Although viruses infect various organs and are associated with diseases, there may be many unidentified pathogenic viruses. The recent development of next-generation sequencing technologies has facilitated the establishment of an environmental viral metagenomic approach targeting the intracellular viral genome. However, an efficient method for the detection of a viral genome derived from an RNA virus in animal or human samples has not been established. Here, we established a method for the efficient detection of RNA viruses in human clinical samples. We then tested the efficiency of the method compared to other conventional methods by using tissue samples collected from 57 recipients of living donor liver transplantations performed between June 2017 and February 2019 at Kyushu University Hospital. The viral read ratio in human clinical samples was higher by the new method than by the other conventional methods. In addition, the new method correctly identified viral RNA from liver tissues infected with hepatitis C virus. This new technique will be an effective tool for intracellular RNA virus surveillance in human clinical samples and may be useful for the detection of new RNA viruses associated with diseases. Viruses are intracellular parasites; they can replicate only within cells. They were first identified from plants at the end of nineteenth century . VirusesNext-generation sequencing technologies have recently been developed and have exerted a strong influence on the field of genomic research. These advanced technologies have enabled us to detect virus genomes more effectively . Most viUrayama and colleagues established a dsRNA sequencing method named FLDS (fragmented and primer ligated dsRNA sequencing) to determine the full-length sequences of intracellular RNA viruses. This method includes the following steps: dsRNA purification with a cellulose column, dsRNA fragmentation, adapter ligation to the fragmented dsRNA, cDNA synthesis using a SMARTer system with a primer complementary to the adapter sequence and cDNA amplification. In a previous study, they applied FLDS to marine microorganisms and successfully determined the sequences of several hundred full-length RNA viral segments. However, it is not clear whether the FLDS method is the best method for high-throughput and/or screening application for human samples . In the Living donor liver transplantation (LDLT) is a choice of treatment for chronic or acute liver failure. There are many causes of liver failure, including cholestatic diseases such as primary sclerosing cholangitis (PSC) or primary biliary cholangitis (PBC). In addition, hepatocellular diseases, including liver cirrhosis related to hepatitis B virus (HBV) or hepatitis C virus (HCV) infection, and alcoholic and nonalcoholic fatty liver disease (NASH) are frequent causes . HoweverThe experiments using liver samples from LDLT recipients were conducted at Kyushu University. Between June 2017 and February 2019, we enrolled 57 LDLT recipients at the Kyushu University Hospital. Liver specimens were obtained from each of these recipients . We obta5 PFU) of recombinant strain Miyazaki-Bali/2007 (rsMB) generated by reverse genetics [The experiments using lung tissue samples from mice were conducted at Osaka University. Lung samples were prepared from mice using a lethal murine infection model for Pteropine orthoreovirus (PRV) . Four-wegenetics . A lung Magnaporthe oryzae strain APU10-199A, containing three RNA viruses, were incubated in YG broth (0.5% yeast extract and 2% glucose) with reciprocal shaking (60 rpm) at 25 °C for two weeks [The experiments using fungi cells were conducted at Tsukuba University. To obtain fungi cells, mycelial plugs of wo weeks . The experiments using liver samples from mice were conducted at Yamanashi University. Five Apodemus speciosus and forty-three Myodes rufocanus bedfordiae were captured in Tobetsu Town, Hokkaido. Liver samples were obtained from each mouse. The mouse experiments were conducted following the approval of the Animal Research Committee of Graduate School of Medical Science, Yamanashi University.2, 1.9 mM ZnSO4, and 189 mM NaCl) and was then incubated at 37 °C for 15 min. DsRNA was purified using a ZymoClean Gel RNA Recovery Kit . DsRNA for each sample was sheared to about 200 bp using a Covaris S220 ultrasonicator (Covaris). After the denaturation of dsRNA at 95 °C for 3 min, each library was prepared using a SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (TaKaRa/Clontech).DsRNA was purified as described by Okada et al. with a few modifications ,20. A mecDNA was obtained as described previously . In brieTotal RNA was isolated using a TRIzol Plus RNA Purification Kit (Invitrogen) according to the manufacturer’s protocol. The RNA fraction was treated with DNase I  and purified using an RNA Clean & Concentrator-5 (Zymo Research). rRNA was removed using a RiboMinus Eukaryote System v2 (Invitrogen). The cDNA was amplified using a SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (TaKaRa/Clontech).Paired-end sequencing (100 bp × 2) was performed on the MiSeq and HiSeq3000 platforms. Sequencing reads were trimmed of their adapter sequences by cutadapt 1.8.1 and then mapped onto a custom database, which includes human and microorganism sequences from NCBI-nt by bwa. The taxonomic information of each sequence was assigned and summarized by BioRuby scripts. Trimmed reads were mapped on the viral reference sequences obtained from a public database with CLC Genomics Workbench version 11.0  using the following parameters: mismatch cost of 2, insertion/deletion cost of 3, length fraction of 0.8 and similarity fraction of 0.8. To detect novel RNA viruses, contigs were obtained de novo exclusively using the CLC Genomics Workbench version 11.0 (CLC Bio) with the following parameters: a minimum contig length of 500, word value set to auto and bubble size set to auto. The contig sequences were compared against the NCBI non-redundant amino acid databases using BLASTX-plus.Total RNA was extracted from recipient liver samples using a PureLink RNA Mini Kit . The synthesis of the first-stranded cDNA was performed by using a PrimeScript RT Reagent Kit (Takara Bio). The HCV NS5B region sequence was amplified from the synthesized cDNA using PrimeSTAR GXL DNA polymerase (Takara). The following primer set was designed for the NS5B gene: F1, CTACTTGCTCCGAGGAG; R1, AGTCTTCCAGGAGGTCCTTC; F2: GCCGTTAACCACATCAAGTC; R2, ATACCTGGTCATAGCCTCCG; F3, ATCTCAGAAAGCCAGGGGAC; R3, ACCTTTCACAGCTAGCCGTGAC. Amplicons were purified after agarose gel electrophoresis using a Gel/PCR DNA Isolation System (VIOGENE). The NS5B sequence was determined by direct sequencing using a BigDye Terminator v3.1 Cycle Sequencing Kit and ABI3130 Genetic Analyzer (Applied Biosystems). The generated NS5B sequences were aligned using Clustal W. Neighbor-joining phylogenetic trees were inferred in MEGA X using the Tajima–Nei model and gamma-distribution of rates among sites. The confidence of the branches was assessed by the bootstrap test using 500 replicates.2. The Huh7-derived cell line Huh7.5.1 was kindly provided by F. Chisari, and was maintained in DMEM  supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin (Sigma), and 10% fetal bovine serum (FBS). Primary human hepatocytes (PHHs) and PXB cells were isolated from urokinase-type plasminogen activator transgenic/SCID mice inoculated with PHH  and cultured in the medium according to the manufacture’s protocol. pHH-JFH1 encoding full-length cDNA of the JFH1 strain [All cell lines were cultured at 37 °C under the conditions of a humidified atmosphere and 5% CO1 strain ,22 was k1 strain , was intThe human hepatoblastoma-derived HepG2-hNTCP-C4 cells were maintained in the above medium supplemented with 400 μg/mL G418 . HepAD38.7 cells were cultured in DMEM/F-12 medium supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 18 μg/mL hydrocortisone (Sigma), 5 μg/mL insulin (Sigma), 400 μg/mL G418, and 400 ng/mL tetracycline . HepG2-hNTCP-C4 cells and HepAD38.7 cells were kindly provided by Dr. T. Wakita. The identification of human sodium taurocholate co-transporting polypeptide (hNTCP) as an HBV receptor led to generation of the HepG2-hNTCP-C4 cell line, which stably expresses hNTCP and therefore readily permits the infection and replication of HBV ,25. For We examined whether this modified dsRNA-seq method was more efficient at detecting virus reads than other conventional methods. Three methods were compared—rRNA-depleted total RNA sequencing , the FLDS method and our modified dsRNA-seq method—using fungus samples and two LDLT recipient liver samples. In the analysis of the fungus samples, for every 100,000 reads, total RNA-seq detected 4526 reads of viral genome (4.53%), the FLDS method detected 55,132 reads (55.13%) and the modified dsRNA-seq method detected 26,185 reads (26.19%) A. The FLAbove, we demonstrated that viral genomes were accurately detected in clinical samples by the modified dsRNA-seq method. Next, we evaluated whether this method would work effectively in animal samples or cultured cells. Lung tissue of a reovirus-infected mouse and Huh7.5.1 cells and PHHs inoculated with HCV (JFH-1 strain) were used to test the feasibility of this method. Surprisingly, 91.8% and 60.8% of total reads in the lung tissue from the mouse and the cultured cells were derived from virus reads, respectively A. ReovirWe next examined the feasibility of using the modified dsRNA-seq method with human clinical samples. For this purpose, we assessed whether the modified dsRNA-seq method could identify new RNA viruses associated with diseases or clinical states in liver samples obtained from LDLT recipients (n = 57) . Three oWe could detect HCV correctly from human clinical samples using the modified dsRNA-seq method. In the analysis of recipient liver samples, we used eleven liver samples previously infected with HCV, nine of which were found to contain HCV-RNA in the preoperative blood test A. We couThe current modified dsRNA-seq method was established to detect RNA viruses. Interestingly, the method allowed us to identify HBV in an analysis of samples obtained from a recipient liver infected with HBV. In this analysis, we used eight liver samples previously infected with HBV, among which five liver samples had been revealed to contain HBV-DNA in a preoperative blood test A. HBV reBunyavirales families and was associated with liver disease in wild rodents. In this analysis, we