We analysed the survival of colorectal cancer (CRC) patients with lung metastasis and lung-only metastasis and determined the risk factors for lung metastasis in CRC patients.Data from colorectal cancer patients with lung metastasis diagnosed from 2010 to 2015 were obtained from the SEER database. Survival was analysed using the Kaplan–Meier method and log-rank test, the Cox proportional hazards regression model, and a competing risk model. The predictive ability of the nomgram was assessed by the concordance index (C-index) and calibration curves. The data from the SEER database for the period 2016–2019 was used as an external validation set. The characteristics of 70 CRC patients treated at Shanghai East Hospital between 2016 and 2019 were retrospectively analysed and data from China was chosen as an external validation set.The median survival time for colorectal cancer patients with lung metastasis was 12 months, while this value was 24 months in patients with lung-only metastasis. Among all CRC patients with lung metastasis, age, grade, T stage, N stage, presence of liver, brain or bone metastasis, anatomic site and surgery were related to overall survival (OS). In CRC patients with lung-only metastasis, age, T stage, marital status, chemotherapy and surgery were independent prognostic factors affecting OS. Two nomograms predicting OS were established, with great discrimination (C-index between 0.67 and 0.81) and excellent calibration. Factors including age, race, sex, tumour grade, T stage, N stage, presence of liver, brain or bone metastasis, marital status, insurance status and anatomic location were related to the occurrence of lung metastasis in CRC patients.We developed two reliable clinical prediction models among CRC patients to predict the OS rates in patients with lung metastasis and lung metastasis only.The online version contains supplementary material available at 10.1186/s12876-022-02547-9. Most people with colorectal caner (CRC) die from the disease, and it is the second leading cause of death in the United States . There aThe lung is the second most common site of CRC metastasis, after the liver, accounting for approximately 10–15% of metastasis . The medSome articles suggest that the site of the primary tumour also affects prognosis. That is, because of a more aggressive phenotype, right colon cancer has a worse prognosis –14. In aDue to the lack of large-scale retrospective studies describing the clinical characteristics of CRC patients with lung metastasis, no prediction of cancer-specific survival (CSS) has been reported, and limited information is available to analyse the prognosis of CRC patients with lung-only metastasis. Therefore, we used information from the Surveillance, Epidemiology, and End Results (SEER) database to analyse the incidence, risk factors, and prognostic factors of CRC lung metastasis. This analysis was performed separately for CRC patients who developed lung metastasis and those who had only lung metastasis. Additionally, we reviewed the data of 70 CRC patients who were diagnosed with lung metastasis and were admitted to our hospital and analysed their clinical characteristics, treatment methods and efficacy.The SEER database is a comprehensive cancer statistics database that contains 17 population-based cancer registries that account for 28% of the US population and record information on individuals with malignant tumours in the United States. We first submitted the data consent form to the SEER administration and then collected the relevant data using SEER*Stat version 8.3.5. The information presented in this study is based on the most recent follow-up  available in the SEER database. Informed consent was not necessary in our investigation since the data gathered from the SEER database were anonymized prior to release.New CRC cases were collected from the SEER database between January 2010 and December 2015. Based on these data, we chose patients using the procedure depicted in Fig. The study included 70 CRC patients with lung metastasis who underwent resection at Shanghai East Hospital between May 2016 and March 2019. The following criteria were used to determine inclusion: stage III-IV CRC; lung metastasis discovered after CRC resection in our institution; CT utilized to confirm lung metastasis; and more than 2 years of follow-up. Sex, age, site, T and N stage, survival months, organ metastasis, lymph node involvement, classification of lung metastasis, interval between operation and lung metastasis, treatment after lung metastasis, interval between lung metastasis and death or last follow-up, carcinoembryonic antigen (CEA) level , and status were collected from each record. Due to the limited sample size, the cause of death for 70 patients at our hospital could not be determined. The 2016 ESMO handbook defines oligometastasis as “the presence of metastasis at up to two or three locations and five or more lesions.” This study followed the Declaration of Helsinki and the ethical principles of Shanghai East Hospital.x2-test was performed to analyse lung metastasis incidence between subgroups for categorical variables to identify risk factors for CRC patients with lung metastasis, and variables with statistical significance were chosen for multiple logistic regression. The Kaplan–Meier technique and log-rank test were used in the training cohort to determine factors that impacted OS; competitive risk analysis was utilized to identify significant variables that influenced CSS; and a multivariate analysis was performed using a Cox regression model. The concordance index (C-index) was used to examine the internal validity of the nomogram, which was calculated by comparing the nomogram-predicted probability with the observed probability. The nomogram was then verified further by comparing the nomogram-predicted likelihood of patients in the validation cohort to their actual survival. SPSS 24.0  and the ggplot and rms packages in R 3.4.3 (https://www.r-project.org/) were used for statistical analyses. All statistical tests were two-tailed, with p < 0.05 deemed statistically significant.For the description of baseline attributes, descriptive statistics are utilized. The As shown in Table p < 0.001); race (p < 0.001); sex (p < 0.001); grade (p < 0.001); primary tumour site (p < 0.001); T stage (p < 0.001); N stage (p < 0.001); the presence of liver (p < 0.001), brain (p < 0.001), or bone (p < 0.001) metastasis at initial diagnosis; marital status (p = 0.004) and insurance status (p < 0.001) were independent prognostic factors that affected the risk of lung metastasis.As illustrated in Supplemental Table Table p < 0.001); race (p = 0.027); tumour grade (p < 0.001); T stage (p < 0.001); N stage (p < 0.001); M stage (p < 0.001); bone metastasis (p < 0.001); brain metastasis (p < 0.001); liver metastasis (p < 0.001); marital status (p < 0.001); insurance status (p < 0.001); chemotherapy (p < 0.001); grade (p < 0.001); radiation therapy (p < 0.001); surgery (p < 0.001); and primary tumour site (p < 0.001) were significant prognostic factors. Moreover, competing risk models were constructed, and the CSS curves of the patients were parsed ; pathological grade (p < 0.001); T stage (p < 0.001); N stage (p < 0.001); bone metastasis (p < 0.001); brain metastasis (p < 0.001); liver metastasis (p < 0.001); primary tumour site (p < 0.001) and surgery (p < 0.001). Additionally, the survival outcomes among CRC patients with isolated lung metastasis were as follows. The results of the Kaplan–Meier analysis are presented in Fig. p < 0.001); T stage (p < 0.001); marital status (p < 0.001); chemotherapy (p < 0.001) and surgery (p < 0.001).As shown in Table As shown in Fig. Supplemental Table This study assessed the independent prognostic factors affecting survival in 6278 CRC patients with lung metastasis and 1065 patients with only lung metastasis from the SEER database records of CRC diagnosed from 2010 to 2015. Two nomograms were separately established, which performed well in predicting survival. The data of 70 hospitalized patients diagnosed between May 2016 and March 2019 were collected and analysed using the nomogram. Data from patients with colorectal cancer lung metastasis and lung-only metastasis diagnosed between 2016 and 2019 selected from the SEER database were used as external validation cohorts.Previous studies have predicted the 1- and 3-year total survival in patients with lung metastasis from CRC, but the variables included in these equations are different due to a lack of univariate analysis . FurtherIn clinical practice, any organ metastasis (such as brain or bone metastasis) corresponds with a relatively poor prognosis . AccordiDifferent primary sites of tumours determine the different biological behaviours of tumours. In our study, the right colon was the most common site of colon cancer (40.9%), while rectal cancer accounted for 22.6% of cases. In terms of lung metastasis, the incidence of lung metastasis and the incidence of lung-only metastasis in the right colon compared to rectal cancer was 31.1% vs. 31 and 28.5% vs. 40.5%, respectively, which indicates a higher risk of lung metastasis in rectal cancer. This result is similar to findings from other studies . PreviouIn terms of treatment, surgery is also a key factor affecting the prognosis of cancer. The choice of primary tumour surgery remains controversial. In fact, more than two-thirds of elderly patients with stage IV CRC are known to have