalaEx by serial ultracentrifugation with an initial centrifugation of 3000 g for 30 min followed by a second at 10000× g for 30 min. Thereafter, MalaEx were pelleted at 100000 × g for 90 min, re-suspended in PBS and pelleted again at 100000× g for 90 min. The resulting pellet was carefully re-suspended in 100 μl PBS. Protein content was measured using a DC protein assay according to the manufacturer’s instructions . The MalaEx preparations were stored at −80 °C.After 48 h culture The particle size and concentration of the MalaEx preparations were measured using a LM10 platform with sCMOS camera from NanoSight Ltd, Amesbury, UK. The system is equipped with a 405 nm laser and was running the NTA 2.3 analytical software package. The samples were analyzed at 1000× dilution in PBS with camera level 14 and detection threshold 6. Two consecutive videos were recorded for each sample at room temperature.Exosomes prepared for electron microscopy were further isolated by sucrose gradient centrifugation as previously describedM. sympodialis cultured pairwise at pH 5.5 or pH 6.1 for 48 h was extracted from 5 different cultures with the miRCURYTM RNA Isolation kit  using the specialized protocol for yeast cells according to the manufacturer’s instructions. RNA-concentration was measured with the Qubit Fluorometer 2.0  and the RNA was stored at −80 °C until further usage.RNA from MalaEx harvested from RNA-sequencing was performed by Exiqon . As starting-material, one μg of RNA from 5 different independent pairwise cultures at pH 5.5 and pH 6.1 was utilized for the library generation. The assessment of the RNA quantity was done with a Bioanalyzer system . An automated gel cutter  was used to excise the band that represents the microRNA-fraction. The cDNA libraries of small RNAs were constructed and then sequenced on a HiSeq 2500 .https://support.illumina.com/sequencing/sequencing_software/igenome.html). rRNA sequences within the M. sympodalis genome were obtained by BLASTing a PacBio assembled and annotated reference genome  against the Saccharomyces cerevisiae reference genome (GCF_000146045.2_R64) downloaded from RefSeq and reads were further filtered against these sequences. Remaining reads were then mapped to the M. sympodalis genome  using the bowtie alignment software packageReads were adapter trimmed using Trimmomatic version 0.34M. sympodialis genome  were then further examined to identify whether they exhibited “miRNA-like” profiles . Length intervals of 15 to 30 nt, 15 to 25 nt and 16 to 25 nt were examined, reads were further filtered by selecting a range of required minimum reads  with the additional constraint that start and stop positions could each only vary by 4 nt. For each of these cases, a feature set was generated and used for counting reads intersecting each feature and then the EdgeR package53To predict potential small RNA features, a modified version of miRParaM. sympodialis  doesn’t include annotation for snRNAs, snoRNAs or tRNAs, we performed the following analysis to investigate whether any of our identified features are mapping to sequences that have high similarity to any of these classes of small RNAs. We selected U. maydis (GCA_000328475.2) as the closest annotated genome to M. sympodialis33et al.C. neoformans - GCA_000149245.3, C. albicans - GCA_000149445.2, P. brasiliensis - GCA_000150735.1 and S. cerevisiae - GCA_000146045.2).As our genome annotation of C. neoformans and P. brasiliensis returned 134 and 103 tRNA features, respectively. The remaining genomes had additional small RNA forms in their annotation: C. albicans ; S. cerevisiae . For all these features, the corresponding sequence was extracted and used to build a BLAST reference small RNA database. The identified feature set from MS with reads >500 and lengths 15 to 30 nt were then BLASTed against this database using the blastn_short setting for short sequence matching.We then extracted the sequences corresponding to identifiers with keywords equal to snRNA, snoRNA, tRNA and misc_RNA. This returned a total of 145 features corresponding to tRNA. Similarly, Homo sapiens and for human cytomegalovirus (HCMV). We then compared the MFE distributions for the four datasets.To investigate whether a sub-population of these features might be achieving a regulatory role in their human host, we attempted to map the features to a human reference genome . We selected the features set identified for a read cut off of 500 nt and a length range of 15 to 30 nt, and allowed for 2 mismatches (to accommodate for similar mismatches that are observed to be present in the seed region of miRNA targets). If these small RNAs were consistent with generation from a miRNA biogenesis-like pathway, we would expect to find they were part of a stable hairpin structure. We therefore selected a 50 bp-flanking region from both sides of each small RNA and predicted the secondary structure. If the predicted structure formed a hairpin, we recorded the MFE. After all small RNAs had been analysed, we plotted the MFE distribution for the set. We also performed a similar analysis for a set of sequences randomly selected from the genome sequence. Finally, we repeated the analysis for miRNA/pre-miRNAs in miRBase release 20 for M. sympodialis . Data were aligned to the MS-PB reference genome using the SMRTAnalysis pipeline to uncover predicted modified bases as previously describedBase modifications were predicted using PacBio data produced for The data have been submitted to the Sequence Read Archive (SRA) database under study accession number BioProject ID PRJNA342612. https://www.ncbi.nlm.nih.gov/bioproject/342612.How to cite this article: Rayner, S. et al. Identification of small RNAs in extracellular vesicles from the commensal yeast Malassezia sympodialis. Sci. Rep.7, 39742; doi: 10.1038/srep39742 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Leishmania pyrimidine salvage is replete with opportunities for therapeutic intervention with enzyme inhibitors or antimetabolites. Their uptake into cells depends upon specific transporters; therefore it is essential to establish whether various Leishmania species possess similar pyrimidine transporters capable of drug uptake. Here, we report a comprehensive characterization of pyrimidine transport in L. major and L. mexicana. In both species, two transporters for uridine/adenosine were detected, one of which also transported uracil and the antimetabolites 5-fluoruracil (5-FU) and 5F,2′deoxyuridine , and was designated uridine-uracil transporter 1 (UUT1); the other transporter mediated uptake of adenosine, uridine, 5F,2′dUrd and thymidine and was designated Nucleoside Transporter 1 (NT1). To verify the reported L. donovani model of two NT1-like genes encoding uridine/adenosine transporters, and an NT2 gene encoding an inosine transporter, we cloned the corresponding L. major and L. mexicana genes, expressing each in T. brucei. Consistent with the L. donovani reports, the NT1-like genes of either species mediated the adenosine-sensitive uptake of [3H]-uridine but not of [3H]-inosine. Conversely, the NT2-like genes mediated uptake of [3H]-inosine but not [3H]-uridine. Among pyrimidine antimetabolites tested, 5-FU and 5F,2′dUrd were the most effective antileishmanials; resistance to both analogs was induced in L. major and L. mexicana. In each case it was found that the resistant cells had lost the transport capacity for the inducing drug. Metabolomics analysis found that the mechanism of action of 5-FU and 5F-2′dUrd was similar in both Leishmania species, with major changes in deoxynucleotide metabolism. We conclude that the pyrimidine salvage system is highly conserved in Leishmania species - essential information for the development of pyrimidine-based chemotherapy.  •Leishmania express 2 adenosine/pyrimidine transporters, 1 oxopurine transporter.•One of the pyrimidine transporters mediates uracil uptake.•Some fluorinated pyrimidines are toxic to Leishmania; resistance could be induced.•The resistant lines lacked capacity to take up fluorinated pyrimidines.