could identify the L and M segments of the Fulton virus, and when viral reads obtained from this liver sample were mapped, we found that they gave a high sequence coverage (RNA virus surveillance was performed using the modified dsRNA sequencing method from liver samples of wild rodents obtained in Hokkaido. Four or five liver samples were mixed and analyzed. We were able to identify the Fulton virus in some liver samples of Myodes rufocanus bedfordiae . The Fulcoverage A,B. We cIn the current study, we established a dsRNA-seq method as an RNA virus surveillance method by improving the FLDS method. RNA viruses infected in human clinical samples or animal samples could be detected accurately by using this method. Moreover, we could almost determine the full-length viral genome with high coverage by analyzing viral reads obtained from this method. In addition, HCV could be detected from a recipient liver sample infected with HCV, and we could perform detailed analyses such as phylogenetic tree analysis or quasi-species analysis. We demonstrated that this modified dsRNA-seq method had the potential to detect viral reads efficiently in a wide variety of samples. As mentioned above, we developed this method based on the FLDS method. In the FLDS method, cDNA synthesis was performed by ligating loop primers on the 3′-terminal end followed by reverse transcription. This protocol had the advantage that loss of both ends due to PCR was minimized and the full-length genome could be analyzed . In the In this analysis, the raw sequence reads generated from NGS were evaluated by BLAST. BLAST compares the similarity between sequences, which means that only known sequences registered in the NCBI database can be evaluated. It is difficult to evaluate unidentified sequences or viruses; this is the limitation of virus surveillance. In our analyses, 30%–40% of unassigned reads were detected from human clinical samples using the modified dsRNA-seq method. It is possible that viruses associated with diseases were present in the unassigned reads. Therefore, it will be necessary to find a better method to search for viruses.Although HBV and HCV infect the human liver, organs such as the liver have their own immune systems, and thus it may be difficult for other viruses to infect human organs while escaping from their immune systems. Unlike the lungs or intestinal tract, the liver has no opportunity to directly contact the outside environment with its many pathogens. Novel RNA viruses may be identified from clinical samples, such as lung or intestinal tract samples. We should apply this modified dsRNA-seq method to these other human clinical samples because there is the potential to detect viruses associated with diseases of unknown etiology, which could lead to the discovery of new treatments and preventive measures. We analyzed about 60 recipient liver samples in this analysis, which is still a relatively small number. Further analyses will be needed to identify RNA viruses from human clinical liver samples. We established the modified dsRNA-seq method to detect RNA viruses more efficiently from clinical samples. We demonstrated that this method detected RNA viruses more efficiently than other conventional methods. In the future, it may be possible to elucidate associations between various viruses and diseases by analyzing the organ-specific viromes obtained by using this method. This method was an effective tool for intracellular RNA virus surveillance using human clinical samples, and it was shown to have good potential for the detection of new RNA viruses.


This review examines the quantitative behavioural studies that have evaluated the effects of Montessori pedagogy on children’s psychological development and school learning. The analyses of only three “Randomized Controlled Trials—RCT” studies published to date reveal varied and contradictory effects. Firstly, these findings are discussed in the light of several methodological limitations: the absence of active control groups, small sample sizes, diversity of measures, or lack of control over the implementation fidelity of both Montessori and conventional pedagogy. Secondly, these findings are discussed in the light of what Montessori pedagogy does not emphasise in its conception of development and the role of the teacher, namely the place given to language and pretend play. Montessori pedagogy has been very successful in recent decades ,2. This It is important to recall the historical and cultural context in which this pedagogy emerged to better understand its characteristics. Maria Montessori, a doctor by training, was called upon in 1902 to take care of children from an underprivileged area of Rome in Italy in the first “Children’s House” of its kind . The chiFinally, more globally, it is necessary to recall that the Montessori pedagogy and most other alternative pedagogies were developed in the first half of the 20th century in the movement of new education, when teaching was very top-down from the teacher to the student. These alternative pedagogies put children’s needs and abilities first. Compared to this conventional top-down view of instruction, alternative pedagogies are based on a more bottom-up vision of knowledge and learning.Currently, several structural and specific characteristics are present in schools that offer Montessori pedagogy : teacherThis review does not aim to discuss in detail whether the eight principles of Montessori pedagogy correspond to current researches in developmental and learning sciences ,5, but aIn the educational field, it is traditional to consider two general types of scientific approaches. The first, called “hypothetical-deductive”, starts from theories or laws that are as general as possible, drawing predictions from them that are compared with data from experience. The second, called “explanatory”, starts from what is observed and questions its determinants by attempting to trace the causal chain. These two approaches are linked, and there is a back-and-forth flow between them. In both approaches, researchers use complex causal systems involving several explanatory factors revealed through statistical analysis. Both approaches are legitimate in the field of education ,14.The “hypothetico-deductive” approach is favoured in research in interventional cognitive sciences. This type of research mainly uses the so-called experimental method. Its main objective is to “administer the evidence”, i.e., to show that a factor  is indeed the main cause of the appearance of observed behaviour , “all things being equal” . To acquTo measure the effectiveness of an “intervention” or “pedagogy”, three (from A to C) levels of methodological rigour can be considered in the type of experimental protocol used by researchers ,16. Two In this perspective, level A corresponds to studies using “intervention-only” protocols. The intervention is conducted with a single group of students, and pre- and post-intervention measures are carried out to evaluate the impact of the intervention but without comparison to a reference control group. This type of protocol makes it possible to test the “feasibility” of the intervention but prevents the interpretation of the results since no control group can ensure that students not benefitting from the intervention do not show the same evolution on the measured variables. In this review, we have chosen not to present the results of the Level A studies given their difficulties of interpretation.Level B corresponds to studies conducting an intervention with a group of students  and comparing the pre- and post-intervention measurements to a control group of students with no random assignment. Studies unable to do so correspond, for example, to a student assignment guided by the teacher’s voluntary commitment to the research and, therefore, the students in his or her class. With this type of study, a distinction can also be made between the “active control group”  and the “passive control group” . In this case, while this type of study allows for comparison with a reference group, it does not prevent possible well-known psychosocial effects (such as positive expectations of the intervention or the placebo effect) in the group benefiting from the intervention, which may influence the measures. However, the effects of passive and active controls on cognitive interventions are in debate. Two meta-analyses to estimate their relative effect sizes showed no meaningful performance difference between passive and active controls, suggesting that current active control placebo paradigms might not be appropriately designed to reliably capture these non-specific effects or that these effects are minimal in this literature . The anaLevel C corresponds to studies conducting an intervention with a group of students  and comparing the pre- and post-intervention measurements to a control group (CG) of students with a random assignment of students. Studies able to carry out a random assignment are called “randomized studies or randomized controlled trials—RCT”. These studies can be “double-blind”, but this condition cannot ensure a pedagogical intervention for the teachers, in particular, or the experimenters. As with Level B, a distinction can be made between the active and passive control groups.Several authors have synthesized some of the studies that have evaluated the effects of Montessori pedagogy on children’s psychological development and learning ,19,20. WLillard and Else-Quest  evaluateAt age 5, Montessori students scored better on the cognitive flexibility test than conventional students (d = 0.61), but the two groups did not differ in inhibition or on the three tests assessing reasoning. Academic skills were higher for Montessori students in math problem solving (d = 0.55), reading (d = 0.44) and phonology (d = 0.63), and equivalent between the two groups in vocabulary. This lack of difference between the two groups on the vocabulary test suggests that the previous results cannot be mainly explained by the socio-economic level of the parents . StudentAt age 12, Montessori students wrote more creative stories (d = 0.71) with more sophisticated sentence structures (d = 0.59). However, there were no differences in spelling, punctuation, or grammar in the stories written by the children in the two groups. The 12-year-olds in the Montessori group also had better social skills (d = 0.73) and expressed better academic well-being (d = 