undergone primary tumour surgery . The reaThe data for 70 patients with colorectal cancer lung metastasis in our hospital were evaluated. The incidence of lung metastasis 1 year after primary resection was 40.7%, and the probability of lung metastasis after 2 years was 20.0%, which suggests that we should identify lung metastasis as soon as possible, especially for patients with high-risk factors, and regular CT reviews should be conducted. In comparison with a previous study, Facciorusso A. et al. . definedOur study has clear limitations. First, the manuscript lacks of granularity in the SEER database as it relates to the therapy sequencing and timing, patient comorbidities, and timing of clinical progression. Numerous data were missing in the SEER registry. Second, the differences in race, treatment methods and some baseline data could lead to bias when using data from 70 patients in China to verify the nomogram established from the SEER database in the US. Third, in addition to clinical baseline data, many biomarkers are associated with prognosis in CRC patients, such as RAS, BRAF, and MMR/MSI –38. SeveIn summary, we developed two nomograms to predict OS in CRC patients with lung metastasis and lung metastasis only. It is recommended to use nomograms as a useful tool to predict prognosis at different time points in CRC patients with lung metastasis and to help clinicians choose appropriate treatment options for different populations. In addition, advanced age, nonwhite race, female sex, poor differentiation, T4 stage, lymph node metastasis, liver metastasis, brain metastasis, bone metastasis, being unmarried, and rectal cancer are associated with an increased risk of lung metastasis.Additional file 1: Supplemental Table 1. Clinical and demographic characteristics of the external cohort.Additional file 2: Supplemental Table 2. Univariate and multivariate analysis for the presence of lung metastasis in CRC patients.Additional file 3: Supplemental Table 3. Clinical and pathological characteristics, treatment modalities, and outcomes.Additional file 4: Supplemental Figure 1. 1 Competing risk analyses for CRC patients with lung metastasis in training cohort according to (A) Grade, (B) Age, (C) Race, (D) Site, (E) Bone metastasis, (F) Brain metastasis, (G) Insurance status, (H) Chemotherapy. 2 Competing risk analyses for CRC patients with lung metastasis in training cohort according to (I) Liver metastasis, (J) Marital status, (K) N stage, (L) Radiotherapy, (M) Gender, (N) Surgery, (O) T stage.Additional file 5: Supplemental Figure 2. Calibration curve of the nomogram predicting 1- and 3-year OS rates of CRC patients with lung metastasis in the external validation cohort (A) Calibration curve of the nomogram predicting 1- and 3-year OS rates of CRC patients with lung-only metastasis in the external validation cohort (B).


Background: Immune checkpoint inhibitors (ICIs) represent one of the most effective treatments for patients with cancer. As their activity relies on host immune system reactivity, the role of concomitant medications such as corticosteroids and antibiotics has been extensively evaluated. Preclinical data suggest that opioids may influence the immune system. Methods: a systematic literature revision was performed using specific keywords on the major search engines. Two authors analysed all the studies and provided a selection of the following inclusion and exclusion criteria, respectively: 1. data collection of patients older than 18 years old affected by solid tumours; 2. description of ICIs efficacy in terms of PFS, OS, TTF, and ORR; 3. concomitant ICIs-opioids treatment and 1. language different from English; 2. not pertinent analyses. Results: 523 studies were analysed, and 13 were selected and included in our series. A possible negative interaction between oral opioids and ICIs efficacy was observed. Most evidence was retrospective, and studies were heterogeneous. Conclusions: Even if oral opioids seem to impact negatively on ICIs efficacy in cancer patients, to date there is not sufficient evidence to avoid their prescription in this population. Given the wide spread of immune checkpoint inhibitors (ICIs) as a treatment for several tumours, the definition of possible interactions between ICIs and concomitant drugs has recently gained importance. While the negative interactions of antibiotics and corticosteroids are now well known ,2,3, othThe mechanisms of opioids on the immune system or immune cells have been studied for over 40 years. Some opioids are associated with immunosuppressive effects, with a developing knowledge that morphine, fentanyl, buprenorphine, and methadone suppress innate immunity while having different effects on adaptive immunity. It is similarly apparent that specific opioids have immunostimulatory effects, some exhibit dual effects, and others have no immunomodulatory effect . Indeed,In this systematic review, we aimed at describing the relationship between the immune system and opioids in patients affected by solid tumours.A systematic literature review was performed by using the following search engines: PubMed, Google Scholar, Cochrane, and Cinahl. The following keywords were used: “opioids” OR “concomitant treatments” AND “neoplasm” OR “tumour” OR “cancer” AND “immunotherapy” OR “immune checkpoints inhibitors” OR “PD-1/PD-L1 inhibitors”. We considered reports published from 1st January 2000 to 1st August 2022. It was decided to include only studies, which analysed adult patients affected by solid tumours. Other inclusion criteria were as follows: (1) the description of ICIs efficacy outcomes in terms of progression-free survival (PFS), overall survival (OS), overall response rate (ORR), time to treatment failure (TTF); (2) concomitant ICIs-opioids treatment. Otherwise, (1) language different than English and (2) not pertinent works were excluded. Two authors (MC and PB) reviewed all the studies and approved the selection following the inclusion and exclusion criteria. In case of disagreement a third author (LB) was asked to make the final decision. The following outcomes considered were: PFS, OS, ORR and TTF. Finally, the reviewing process followed PRISMA guidelines  even if Five hundred and twenty-three studies were analysed, including abstracts, posters, and oral presentations for international meetings [such as the European Society of Medical Oncology (ESMO) and the American Society of Clinical Oncology (ASCO) meetings]. Thirteen studies were finally selected . Of thesp = 0.018) [p < 0.001) [p < 0.001). These associations were maintained at a multivariate analysis that included performance status, clinical stage, and a number of metastatic sites [p = 0.007), together with subsequent lines of treatment and higher ECOG PS [p = 0.0515) [p = 0.001) [Studies were heterogeneous also when accounting for main outcomes, including progression-free survival (PFS), overall survival (OS) and in some cases, time to treatment failure (TTF) or response rate (RR). However, all reports showed a negative correlation in terms of OS, PFS or TTF between opioids and ICIs. Taniguchi et al., by analyzing 38 patients treated with opioids matched with others 38 opioids naïve, showed a decreased mOS for the first group  . Another ECOG PS . Other s 0.0515) . Similar= 0.001) .Up to 90% of cancer patients experience pain at some stage of their disease journey, with a third rating the intensity of their pain as moderate to severe, and up to half being undertreated . In the Regarding the possible influence of opioids on ICIs efficacy, our systematic review suggests that opioid use may be associated with worse outcomes. None of the studies evaluated specific safety issues when dealing with the concomitant administration of opioids and ICIs. As already stated, our study has several limitations. One of these consists of the retrospective nature of all the included studies. Therefore, concomitant medications have been extracted from prescription files, and some authors did not report the type and/or dosage of opioids. Furthermore, 7 out of 13 studies were designed to include all concomitant drugs during ICIs treatment. Another major limitation bias is the heterogeneity of included population. Indeed, 6 studies enrolled patients affected by different tumours, while 11 studies the included patients treated with different ICIs in different treatment lines. In almost all the studies both the performance status and the tumour burden were assessed and evaluated in multivariate analyses, generally maintaining statistical significance, with the exception of the study of Gaucher et al. . It shouMoreover, poli-pharmacy treatments identify patients characterised by several comorbidities, a higher tumour burden and already treated with several lines of treatments, with a well-known reduced response to ICIs . In the Oral opioids seem to impact ICIs efficacy in cancer patients in different and not completely known ways. Possible mechanisms rely on the presence of morphine receptors on cancer cells and the influence of opioids on the immune system; others consider the role of the microbiome. To better understand the relationship between ICIs and concomitant drugs such as opioids, prospective studies with large sample sizes should be encouraged. Concomitant drugs during chemotherapy and ICIs treatment should be carefully prescribed even if, as of today, there is not enough evidence to avoid the prescription of opioids in patients treated with ICIs.