•Leishmania metabolism of fluorinated pyrimidines was different from Trypanosoma. The disw World) . Due, inw World) . Nucleotutrients . Nonetheutrients . For theutrients . In contutrients . It has utrients .Leishmania purine or pyrimidine transporter can be considered essential  nucleoside transport is highly similar in multiple Leishmania species; (2) that the substrate binding of the LmajNT1 transporter depends on interactions with the 2-keto and N3 positions of the pyrimidine ring and the 3′ and 5′ hydroxyl groups of the ribose moiety; and that (3) the antimetabolite 5-fluoro-2′-deoxyuridine (5F-2′dUrd) is principally converted to 5F-dUMP, by thymidine kinase, causing the inhibition of thymidylate synthase and the consequent disruption of deoxynucleotide metabolism; 5-fluorouracil is first converted to 5F-2′dUrd and thence to 5F-dUMP.There remain many caveats to the pharmacological exploitation of the purine and/or pyrimidine salvage pathways for antileishmanial chemotherapy, including whether there are significant differences in nucleoside transport activities between the various 22.1L. mexicana (MNY/BZ/62/M379 strain) and L. major (Friedlin strain) were grown in HOMEM medium  (pH 7.4) supplemented with 10% fetal bovine serum (FBS) (Gibco) 1% Penicillin/Streptomycin antibiotic (Gibco) at 25 °C as described  under a 5% CO2 atmosphere at 37 °C  were washed into Human T Cell buffer for transfection using the desired cassette with an Amaxa Nucleofector using program X-001. Cells were transferred into pre-warmed HMI-9 medium and allowed to recover for 8–16 h at 37 °C and 5% CO2. Following recovery, transfectants were grown and cloned in selective medium containing 5 μg ml−1 blasticidin S using limiting dilution.Plasmid construction and transfection was performed according to and NT1B . The priand NT1B  were desand NT1B  and then2.3®  using a Reverse-Transcriptase (RT) kit . For each sample the cDNA was diluted with RNase free water to 20 ng/μl for qRT-PCR. Amplification of cDNA was performed in a 7500 Real Time PCR System . The dissociation curve was used to ensure the amplification of only one product; samples without RT or cDNA were used as controls. The constitutively expressed gene GPI8 was used as an internal control  (Sigma-Aldrich) were performed using a protocol adapted from 6 cells/ml, of which 100 μl was added to wells of 96-well plates pre-loaded with 100 μl of doubling dilutions of test compounds, resulting in a final density of 1 × 106 cells/ml; the dilutions were over 2 rows of the plate (23 concentrations), with the last well containing only medium and serving as the no-drug control. The plates were incubated at 25 °C for 72 h before adding the Alamar Blue dye  in 100 ml phosphate buffered saline ). Since Leishmania parasites metabolize the Alamar Blue dye slower than trypanosomes (50) were calculated using the equation for a sigmoidal curve with variable slope using Prism 5.0  software; extrapolation of incomplete curves was used when >50% inhibition was achieved, using the minimum fluorescence in the curve with the control drug (pentamidine) as the sole constraint. Each experiment was performed independently at least 4 times; statistical significance was determined using Student's unpaired t-test.Sensitivity assays of anosomes , the cel2.5T. brucei and Leishmania species ; [3H]-adenosine ; [3H]-uridine ; [3H]-uracil ; [3H]-inosine ; [3H]-5-fluorouracil .Using the standard uptake technique as described for  species , the dosg. The cells were washed twice with transport assay buffer , resuspended at a density of 108 cells ml−1 in AB, and left for 20–30 min at room temperature to recover from centrifugation stress. One hundred microliters of cell suspension was incubated at ambient temperature for a predetermined time with 100 μl radiolabeled test compound, in the presence or absence of unlabeled substrate or other competitive inhibitors. The incubation was terminated by the addition of ice-cold stop solution  and centrifugation through oil for 1 min at 13,000 × g. Trapped extracellular radioactivity was determined as the amount of radiolabel associated with the cell pellet in the presence of 1 mM permeant , and subtracted. Radioactivity was determined by liquid scintillation counting in a Beckman LS6000 TA scintillation counter. Saturation data, inhibition data, and time courses were plotted to equations for linear or non-linear regression (hyperbolic or sigmoid curves), as appropriate. All experiments were performed in triplicate and on at least three independent occasions.Briefly, cells in the mid-to-late logarithmic stage of growth were harvested by centrifugation for 10 min at 1500×2.6L. mexicana M379 and L. major Friedlin strains were exposed to non-lethal concentrations (0.5 × EC50) of 5-fluorouracil and 5-fluoro-2′-deoxyuridine. The cells were then visually observed for viability and sub-passaged to tolerated concentrations of the drugs. The procedure was repeated until a high level of tolerance to the drug was obtained, essentially as described for the adaptation of T. brucei to pentamidine  before transfer to a 50-ml centrifuge tube for instantaneous cooling  and centrifugation . The pellet was lysed with 200 μl of chloroform/methanol/water (1:3:1 v/v/v) containing mass spectrometry standards, and vigorous mixing ; cell debris was removed by centrifugation and the metabolite extracts were stored in HPLC vials at −80 °C. Control samples were prepared in parallel and included untreated cells, unused growth medium, test compound solution and extraction solvent blanks. The analysis used a hydrophilic interaction liquid chromatography (HILIC-LC) fitted with a zwitterionic ZIC-pHILIC column (Merck Sequant), coupled to high resolution mass spectrometry (MS) using a Thermo Q-Exactive, and metabolomic data outputs were analyzed using the IDEOM application (http://mzmatch.sourceforge.net/ideom.php) with default parameters  over 30 s with a rate of 0.082 ± 0.005 pmol(107 cells)−1s−1, and was clearly saturable, as transport in the presence of 1 mM unlabeled thymidine was not significantly different from zero (P = 0.95) (3H]-thymidine was linear over 2 min (i) or Michaelis-Menten constants (Km) were determined the conditions used had been verified to be well within the linear range of uptake. Indeed, we did not observe non-linearity when studying nucleoside or uracil transport in Leishmania promastigotes, even at longer times and where there was a net-accumulation of radiolabel over the external concentration. In no time course experiments (whether shown in this paper or not) did runs tests show a significant deviation from linearity. This is likely because the rate of metabolism of nucleosides and nucleobases is very fast in kinetoplastids and therefore does not become rate limiting for uptake, especially over very short intervals. Moreover, the uptake of both nucleobases and nucleosides has been shown to be a secondary active transport mechanism, using proton symport to accumulate purines and pyrimidines highly efficiently (denosine . In orde = 0.95) A. Similaer 2 min B, allowiiciently .Fig. 1Trm) for thymidine was determined to be 11.2 ± 2.4 μM (i = 0.25 ± 0.4 μM), by uridine with low micromolar affinity (Ki = 9.1 ± 0.6 μM) and by cytidine with mid-micromolar affinity (Ki = 82 ± 5 μM)  D. The tr ± 5 μM)  and is c3.1.23H]-adenosine in L. mexicana promastigotes, which according to the efficient inhibition of [3H]-thymidine by adenosine, should be taken up with high affinity. Transport of 0.1 μM [3H]-adenosine was linear over at least 30 s with a rate of 0.21 ± 0.01 pmol(107 cells)−1s−1 −1s−1 A and the 0.16 μM B. Fig. 2 0.16 μM . The kin3.1.33H]-uridine was taken up efficiently and linearly over 120 s, with a rate of 0.0042 ± 0.0002 pmol(107 cells)−1s−1. The Km for the uridine flux was determined to be 7.15 ± 0.90 μM (n = 5). As shown in 3H]-uridine uptake by uridine was close to −1 , and thus consistent with a one-transporter model, but uracil inhibited only part of the flux, revealing the existence of a uracil-sensitive transporter (Ki = 25.7 ± 6.6 μM (n = 5)) and a uracil-insensitive uridine transporter. From the [3H]-uridine Hill slope it follows that both transporters have a similar affinity for uridine and 50 for thymidine was similar for the uracil-sensitive and –insensitive uridine transporters. Adenosine clearly inhibited both transporters with near-equal affinity, with a Hill slope close to −1 −1s−1. Transport of uracil over 4 min could thus be measured accurately and the Km was determined as 29.7 ± 4.4 μM (n = 3), with 5-fluorouracil displaying a somewhat lower affinity with a Ki of 56.3 ± 4.4 μM  (3H]-uracil transport was dose-dependently inhibited by uridine (Ki = 2.0 ± 0.5 μM), as well as by 2′-deoxyuridine  (3H]-uracil transport (n = 3). However, the flux through a presumed uridine-insensitive uracil transporter was too small to be characterized; it could be speculated that it is barely expressed in the promastigote forms, at least under the culture conditions used. We propose the designation L. mexicana uridine-uracil transporter 1 (LmexUUT1) for the uracil-sensitive uridine transporter here described.In order to study the uracil-sensitive uridine transporter we next employed 1 μM -thymidine was linear over 10 min, albeit with a very low rate of 0.0043 ± 0.0002 pmol(107 cells)−1s−1, and completely inhibited by 1 mM unlabeled thymidine  (inset), an average Km value of 30.7 ± 2.1 μM (n = 5) was calculated. This compared to a Ki value of 61.1 ± 7.4 μM (n = 3) for uridine. The figure further illustrates that the Hill coefficient of both sigmoid curves was greater than the value of −1 