0.54). However, there was no difference between the groups in the reasoning tasks. In addition, there were no differences between the groups in the various math and language  tasks.Science’ subsequently published critical comments. Lindenfors (2007) [As this study generated a lively debate, the journal ‘s (2007)  criticizs (2007) , for hisn = 70) was divided into 11 triple-level classes from two AMI-recognized Montessori public “magnet” schools. The students in the control group (n = 71) were divided into 71 conventional schools. Thirty of these schools were public , and 41 were private. The “magnet” schools are public schools that receive additional funding from the state and offer specialized education. Their admission system operates by drawing lots, and their objective is to promote social mixing by attracting students from both well-off and disadvantaged backgrounds. Head Start programs aim to prepare children from birth to 5 years old from low-income families for school by supporting their overall development. “Head Start” thus includes “Head Start” preschool programs for 3–4-year old and “Early-Early Head Start” programs for toddlers and pregnant women. The sample included a wide range of income levels and parental education, but the two groups did not differ in these demographic variables. Children were tested at least three times over the four testing sessions organized over their three years of kindergarten: once at the beginning (T1), then once at the end of each school year . The tests repeated those of 2006 with a few additions, such as an evaluation of perseverance in the face of effort  and creativity. The authors grouped performance on the language (vocabulary and reading) and math (problem-solving and numeracy) tests into a single measure called “academic skills”. They then grouped a task assessing behavioural inhibition and visuospatial ability (figure copying) into a factor called “executive functions”. Unfortunately, the descriptive data are not reported in the article.Subsequently, Lillard et al.  comparedAnalyses conducted at T1 revealed no initial differences between the groups in the different tests assessed. The authors then observed that, compared to the control group, Montessori students progressed more in the overall academic skills score (d = 0.41 at T4) and the theory of mind test (d = 0.32 at T4). In addition, they were more persistent in their efforts in T3 and T4 (65% Montessori students versus 47% control students). Montessori students at all grade levels reported on average more enjoyment of academic tasks than students in the passive control group. Nevertheless, the creativity test revealed similar results between the two groups at each assessment time. Furthermore, contrary to the results of their previous cross-sectional study, there was no difference between the two groups in the proportion of references to justice in solving a social problem and executive functions. Since the different academic skills were not examined individually, it is difficult to compare these results with those of the previous study regarding reading and mathematics learning.Recently, Courtier et al.  conducteStatistical analyses reveal an absence of difference between the two groups in the cross-sectional study at the end of the year for almost all the classic or original tests or trials: Evaluation of oral language; phonological awareness; pragmatic reading test; math problem solving; digital skills; self-regulation; short-term memory and visuospatial working memory; labyrinth test; social problem-solving test-revised; theory of mind; allocation of resources to a third party; dictator game test; feeling about school test. The results revealed better performance for the Experimental Group (EG) regarding a French reading test . The analysis of the progress made between the two groups and between the 3–4-year-olds and the 5–6-year-olds confirms the previous results.Regarding the literacy curriculum in both Montessori and non-Montessori public schools in France, both made use of exercises intended to develop phonological  awareness, letter recognition, and knowledge of letter/sound correspondences on the understanding and use of the alphabetic principle in pre-reading kindergarten children ,32. ThesThe results reveal varied and contradictory effects of Montessori pedagogy on the psychological development of children and school learning. These results may be explained by several methodological limitations present in most of these studies, which can sometimes result from the complexity of the task.Firstly, Montessori schools can be public or private institutions of very different sizes, ranging from small family-based care structures to schools with several hundred students. Moreover, the integration of children in Montessori schools often results from a parental choice, which indicates a family sensitivity to pedagogy. Analysis of the studies reveals that most of them are not randomized studies and, even when this is the case, the results are contradictory in 5-year-old children for certain skills.n = 36) from the so-called supplemented Montessori classes (n = 41). These two types of classes respected the fundamental criteria of Montessori pedagogy, such as multi-aging, individualization of teaching, or lack of rewards. Nevertheless, the supplemented Montessori classes offered non-Montessori materials  and the interruption of working time by special lessons  two to three times a week. Supplemented Montessori classes also sometimes involved a second teacher in the class. Thus, 95–100% of the students used Montessori materials in the three so-called high-fidelity classes, compared to 38–56% in the nine supplemented classes. Employing the tests used in 2006, the researchers observed greater progress in the high-fidelity group for executive functions, reading, vocabulary, and mathematics, but not for the other tests of social skills and mind theory .Another argument favouring the heterogeneity of the results could be the degree of fidelity to which the Montessori method was applied. Following the observation that the fidelity of implementation of the Montessori pedagogy was not respected or was not reported in most of the previously published articles, Lillard  differenIn this vein, it is important to recall that the three selected RCT studies do not include a fully active control group with an alternative pedagogy that would be measurable on the level of its implementation. This aspect is also another limitation that should be considered in future research evaluating the effectiveness of an integrated curriculum in the daily classroom reflecting a certain type of pedagogy   highlighA final limitation is the publication bias of this type of study. This bias means that the majority of the published studies show an effect, which potentially prevents the dissemination of non-significant results. This publication bias mechanically leads to the over-representation of false positives and the overestimation of effect sizes in meta-analyses . MoreoveFinally, if we analyse the latest studies published very recently, most beneficial effects are observed when researchers test students from high socio-economic backgrounds voluntarily enrolled in private schools   defined In conclusion, many features in the Montessori pedagogy positively take children’s developmental needs into account and should be an integral part of teachers’ working modalities in so-called “conventional” public schools . However, Montessori’s view of the role played by language as well as by pretend-play should be analysed more carefully and could also partly explain the contrasting results of the different studies highlighted in this article.Finally, it is critical not to ignore the role played by parents in their childrens’ global development . The fact that children in Montessori pedagogy are globally at least as competent as other children in more conventional pedagogy is likely due to the parent’s conception of education finality . Maybe, these parents are already sensitive to the importance of scaffolding their children’s language skills from birth, allowing more time for certain forms of play, and maybe are more concerned about their child’s academic future than other parents who are less inclined or aware of the importance of these stimulations. They may also have at least partially compensated the results obtained on some measures. In the two first RCTs studies described above, the recruitment process leads us to believe that the children selected are likely to have parents who are maybe more academically involved and supportive. In the first RCT study, all parents volunteered to have their child follow Montessori pedagogy in an AMI-affiliated school. In the second RCT study, parents “had submitted a lottery application (…) selecting one of the two Montessori schools as their first of five school choices”  (Lillard