Biological condensates are membraneless organelles with different material properties. Proteins and RNAs are the main components, but most of their interactions are still unknown. Here, we introduce PRALINE, a database for the interrogation of proteins and RNAs contained in stress granules, processing bodies and other assemblies including droplets and amyloids. PRALINE provides information about the predicted and experimentally validated protein–protein, protein–RNA and RNA–RNA interactions. For proteins, it reports the liquid–liquid phase separation and liquid–solid phase separation propensities. For RNAs, it provides information on predicted secondary structure content. PRALINE shows detailed information on human single-nucleotide variants, their clinical significance and presence in protein and RNA binding sites, and how they can affect condensates’ physical properties.http://praline.tartaglialab.com.PRALINE is freely accessible on the web at  Although the exact composition and functions of the different condensates are unknown, they are enriched in protein and RNA molecules that interact through protein–protein, protein–RNA and RNA–RNA networks. Solid-like condensates, and in particular amyloids, are generally considered to be inherently irreversible aberrant clumps https://www.ensembl.org/) and UniprotKB ID , while experimental and predicted protein–RNA interactions can be retrieved from RNAct (https://rnact.crg.eu/). Protein–RNA interactions are calculated using catRAPID, an algorithm trained on NMR and X-ray structures (area under the curve (AUC), of 0.77 on eCLIP interactions)  and the organelle in which it has been found (SG/PB). The predicted liquid–liquid phase separation (LLPS) and LSPT propensities and profiles of the wild-type sequence are provided, calculated with tGRANULE . Experimactions) .http://s.tartaglialab.com/page/cross_group), the experimentally validated RNA interactions  and the predicted or experimentally validated protein interactions reported in RNAct (https://rnact.crg.eu/) for both the reference sequence and SNVs (https://www.encodeproject.org/eclip/) that involve the SNV containing region , the RNA secondary structure content (table and profile predicted using CROSS, and SNVs . The RNAand SNVs . The SNVg region .For most genes, information is available at both the protein and RNA levels, so it is possible to navigate from one molecule to the other, revealing the links between them.PRALINE is a database that provides a comprehensive view of protein and RNA interactions and SNVs in human liquid-like and solid-like condensates. Information about experimentally validated and predicted molecular interactions, including protein–protein, protein–RNA and RNA–RNA, is provided, as well as the predicted RNA secondary structure content and both LLPS and LSPT propensities of proteins.catGRANULE predictions could not be benchmarked against a database of individual SNVs causing LLPS, due to a lack of adequate published resources. The availability of such databases will lead to a more precise understanding of the relationship between SNVs, structural conformations, protein–RNA assembly and diseases.For each SNV, we provide a description of the associated diseases, the binding sites and the change in RNA secondary structure, LLPS and LSPT propensities. Combining physico-chemical properties of molecules and disease-related annotations, PRALINE helps to unravel macromolecular connections that sustain different types of condensates and how variants can affect their equilibrium. PRALINE is the first database providing LLPS and LSPT predictions for SNVs, and we envisage that it would greatly facilitate the design of experiments to study condensates’ formation and implication in human diseases. Although tested extensively and validated experimentally,


Fusarium graminearum small RNA (sRNA) pattern was regulated by Fusarium graminearum hypovirus 1 (FgHV1) infection, indicating the activation of RNA silencing in virus defense. In this study, we focused on the function of an uncharacterized protein sized at 20 kD (p20) encoded by FgHV1. In the agro-infiltration assay, p20 was identified as a novel fungal RNA silencing suppressor. p20 can block systemic RNA silencing signals besides local RNA silencing suppression. We further elucidated the RNA silencing suppression mechanism of p20. The single-strand sRNA, instead of double-strand sRNA, can be incorporated by p20 in electrophoretic mobility shift assay. p20 binds sRNA originating from virus and non-virus sources in a non-sequence-specific manner. In addition, The F. graminearum 22 and 23-nt sRNA abundance and pathways related to RNA processing and redox regulation were regulated by p20. Our study revealed the first fungal virus-encoded RNA silencing suppressor with sRNA binding capability.Fungal viruses are widespread in fungi infecting plants, insects and animals. High-throughput sequencing has rapidly led to the discovery of fungal viruses. However, the interactive exploration between fungi and viruses is relatively limited. RNA silencing is the fundamental antivirus pathway in fungi.  Among them, sense single strand RNA fungal viruses are the largest group. Most fungal viruses exist as latent or asymptomatic infections. With the development of high-throughput sequencing technology, fungal viruses have been excavated and characterized more rapidly. Currently, some fungal viruses have the ability to attenuate the pathogenicity of host fungi, such as Cryphonectria parasitica, Sclerotinia sclerotiorum and Fusarium graminearum [A fungal virus (mycovirus) is a kind of virus that replicates inside fungi . The genminearum ,4,5,6. Iminearum .Fusarium spp. Causing Fusarium head blight threatens wheat, barley and small grain cereals worldwide [Fusarium spp [F. graminearum. FgHV1 is closely related to Cryphonectria hypovirus 1 (CHV1) [S. sclerotiorum morphology, pathogenicity, pigmentation and sporulation, while FgHV1 had minor effects on fungal morphology, growth and spore production [F. graminearum. These genes are mainly related to transcription factors, cellular redox regulation and ubiquitination system. Moreover, p20 encoded by FgHV1 can induce hyper antisense response and H2O2 accumulation in Nicotiana benthamiana leaves [orldwide . More thrium spp . Among t1 (CHV1) . Howeveroduction ,10. Addia leaves .F. graminearum, and it was revealed that there were 1,831,081  sRNA reads mapped to FgHV1 genome, accounting for 16.51% of F. graminearum total sRNA [RNA silencing is the fundamental pathway for gene expression regulation and virus defense . RNA siltal sRNA . In addiCpDCL2 and CpAGO2 [CpDCL2 was also suppressed by p24 from CHV4 [FgDICER2 and FgAGO1, the ORF2 protein of Fusarium graminearum virus 1 protect the virus from host RNA silencing elimination [To counter-dense host RNA silencing, viruses encode various RNA silencing suppressors (RSS) . Until nd CpAGO2 ,18,19. Srom CHV4 . By bindmination . For themination . To explF. graminearum strain 4, which was collected and kept in our lab. After sequencing, the fragment was inserted into multiple clone sites (MCS) of PGTN vector for fungal transformation. In the agro-infiltration assay, p20 was inserted into pGD construct for transient expression. P19-pGD  and GFP-pGD construct were used and kept in our laboratory.The cDNA of p20 were reverse transcripted and amplified from RNA extracted from FgHV1 infected F. graminearum PH-1 strain, stored in our laboratory, was used for constructs transformation. All fungal strains were maintained at 4 °C and −80 °C in 25% glycerin. The growth condition of F. graminearum was at 25 °C in the dark on the PDA plate. The fresh mycelium used for DNA or RNA extraction were obtained from 3-day-old cultures grown on PDA overlaid with cellophane membrane . N. benthamiana wild type and transgenic N. benthamiana (line 16 c) seeds were a gift of Dawei Li from China Agricultural University. Plants were grown in a greenhouse at 25 °C with 16/8 light and dark cycles. Four-week-old plants were used for agro-infiltration.The sequence of 21 nt eGFP-sense small single-strand RNA was 5′-GCUGACCCUGAAGUUCAUCUU-3′. 