associated with a one-transporter model. The average Hill slopes were −0.67 ± 0.06 and −0.87 ± 0.10 for thymidine and uridine, respectively – indicative of a two-component transport system with distinct but not greatly different affinities. In order to separate out the two transporters, the thymidine inhibition data were redrawn using a Lineweaver-Burke double reciprocal plot (m of 4.2 ± 1.1 μM and Vmax of 0.023 ± 0.003 pmol(107 cells)−1s−1, and a lower affinity thymidine transporter with a Km of 26.8 ± 5.4 μM and Vmax of 0.14 ± 0.03 pmol(107 cells)−1s−1 (both n = 5). The approximately 6-fold difference in apparent Km between the two transporters would be consistent with the observed Hill slopes. Indeed, we were unable to separate the two transporters by inhibitor profile, with several inhibitors displaying 100% inhibition and Hill coefficients near −1, or incomplete inhibition at the highest concentration tested  and in most cases inhibited 100% of [3H]-thymidine transport, although in some cases a small percentage of the flux appeared to resist adenosine inhibition (Transport of 0.25 μM -thymidine transport in L. major promastigotes, which we treat as inhibitors of the total thymidine transport activity constituted of LmajNT1.1 and LmajNT1.2, as we were unable to measure each separately in wild-type promastigotes and the kinetics strongly suggested that both transporters were similarly sensitive to each inhibitor. Apart from adenosine, i value of 765 ± 68 μM versus 55.7 ± 5.4 μM for uridine. When these values are converted to the Gibbs free energy of binding (ΔG0), it can be found that the difference in binding energy (δ(ΔG0)) is 6.5 kJ/mol (0) = 10.4 kJ/mol). Furthermore, cytidine also displayed low affinity, but, since the 4-position keto group is not involved in binding, this can be attributed to the change in N(3) protonation state in cytidine versus uridine, consistent with a role for N(3). Substitutions at position 5 of the pyrimidine ring  appear to be slightly favorable (0) of 14.1 kJ/mol comparing 3′-deoxythymidine with thymidine, and 4.9 kJ/mol comparing 5-fluorouridine with 5-fluoro, 5′-deoxyuridine, respectively. The contribution of 2 strong interactions from the ribose moiety to pyrimidine nucleoside binding to the NT1 transporters provides the rationale for the complete lack of inhibition by up to 1 mM of the nucleobases adenine, uracil and thymine  were also observed in the binding mode for uridine for the Toxoplasma gondii AT2 and Trypanosoma brucei P1 transporters −1s−1. Like [3H]-thymidine transport, two distinct components for [3H]-uridine transport were in evidence, leading to a Hill coefficient of −0.79 ± 0.11 for inhibition with unlabeled uridine (m of 7.3 ± 1.3 μM (n = 3)) (inset), but a double reciprocal plot separated two components  (3H]-uridine by inosine was further confirmed by a time course over 20 min (P = 0.017), compared to the complete inhibitory effect of 1 mM unlabeled uridine (P = 0.22).Transport of 0.25 μM -uridine transport. This is consistent with our previous characterizations of purine and pyrimidine transporters in T. brucei, showing that uridine can be taken up, although very inefficiently, by the U3 uracil transporter (i = 830 μM) for the P1 adenosine/inosine transporter s−1 s−1 B; the trells)s−1 .Fig. 7UrLeishmania NT1-type transporters, LmexNT1A/B and LmajNT1A/B, were separately transfected into B48 and [3H]-uridine transport . As uridine had only a marginal effect on [3H]-inosine transport in the cells expressing LmajNT1A, this transporter did not mediate any substantial amounts of inosine uptake.Each of the ransport A–D. In eransport A–D, and ransport . In the -inosine E. In thi3.3.2Leishmania donovani, and was found to be an adenosine-insensitive inosine/guanosine (oxopurine nucleoside) transporter  in untransfected B48 cells  is given in the legend to The effects of the fluorinated pyrimidine analogs was similar against ow 20 μM . This mansferase . A list 3.5L. mexicana and L. major were adapted by in vitro exposure to 5-FU and 5F-2′dUrd. It was noted that Leishmania cells were adapted to 5F-2′dUrd more quickly than to 5-FU; while Leishmania cells become insensitive to high concentrations of 5F-2′dUrd in a few months, the resistance induction to 5-FU required approximately one year , but for L. mexicana the level was below the level at which it could confidently be detected with this metabolomics workflow  were observed in extracted 5-FU-treated promastigotes, in complete contrast to 5-FU metabolism in T. brucei, where numerous such metabolites were found after exposure of bloodstream forms to 5-FU (Leishmania uracil phosphoribosyl transferase (UPRT), and that 5F-2′dUrd is its only direct metabolite, analogous to the deoxyribosylation of thymine to thymidine, by uridine phosphorylase and/or by a separate thymidine phosphorylase activity. This also implies that the detected deoxy 5-FU nucleotides cannot have been derived from 5F-UDP via ribonucleotide reductase.As expected from the characterization of the lar 5-FU A. Neitheworkflow   , evidenced by the massive build-up of dUMP in the cell, that may be the most relevant as it is an essential enzyme in Leishmania  E and F. ishmania , and theishmania . It is fishmania , explainL. mexicana and L. major treated with 5-FU were an elevation of the levels of 2′deoxyuridine  and 2′deoxyadenosine (L. major) compared to untreated controls. We also observed large increases in the intensity of 2′deoxynucleotides  relative to respective untreated controls  of 5F-2′dUrd in 5-FU-treated L. mexicana , and an increase in the 2′deoxyadenosine intensity . It was further observed that treatment with 5F-2′dUrd, like 5-FU, lead to similar increases in the levels of 2′deoxycytidine nucleotides  and 2′deoxyadenosine nucleotides   .3.6.3L. major; L. mexicana; L. mexicana was also observed after treatment with 5F-2′dUrd  Are the nucleoside transporters in various clinically important Leishmania species similar enough to allow the efficient uptake of the same analogs? (2) Would the Leishmania nucleoside transporters, like their T. brucei counterparts, allow the uptake of modified nucleosides and if so, which modifications might be admissible? (3) How susceptible is the pyrimidine salvage system to pyrimidine analogs, including 5-halogenated pyrimidines that have been widely used in anti-cancer chemotherapy, and could these be repurposed? (4) How are such pyrimidine analogs metabolized in Leishmania, are there significant differences between species, and how does this compare to the same process in African trypanosomes?Nucleotide metabolism in protozoa is replete with promising drug targets, and nucleoside analogs have become key players in anticancer and anti-viral chemotherapy. In tabolism . This intabolism . DevelopLeishmania species contribute to the various clinical manifestations of leishmaniasis world-wide, it is virtually essential that any new treatments developed should be efficacious to at least the main pathogenic species involved, and this issue should be addressed at the very onset of a program, before major resources are invested in a strategy. In that context we address here whether nucleoside transport in L. mexicana and L. major are substantially different from each other, and from the L. donovani transporters reported earlier (see Introduction). The three ENT-family nucleoside transporters  seem to be syntenically preserved throughout the genus. We cloned these genes from L. mexicana and L. major, and expressed them heterologously in the related trypanosomatid T. brucei. The results show that, as in L. donovani , ‘old world’ cutaneous (L. major) and diffuse cutaneous and muco-cutaneous ‘new world’ leishmaniasis (L. mexicana).Since more than 20 different donovani , the NT1L. donovani promastigotes and individual L. donovani nucleoside transporters expressed in Xenopus laevis oocytes established that LdNT1.1 was a higher affinity transporter than LdNT1.2 , rather than NT1.1. However, the UU1 transporters of L. mexicana and L. major clearly are high affinity adenosine/uridine transporters, with a secondary ability to transport uracil , and are thus nucleoside transporters rather than nucleobase transporters. For L. donovani it was never tested whether either of the NT1-type transporters might be sensitive to inhibition by uracil, but by extension it could be speculated that LdNT1.1 might be homologous to LmexUUT1 and LdNT1.2 the equivalent of LmexNT1; this remains to be established. LmexUUT1 was sensitive to both thymidine and adenosine, as shown above for the equivalent activity LmajUUT1. In L. major, it was possible to detect two separate thymidine transport activities with Km values of 4.2 μM and 26.9 μM, respectively. Thus, all three Leishmania species express 2 uridine transporters, which are also transporters for adenosine and