RNA sequencing enables high-content/high-complexity measurements in small molecule screens. Whereas the costs of DNA sequencing and RNA-seq library preparation have decreased consistently, RNA extraction remains a significant bottleneck to scalability. We evaluate the performance of a bulk RNA-seq library prep protocol optimized for analysis of many samples of adherent cultured cells in parallel. We combined a low-cost direct lysis buffer compatible with cDNA synthesis (in-lysate cDNA synthesis) with Smart-3SEQ and examine the effects of calmidazolium and fludrocortisone-induced perturbation of primary human dermal fibroblasts. We compared this method to normalized purified RNA inputs from matching samples followed by Smart-3SEQ or Illumina TruSeq library prep. Our results show the minimal effect of RNA loading normalization on data quality, measurement of gene expression patterns, and generation of differentially expressed gene lists. We found that in-lysate cDNA synthesis combined with Smart-3SEQ RNA-seq library prep generated high-quality data with similar ranked DEG lists when compared to library prep with extracted RNA or with Illumina TruSeq. Our data show that small molecule screens or experiments based on many perturbations quantified with RNA-seq are feasible at low reagent and time costs. The typical RNA-seq workflow starts with RNA extraction from samples, followed by cDNA synthesis, adaptor ligation, size selection and high-throughput sequencing. The sequences are aligned to a reference genome and/or transcriptome to generate a genome wide transcription map which includes the transcriptional structure and/or expression profile for each gene3.RNA sequencing (RNA-seq) is a popular tool in modern biology that has expanded our understanding of the regulation and complexity of the transcriptome of many species. Primarily used for differential gene expression analysis, other applications include transcript variants detection, alternative splicing, pathway analysis and exploring cellular heterogeneity and diversity in stem cell biology2. By studying the effects of many perturbations  in parallel on cellular gene expression, efforts such as the Connectivity Map4 have uncovered novel mechanisms of disease and therapeutic targets. However, significant bottlenecks impede adoption of highly parallelized methods in the absence of robotics equipment. Highly efficient RNA extraction is simple to perform in a small number of samples but becomes costly and laborious at the scale of hundreds of samples, particularly for adherent cells that may require dissociation prior to processing. Moreover, highly multiplexed RNA-seq library prep generally requires costly commercial kits.RNA-seq workflows have become progressively more efficient over time. There are at least 100 distinct RNA-seq library prep protocols9. Recently, Foley et al.10 developed a simple method (Smart-3SEQ) for rapid RNA-seq library prep; this was tested with laser micro dissected tissue applied to cDNA synthesis and with purified RNA, taking up to 4 h on average and with low reagent costs.Prior work has demonstrated that qPCR can be accurately performed using bulk human primary and transformed/cancer cell samples without RNA extraction. These methods are based on the use of a lysis buffer compatible with in-lysate cDNA synthesis and downstream sample processing and have been applied to single cell RNA-seqWe hypothesized that in-lysate RNA-seq library prep with Smart-3SEQ would reduce reagent and time costs while maintaining quality comparable to a gold standard. We did side-by-side comparisons of library prep in-lysate vs. using purified RNA, followed by either Smart-3SEQ or Illumina TruSeq. We found that in-lysate cDNA synthesis allows generation of high-quality sequencing data without RNA input normalization. The gene expression profile of in-lysate RNA highly correlated with purified RNA. When compared to our gold standard, both methods showed similar performance for detection of DEGs and for pathway analysis. Thus, our data suggests that in-lysate RNA-seq library prep with Smart-3SEQ facilitates high-quality RNA-seq analysis of cultured adherent cells.10. We further used purified RNA for TruSeq RNA-seq library prep as a gold standard  of RNA input at scale is costly and laborious, so we examined the performance of normalization free in-lysate cDNA synthesis. For in-lysate samples, RNA input was neither normalized for cDNA library prep nor for sequencing. For purified RNA samples and for TruSeq (gold standard) samples, RNA input was normalized for cDNA library prep based on Qubit RNA quantification. Figure Use of in-lysate RNA for RNA-seq library prep may result in unexpected biases in the type  of mRNA molecules recovered that could either mask or accentuate variation between samples. We hypothesized that in-lysate and purified RNA library prep for RNA-seq would result in highly correlated data from matching samples. We transformed the uniquely aligned gene reads into log2 counts per million (CPM) and performed Pearson’s correlation within each treatment group: DMSO (vehicle), calmidazolium and fludrocortisone Fig. . Out of 11, design: ~ donor + treatment) analysis for each method using uniquely aligned gene reads. Figure Variable sequencing depth can affect quantification of low-expressed genes for accurate differential expression analysis. Since the gene counts in matching samples between methods highly correlated with each other Fig.  we hypot2 for these genes between methods was 0.796 and 0.919 for calmidazolium and fludrocortisone treatment groups, respectively. The data show high positive correlation between the LFC of genes that are expressed in high levels and considered significant in both methods. For genes significant in one method only (In-lysate- or Purified-only), we analyzed the distribution of q-values in the corresponding non-significant method  and Precision-Recall (PR) curves for each treatment group Fig. . To prod16. We hypothesized that data from in-lysate RNA and purified RNA library preps ranked by q-value and absolute fold change would highly correlate near the top of the ranked list when compared to data from TruSeq libraries as a gold standard. We found that there was a high (50–75%) overlap in lists of DEGs when the threshold used was between 300 and 600 top DEGs  all quality control criteria were achieved, and (2) gene response profiles and pathway analysis reported by in-lysate and purified RNA library highly correlate with each other. Thus, the approach presented here enables rapid, robust and highly cost-efficient RNA-seq of adherent cell samples in parallel. The reagent costs for generating lysates used for library prep are negligible compared to RNA extraction using silica columns or magnetic beads. Moreover, this approach takes only approximately 4–6 h from sample processing to final sequencing-ready library  the expression profile of human fibroblasts may become more similar between individuals upon stimulation, (2) since the lysis buffer leaves nuclei intact9, in-lysate RNA library prep captures mRNA exported to the cytoplasm which may occur differentially between individuals, or (3) perturbation with calmidazolium may cause changes undetected by our methods in the ability to correctly fragment and process mRNA.However, there are some limitations to the approach presented here. The amount of RNA loaded for in-lysate cDNA synthesis was not normalized which resulted in variable and low-sequencing depth across samples  is another low-cost approach for genome wide profiling which highly compares with other large-scale profiling, however it is based on RNA purification4. Another approach, Digital RNA with perturbation of Genes (DRUG-seq) skips RNA extraction by using the direct lysis approach and cuts down the library prep time and cost. Here, we chose SMART-3SEQ as it relies on chemical fragmentation of RNA rather than tagmentation of pooled libraries which is costly and can be variable. The approach presented here is therefore highly compatible with high throughput drug screening at a low cost.Other transcriptional profiling platforms have been developed that facilitate processing of hundreds of samples in parallel. Luminex L1000, used for the Connectivity MapHighly-parallel assessment of perturbation-transcriptome responses in biological samples can be an unbiased and data-rich approach to screen compounds and to test hypothesized mechanisms of disease. Other novel approaches such as single cell RNA-seq-based genetic perturbation analysis together with small-molecule-based approaches as presented here provide a nimble toolbox to uncover novel biology.20. Cells were maintained in DMEM  with 20% FBS  and Pen/Strep 1:100  and gentamycin 25 μg/mL , at 37 °C with 5% CO2 and were used at passage #4, near confluency, in 24 well plates. Samples were treated for 6 h as indicated with 10 μM Calmidazolium chloride  or Fludrocortisone acetate  or DMSO (10 μM).Human primary dermal fibroblasts  were obtained using the method described inTotal RNA was directly extracted and purified from cell cultures using RNeasy plus mini kit from QIAGEN . In-lysate RNA was prepared by washing cell cultures with PBS and incubating them in lysis buffer for 5 min while on ice. The lysis buffer was adapted from 5 to 9 and consists of 0.1% (1 mg/mL) BSA  and 0.3% Igepal CA 630 , near confluent cultures correspond to an average of 100,000 cells/well. We chose a ratio of 200 cells/μL buffer, so 500ul of Igepal/BSA buffer was used per well. The lysate was aspirated and was only mixed with the pipette tip once in a collection tube. Total RNA concentrations were measured using Qubit HS RNA, Trinean DropSense 16 and Nanodrop  and, RNA quality was measured by Agilent BioAnalyzer  was used for preparing sequencing libraries according to the Smart-3SEQ protocol v1.93 ng/ul of pooled cDNA library was used as sequencing input for each Smart-3SEQ libraries. Smart-3SEQ cDNA libraries were prepared with a 40% spike-in of phiX and sequenced on Illumina NovaSeq 6000 SP Flowcell instrument (approximately 400M PE Reads per lane) configured for 100nt read 1 and 8nt for the P7 index read. TruSeq mRNA-seq libraries were sequenced on two lanes of an Illumina HiSeq 4000 in 75PE configuration. 40% PhiX was added out of an abundance of caution during method implementation. This is due to our use of the two-color Illumina Novaseq 6000 system, which may generate errors when sequencing SMART-3SEQ libraries due to a trinucleotide G repeat in the second strand template-switching reverse transcription primer. We did not systematically investigate lower amounts of PhiX, which would further decrease sequencing costs.10. Quality control analysis was done with FastQC and MultiQC21. The processed fastq files were aligned using HISAT2-2.1.022 and human reference genome-build GRCh38.p13 . Uniquely aligned reads were counted by featureCounts from the Subread package-1.6.423. Overall alignment rate and featureCounts summary can be found in Supplementary Tables The base calls were demultiplexed and converted to FASTQ format by sequencing core facility. For Smart-3SEQ data, adapters were trimmed, UMI sequence and G-overhang were extracted and polyA tails were removed according to the suggested pipeline in data processing protocol11. Pearson’s Pairwise correlation and F-F plot was created in R using various packages and functions. ROC and PR curve were plotted using ROCR-1.0-11 package24. For additional software packages versions and parameters, please see Supplementary Table Uniquely aligned gene reads were then used as input for differential gene expression analysis with DESeq2-1.3-0.1Human dermal fibroblast samples were obtained in compliance with the guidelines of the Declaration of Helsinki as revised in 2013. Approval was obtained from the University of Iowa Institutional Review Board, IRB# 201311783. Informed consent was obtained from the participants of the study.Supplementary Information 1.Supplementary Table S3.Supplementary Table S8.