21 nt eGFP-antisense small single-strand RNA was 5′-GAUGAACUUCAGGGUCAGCUU-3′. 24 nt eGFP-sense small single-strand RNA was 5′-CGUACGCGGAAUACUUCGAAAGUU-3′. 24 nt eGFP-antisense small single-strand RNA was 5′-CUUUCGAAGUAUUCCGCGUACGUU-3′. The sense and antisense single-strand sRNA were designed as complementary with a 2 nt 3′ overhang for duplex formation. Double-strand small RNA was synthesized with the above sense and antisense single-strand small RNA. The concentration of single-strand small RNA was 100 μM, and the reaction mix together with 5× annealing buffer  was denaturated at 94 °C for 5 min and then incubated at 37 °C for 1 h. The FgHV1 derived sRNA for specificity binding test was siRNA-t1130926 (5′-CTCCAGTAGCATGTTCTTCGT-3′) targeting FGSG_09025 (Dicer 1), siRNA-t0999532 (5′-CTCCAGTAGCATGTTCTTCGTG-3′), targeting FGSG_09025 (Dicer 1) and siRNA-t0031901 (5′-TGGAAGAGAATGACGATATT3′), targeting FGSG_09076 (RDRP 5).Agrobacterium tumefaciens (strain EHA105) with electroporation. An equal volume of p20-PGD, p19-PGD (positive control) and pGD (negative control) transformed A. tumefaciens were mixed with A. tumefaciens containing GFP-PGD. All A. tumefaciens strains were adjusted to OD600 = 1 before mixing. The mixtures were stood for at least 3 h before infiltration. The middle leaves of 4-week-old wild type or GFP transgenic N. benthamiana were injected with 1 ml of the mixture with sterile syringes. The injection point was in the middle of the leaf in half. The GFP fluorescence was checked by handheld UV light. Leaves and plants were photographed at 3 and 15 dpi. Each agro-infiltration experiment was conducted on at least 20 leaves of 10 plants and repeated three times.p20-PGD, p19-PGD, GFP-PGD and pGD were transformed into Escherichia coli BL21, tagged with histidine. The transformed BL21 strain was cultured in LB medium with 1 mM IPTG at 16 °C for 16 h. Then the bacteria cells were collected by centrifugation. The his-tagged protein was purified with affinity chromatography by ÄKTA. p20 purity was separated on 12% polyacrylamide gel electrophoresis and stained with coomassie blue dye. The final protein was adjusted to 1 mg/mL for electrophoretic mobility shift assay (EMSA), and the reaction concentration of chemically synthesized siRNA was 100 μM. Four EMSA reactions were conducted with 0, 1, 3, 9 μL of p20, respectively. The reaction mix was incubated at room temperature for 30 min and loaded in 8% native polyacrylamide gel for electrophoresis. The gel was run for 10 min at 30 mA, followed by 40 min at 10 mA. The gel was transferred for nucleic acid and protein staining, respectively, using an EMSA kit .p20 gene was reverse transcribed and PCR amplified from FgHV1 genome RNA. The segment was purified and cloned into MCS sites of the protein expression vector, pET30 a in http://www.mirbase.org/, accessed on 14 October 2022) database contain a large number of miRNA information and target gene of animals and plants. RNAcentral V16.0  is a non-coding RNA database, and it provides the most comprehensive and up-to-date non-coding RNA information on different species. The Rfam V13.0 database  is a collection of RNA families, each represented by multiple sequence alignments, consensus secondary structures and covariance models. Based on the taxonomy ID of the species, we downloaded the non-coding RNA sequence of this species from miRBase and RNAcentral, and then map reads to these sequences to identify known non-coding RNAs. After these, the unannotated reads were mapped to Rfam. However, if there were no non-coding RNA in miRBase and RNAcentral, reads were mapped to Rfam directly. Based on the AASRA theory , we chose the best mapping of each read, and then stat the TPM (tags per million reads) expression of each non-coding RNA . After obtaining the expression of all non-coding RNAs, principal component analysis and correlation analysis were carried out. According to the results of correlation analysis and principal component analysis, the similarity between samples can be judged. The better the repeatability, the greater the correlation coefficient, and the closer the cluster in the principal component analysis diagram. The sRNA sequencing raw data was submitted to the national center for biotechnology information website  under project No. PRJNA879941.Total RNA was extracted, and quality was determined by HPLC. Additionally, the qualified RNA was used for high-throughput sequencing using the DNBSEQ-T7 system . The original data was obtained by sequencing (Raw Reads); before data analysis was carried out, low-quality data and adapters were removed. By mapping the sequencing data with the reference genome sequence, the contamination or confusion in the sample was judged. Samples with very high genome mapping rates were used for the following analysis. Three databases were used for non-coding RNA identification. miRBase V22 . DEseq2 was used for differentially expressed gene analysis with p ≤ 0.05. Based on GO and KEGG annotation, differentially expressed genes (DEGs) were enriched into different terms with pHYPER function in R statistical software. The RNA sequencing raw data was submitted to the national center for biotechnology information website  under project No. PRJNA879941.RNA for transcriptome sequencing was prepared as follows: mRNA was enriched by oligodT beads. rRNA was eliminated by RNase H digestion after hybridization with a DNA probe. The probe was digested with DNase. The RNA was cut into appropriate lengths and reverse transcripted with a random N6 primer. Then the second strand was synthesized. The products were phosphorylated and ligated with an adapter. After amplification, the heated single strand was cyclized and sequenced. The clean reads were mapped to Nicotiana benthamiana wild type was co-infiltrated with Agrobacterium strain containing pGD-GFP construct under 35 s promoter for GFP expression (35 s-GFP). After mix-infiltration with pGD-p19, p20 and empty vector, green fluorescence was still stable and clear in p20 and p19 co-infiltrated leaves at 3 dpi, while the GFP signal was disappearing in the empty control  [N. benthamiana line 16 c were agro-infiltrated with the three mixture groups, respectively. At 3 dpi, the agro-infiltrated leaves were observed for green fluorescence under a handheld UV trigger with an orange filter. As shown in N. benthamiana plant as a result of RNA silencing stimulating. In contrast, the RNA silencing suppressor, p19 construct agro-infiltrated leaves exhibited obvious green fluorescence. The green fluorescence intensity was also obvious on p20 agro-infiltrated leaves, which