thymidine  uracil transporter, LmajU1  by LdNT1.1 . Indeed, we found that 4-thiouridine and 5F-5′dUrd (which like 5F-Urd are inhibitors of LmajNT1) had no effect on promastigotes even at millimolar levels. This highlights a key challenge to developing a rational nucleoside therapy to protozoa, i.e. analogs must be compatible both with the transporters that facilitate their entry into the cell, and with the metabolic enzymes that are required to activate these prodrugs. Nonetheless, 5-FU, 5F-2′dCtd, and in particular 5F-2′dUrd, showed activity against both Leishmania species in the low-micromolar range. In this the Leishmania promastigotes were surprisingly similar to bloodstream form T. brucei: 5-FU, 5F-2′dUrd and 5F-2′dCtd were much more active against the trypanosomes than any of the other halogenated pyrimidines tested against those parasites .T. gondii  uptake capacity, whereas uracil uptake in Lmex-5F2′dURes was unaffected, but 80% lower in Lmaj-5F2′dURes. We conclude that loss of NT1-like transport activity can account for much of the resistance phenotype. It has previously been shown that the Leishmania nucleoside transporters are not essential proteins under standard in vitro or in vivo conditions  to 5F-2′dUMP, as also reported in T. brucei (Apart from the details of pyrimidine transport, the pyrimidine salvage pathways are believed to be identical in the various pathogenic kinetoplastids . However. brucei , 5-FU wa. brucei . In contctivity” . We prop. brucei , which iL. infantum. They found the amplification of DHFR-TS as one main adaptation, in addition to point mutations in UP and TK, all consistent with our model. However, they also found point mutations in LinfUPRT, although we found no evidence of this enzyme being involved in 5-FU metabolism in L. mexicana and L. major. One explanation might simply be that 5-FU metabolism in L. infantum is different in this respect, but unlike UP and TK, the reintroduction of a wild-type copy of LinfUPRT only marginally reversed the resistance phenotype, and the authors point out that the same resistant strain had also lost all capacity for 5-fluorouracil uptake, implying that the UPRT mutation may have been an early mutation during the adaptation, conferring a marginal advantage that was superseded by a subsequent loss of transport activity (Leishmania species covering most of the spectrum of leishmaniasis pathologies. Finally, we propose that 5F-2′dUrd is a more promising therapeutic lead against leishmaniasis, as the nucleoside display a more potent anti-leishmanial activity, is efficiently quickly taken up the parasites, and does not require the UP activation step.This model for the trypanocidal activity of fluorinated pyrimidines is compatible with observations made by Ritt and coworkers , who seqactivity . Altoget


Sedentary endoparasitic cyst nematodes form a feeding structure in plant roots, called a syncytium. Syncytium formation involves extensive transcriptional modifications, which leads to cell modifications such as increased cytoplasmic streaming, enlarged nuclei, increased numbers of organelles, and replacement of a central vacuole by many small vacuoles. When whole root RNA is isolated and analyzed, transcript changes manifested in the infected plant cells are overshadowed by gene expression from cells of the entire root system. Use of microaspiration allows isolation of the content of nematode infected cells from a heterogeneous cell population. However, one challenge with this method is identifying the nematode infected cells under the microscope at early stages of infection. This problem was addressed by staining nematode juveniles with a fluorescent dye prior to infection so that the infected cells could be located and microaspirated.Globodera pallida in Solanum tuberosum root cells. This enabled microaspiration of nematode-infected root cells during the early stages of parasitism. To study the transcriptional events occurring in these cells, an RNA isolation method from microaspirated samples was optimized, and subsequently the RNA was purified using magnetic beads. With this method, we obtained an RNA quality number of 7.8. For transcriptome studies, cDNA was synthesized from the isolated RNA and assessed by successfully amplifying several pathogenesis related protein coding genes.In the present study, we used the fluorescent vital stain PKH26 coupled with a micro-rhizosphere chamber to locate the infected nematode The use of PKH26 stained nematode juveniles enabled early detection of nematode infected cells for microaspiration. To investigate transcriptional changes in low yielding RNA samples, bead-based RNA extraction procedures minimized RNA degradation and provided high quality RNA. This protocol provides a robust procedure to analyze gene expression in nematode-infected cells. Globodera pallida (Stone) Behrens  is a sedentary endoparasitic nematode that causes severe yield loss of potatoes (Solanum tuberosum L.). Infective second stage juveniles (J2s) of G. pallida invade the roots of the host plant and establish a feeding site by fusing several cells into a single syncytium [G. pallida infection is important for the development of resistance as is needed for potato including russet-type varieties.yncytium , 22, 28.yncytium , 16. UndGlobodera rostochiensis-infected Solanum lycopersicum. On the other hand, Ithal et al. [Glycine max roots were infected by Heterodera glycines. In a recent study, we have reported localized cell death as early as 2 days post infection in Solanum sisymbriifolium roots [G. pallida infection stages.Genes upregulated in nematode infected plants have been classified into primary (genes induced up to 1-day post infection) or secondary upregulated genes (genes induced after 3-days infection) . Primaryl et al.  found a um roots . In the Meloidogyne javanica 4 days post infection. Anjam et al. [Arabidopsis roots infected by Heterodera schachtii and compared pathogenesis related gene expression in 5-day-old syncytial cells to uninfected cells using qPCR.In recent years, several investigators have conducted gene expression studies from nematode infected roots , 12, 15.M. javanica in tomato roots 25 days post infestation. For studies of 5 day old H. schachtii infected Arabidopsis cells, Szakasits et al. [S. tuberosum roots that were grown in micro-rhizosphere chambers (micro-ROCs). In addition, a protocol was optimized for RNA isolation from microaspirated samples and utilized for downstream applications such as qPCR analyses.At early stages of infection, nematode infected plant cells are structurally similar to other cells in the root system which makes them difficult to differentiate using microscopy techniques. To alleviate difficulties in recognition of infected tissue, many investigations on cell specific transcriptome analyses of nematode infected plant cells have been performed on tissues in later stages of parasitism when cells are morphologically distinguishable. For instance, to investigate the differential expression of several pathogenesis related genes, Wang et al.  microasps et al.  found a s et al. , 14, 24.To investigate the cell specific transcriptome of nematode infected plant cells at early stages of infection, we developed a non-destructive microaspiration method. Plants were grown in micro-ROCs, which allows non-destructive detection and observation of nematodes within plant roots . To prevG. pallida with the rhodamine filter (in the absence of bright field illumination) has been inserted in Fig. ® PicoPure® RNA isolation kit) to prevent RNA degradation, our early efforts resulted in degradation of the microaspirated RNA. Fragment analysis revealed higher levels of RNA once the capillary needle was filled with buffer prior to aspiration of cell content.In the past, locating infected plant cells to study cellular content has been difficult because detection methods were not available. To facilitate this process, hatched J2s were stained with PKH26 prior to inoculation on their host. With a rhodamine filter (dye excitation max. 551 nm and emission max. 567), PKH26 stained nematodes fluoresce red, and their movement and position was easily observed. To microaspirate cell content both nematode and plant cells need to be visualized at the the same time. To achieve this, brightfield was turned to a low setting which allowed visualization of the plant cell while also maintaining the fluorescence to visualize the site of the nematode within the plant root  of 4.0 and the total concentration of 0.85 ng/µL  genes (genes contributing to disease resistance), NADH-quinone oxidoreductase subunit C, CHTB4 (Chitinase), cinnamyl alcohol dehydrogenase, and 6-phosphogluconate dehydrogenase, were amplified using specific primers Table . The PR All genes were successfully amplified from the microaspirated cDNA derived samples and