RNA modification represents one of the most ubiquitous mechanisms of epigenetic regulation and plays an essential role in modulating cell proliferation, differentiation, fate determination, and other biological activities. At present, over 170 types of RNA modification have been discovered in messenger RNA (mRNA) and noncoding RNA (ncRNA). RNA methylation, as an abundant and widely studied epigenetic modification, is crucial for regulating various physiological or pathological states, especially immune responses. Considering the biological significance of T cells as a defense against viral infection and tumor challenge, in this review, we will summarize recent findings of how RNA methylation regulates T cell homeostasis and function, discuss the open questions in this rapidly expanding field of RNA modification, and provide the theoretical basis and potential therapeutic strategies involving targeting of RNA methylation to orchestrate beneficial T cell immune responses. N6-methyladenosine (m6A), N6-2’-O-dimethyladenosine (m6Am), N1-methyladenosine (m1A), and 5-methylcytosine (m5C)  . With thne (m5C) , 5.C . With The methylation of RNA represents one of the most ubiquitous modifications in mammalian cells and modulates multiple biological functions, especially innate and adaptive immune responses , 7. As cIn this review, we will outline recent findings regarding the role of RNA methylation in regulating T cell function, highlight current challenges in these areas, and clarify the potential application of RNA methylation in orchestrating T cell immune responses.6A and identification of its critical role in regulating immune responses, emerging evidence has stimulated researchers to explore how other major forms of RNA methylation may modulate the host immune system, especially T cell immunity, to expand the basic understanding of the epitranscriptomic code of RNA.Methylation of RNA is indispensable for the biogenesis and function of prokaryotes and eukaryotes . This mo6A refers to methylation of adenosine (A) at the nitrogen-6 position and represents one of the most frequent RNA modifications. It is preferentially enriched in 3’untranslated regions (3’UTR), long internal exons, and near stop codons of linear RNAs  and demethylases (“erasers”) , RNA binding motif protein 15/15B (RBM15/15B), cbl-proto-oncogene-like protein 1 , and Zinc finger CCCH-type containing 13 (ZC3H13), are essential for the nuclear localization and stabilization of the “writer” complex, or m6A deposition specificity . To date, three different groups of “readers” have been identified: YTH-RNA binding domain family, including three YTH-domain-family (YTHDF1-3) and two YTH domain-containing (YTHDC1-2) proteins  . The “wrrasers”) , 19, METrasers”) , 21, andrasers”) , 23. Othcificity  and alkycificity . FTO not or mRNA . Multiplproteins –29, the proteins –32 and iproteins , 34. Allnd decay –29  cell subsets upon stimulation by various microenvironmental signals  for m6A writer protein-METTL3 in T cells, in the first attempt to elucidate the in vivo role of METTL3 and consequently the m6A RNA modification in regulating mouse T cells homeostasis and differentiation  induction compared with WT naïve CD4+ T cells (+ T cell homeostatic expansion in vivo during the adoptive transfer colitis model (6A underwent rapid mRNA degradation upon interleukin (IL)-7 stimulation, thus initiating the homeostasis and differentiation of naïve T cells by relieving the block on the IL-7-signal transducers and activators of transcription 5 (STAT5) pathway -dependent T cell induction system  formation and promote humoral immunity (6A modification remains to be identified. One study indicated that the knockdown of METTL3 or METTL14 in CD4+ T cells with short hairpin RNA (shRNA) could promote Tfh development upon lymphocytic choriomeningitis virus (LCMV) infection  mRNA can stabilize the transcript, and therefore maintain protein expression of TCF7 to initiate and secure the differentiation of Tfh cells  cells, as a specialized CD4immunity , whethernfection . Howeverhallenge . Notablyfh cells . This relization , 40. The6A methylation may modulate T cells either directly or indirectly by affecting the function of antigen-presenting cells (APCs)   , 45. In s (cDCs) . Further6A in governing T cell functions, it seems that these have only focused on better defining the role of the “writers” or “readers”   in mitochondrial messenger RNA (mt-mRNA) blocks the effective translation of modified codons 1A was fieraction –62. m1A am genes , 64. Subreported , 66. Recd codons . Moreovenslation . However1A, as a reversible RNA modification, is catalyzed by methyltransferases including tRNA methyltransferase 6/61A (TRMT6/61A), TRMT61B, TRMT10C, and Nucleomethylin (NML), and by demethylases FTO, ALKBH1, and ALKBH3  stem and a 7-bp loop  family proteins (including NSUN1-7) and DNA methyltransferase (DNMT) homologue DNMT2  compared with CD4+ T cells of healthy controls (HCs) , N1-methylguanosine (m1G), 5-methyluridine (m5U) have also been mapped in CD4+ T cells (6A in maintaining T cell homeostasis in the future.A recent study has reported reduced mRNA mls (HCs) . NSUN2 insferase  and whose to HCs . Importahylation . In addie system . This st T cells , and it 6A methylation as a “brake” to modulate transcription during T cell activation , Shanghai Science and Technology Committee (20JC1417400/201409005500/20JC1410100 to HBL), the Postdoctoral Innovation Talent Support Program (BX20190214 to JZ), the Shanghai Super Postdoctoral Program (JZ), China Postdoctoral Science Foundation (2020M671149 to JZ), the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning (HBL), the start-up fund from the Shanghai Jiao Tong University School of Medicine (HBL).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.


A large number of RNA molecules have been found in the phloem of higher plants, and they can be transported to distant organelles through the phloem. RNA signals are important cues to be evolving in fortification strategies by long-distance transportation when suffering from various physiological challenges. So far, the mechanism of RNA selectively transportation through phloem cells is still in progress. Up to now, evidence have shown that several RNA motifs including Polypyrimidine (poly-CU) sequence, transfer RNA (tRNA)-related sequence, Single Nucleotide Mutation bound with specific RNA binding proteins to form Ribonucleotide protein (RNP) complexes could facilitate RNA mobility in plants. Furthermore, some RNA secondary structure such as tRNA-like structure (TLS), untranslation region (UTR) of mRNA, stem-loop structure of pre-miRNA also contributed to the mobility of RNAs. Latest researchs found that RNA methylation such as methylated 5′ cytosine (m5C) played an important role in RNA transport and function. These studies lay a theoretical foundation to uncover the mechanism of RNA transport. We aim to provide ideas and clues to inspire future research on the function of RNA motifs in RNA long-distance transport, furthermore to explore the underlying mechanism of RNA systematic signaling. Cucumis sativus L.) (Cucurbit maxima) (Nicotiana benthamiana/tomato (Solanum lycopersicum)  (Vitis vinifera) (in vivo have indicated that the regulatory function of RNAs (non-coding RNA and mRNA) were involving plant growth, defense, biological/economic yield, and certain morphological characteristics  , pumpkin maxima) , Nicotiaersicum) , Arabidohaliana) , and grainifera) . Evidenceristics .It has been reported that RNA local mobile from CC to SE are following a selective way. Properties affect RNA movement or upload to SE including transcript length, shape, charge, and genetic sequence or structure motifs . One assHere, we summarize some of the findings of RNA transport mechanisms, mainly related to RNA motifs including Polypyrimidine (poly-CU) sequence, transfer RNA (tRNA)-related sequence, Single Nucleotide Mutation. Furthermore, some RNA secondary structures such as tRNA-like structure (TLS), untranslation region (UTR) of mRNA, stem-loop structure of pre-miRNA and RNA methylation such as methylated 5′ cytosine (m5C) also contribute to the mobility of RNAs in plants. All the evidence of these RNA motifs that assist RNA transportation provides us a better understanding of the long-distance transport of plant endogenous RNA mechanism.The pathogenicity of viroid in plants was determined by their single-nucleotide motif, secondary, and tertiary structure . Being cPSTVd could affect the systemic infection and pathogenicity of PSTVd. PSTVd consists 359 RNA nucleotides folded into a secondary structure, which contains 26 base pair stems and 27 loops, at least 11 loops were essential for the systemic transport of PSTVd in N. benthamiana  both in vitro and in vivo  motifs through non-Watson Crick (non-WC) base pairing, base stacking and other base-base interactions , potato (Solanum tuberosum), and pear (Pyrus betulaefolia) were confirmed binding the CU-rich motifs of CmGAIP, StBEL5, and PbWoxT1, thereby facilitating long-distance movement of mRNAs (P. betulaefolia), was identified interaction with PbPTB3. The protein complex PbTTG1 and PbPTB3 promote PbWoxT1 mRNA transport in grafted pear  move from lower leaves to upper leaves  containing imperfect hairpin structures  KNOTTED transcription factor family, which accumulates with the increases of the short-day light cycle. It has proved that 5′UTR of StBEL5 promote mRNA transport from leaves to the stolon tip of the underground through the phloem, and increase the yield of tubers  showed that phloem-specific tRNA fragments could interfere with ribosomal activity, further, block the transcription of mRNA during its transportation in the phloem  transcripts into meiosis tissues  and HEAT SHOCK homologous protein 70.1 (HSC70.1) were insufficiently methylated due to 5mC mutation with reduced transport, 5mC modified TCTP1 can transport to root cells and increase root growth -mediated m5C methylation in elopment . TherefoArabidopsis TCTP1 mRNA through SE. In this process, specific motifs in the mRNA were recognized by methyltransferase to achieve methylation of TCTP1 mRNA  and monosome (80S) rather than polysome, proving the abundance of m5C was negatively correlated with mRNA translation activity  Methylation mediates mRNA nucleocytoplasmic shuttling and marking as transport mRNA. (ii) The motifs surrounding methylation are recognized to form a protein–RNA complex. (iii) Mediate size-exclusion limit of PD to achieve CC-SE transport. (iv) Stabilize RNA molecules transport in SE and reduce translation ability. (v) Mediates the unloading of CC-SE. (vi) Reduce methylation level and restore translation ability . NeverthThere are still many questions to be answered about the mechanism of RNA transportation. For example, will all RNA be transport? What is the relationship between RNA motifs and transport between different cells during regulated transportation? How these RNA motifs participate in guidance, and whether they have corresponding changes in this process, requires further research? Although we have drawn some insights from the research, there is still a long way to go for the comprehensive answer.In this paper, the possible mechanism of long-distance RNA transport was discussed from four aspects: single nucleotide mutation, secondary structure of pre-miRNA, tRNA-related sequence and TLS, and RNA methylation. These studies lay a theoretical foundation to uncover the mechanism of RNA transport. The importance of this review is not only to explore the mechanism of RNA signaling, but also to provide an insight for plant growth, development and stress resistance regulation in RNA levels.LG and WZ revised the final manuscript. TW and ZL wrote viroid and polypyrimidine part. XLi and ML wrote methylation part. XZ, XLu, and WL wrote pre-miRNA and TLS part. QW and SG revised the whole draft. All authors contributed to the article and approved the submitted version.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.