indicates that p20 protect GFP from degradation by suppressing the host RNA silencing system. This phenomenon supported that p20 inhibited local RNA silencing on infiltrated leaf portions. To further examine the local suppression of the RNA silencing capability of p20, RSS candidates together with a sense GFP transgene (35 s-GFP), RNA silencing inducer, were transiently co-expressed in GFP-transgenic c (16 c) ,25. The RNA silencing signal can move systemically to other tissues in an organism . Next, wF. graminearum and found that 21 and 24-nt were the sRNA peaks and most regulated sRNA lengths. As it is demonstrated above, p20 suppresses GFP silencing in 16 c plants. Hence, 21 and 24-nt sense and antisense single-strand GFP RNA were chemically synthesized. In addition, 21 and 24 bp double-strand GFP RNA were acquired by single-strand RNA annealing from sense and antisense single-strand GFP RNA, respectively. In the binding reactions, 1 μL of sRNA (100 μM) was incubated with 0, 1, 3 and 9 μL of purified p20 (1 μg/μL). After incubation, p20 protein and sRNA were visualized on the same gel. If the sRNA were trapped in p20, the protein-sRNA complex moved much slower than free sRNA in gel. As for 21 and 24-nt sense and antisense single-strand sRNA binding, there were less free sRNA observed in gel with an increasing concentration of p20  was found to be between 26–32 nt, small interfering RNA was found to be between 20–25 nt. The genome mapping rate ranged between 90.30 to 93.12%. The clean sRNA reads number for PGTN, PGTN-r1, p20 and p20-r1 was 23,772,921, 22,955,622, 22,844,246 and 24,607,579, respectively. The number of sRNA reads in the p20 group did not decline significantly. This indicated that p20 could not bind a large number of sRNA. Length distribution analysis allowed us to check the abundance of each sRNA length. Thus, we also analyzed the influence of p20 on the length distribution of sRNA in F. graminearum. As shown in As demonstrated above, p20 had single-strand small RNA incorporating capability. Small RNA profiles of p20 transformed Further, we wondered if FgHV1-encoded p20 differs in its ability to bind exogenous and autologous sRNA (virus-originated and non-itself sRNA). Three virus-derived 20, 21 and 22-nt sRNA (vsiRNA) were synthesized and used for the protein-RNA binding capacity test. We also carried out EMSA to investigate whether they can be incorporated into p20. As shown in F. graminearum host, we examined the DEGs via RNA sequencing. Compared with the empty vector control, 1764 genes were differentially expressed upon p20 expression in F. graminearum, which accounted for about 10% of total host genes  exists in all kinds of fungi . Mycovirminearum . In the minearum ,15. MoreN. benthamiana line 16 c, our experiments demonstrated that the p20 expression construct could inhibit RNA silencing induced by GFP mRNA co-infiltration. In addition, we wondered whether p20 could suppress systemic RNA silencing signal transmission. It was quite exciting to find that the systemic RNA silencing signal was blocked by p20. In fungi, further experiments can be designed to confirm whether p20 can facilitate virus spread and benefit other viruses’ co-infection for its systemic RNA silencing suppression capability.Using the GFP-transgenic FgDCL2 and FgAGO2 [FgDCL2 and FgAGO1 could be bounded by ORF2, which resulted in their expression suppression. Further experiments about other RSS mechanisms of p20 can be explored.Further, we elucidated the RNA silencing suppression mechanism of p20. Based on protein structure, p20 might have sRNA-incorporating channels. EMSA indicated that single-strand sRNA could be loaded into p20, while double-strand sRNA cannot be incorporated inside. The mechanisms of RSS are also various in different viruses infecting plants and animals. Until now, there have been four fungal virus RSS, and three of them have been characterized by their suppression mechanism. p29 and p24 from CHV1 and CHV4 could interact with RNA silencing-related protein, thus suppressing the expression of RNA silencing critical genes, d FgAGO2 ,18,19,20d FgAGO2 . The proF. graminearum. However, the total sRNA reads were nearly equal in p20 transformed and empty construct control groups. This result indicated that p20 could not bind a large number of sRNA in the host, even with sRNA binding ability. However, it was also possible that FgHV1 infection induces RNA silencing and sRNA production increase. Partial sRNAs were incorporated into p20, and then the accumulation of sRNA was returned to a normal level. We also analyzed the data in detail and found that 22 and 23-nt sRNA abundances were regulated. Then we were curious about the sequence selectivity of p20. It is well known that the targets of some virus-derived sRNA were host genes, which mediated host gene suppression. Two RNA silencing critical genes, FgDicer1 (FGSG_09025) and FgRdRp5 (FGSG_09076), have been predicted as targets of three FgHV1-derived sRNAs. The incorporation of these three vsiRNA is not beneficial for RNA silencing suppression and virus accumulation. Thus, we tested the binding of p20 with these three vsiRNA. It is unexpected that p20 incorporated them un-selectively. This result indicated that p20 bound sRNA in a non-sequence-specific manner.Because of the sRNA binding capacity of p20, it was reasonable to speculate that the total sRNA amount in the host would be reduced. Therefore, we sequenced the sRNA in p20-transformed 2 O2 accumulation and hypersensitive reaction in N. benthamiana. Future studies about the functions of p20 during host antivirus and virus counterdefense are needed.In this paper, it was interesting to figure out the sRNA binding capacity of p20, as this is the first report of a fungal virus with an sRNA-binding RSS. However, the sRNA incorporating capability might not be the only manner for RNA silencing suppression by p20. Many genes and pathways were regulated by p20. DNA repair (methylation), RNA processing and redox regulation were the most enriched pathways among p20-regulated terms. Previously, we also found that p20 can induce H


There are several theories on the origin of life, which differ by choosing the preponderant factor of emergence: main function , initial location (black smokers versus ponds) or first molecule (RNA versus DNA). Among the two last ones, the first assumes that an RNA world involving a collaboration of small RNAs with amino-acids pre-existed and the second that DNA–enzyme–lipid complexes existed first. The debate between these classic theories is not closed and the arguments for one or the other of these theories have recently fueled a debate in which the two have a high degree of likelihood. It therefore seems interesting to propose a third intermediate way, based on the existence of an RNA that may have existed before the latter stages postulated by these theories, and therefore may be the missing link towards a common origin of them. To search for a possible ancestral structure, we propose as candidate a small RNA existing in ring or hairpin form in the early stages of life, which could have acted as a “proto-ribosome” by favoring the synthesis of the first peptides. Remnants of this putative candidate RNA exist in molecules nowadays involved in the ribosomal factory, the concentrations of these relics depending on the seniority of these molecules within