clear bands were observed on the electrophoresis gel Fig. . NematodCell specific transcriptome analysis using microaspiration and LCM have been successfully used to isolate cellular materials from the syncytium for plant-nematode interaction studies , 9, 18. Despite being an inexpensive technique compared to LCM, microaspiration is laborious for obtaining sufficient amounts of input sample for RNA isolation , and canRecent studies using microscopy assisted techniques are conducted, in most cases 5 days or later after infection , 26, 29.To study the nematode-plant interaction, we used PKH26 coupled with micro-ROCs , 14, whi® PicoPure® RNA isolation kit has been used for isolation of RNA from low yielding samples [® PicoPure® RNA isolation kit. Despite several attempts using the manufacture’s protocol for the ARCTURUS® PicoPure® RNA isolation kit we did not get sufficient amounts or quality of RNA for downstream applications. However, when we reduced the incubation time from the initial 30 to 15 min an RQN of 4.0 was obtained. Most of the commercially available kits use column separation methods, which, in our experiments, did not yield high quality RNA for downstream applications such as RNAseq. In order to obtain RNA of adequate quality, we replaced columns with magnetic beads  and were able to obtain RQN values of 7.8. Karrer et al. [After aspirating the cell contents from infected cells, we attempted several methods to isolate pure RNA from this sample. Although, the ARCTURUS samples , 8, 19, r et al.  used olir et al. . In our To evaluate whether the RNA we obtained could be used for downstream applications, the isolated RNA was converted into cDNA and used for RNAseq and qPCR. Elongation factor and PR protein coding genes were amplified from these cDNA samples. qPCR was also successfully conducted for gene expression analysis from infected and non-infected cells of the same plant. RNAseq libraries were also prepared from this study and the results will be published elsewhere.® PicoPure® RNA isolation kit and by using a bead-based RNA purification kit . As evidenced by qPCR of genes from infected and non-infected material, this RNA was of sufficient quality for gene expression studies. The approach presented here will provide suitable methods for early parasitism studies in nematode-plant interactions at the cellular level.Because of the ability to study a single cell-type from a heterogeneous population, cell specific transcriptome analysis has become increasingly popular in animal, human and plant systems. Sedentary endoparasitic nematodes, such as cyst and root knot nematodes, form a feeding structure in the vascular tissues of the plant roots, and these tissues are metabolically different from other plant tissues due to the interaction between the host and the pathogen. It is crucial to understand the nematode-plant interaction at early stages of infection because early responses that may be suppressed in later stages, may be important for parasitism and host defense. PKH26 stained juveniles were used to locate infected sites for microaspiration. The RNA quality was improved significantly by adjusting the incubation time of the ARCTURUSSolanum tuberosum cv. ‘Desiree’ grown in the tissue culture medium was transplanted into micro-ROCs and kept under greenhouse conditions . Globodera pallida encysted eggs were hatched in potato root diffusate. The hatched second stage juveniles (J2s) were surface sterilized and stained using PKH26  as per the manufaturer’s protocol, and then suspended in 0.01% sterile agarose. Two weeks after planting in micro-ROCs, pproximately 200 J2s suspended in agarose were inoculated onto the roots of S. tuberosum [Planting, nematode inoculum preparation and inoculation were performed as described in Kooliyottil et al. . In brieuberosum , 14.G. pallida J2s were located within the root by use of a rhodamine filter under low magnification (20X objective lens).Twenty-four hours after inoculation, the plant was carefully removed from the micro-ROCs by breaking the glass plate and gently washing the roots with sterile water for 15 s. The plant was then placed on to a glass plate (7.5 cm L × 5.0 cm W) previously lined with 1% agarose  of the micro manipulator  filled with RNA extraction buffer  was used to pierce the plant root with assistance from TransferMan® . While entering the root, the CellTram® vario was turned to the injection mode to avoid the entry of any unwanted plant cell material into the needle due to the turgor pressure of the plant cell. Once the infected cell was reached, the CellTram® vario was turned to the aspiration mode to aspirate plant cellular material. The microaspirate contents were immediately transferred to 10 µL RNA extraction buffer provided in the ARCTURUS PicoPure RNA Isolation Kit , frozen in liquid nitrogen, and stored at −80 °C. Cell content of uninfected cells from the infected roots were also aspirated for gene expression comparison.To aspirate cell content, the capillary needle . Frozen microaspirated samples were thawed by incubating at 42 °C for 30 min. However, the quality of RNA extracted in this way was not sufficient for further analysis despite several attempts. Reduction in the initial incubation time was attempted by first using 30 min (as recommended manufacture’s protocol), and then at intervals of 5 min from 0 to 20 min; 15 min interval provided the best quality RNA. However, to further improve the quality, samples were processed by using a magnetic bead-based method. In brief, the samples incubated at 42 °C for 15 min (from the above mentioned protocol), were supplemented with the magnetic bead buffer provided in the Agencourt RNAdvance Tissue total RNA extraction kit  and further steps of magnetic separation and washing was performed as per the protocol provided by the manufacturer. To avoid any genomic DNA contamination in the RNA, each sample was treated with DNAse following the recommended protocol from the Agencourt RNAdvance Tissue total RNA extraction kit.® II reverse transcriptase (Invitrogen) using oligo (dT) primer  and dNTP mix. The quality and quantity of cDNA was determined by using a NanoDrop™ 2000c instrument . The cDNA from infected and uninfected samples were amplified using conventional PCR, with a housekeeping gene specific primer for elongation factor, and pathogenesis related (PR) genes namely NADH-quinone oxidoreductase subunit C, CHTB4 (Chitinase), and 6-phosphogluconate dehydrogenase [Qualitative and quantitative analysis of the purified RNA was determined by using a FRAGMENT ANALYZER™ . RNA was reverse-transcribed into cDNA according to the procedure described in the SUPERSCRIPTrogenase , 20, 23.2 = 0.99) each of forward and reverse primers of elongation factor and 100 nM  each of forward and reverse primers of CHTB4, and nuclease free water was added. The PCR condition was as follows: 50 °C for 2 min, 95 °C for 10 min, 60 °C for 1 min, 95 °C for 15 s 15 °C for 1 min and 95 °C for 15 s with a total of 40 cycles. The relative expression level of CHTB4 was calculated in comparison to elongation factor by means of 2(control sample Ct of target−infected sample Ct of target) ÷ 2(control sample Ct of reference−infected sample Ct of reference). The results presented are the average of three independent infections and sample collections.To confirm gene expression levels in microaspirated infected and uninfected cells, cDNA from both samples were analyzed for expression of CHTB4 using qPCR . The reaction was prepared in a total of 20 µL in which 10 µL master mix , 1 µL template cDNA (10 ng), 150 nM (primer efficiency = 102.30, R


Scientific Reports 10.1038/s41598-017-10134-2, published online 29 August 2017Correction to: In the original version of this Article there were errors with the affiliations which were incorrectly listed as ‘Department of Urology, Fudan University Shanghai Cancer Center, Shanghai, 200032, China’, ‘Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, China’, ‘Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200032, China’ and ‘Department of Pathology, Fudan University Shanghai Cancer Center, Shanghai, 200032, China’.The correct affiliations are listed below.Affiliation 1:Department of Urology, Fudan University Shanghai Cancer Center, Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, China.Affiliation 2:Department of Urology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200032, China.Affiliation 3:Department of Pathology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200032, China.Affiliation 4:Department of Urology, Fudan University Shanghai Cancer Center, Institutes of Biomedical Sciences, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200032, China.These errors have now been corrected in the PDF and HTML versions of the Article, and in the accompanying Supplementary Information file.