Ceratitis capitata, is an agricultural pest of a wide range of fruits. The advent of high-throughput sequencing has boosted the discovery of RNA viruses infecting insects. In this article, we aim to characterize the RNA virome and viral sRNA profile of medfly. By means of transcriptome mining, we expanded the medfly RNA virome to 13 viruses, including two novel positive ssRNA viruses and the first two novel dsRNA viruses reported for medfly. Our analysis across multiple laboratory-reared and field-collected medfly samples showed the presence of a core RNA virome comprised of Ceratitis capitata iflavirus 2 and Ceratitis capitata negev-like virus 1. Furthermore, field-collected flies showed a higher viral diversity in comparison to the laboratory-reared flies. Based on the small RNA sequencing, we detected small interfering RNAs mapping to all the viruses present in each sample, except for Ceratitis capitata nora virus. Although the identified RNA viruses do not cause obvious symptoms in medflies, the outcome of their interaction may still influence the medfly’s fitness and ecology, becoming either a risk or an opportunity for mass-rearing and SIT applications.The Mediterranean fruit fly (medfly),  Iflaviridae, Nodaviridae, Negeviridae, Togaviridae, Rhabdoviridae, Reoviridae, or Totiviridae [Insect specific viruses (ISVs) specifically infect insect hosts. Some ISVs are highly pathogenic , while iviridae ,6,7,8.Ceratitis capitata, fitness studies have shown that Ceratitis capitata nora virus (CcaNV) infection is correlated with a reduced adult lifespan [Covert RNA viruses represent a subset of ISVs that are found to co-exist in an equilibrium with their host, in which virus vertical transmission is assured while no major detrimental fitness costs are caused to the host . Howeverlifespan . These eCeratitis capitata (Diptera: Tephritidae) is a polyphagous species causing economic losses in agriculture worldwide. The larvae of this species can feed on a wide range of fruits including citrus fruits (Citrus spp.), apples , or pears (Pyrus communis) . Sterile insect technique (SIT) programs have been applied for medfly control in North America, Central America, South America, Europe, the Middle East, Asia, Africa, and Australia [ustralia . This stustralia ,13.C. capitata and all of them seem to be present in a covert state, not causing clear symptoms [Rhabdoviridae family [Picornavirales order: Ceratitis capitata iflavirus 1 (CcaIV1), Ceratitis capitata iflavirus 2 (CcaIV2), Ceratitis capitata iflavirus 3 (CcaIV3), Ceratitis capitata iflavirus 4 (CcaIV4), and CcaNV [C. capitata.In recent years, up to nine RNA viruses have been identified in symptoms ,8,10,14.nd CcaNV ,10. Up uAedes vexans arabiensis and the oriental fruit fly Bactrocera dorsalis [D. melanogaster [In contrast to vertebrates, no evidence of adaptative immunity in insects has yet been provided . Insteaddorsalis ,20. The nogaster . The potnogaster ,22.C. capitata, identifying new viral species. Further analysis of viral prevalence as well as viral small RNA profiling in reared and field-collected medfly samples additionally revealed a high diversity in viral abundance in different populations, and presented important insight into mechanisms of insect defense against RNA viruses in this pest.In this study, high throughput sequencing and data mining were used to characterize the RNA virome of Two medfly laboratory strains (control and W-1Kλ) were used for transcriptomic analysis by RNA-seq and further virus discovery. Moreover, viral detection and abundance were determined in vivo in the pupae of two long-term laboratory-reared medfly strains (control and IVIA), one recently field-derived strain (Wild-F4), and one mass-reared strain (V8A). In total, five different medfly strains were used, and their origin and rearing are described below.The control strain (C) originates from flies collected from untreated experimental fields at the “Instituto Valenciano de Investigaciones Agrarias”  in 2001, and it has been reared for more than 100 generations under laboratory conditions  at the “Centro de Investigaciones Biológicas Margarita Salas”  .The IVIA strain (I) originates from the control strain and is regularly renewed with field-captured flies from the experimental fields at the IVIA. The IVIA strain has been reared under the above-mentioned laboratory-rearing conditions for more than 30 generations.Lambda-cyhalothrin resistant strain (W-1Kλ) started with flies collected from citrus orchards treated with malathion in Castellón  in 2004, and first were selected for resistance to malathion for 39 generations and then for resistance to lambda-cyhalothrin for 43 generations after. The W-1Kλ strain is maintained under the above mentioned laboratory-rearing conditions at the facilities of CSIC (Madrid) .C. capitata pupae obtained from infested figs fruits (Ficus carica) collected from isolated trees in commercial citrus orchards in Alcira  in August 2020. The pupae used in this study were obtained from the F4 generation in December 2020. This colony was maintained daily at the facilities of the IVIA by TRAGSA .The recently field-derived Wild-F4 strain (W) originates from field-collected The Vienna 8A (V8A) strain is a naturalized V8-strain obtained by crossing the temperature sensitive lethal genetic sexing strain Vienna-8 mix 2002 with individuals collected in citrus orchards located in the province of Valencia (Spain). This medfly strain is currently mass-reared for application in SIT control in the province of Valencia, and was kindly provided by TRAGSA.C. capitata fertilized eggs [®, St Louis, MO, USA) with 10% FBS (PAA) and 100 U/mL streptomycin/penicillin at 25 °C.The Cce/cc128 cell line, which was developed in 1981 from zed eggs , was kinhttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 1 December 2020). The adaptors were removed, and the low-quality sequences were trimmed using Trimmomatic [The presence of RNA viruses was analyzed in six transcriptome data sets obtained from the control and W-1Kλ strains . The total RNA was extracted and used for cDNA synthesis and high-throughput sequencing by Illumina HiSeq 2000 paired-end sequencing, as described before [−5. As a query, we used the protein viral sequences deposited in GenBank, classified within the Riboviria domain (RNA viruses), and having “invertebrates” as the host. After tBLASTn, the putative viral contigs resulting from the analysis were manually filtered to remove (a) the contigs less than 2000 bp in length, (b) the contigs found in the reference genome of C. capitata after BLASTn , and (c) the contigs without open reading frames (ORFs) after evaluation with ORFfinder . Viral length was further confirmed by mapping all reads against the putative viral candidates using BWA [Viral presence in the assembled contigs of each medfly strain was assessed using tBLASTn (protein query against translated nucleotide database) with a threshold value of 1 × 10sing BWA , revealiViral candidates were annotated running BLASTx against the non-redundant protein sequence database (NCBI). The ORFs contained in the viral fragments were determined and their translated sequences were queried against the non-redundant protein database using BLASTp and the Conserved Domain Architecture Retrieval Tool (CDART) in GenBank. The phylogenetic analysis was performed at the amino acid level. The RNA dependent RNA polymerase (RdRp) conserved domain was selected to construct the phylogeny. When no conserved domains were defined, the protein sequence of the ORF containing the RdRp was selected. The closest viral relatives for the putative viruses under analysis were identified in the NCBI nr database using BLASTp. Other viruses recently described for medfly or similar dipteran species and belonging to the same viral families were added to the analysis .Protein sequences were aligned using T-coffee mode “psi-coffee”, which aligns distantly related proteins using the homology extension . FragmenTM DNAse I) to digest the DNA residues. The purified RNA was reverse transcribed into cDNA using oligo (dT) primers and random hexamers as specified in the Prime-Script RT Reagent Kit . Viral presence was detected through RT-qPCR . All reactions were prepared in a total volume of 20 µL using 5× HOT FIREpol EvaGreen qPCR Mix Plus (ROX) from Solis BioDyne . Primer pairs for virus detection were designed using Primer 3 software [−Ct (virus)/E−Ct (L23a), where E represents an amplification factor dependent on primer efficiency, which is 2 for 100% efficiency.The presence and relative abundance of RNA viruses in laboratory-reared, mass-reared, and field-collected flies were determined using molecular methods. For this purpose, the total RNA was isolated from individual pupa using the TriPure isolation reagent  according to the manufacturer’s protocol. Then, 1 µg of total RNA was treated with 1 µL of DNAse I  were suspended in 5 ml of TE buffer  with 0.06% SDS, and were homogenized using T 50 digital ULTRA-TURRAXThe control and Wild-F4 medfly strains were used for the small RNA analysis. Those strains were representatives of the highest (Wild-F4) and lowest (Control) number of different viruses. For each population, virgin adult females were isolated after emergence and were maintained in a plastic box separately from the males for 8 days. Water and food were provided ad libitum. On day 8 of adulthood, virgin females were collected and dissected with entomological tweezers under the binocular. First, the head and thorax of the flies were separated from the abdomen and conserved in RNA later  as a somatic tissue sample. Then, the abdomen was opened vertically, visualizing the ovaries. Ovaries were carefully separated from other debris using sterile entomological pins and were conserved in RNA later .RNA was extracted from pools of 10 head/thorax and 20 ovaries using TriPure isolation reagent . RNA integrity and quantity were assessed by 1% agarose gel electrophoresis and spectrophotometry. The viral prevalence and abundance in these samples were determined by RT-qPCR as described above.Small RNA library construction and sequencing were carried out by Macrogen . Libraries were prepared using the TruSeq Small RNA Library Prep Kit . Then, sequencing was performed in the HiSeqX platform (Illumina) generating paired-end reads of 150-nt long. Approximately 1 Gb of raw data were obtained from each of four the libraries