the translation process. Xanthoria elegans remained viable after 18 months of space exposure outside the International Space Station (ISS) [When the Latin poet Ovid was writing about metamorphoses two thousand years ago, things were very simple: living beings were spontaneously generated from non-living matter . It was on (ISS) , it is eAzoarcus ribozyme [Several theories have dealt with the problem of the origin of life. They differ in focusing each on a preponderant factor of emergence: the main primitive function , the initial location of the first living systems (black smokers versus ponds) or the first molecule involved in the origin of life (RNA versus DNA). In the last couple of opposite theories, the first assumes that a primitive “RNA world” has existed. It was based on the collaboration of nucleic and amino-acids polymers, the organic synthesis of which is obtained in absence of any living process ,14,15,16ribozyme ,19,20, bIn the second theory, it was assumed that a primitive DNA–enzyme–lipid complex pre-existed, which allowed storage of information in DNA, replication by polymerases and individualization by lipids of a living machine, which were then subject to Darwinian selection. The main criticism about the RNA world was that it should contain reflexive information together with information, i.e., information which, decoded by the system, produces the components that perform exactly that particular decoding. Moreover, it was proposed that protein-like molecules were better candidates than RNA for having been the first self-replicators functioning on the planet.To solve this problem, authors recently used, in the spirit of Stanley Miller’s pioneering experiment, a hydrogen cyanide-based chemical medium mimicking early Earth conditions to synthesize the four purine and pyrimidine bases ,22: the More recently, it was suggested to integrate the two theories with each other. DNA, enzymes and lipids were assumed to have interfered early with the engineering of the primitive RNAs  in orderWe will present successively, in GCC AC-loop.We start by searching in the oldest living organisms, the Archaea, the most conserved sequences in the machinery devoted to the protein building, that is, the ribosome and associated molecules as tRNAs, amino-acyl tRNA ligases, nucleolin, etc. Concerning the ribosome, we will examine, namely, its smallest component, the 5S rRNA as well as the most ancient ribosomal RNAs and proteins following the classification by authors such as G. Caetano-Anollés ,43,44,45® [If we search similar sequences among the 5S rRNAs , we find®  the hair® ,29,30,31-The subsequences of AL are the most frequent in 5S rRNAs and in Gly-tRNAs.-All dinucleotides appear at least once .-12 of possible solutions) containing only one codon AUN repeated, N being G in 52% of the cases.Among the rings satisfying the minimality principle “to be as short as possible and contain at least one codon of each amino acid class of synonymy” of the genetic code b, there -® for the most stable hairpins gave 25 rings of length 9, with a head of length 3, the most stable being AGA , one singular ring (the AL ring) exhibits a minimum average distance as compared to the others. Therefore, only this sequence can be that acting as a barycenter of the set of the 18 others.If we consider the AL sequence as a source of primitive information on the genetic code ,49,50, iAmong its pentamer subsequences, we have selected the eight pentamers resulting from the opening of its hairpin head conformation c, i.e., The ancestral molecules that participated in the first prebiotic assemblies might have been RNA oligomers and small peptides formed from nucleotides on the one hand and amino-acids on the other hand, both being present in hot springs and/or in clay matrices . We may The small RNAs would be good candidates to be actors and witness of this evolution from a purely mineral behavior to the organic world. If we consider that the current most effective machinery for protein synthesis is the ribosome, we may wonder whether a “memory” of the primitive machinery possibly exists in that particle. Such a memory should be almost universal, i.e., it should be found from the origin in a large majority of ribosomal mechanisms of different species, which is the case in Methanococcus vanniellii or Maripaludis voltae : AL mee® [All the material is coming from the public databases GtRNAdb  and 5S R® .Less than a decade later, Stanley Miller  announceAlthough this ambitious goal has still not been achieved, efforts are continuing, eliminating step by step the remaining gaps in the knowledge of prebiotic chemistry. One step could be represented by the present effort to solve the challenge of the origins, DNA first or RNA first. The ring AL we have exhibited could be an element of an answer, because its purely informative, combinatorial and chemical properties, could help to: (i) build the first peptides by promoting the coming together of amino acids synthesized in the primitive atmosphere (as demonstrated by S. Miller), thus acting as a proto-ribosome. These amino acids could have created more stable peptide bonds between them than their weak electromagnetic bonds to AL and then, (ii) promote a primitive metabolism from these peptides, the first role of which would have been to promote the replication of AL and its spatial individuation by organizing a first water-repellent peptide or lipid–peptide structure serving as a proto-membrane (as postulated by Dyson ).The major criticisms that can be made to our approach are three-fold: (i) the proposed structure is too small in size to be functional, but recent works show that very small RNAs of size 12 nt can have an important function, such as being able to modulate the microRNA from which they derive , (ii) thIf the birth of life really took place shortly after Earth’s creation, it is likely that it will remain not-understood forever. It might have been anything, perhaps a very improbable event occurring in an extremely brief instant, such as the Late Heavy Bombardment (LHB) on Earth. However, even in that case, it is a goal of major interest to continue deciphering the history of life on Earth, as close as possible to the non-understandable discontinuity “non-living/living”. New data can be gathered in different ways to check the theories and the present proposal of a primitive ring/hairpin RNA structure spontaneously generated among random sequences and the shortest to present a great coherence with the genetic code could help the theoretical and experimental understanding of this primordial founding event of life on Earth. Concerning the experimental validation, we have proposed in  a test i