Therefore, knowledge about the A-to-I editing sites in RNA is crucially important for both basic research and drug development. Given an uncharacterized RNA sequence that contains many adenosine (A) residues, can we identify which one of them can be of A-to-I editing, and which one cannot? Unfortunately, so far no computational method whatsoever has been developed to address such an important problem based on the RNA sequence information alone. To fill this empty area, we have proposed a predictor called iRNA-AI by incorporating the chemical properties of nucleotides and their sliding occurrence density distribution along a RNA sequence into the general form of pseudo nucleotide composition (PseKNC). It has been shown by the rigorous jackknife test and independent dataset test that the performance of the proposed predictor is quite promising. For the convenience of most experimental scientists, a user-friendly web-server for iRNA-AI has been established at  RNA editing is a post-transcriptional modification that changes the genomic template through the insertion, deletion, deamination or substitution of nucleotides within the edited RNA molecule. Among the five types of RNA editing reported so far, the modification from adenosine to inosine, the so-called “A-to-I” editing, is the most common one , 2. ThisBesides altering the genetic code that can expand the transcriptome and proteome, the A-to-I editing may also involve in various important biological processes ranging from alternative splicing , nonsensAlthough the experimental method called “RNA-Seq” is a powerful tool for determining the RNA editing candidates , 14, it Therefore, it would be very useful for in-depth genome analysis or drug development to develop a sequence-based computational method that can effectively predict which adenosine sites in a RNA sequence can be “A-to-I” edited, and which ones cannot.The present study was devoted to address this problem.A computational method called “iRNA-AI” has been developed. It is the first predictor ever established by using the computational approach and sequence information alone to identify human A-to-I editing sites.Rigorous cross-validations on a well-established benchmark dataset  have shon, Sp, Acc, and MCC, see Eq.16 given later.For the rigorous but intuitive definitions about SSince so far there is no other existing computational method whatsoever for predicting the A-to-I editing sites in RNA of human transcriptome, it is not possible to show the predictor's power by the comparison manner between counterparts. Nevertheless, the power of iRNA-AI and its quality can be examined via a practical application on a set of experiment-confirmed independent dataset , which cThe above results indicate that the success rates achieved by the predictor iRNA-AI on the independent dataset of http://lin.uestc.edu.cn/server/iRNA-AI/.For the convenience of most experimental scientists, the web-server of iRNA-AI has been established at Moreover, to maximize the users’ convenience, a step-by-step guide has been provided in Because knowledge about the positions of A-to-I editing sites would be of great help for in-depth understanding the biological functions and processes concerned. It is anticipated that iRNA-AI will become a useful high throughput tool for understanding the biological significance of A-to-I RNA editing, or at the very least, a complementary tool to the existing experimental methods in this regard.Although the detailed ADAR's action mechanism is not well understood yet, the crystal structure of human ADAR has been solved (PDB code: 3IAR) that provides structural basis for the elucidation of its catalytic mechanisms and its specific recognition of the target sequence. Since both computational biology and structural biology have made great contributions to understand enzyme activities and catalytic mechanisms , 17, it Prediction is always difficult, particularly in dealing with a complicated biological system as studied here. Nevertheless, a prediction method would be deemed rewarding or successful if it could timely help getting some useful information or stimulate and inspire some other relevant methods. To realize this, we should make the following five procedures very clear as done in a series of recent publications –29 accor– ξ represents the ξ-th upstream nucleotide from the center, the N+ ξ the ξ-th downstream nucleotide, and so forth. The (2ξ + 1)-tuple RNA sample For facilitating formulation, the Chou's sequential scheme  was adopThe benchmark dataset is derived from DARNED database  that con program  to removIn Eq.5 the symbol After going through the above procedures, we obtained 6,243 positive label samples, from which we randomly picked out 3,000 to form the positive subset for the benchmark dataset. But keep it in mind that the remaining 3,243 samples would be used later for other purpose.The corresponding negative label samples were substantially more than the positive ones. Although this reflects the fact that in the real world most adenosine nucleotides in RNA cannot be edited to inosine, a machine learning predictor trained by an imbalanced or highly skewing benchmark dataset may negatively affect its performance , 45. To t theory .The detailed sequences for the samples in the benchmark dataset are given in In literature the benchmark dataset usually consists of a training dataset and a testing dataset: the former is for the usage of training a model, while the latter for testing the model. But as elucidated in a comprehensive review , there iThe detailed sequences for the samples in the independent dataset are given in With the avalanche of biological sequences emerging in the post-genomic era, one of the most challenging problems in computational biology is how to formulate a biological sequence with a discrete model or vector that can, however, reflect its essential sequence pattern or feature. This is indeed indispensible since nearly all the existing machine-learning algorithms were devised to handle vectors but not sequences, as elucidated in a recent review . UnfortuT is a transpose operator, while the subscript Z is an integer and its value as well as the components According to , the genRNA is formed by four types of nucleotides: A (adenosine), C (cytidine), G (guanosine), and U (uridine). Among the four types: 1) A and G have two rings, whereas C and U only one; (2) from the angle of chemical functionality, A and C can be categorized as amino group, while G and U as keto group; 3) in forming the secondary or tertiary structure, there are three hydrogen bonds between C and G but only two between A and U , C as , G as , and U as .iD is the density of the nucleotide Ni at the site i of a RNA sequence, Li the length of the sliding substring concerned, @ denotes each of the site locations counted in the substring, andTo reflect the occurrence frequency of a nucleotide and its distribution along the sequence of a RNA sample, we use the following equationsFor instance, suppose a RNA sequence “ACGUA”. The density of “A” at the sequence position 1, 2, 3, 4, or 5 is i-th nucleotide of Eq.3 can be uniquely defined by a set of four variables; i.e.,By combing Eq.9 and Eq.11, the Accordingly, the RNA sequence “ACGUA” can be successively expressed by the following five sets of digital numbers: , , ,  and . Or, according to the general form of PseKNC (Eq.8), it can be expressed asmeaning that the 5-tuple nucleotide example can be defined by a 5 × 4 = 20-D  PseKNC vector.Consequently, a 51-nt RNA sample in the current benchmark dataset can be formulated with a 51 × 4 = 204- D vector.Being a machine learning algorithm based on statistical learning theory, SVM has been widely and successfully used in the realm of bioinformatics , 73–75 ahttp://www.csie.ntu.edu.tw/~cjlin/libsvm/. Because of its effectiveness and fast speed in training process, the radial basis kernel function (RBF) was used to obtain the best classification hyperplane here. In the SVM operation engine, the regularization parameter C and the kernel width parameter γ were optimized via an optimization procedure using the grid search approach as described byC and In the current study, the LibSVM package 3.18 was used to implement SVM, which can be freely downloaded from The predictor obtained via the above process is called iRNA-AI, where “i” stands for “identify”, and “AI” for “A-to-I editing” sites in RNA sequence.How to objectively evaluate the anticipated success rates is an indispensible step in developing a new predictor . To addrTo quantitatively evaluate the quality of a binary classification predictor, four metrics are generally needed . They arNow it is crystal clear to see the following from Eq.16. When Accordingly, it has rendered the meanings of sensitivity, specificity, overall accuracy, and stability much more intuitive and easier-to-understand by using Eq.16, particularly for the meaning of MCC, as concurred recently by many investigators .Note that, however, the set of metrics as defined in Eq.16 is valid only for the single-label systems. As for the multi-label systems whose emergence has become more frequent in system biology –91 and sWith a set of good metrics to measure the quality of a predictor, the next thing we need to consider is what validation approach should be adopted to score these metrics. In statistical prediction, the following three cross-validation methods are usually applied: (1) independent dataset test, (2) subsampling  test, and (3) jackknife test . Of thesEven though, however, in order to reduce the computational time, the K-fold cross-validation approach has still often been used, as done by many investigators with SVM as the prediction engine . A