analyzed .https://github.com/SamuelHLewis/sRNAplot, accessed on 12 May 2021).Adapters, empty sequences, low complexity sequences, and reads with more than 20% low-quality were removed from the raw RNA-Seq reads. Moreover, clean reads were filtered by length, maintaining only the sequences between 18 and 32 nt. Viral-derived sRNA sequences (vsRNAs) were obtained after mapping the clean reads against the genome sequences of the 13 RNA covert viruses described in medfly using Bowtie 2 v 2.3.5.1 . The perIn our aim to explore the RNA virome of medfly, we analyzed two different strains: control and W-1Kλ . By idenTotiviridae family, with two ORFs encoding the capsid proteins and the RdRp, respectively  and Ceratitis capitata reo-like virus 1 (CcaRLV1). CcaTV1 maintains the typical genome length (6132 bp) and organization of viruses in the ectively . The phyectively . For Ccaectively . Presumald virus .Nodaviridae) and a new member of the Narnaviridae family were retrieved and annotated as Ceratitis capitata nodavirus 1 (CcaNdV1) and Ceratitis capitata narnavirus 1 (CcaNaV1), respectively. CcaNdV1 has a small segmented RNA genome with methyltransferase and RdRp encoded on the longer 3092-nt segment . Similarly, we suggest the addition of CcaTSA2 and CcaTSA3 [Other viruses discovered in our analyzed RNAseq data concern viruses recently described by other studies ,8, altho CcaTSA3 ,8 into tC. capitata Cce-cc128 cell line. Viral pre-purifications were obtained from pools of 50 pupae to increase the concentration of viruses, as infections with certain RNA viruses could exhibit low viral titers.We assessed the prevalence and relative abundance of the 13 RNA viruses in individuals and viral pre-purifications from two laboratory-reared medfly strains (“control” and “IVIA”), a mass-reared strain (“Vienna 8A”), a recently field-captured strain (“Wild-F4”) and in the . Interestingly, the highest number of viral species, and the largest variation between individuals in viral titers and viral abundance were observed in the samples derived from the Wild-F4 strain , EgyptII (Egypt), Hawaiian (Hawai), Ispra , Madrid (Spain), Toliman , Vienna 7 (Austria), and Wp (Greece)  . Our res(Greece) . CcaIV2 (Greece) . On the (Greece) . In addi(Greece) .Two medfly strains (control and Wild-F4) were selected for sRNA sequencing, and sRNA profiles were obtained for the ovaries  and the remaining somatic tissues. Viral relative abundance was evaluated in the four samples prior the sRNA library construction A. Five RThe sRNA profiles showed specific targeting of sRNAs to those viruses detected in the control and Wild-F4 strains. The abundance of the sRNA reads mapping to the different viral sequences ranged from 0% to 8.88% of the total sRNA reads B, and weBactrocera tryoni and Bactrocera cacuminata [By means of transcriptome mining and viral genome annotation, we have obtained and characterized the largest RNA virome of medfly described to date ,8,10,14.cuminata  strikingS. exempta fitness by increasing its resistance against baculovirus infection [The viral prevalence and abundance of the 13 identified RNA viruses corroborated the presence of multiple and simultaneous viral infections in medflies  and revenfection . In anotnfection .Ae. vexans arabiensis mosquitoes and the oriental fruit fly B. dorsalis [While the lack of medfly strains free from specific viruses limits the study of functional associations, the characterization of the immune responses elicited in the host against viral infections may contribute to elucidating the outcome of the virus–host interactions. According to our results, the siRNA pathway is active against RNA viruses in medflies, as was previously observed in dorsalis ,20. Excedorsalis . Interesdorsalis . The proIn summary, we have expanded the RNA virome of medfly and revealed the ubiquitous distribution of two medfly viruses (CcaIV2 and CcaNeLV1) in different medflies worldwide. Unraveling the impact of these viruses on medfly fitness, including their influence on medfly interactions with third trophic level insects, will allow us to determine the impact of this viruses on shaping medfly ecology and contribute to decipher their potential pathogenic risk for medfly mass-rearing and their impact on sterile males released into the field for SIT programs.


Drosophila master sex-switch protein Sex-lethal (SXL) is an RNA-binding protein that controls splicing, polyadenylation, or translation of certain mRNAs to mediate female-specific sexual differentiation. Whereas some targets of SXL are known, previous studies indicate that additional targets of SXL have escaped genetic screens.Downstream targets for a large number of RNA-binding proteins remain to be identified. The GEnomic Selective Enrichment of Ligands by Exponential enrichment (GESELEX) using both the genomic DNA and cDNA pools from several Drosophila developmental stages to identify new potential targets of SXL. Our systematic analysis provides a comprehensive view of the Drosophila transcriptome for potential SXL-binding sites.Here, we have used an alternative molecular approach of Drosophila transcriptome. We discuss the significance of our analysis and that the newly identified binding sites and sequences could serve as a useful resource for the research community. This approach should also be applicable to other RNA-binding proteins for which downstream targets are unknown.We have successfully identified new SXL-binding sites in the  For several decades, genetic analyses and biochemical characterizations have offered complementary approaches to investigate developmental processes. Such studies have led to the identification of regulatory proteins in numerous cellular pathways. For a subset of these regulatory proteins, subsequent molecular genetic analysis and biochemical procedures have revealed downstream regulated genes. Nonetheless, while these approaches have hinted existence of additional downstream targets of numerous regulatory proteins, inherent limitations of these techniques have left additional putative targets to be identified. The approach of genomic SELEX is an attempt here to uncover potential new downstream targets which could serve as reagents for future detailed investigations into cellular and developmental processes.Drosophila melanogaster, Sex lethal (SXL), which is an example of a sequence-specific RNA-binding protein [Sxl -> transformer (tra) —> doublesex (dsx)]  , 4.dsx (DSXM) [F isoform and absence of the FRU protein. Moreover, SXL prevents synthesis of the male-specific-lethal-2 (MSL2) protein to controls dosage compensation, which equalizes the expression of X-chromosome genes in both sexes.SXL controls three important regulatory pathways—somatic sexual differentiation, dosage compensation, and the female germline stem cell differentiation. In XY somatic cells, the SXL protein is not synthesized due to an in-frame translation stop codon(s) in its male-specific exon. This results in failure to synthesize the TRA protein, leading to the synthesis of the fruitless (FRU) protein and of the male-specific isoform of x (DSXM) , 6. In cSxl, tra, and msl2 pre-mRNAs [Sxl pre-mRNA, 3’ splice site switching in the tra pre-mRNA, and intron retention in the msl2 pre-mRNA. In addition, SXL represses the translation of its own mRNA and that of the msl2 mRNA through SXL-binding sites in untranslated regions (UTRs) [At the molecular level, SXL regulates splice site choice in somatic cells by binding to specific uridine-rich RNA sequences and blocking adjacent splice sites in re-mRNAs , 8. It fs (UTRs) –12. For s (UTRs) .enhancer of rudimentary, Notch signaling (N mRNA), and Antizyme/gutfeeling [Previous genetic studies suggested that SXL associates with numerous loci or nascent transcripts on polytene X-chromosomes in females  and liketfeeling , 21, 22.Genomic Selective Enrichment of Ligands by Exponential enrichment (GESELEX) strategy. To accomplish this, we prepared the selection pool from the Drosophila genome as well as cDNA pools from developmental stages. Our unbiased and comprehensive analysis provides a transcriptome-wide view of potential SXL-binding sites in Drosophila.Therefore, in our attempt to identify additional targets of SXL, we used Drosophila genome or to the cDNA pools from different stages of development were attached to a T7 RNA polymerase promoter using a two-step random priming process with appropriate primers  affinity selection. A reverse primer (SW3N7N9) with a randomized 3’ end was used for reverse transcription. The cDNA product was next subjected for second-strand synthesis using a forward primer (SW5N7N9) with a randomized 3’ end. The DNA fragments were size selected using polyacrylamide gel electrophoresis to obtain approximately 220 to 350 basepairs products. Alongwith another reverse primer (3N7.1) corresponding to the fixed sequence of the SW3N7N9 reverse primer, a second forward primer (5LN7.1) was used for PCR to add a T7 RNA polymerase promoter to the PCR product. These PCR products, or the cDNA libraries from various developmental stages, served as templates for synthesis of the pool 0 RNA corresponding to each of the Drosophila cDNA SELEX libraries.To obtain cDNA SELEX libraries from different developmental stages, total RNA was isolated from various developmental stages, for example, embryonic, larval, pupal, and adult flies. Poly(A)Size-selected libraries (approximately 150–250 basepairs) using the genomic DNA and cDNA pools from different stages of development are shown in in vitro. The RNA was gel purified and this pool 0 RNA from each of the pools was incubated with appropriate concentrations of the purified recombinant SXL protein. Following six rounds of selection-amplification with these libraries, we cloned and sequenced SXL-enriched sequences. We note that the protein-bound RNA fraction was obtained using a filter-binding assay for the initial rounds of selection-amplification and then by electrophoretic gel mobility shift assay for the last two rounds. The process of SELEX, which was similar for both the genomic and the cDNA libraries, is schematized in Both the genomic and the cDNA SELEX libraries were subjected to iterative selection-amplification protocol, as described for the previous random SELEX protocol we have used . In brieSxl, tra, msl2, and e(r) are shown. Moreover, the consensus sequence for SXL-binding site is shown.As an evidence of successful