Nicotiana benthamiana genome contains hundreds of retrozyme loci, of which nine represent full-length retrozymes. The LTR contains a promoter directing retrozyme transcription. Although retrozyme RNA is easily detected in plants, the LTR region is heavily methylated, pointing to its transcriptional silencing, which can be mediated by 24 nucleotide-long retrozyme-specific RNAs identified in N. benthamiana. A transcriptome analysis revealed that half of the retrozyme-specific RNAs in plant leaves have no exact matches to genomic retrozyme loci, containing up to 13% mismatches with the closest genomic sequences, and could arise as a result of many rounds of RNA-to-RNA replication leading to error accumulation. Using a cloned retrozyme copy, we show that retrozyme RNA is capable of replication and systemic transport in plants. The presented data suggest that retrozyme loci in the N. benthamiana genome are transcriptionally inactive, and that circular retrozyme RNA can persist in cells due to its RNA-to-RNA replication and be transported systemically, emphasizing functional and, possibly, evolutionary links of retrozymes to viroids—noncoding circular RNAs that infect plants.Retrozymes are nonautonomous retrotransposons with hammerhead ribozymes in their long terminal repeats (LTRs). Retrozyme transcripts can be self-cleaved by the LTR ribozyme, circularized, and can undergo RNA-to-RNA replication. Here, we demonstrate that the  Circular RNAs (circRNAs) represent a distinct type of long noncoding RNA . In mostArabidopsis thaliana plants significantly suppresses the expression of these genes [Tomato yellow leaf curl virus infection in tomato plants [The investigations of plant-encoded circRNAs are at their very beginning. High-throughput sequencing of circRNA-enriched fractions of plant RNA and data analyses aimed at searching for back-spliced sites have resulted in the identification of tens to thousands of circRNAs in different plant species . Howeverse genes . The mecse genes . Importase genes . In addio plants , suggesto plants .retrotransposons with hammerhead ribozymes) are retroelements found in the genomes of many plant and metazoan species [A specific class of plant genome-encoded circRNAs is represented by retrozyme RNA. Retrozymes  complementary to the tRNA-Met 3′-terminal sequence and a polypurine tract (PPT) . The RNAN. benthamiana genome contains hundreds of retrozyme loci, of which nine represent full-length retrozymes. We demonstrate that the retrozyme LTR region contains a promoter capable of directing retrozyme RNA synthesis and that the retrozyme LTR is heavily methylated in N. benthamiana leaves, suggesting that it is transcriptionally inactive. Accordingly, linear retrozyme-specific RNA is undetectable in plant leaves. We further show that the fraction of N. benthamiana small RNA contains 24-nucleotide-long retrozyme-derived RNAs that can be involved in the transcriptional silencing of the LTR promoter. Using transcriptome analysis, we demonstrate that 49% of retrozymes in the RNA population of N. benthamiana leaves have no exact matches in the genome and could arise as a result of RNA-to-RNA replication, leading to the accumulation of errors. Additionally, we provide evidence that retrozyme RNA is capable of replication and systemic transport in plants.In this paper, we report that the N. benthamiana has been reported to contain a single hammerhead ribozyme sequence and no retrozymes [N. benthamiana genome scaffolds  using, as a query, the 352-nucleotide-long LTR sequence of a previously characterized Fragaria ananassa retrozyme [10) that corresponded to full-length retrozyme LTRs or their fragments containing the LTR-embedded ribozyme sequence  in 191 genomic scaffolds , of which nine corresponded to previously identified complete retrozymes NbRZ1–NbRZ9, whereas others corresponded to incomplete retrozyme sequences. Collectively, these observations show that the N. benthamiana genome contains two groups of retrozymes, with each group being represented by full-length retrozymes, incomplete CR-containing retrozymes, and smaller retrozyme fragments containing mostly one LTR sequence.The individual analysis of to NbRZ9 . Accordie length . Howevere length . BLAST sInterestingly, NbRZ3 and NbRZ9 represented incomplete tandem repeats of the retrozyme sequence , supportN. benthamiana retrozymes were found to comprise characteristic sequence elements conserved in retrozymes of other species. As illustrated for NbRZ1 . In the HTS raw data, reads that could be aligned to NbRZ1–NbRZ9 were identified. Initial analysis revealed that 14.8% of the retrozyme-specific reads aligned to the CR of the Group 1 retrozymes, 22.2% to the Group 2 CR, and 63.0% to LTR regions, confirming that both groups of N. benthamiana genome contains many retrozymes that are close in terms of sequence, special precautions have been taken to avoid the assembly of reads corresponding to different RNAs into a single contig  contigs were identical to genomic sequences, whereas others had the closest genome matches with 88.4% to 99.7% identity. To verify that the observed contig differences from the genomic retrozyme copies did not result from an erroneous contig assembly, BLAST searches of the N. benthamiana genome were carried out using every retrozyme-specific read as a query. As a result, only 51.0% of the reads were found to have perfect matches in the N. benthamiana genome, whereas others had, relative to the closest genomic sequences, 1% mismatches (21.8% reads), 2% mismatches (12.4% reads), or 3 to 13% mismatches (14.8% reads)  B. Thus, N. benthamiana. Therefore, the rapid amplification of cDNA ends (5′-RACE) with Group 1 retrozyme-specific primers  was generated. A reverse transcription-PCR analysis of the N. benthamiana leaves infiltrated with agrobacteria carrying the constructs LTR-GFP, 35-GFP, and NoPr-GFP revealed the presence of a GFP transcript directed by the NbRZ1 LTR  strand-specific product confirmed its identity and revealed that all sequenced clones had no exact matches to retrozyme genomic loci  and (−) strands of retrozyme RNA was analyzed in the trozymes E. In thitrozymes E, suggesN. benthamiana retrozymes, and we suggest that retrozyme RNA transported to distant plant parts can replicate after unloading from the phloem.In line with this assumption, the reverse transcription-PCR analysis of the upper leaves of the plants agroinfiltrated for the expression of the NbRZ1-Tag carried out with a pair of primers, in which one was complementary to the tag sequence C, revealN. benthamiana genome contains hundreds of retrozyme loci representing full-length retrozymes, incomplete retrozymes with truncated terminal regions, and retrozyme fragments containing mostly one LTR sequence. Interestingly, the retrozymes in the N. benthamiana genome fall into two distinct groups, in which central regions located between two LTRs are conserved within each group and dissimilar between groups. As both types of central regions have no sequence counterparts in other plants, the two retrozyme groups have likely evolved in the N. benthamiana genome.In this paper, we reveal that the N. benthamiana plants. However, we report data suggesting that N. benthamiana retrozyme-specific RNAs are largely products of RNA-to-RNA replication rather than transcripts of genomic retrozyme copies.The detection of retrozymes in the plant RNA preparations has led to the conclusion that genomic retrozyme loci are actively transcribed . Indeed,Jatropha curcas retrozyme cDNA clones, none were found to perfectly match known genomic retrozymes [First, using both genome- and transcriptome-wide analyses, we demonstrated that half of the retrozyme-specific RNAs in the plant leaves do not match the genomic retrozyme copies. Similarly, in a limited set of 16 analyzed trozymes . We assuN. benthamiana genomic DNA, we showed that the LTR region of the prototype retrozyme NbRZ1 is heavily methylated. As the retrozyme LTR is shown to contain a promoter, LTR methylation likely suppresses the transcription of retrozyme