Eggplant latent viroid (ELVd) is a relatively small non-coding circular RNA that induces asymptomatic infections in eggplants (Solanum melongena L.). Like other viroid species that belong to the family Avsunviroidae, ELVd contains hammerhead ribozymes in the strands of both polarities that self-cleave RNAs producing terminal 5′-hydroxyl and 2′,3′-cyclic phosphodiester groups. Available experimental data indicate that ELVd replicates in the chloroplasts of infected cells through a symmetric rolling-circle mechanism, in which RNA circularization is catalyzed by the chloroplastic isoform of the tRNA ligase. In this work, a mutational analysis was performed to gain insight into the sequence and structural requirements of the tRNA ligase-mediated circularization of ELVd RNAs. In the predicted minimum free energy conformation of the monomeric linear ELVd RNA intermediate of plus (+) polarity, the ligation site is located in the lower part of an opened internal loop, which is present in a quasi-rod-like structure that occupies the center of the molecule. The mutations analyzed herein consisted of punctual nucleotide substitutions and deletions surrounding the ligation site on the upper and lower strands of the ELVd quasi-double-stranded structure. Computational predictions of the mutated ELVd conformations indicated different degrees of distortions compared to the minimum free energy conformation of the wild-type ELVd linear monomer of + polarity. When these mutant RNAs were expressed in Escherichia coli, they were all circularized by the eggplant tRNA ligase with approximately the same efficiency as the wild-type ELVd, except for those that directly affected the ribozyme domain. These results suggest that the viroid ribozyme domains, in addition to self-cleavage, are also involved in the tRNA ligase-mediated circularization of the monomeric linear replication intermediates. Pospiviroidae and Avsunviroidae, depending on the presence of particular domains in the viroid molecule; more specifically a central conserved region (CCR) that is present in all members of the family Pospiviroidae, but is missing in those of the Avsunviroidae, and hammerhead ribozymes that are exclusive of this last family  non-coding circular RNA. Despite the small size and with no help from self-encoded proteins, when these RNAs manage to enter compatible host cells they are able to move to appropriate cellular structures and to recruit the right host proteins to start their replication. Then the viroid progeny moves cell-to-cell through the plasmodesmata, the channels that traverse the cell walls of plant cells, and long-distance through the phloem to establish a systemic infection in the host plant frequently inducing a disease. The more than 30 viroid species known to date are classified into two families, t family .Potato spindle tuber viroid (PSTVd), and possibly all members of the family Pospiviroidae, follow the asymmetric variant in which the minus (-) multimeric RNAs directly serve as templates for the transcription of multimeric + RNAs, which are then cleaved into monomeric linear viroid RNAs, and are finally circularized acquiring their mature form (Avocado sunblotch viroid (ASBVd) and members of the family Avsunviroidae follow the symmetric variant in which the multimeric - RNAs self-cleave after the transcription through embedded hammerhead ribozymes, and the resulting monomers are ligated into monomeric circular RNAs. Next these viroid molecules, which are distinctive of this family, serve as templates in a second (symmetric) rolling circle for the transcription of multimeric + RNAs, which once again, are processed into monomeric circular RNAs of + polarity  polarity is arbitrarily assigned to the circular RNA strand that most abundantly accumulates in the host plant. Analyses of the replication mechanisms in different viroid species have indicated that members of both families follow two different variants of the rolling circle, termed asymmetric and symmetric . Potato ure form . In contpolarity .Potato spindle tuber viroid, and apparently its family members, replicate in the nucleus of infected cells  is able to traffic from the cytoplasm to the nucleus and from there to the chloroplasts  (Solanum melongena L.) and has been recently suggested to be a friendly experimental system to research many aspects of the Avsunviroidae molecular biology (Chlamydomonas reinhardtii (phylum Chlorophyta) are efficiently processed to monomers and circularized  , induces biology . Experim biology , viroid  biology , viroid  biology , 2012 an biology . Regardiularized . Furthernal loop . A combiA ligase  is the hA ligase . In vitrularized . A circuligation , 2018.E. coli co-expression system to analyze the effect of the mutations surrounding the ligation site in this reaction. Surprisingly, all the assayed ELVd mutants were efficiently circularized by the eggplant tRNA ligase in E. coli, provided they did not directly affect the ribozyme domain, which supports the notion that this RNA domain, in addition to viroid RNA cleavage, is also involved in the circularization step that is mediated by the tRNA ligase.To gain further insight into the sequence and structural requirements of the tRNA ligase-mediated circularization of ELVd, we herein used the AJ536613; note that ELVd is circular and A333 is followed by G1), which included the repetition of the + -strand hammerhead ribozyme domain under the control of the E. coli murein lipoprotein promoter and the 5S rRNA (rrnC) terminator (Table 1). Most of these pairs of primers, which harbored the desired mutations, contained a recognition site for type-IIS restriction enzyme BpiI at the 5′ ends. The PCR products of full plasmid size were eluted after separation by electrophoresis in 1% agarose gels. Next they were digested with BpiI (Thermo Scientific) and subjected to ligation with T4 DNA ligase (Thermo Scientific). In those cases in which primers did not harbor BpiI sites (Table 1), deletions simply resulted from ligation of phosphorylated  blunt end PCR products. In both cases, E. coli DH5α were electroporated with the products of the ligation reaction and the recombinant clones selected on plates with Luria-Bertani (LB) medium that included ampicillin. The plasmids that contained the desired mutations were selected after sequencing .Plasmid pLELVd contains a longer-than-unit ELVd cDNA  and p15LtRnlSm, a plasmid with a compatible p15A replication origin to express a recombinant version of the chloroplastic isoform of the eggplant tRNA ligase (GenBank accession no. JX0225157) under the control of the E. coli murein lipoprotein promoter and the rrnC terminator . The K219A mutant of eggplant tRNA ligase was obtained by mutagenic PCR on template p15LtRnlSm using primers 5′- CAAGTGTGACCTCGACTATAG-3′ and 5′-CGCATTCTGGATCTCTTTTTATG-3′, as previously described for ELVd mutants.rminator , 2018. TE. coli RNA was recovered after centrifugation and was frozen.Cells in the 2-ml cultures were harvested by centrifugation and resuspended in 50 μl of TE buffer . To break cells, one volume (50 μl) of a 1:1 mixture of buffer saturated phenol  and chloroform was added and the mix was vigorously vortexed. The aqueous phase that contained the N,N’-methylenebisacrylamide) that contained 8 M urea in TBE buffer . In some experiments, in order to further separate circular molecules, RNA preparations were subjected to a two-dimension electrophoresis. After separating the RNA as explained above, the whole lane was transversally laid on top of a second urea gel at a lower buffer concentration (0.25 × TBE) and electrophoresis continued.Aliquots of 10 μl of the RNA preparations were mixed with one volume of loading buffer that contained 98% formamide, and was denatured by heating for 1.5 min at 95°C, followed by snap cooling on ice. After denaturation, RNA was separated by electrophoresis in 5% polyacrylamide gels . Hybridization was performed overnight at 70°C in 50% formamide, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 100 ng/ml salmon sperm DNA, 1% sodium dodecyl sulfate (SDS), 0.75 M NaCl, 75 mM sodium citrate, pH 7.0, with approximately 1 million counts per minute of a 32P-labeled ELVd probe of - polarity. The hybridized membranes were washed three times for 10 min with 2 × SSC, 0.1% SDS at room temperature and once for 15 min at 55°C with 0.1 × SSC, 0.1% SDS . Hybridization signals were recorded by autoradiography using X-ray films (Fujifilm). The radioactive RNA probe consisted of a full-length ELVd RNA monomer of complementary polarity. This probe was obtained by in vitro transcription of a linearized plasmid with 20 U of T3 bacteriophage RNA polymerase (Roche) in 20-μl reactions that contained 40 mM Tris-HCl, pH 8.0, 6 mM MgCl2, 20 mM DTT, 2 mM spermidine, 0.5 mM each of ATP, CTP and GTP, and 50 μCi of [α-32P]UTP (800 Ci/mmol), 20 U RNase inhibitor  and 0.1 U yeast inorganic pyrophosphatase (Thermo Scientific). Reactions were incubated for 1 h at 37°C. After transcription, the DNA template was digested with 20 U DNase I (Thermo Scientific) for 10 min at 37°C. The probe was purified by chromatography in a Sephadex G-50 column .After electrophoresis, RNAs were electroblotted to positively charged nylon membranes  and were cross-linked by irradiation with 1.2 J/cm1.The Mfold algorithm  was usedquasi-double-stranded structure that is present in the central part of the molecule . In this region, this predicted conformation mostly agrees on those of two independent experimental determinations of circular