selection-amplification, While representative sequences from genomic and cDNA SELEX are shown in Tables tra pre-mRNA (Kd~ 1 nM)  with high affinity. We note that the binding affinity for sequences in SXL-selected pool from the genomic DNA was comparable to that for one of the well-characterized SXL-binding site (non-sex-specific polypyrimidine tract) in the ~ 1 nM) .tra pre-mRNA (our positive control)  with high affinity. We reiterate that binding affinities for sequences in SXL-selected pools for the cDNA libraries were also similar to that for the well-characterized SXL-binding site, the non-sex-specific polypyrimidine tract, in the control) .We conclude that SXL selected high-affinity sequences from both the genomic and the cDNA SELEX libraries.Drosophila Shaker/TnI gene and sequence 14 corresponded to the Glutamine Synthetase (Gs2) gene. Both genes are located on the X chromosome. These binding sequences are present in the 5’ UTR of the TnI and the 3’ UTR of the Gs2 transcripts. To experimentally show that SXL-binding sites identified in TnI and Gs2 mRNAs bind SXL, we individually analyzed these sequences by the electrophoretic gel mobility shift assay. TnI and Gs2 transcripts with high affinity (Kd~ 1 nM), which is similar (within 2-3-fold difference) to that for the non-sex-specific Py-tract of tra, which is a known biologically relevant and high affinilty SXL-binding site [Since the genomic and cDNA SELEX was intended to be able to identify potential targets of SXL using the sequence tags that flank the SXL-binding site within the 150–250 long sequence in our library, we were able to identify many genes that contain these SXL binding sites. We randomly picked two of the selected sequences from the cDNA library prepared from embryonic stages 10–15 hours, sequence 5 corresponded to the ing site .TnI and Gs2 mRNAs, respectively.We conclude that SXL binds with high affinity to the newly identified SXL-selected binding sites in the 5’ and 3’ UTRs of the TnI and Gs2 using poly(A)+ RNA obtained from male and female adult flies. TnI and Gs2 showed sex-specific expression and these transcripts were present primarily in male flies and very little expression in females.Since the genomic and cDNA SELEX was intended to lead us to potential targets of SXL and that SXL is present in females and absent in males, we asked if these candidates showed sex-specific differences in their expression profile or mRNA isoforms. To test this hypothesis, we performed Northern analysis for these randomly selected candidates TnI and Gs2 mRNAs have potential SXL-binding sites and show sexual polymorphism in their expression pattern.We conclude that In this study, we have identified potential new targets of SXL, a subset of which may be regulated by SXL. Below we discuss advantages of GESELEX, and the biological significance of these findings. The sequences reported here would serve as a resource for those in scientific community studying sexual differentiation.8 versus 1014 complexity). Thus, fewer rounds of selection-amplification could exhaust the genomic library, and would avoid artefacts from additional PCR amplifications [The genomic and cDNA SELEX approaches offers several important advantages. First, the genomic and cDNA SELEX libraries are much simpler than a random library  than randomized sequence libraries , but provide a sequence space that is functionally and biologically more relevant to search for targets for an RNA-binding protein. We posit that our combined analysis appears to have approached saturation in terms of finding SXL-binding sites that are present in the Drosophila transcriptome.Specifically to SXL regulation, we note that the sequences identified here from genomic and cDNA SELEX are similar to the uridine-rich sequences selected from a random pool of RNA  and to tDrosophila genome. These repeat sequences happen to be among the high-affinity SXL-binding sites. This was part of the inspiration why we undertook cDNA SELEX, which complements SELEX with genomic DNA, so that the library was not overpopulated with the GU repeats. In this sense, both genomic and cDNA libraries complement each other.In the past, the consensus-binding site for SXL was characterized by using several approaches, including SELEX from a random RNA library , 31, 32.TnI and Gs2, which show sexually dimorphic expression, are potentially regulated by SXL. Further detailed molecular genetic analysis in the future will be required to link these genes to SXL regulation. Both TnI and Gs2 genes are located on the X chrosomosome and thus are potential targets for regulation [Gs2, could mediate MSL2-independent dosage compensation [TnI and Gs2 are related to the dosage compensation of X-linked genes during developmental. It is intriguing to note that the TnI gene belongs to a haplolethal locus of the Shaker complex [TnI in XX flies observed here is somehow related to its haplolethal phenotype.We favor that mRNAs such as gulation , 35. Preensation . Howeverensation . Further complex , implyinTnI, Gs2, are biologically relevant for SXL-mediated gene regulation. SXL controls its known targets at the level of splicing, polyadenylation, or translation. It will be interesting to determine if the new potential SXL targets are regulated by these known mechanisms or differently [Drosophila development.Future studies will determine whether genes containing the SXL-binding sites, including ferently , if theyferently , and howIn summary, this analysis emphasizes that the genomic and cDNA SELEX can reveal potential candidate genes that have escaped earlier attempts. Finally, such approach should be useful for the functional analysis of hundreds of the other RNA-binding proteins for which downstream targets are unknown.Drosophila melanogaster adult flies using Trizol method (Sigma-Aldrich Co.). This genomic DNA (~30–50 μg) was sheared by passing through a 23-gauge needle, followed by incubating in boiling water for 15 minutes and chilling quickly in ice bath. The denatured genomic DNA was then primed for first strand synthesis using Klenow fragment of DNA polymerase 1 and 3’ random primer (SW3N7N9) followed by purification to remove excess 3’ primer. Second strand synthesis was carried out using a 5’ random primer (SW5N7N9) and Klenow fragment of DNA polymerase 1. The genomic DNA with annealed 5’ and 3’ random primers is then size fractionated on a denaturing gel and 150 nucleotide long fragments were eluted. Next, the DNA fragments from above were PCR amplified using Taq DNA polymerase and a 5’ primer (5LN7.1 220 μM), which adds a T7 promoter to 5’ end of the DNA, and a 3’ primer (3N7.1 220 μM) that binds to constant region of the 3’ random primer. PCR reaction was carried out as follows: 93°C for 30 seconds, 55°C for 30 seconds and 72°C for 45 seconds for a total of 12 cycles, followed by 7 minutes at 72°C. The PCR amplified genomic DNA with 5’-T7 promoter was purified by treating with Proteinase K, phenol:chloroform extraction and precipitation using absolute ethanol. This is the pool O genomic DNA library. The purified PCR DNA was then used for T7 transcription to generate RNA using T7 RNA polymerase by incubating the reaction mix at 37°C for overnight. After incubation, the reaction mix was treated with DNase RQ RNase free (to digest the template PCR DNA). The transcribed RNA was separated on a denaturing gel and stained with 0.1% methylene blue. The visible RNA bands were eluted from the gel using 1X Proteinase K buffer, phenol:chloroform purified and precipitated using absolute ethanol. The purified RNA (300 ng) was then used for binding to the recombinant GST-SXL protein. RNA was heat denatured and allowed to bind the GST-SXL protein  in a 100 μl binding reaction at 30°C for 30 minutes [The genomic DNA was isolated from  minutes . The bou32P labeled RNA at 20K cpm was used for binding with 160 ng of GST-SXL protein, and for the final round of binding 3000 cpm RNA and 55 ng, 18 ng and 6 ng of GST-SXL protein. The pool of cDNAs from the last round of SELEX were cloned in pAMP plasmid vector (GIBCO-BRL/Life Technologies) and sequenced.The purified RNA was then allowed to bind 3’ primer at 42°C overnight and the annealing reaction was precipitated using Isopropanol. This 3’ primer annealed RNA fragment was then reverse transcribed into a cDNA using AMV reverse transcriptase at 42°C for 60 minutes. The cDNA was then purified using phenol:chloroform and precipitated using absolute ethanol. The cDNA was then used in SELEX PCR using 5’(5LN7.1) primer and 3’ (3N7.1) primer as described above. The amplified PCR DNA represented the pool 1 DNA. Purified PCR DNA (pool 1) was then used for T7 transcription to generate RNA using T7 RNA polymerase by incubating the reaction mix at 37°C for overnight. The transcribed RNA was purified and ~300 ng was used for second round of binding to GST-SXL protein (~160ng). For the next round of RNA-GST-SXL binding the RNA concentration was reduced to 100 ng and GST-SXL protein a ~160 ng. From round 4–7 the RNA was transcribed using radiolabeled nucleotide and Drosophila melanogaster ; Larval ; Pupal ; Adult) using Trizol reagent (Sigma-Aldrich Co.) [Total RNA was isolated from the various life cycle stages of ich Co.) . SubsequElectrophoretic mobility shift assay was performed as described in , 40.TroponinI and Glutamine Synthetase 2 gene sequences were amplified from the EST clones obtained from Research Genetics .Drosophila melanogaster Oregon-R flies were raised in corn-meal Agar medium at 25°C incubator. etc.Total RNA from the flies were isolated using Trizol reagent (Sigma) according to Chomczynski, P, 1993. From this total RNA, PolyA + RNA was isolated by passing them through an oligo dT column. Northern blots containing the poly A+ RNA from various flies were generated using standard protocols , and blo5’-AGGGAGGACGATGCGGATCCNNNNNNNNN-3’SW5N7N9 (A-ran) BamH1–5’-TCCCGCTCGTCGTCTGCAGNNNNNNNNN-3’SW3N7N9 (B-ran) Pst 1–5’-GAAATTAATACGACTCACTATAGGGAGGACGATGCGG-3’5LN7.1–5’-TCCCGCTCGTCGTCTG-3’3N7.1–S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 TableDrosophila embryos [Transcripts, if any, immunoprecipitated with SXL protein in primordial germ cells of  embryos  are indi(DOCX)Click here for additional data file.S4 TableDrosophila embryos [Transcripts, if any, immunoprecipitated with SXL protein in primordial germ cells of  embryos  are indi(DOCX)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.