ELVd + RNAs obtained by high-throughput selective 2′-hydroxyl acylation analyzed by primer extension (hSHAPE) , the first set of mutations were placed on the upper strand of the loop.In the predicted conformation of minimum free energy of the monomeric linear + ELVd RNA (sequence variant AJ536613), the ligation site (positions 333-1) lays on the border of an internal opened loop in a long (hSHAPE) . To studFigure 2). Substitution C200G closes the loop on the right-hand side, whereas G201A causes a strong restructuring of the whole region . The longer-than-unit ELVd transcripts that contained these mutations where co-expressed in E. coli along with the eggplant tRNA ligase. Total RNA was extracted from E. coli cells and separated by denaturing PAGE. Finally, ELVd + strands were detected by Northern blot hybridization using a complementary radioactive probe. Two prominent bands that apparently corresponded to the monomeric linear and monomeric circular ELVd RNAs of + polarity were detected in the lanes that corresponded to all four mutants . The intensity of these bands did not differ from those in the wild-type ELVd control . The controls of this experiment also included the single expressions of the wild-type ELVd or the tRNA ligase. In these controls, E. coli cells were co-transformed with the corresponding empty expression plasmids. Only faint bands were detected when the tRNA ligase was not expressed , and no band was observed when ELVd RNA was not expressed . As previously shown, ELVd RNAs only accumulate efficiently in E. coli cells in the presence of eggplant tRNA ligase  very precisely matches the expected position of the ELVd linear monomers of 333 nt, while the upper one  corresponds to a species that displays the electrophoretic behavior expected for the ELVd circular monomers, which must be delayed from the linear counterparts under denaturing conditions. To ensure that the upper bands corresponded to the ELVd circular monomers, equivalent aliquots of the above samples were separated by two-dimension denaturing PAGE at two different ionic strengths and the ELVd + strands were detected by Northern blot hybridization. As expected for circular RNA molecules, the slow migrating species further delayed in the second dimension at a low ionic strength, and deviated from the diagonal of the linear RNAs . Taken together, these results indicate that the four assayed punctual nucleotide substitutions, which induced different alterations in the secondary structure of the loop on which the + ELVd ligation site is located have no effect on the eggplant tRNA ligase-mediated circularization.Of the two prominent bands, the lower . The ELVd longer-than-unit transcripts that contained these deletions were co-expressed with eggplant tRNA ligase in E. coli and total RNA was extracted. RNA was separated by denaturing PAGE and the + ELVd strands were revealed by Northern blot hybridization. Two prominent bands, whose position was consistent with the migration of the monomeric linear and circular ELVd RNAs, were once again detected . Like the above experiment, the intensity of these bands did not substantially differ from those in the control in which the wild-type ELVd was expressed . The same results were observed in the analysis of two additional independent E. coli clones of each mutant. These results indicate that the two assayed deletions, despite the strong impact on the secondary structure of the ligation domain, have no effect on the efficiency of the tRNA ligase-mediated circularization of ELVd.We reasoned that the assayed punctual nucleotide substitutions may not have sufficiently altered the secondary structure of the loop that contains the ligation site to affect circularization. To test this hypothesis, two additional ELVd mutants were created, which consisted in the deletion of five (from position C197 to G201) or eleven (from position U194 to G204) nucleotides of the upper RNA strand. The Mfold prediction of the minimum free energy conformation of these deleted monomeric linear ELVd + RNA forms showed a profound alteration of the secondary structures around the ligation site in these two cases  and some of these deletions were expected to have a deleterious effect on the self-cleavage of the ELVd + RNA precursor. The two deletions that directly affected the ribozyme domain were the 7-nt deletion from G1 to U7 that completely eliminated the upper strand of the hammerhead helix I and the 5-nt deletion from G38 to U42 that eliminated a key hammerhead conserved motif. The 10-nt deletions from U47 to G56 and from G317 to U326 were created in both borders of the ribozyme domain . We co-expressed longer-than-unit transcripts containing these deletions with the eggplant tRNA ligase in E. coli and analyzed the ELVd + RNAs that accumulated in the bacteria by Northern blot hybridization. Bands corresponding to the monomeric linear ELVd + RNA were detected for all four mutants, but bands corresponding to monomeric circular ELVd + RNAs were only observed for deletion mutants not directly affecting the ribozyme domain . Same results were obtained in the analysis of two additional independent E. coli clones of each mutant.Since punctual mutations and deletions on the upper strand of the E. coli experimental system, the hammerhead ribozyme in addition to ELVd RNA cleavage was also catalyzing the RNA circularization. To do that, we compared the circularization of the wild-type ELVd transcript in the presence of the eggplant wild-type tRNA ligase and the K219A catalytic mutant. This mutation in a nucleotidyltransferase key lysine completely prevents the binding of an ATP molecule, which is critical for the function of different ATP-dependent ligases . This result supports the notion that, despite the observed importance of the viroid hammerhead ribozyme domain, the catalytic activity that circularizes the ELVd RNA resides in the co-expressed eggplant tRNA ligase.In view of these last results, we decided to rule out the possibility that, in our  ligases . By NortAvsunviroidae, like ELVd  of the RNAs of the family oroplast . With th species . In thisnal loop . This rel groups .C. reinhardtii system having many interesting properties to study viroid processing, it also has some limitations. First, C. reinhardtii chloroplast transformation is labor-intensive and time-consuming. Second, and possibly most importantly, viroid ligation is catalyzed by an undefined enzyme in C. reinhardtii chloroplasts. To overcome these limitations, and once the host enzyme involved in viroid circularization was known, we set up a new experimental system that consisted of E. coli recombinant clones in which ELVd longer-than-unit transcripts and the chloroplastic isoform of the eggplant tRNA ligase were co-expressed. In these cells, ELVd transcripts self-cleaved to monomers, which were recognized by the eggplant tRNA ligase and efficiently circularized. However, no viroid RNA-to-RNA replication was detected . In the upper strand, we assayed four punctual nucleotide substitutions that have different effects on the secondary structure of this domain, as well as two deletions with a strong effect. In the lower strand, we assayed two deletions that directly affected the viroid ribozyme and only one of them had a strong effect on the secondary structure of the ligation domain, as well as two deletions outside the ribozyme domain, again with only one having a strong effect on the secondary structure of the ligation domain . Interestingly, both ELVd deletion mutants directly affected in the ribozyme domain were still able to self-cleave in E. coli, although with a low efficiency, most likely through the trans-complementation activity of the second wild-type ribozyme. Note that the + ELVd longer-than-unit transcripts expressed in E. coli consisted of viroid monomers flanked by two complete ribozymes. In these transcripts, the full-length viroid monomers (from G1 to A333) were preceded by ELVd nucleotides C327 to A333 and followed by ELVd nucleotides G1 to G46 to form two complete ribozymes that release the full-length monomeric linear intermediates.In the present work, we used this E. coli, except for those (ΔG1-U7 and ΔG38-U42) that directly affected the ribozyme domain . With the only concern that the precursors of these two mutants self-cleave in E. coli with low efficiency, due to mutations in one of the ribozymes, lack of circularization of the corresponding monomeric linear intermediates suggests the hypothesis that the eggplant tRNA ligase may not recognize the conformation of minimum free energy, rather a hypothetical transient structure formed by the lower strand of ELVd. This hypothesis would reconcile previous in vitro results in which + ELVd monomers opened at sites different to that which corresponded to the hammerhead ribozyme were not circularized by a purified eggplant tRNA ligase  or when a ligase catalytic mutant was expressed . A deeper analysis with additional mutants in the ribozyme domain will be necessary to shed some more light about the contribution of the viroid hammerhead ribozyme domain in ELVd circularization.Despite having more or less affected the secondary structure of the ligation domain, most of the ELVd mutants analyzed in this work  were efficiently circularized by the eggplant tRNA ligase in A ligase . The hamreaction  because J-AD conceived the work and designed the experiments in close collaboration with TC and BO. TC and BO performed the experiments. TC, BO, and J-AD analyzed the data. J-AD wrote the manuscript with inputs from TC and BO. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.