genomic loci. This observation is in agreement with the view that the transcription of transposable retroelements in somatic cells is normally repressed by the methylation of respective genomic regions [N. benthamiana small RNAs. Therefore, retrozyme transcription appears to be controlled by a negative feedback loop when the transcribed retrozyme RNA is processed into small RNAs directing the transcriptional silencing of retrozyme loci. The retrozyme RNA produced by RNA-to-RNA replication can be an additional source of retrozyme-specific small RNAs, which contribute to the further down-regulation of retrozyme genomic loci transcription.Second, using the bisulfite sequencing of  regions . The traN. benthamiana genome, circular retrozyme RNAs and/or linear concatemer products of their rolling-circle replication can serve as templates for reverse transcription, giving rise to integration-competent DNA copies. The genomic integration of retrozymes carrying mutations accumulated during multiple cycles of RNA-to-RNA replication may (i) considerably expedite the evolutionary rate of these retroelements and account, at least in part, for the observed diversity of the genomic retrozyme copies and (ii) provide a mechanism for the fixation of mutations emerging in retrozyme RNA that might contribute to plant fitness if retrozyme RNA has an adaptive function.We hypothesize that the population of retrozyme RNA may persist in plants and propagate through RNA-to-RNA replication, while the transcription of retrozyme DNA is suppressed. Conceivably, retrozyme transcription can be activated when the transcriptional suppression of transposable elements is released . As a reN. benthamiana plants. This finding is consistent with reports showing that many plant RNAs, including both mRNAs and noncoding RNAs, are transported through the phloem to distant plant parts [Avsunviroidae family, of Type III hammerhead ribozymes [Pospiviroidae) or the RNA polymerase NEP in the case of viroids replicating in chloroplasts (family Avsunviroidae) [Using a cloned copy of a full-length retrozyme, we have demonstrated that retrozyme RNA is capable of systemic transport from the lower leaves into the upper leaves of nt parts  and thatnt parts . Retrozynt parts ,41, in tibozymes ,18. Theribozymes ,18. As pibozymes . We hypoiroidae) ,41.Carnation etched ring virus  genome, which exists in infected cells as a circular episome [While viroids, unlike retrozymes, have no genome-integrated DNA copies, a DNA counterpart has been found for carnation small viroid-like RNA (CarSV RNA), a ribozyme-containing circular RNA that is not transmitted from plant to plant and presumably replicates through a rolling-circle mechanism . Multime episome , and, in episome . Remarka episome , suggestN. tabacum miRNAs available in miRBase  taken for analysis in the absence of annotated N. benthamiana miRNAs. Therefore, we hypothesize that the potential function(s) of retrozyme RNA in plants can be linked to its systemic transport abilities. If retrozyme RNA can serve as a signaling molecule, a target of such regulation remains to be identified.Currently, the functions of retrozyme RNA in plants are unknown. Retrozyme RNAs might serve as molecular ‘sponges’ sequestering certain plant miRNAs and thus affecting miRNA-regulated pathways, as shown for circRNAs in animals ,8. HowevN. benthamiana plants, as well as detection of GFP and NbRZ1-Tag RNA in agroinfiltrated plants, was carried out by reverse transcription-PCR with specific primers  and devoid of contaminating genomic DNA were used. Depletion of ribosomal RNA was carried out with Ribozero Plant kit , while cDNA synthesis was carried out with Illumina TruSeq Stranded Total RNA kit using random primers. The quality of cDNA libraries was verified on Fragment Analyzer, while cDNA quantity was estimated by qPCR. cDNA libraries were sequenced on Illumina NovaSeq 6000 producing 100 nucleotide-long single reads. In total, 270 M reads were obtained.The 5′-RACE analysis was carried out using Mint RACE kit (Evrogen) suitable for 5′-RACE on both capped and non-capped transcripts. This protocol employs the Step-Out RACE procedure described in detail earlier . ReverseN. benthamiana genomic DNA was isolated using a modified CTAB method [The B method . BisulfiN. benthamiana genomic DNA with specific primers  cells with recombinant binary vectors was performed using the freeze–thaw method. For agroinfiltration, 5–6-week-old Nicotiana benthamiana plants kept in growth chambers  were used. In experiments with long-distance transport, plants with six leaves were used. The 2nd and 3rd leaves were agroinfiltrated for the NbRZ1-Tag expression; the 5th and 6th leaves were analyzed for the presence of NbRZ1-Tag at 3 days post infiltration. Agrobacteria for infiltration were prepared essentially as described . Agrobacterial cultures were grown overnight at 28 °C in LB medium containing 20 mM acetosyringone, 10 mM 2-(N-morpholino)ethanesulfonic acid , and appropriate antibiotics. Agrobacterial cells were centrifugated, resuspended in infiltration buffer , and incubated at room temperature for 3 to 4 h. Agrobacterial cell suspensions were diluted to the final optical density of 0.3 at 600 nm and infiltrated into abaxial surface of fully expanded leaves using a 2 mL needleless syringe.Transformation of N. benthamiana genome scaffolds were carried out at https://solgenomics.net (accessed on 16 September 2022). Multiple sequence alignments were generated with Clustal Omega . RNA secondary structure predictions were made using RNAfold .BLAST searches of https://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 16 September 2022) and potential contamination using FastQ Screen [N. benthamiana retrozymes (NbRZ1–NbRZ9) with Bowtie-2 [N. benthamiana retrozymes are close in terms of their sequences, we developed an algorithm for contig assembly that did not allow read mismatches and limited contig extension to 25 nucleotides at the addition of each following retrozyme-specific read identified as described above to prevent further anchoring of foreign reads to long single-read overhangs at contig ends. The generated contigs and the primary retrozyme-specific reads were aligned to N. benthamiana genome scaffolds using BLASTN [The HTS libraries were analyzed for read quality using FastQC . The initial processing of the reads included quality control with FastQC and removal of adapter sequences with Trimmomatic [N. benthamiana retrozymes (NbRZ1–NbRZ9) using Bowtie-2 [For identification of retrozyme-specific small RNAs, publicly available small RNA HTS libraries  were used. The libraries were downloaded using NCBI SRA Toolkit (mmomatic . The libBowtie-2 , alignmeBowtie-2 .Retrozymes have a dual nature, being both nonautonomous retrotransposons, which transiently go through the RNA phase of their life cycle, and replicating circRNAs. The findings reported in this paper, including the long-lasting persistence of retrozyme RNA in plants and its ability to systemically spread over the plant body, suggest that retrozyme RNA has potential undiscovered functions.


It was estimated that 70% of patients with colorectal cancer were found to have viable exfoliated malignant cells in adjacent intestinal lumen. Exfoliated malignant cells had been reported to implant on raw surfaces, such as polypectomy site, anal fissure, anal fistula, hemorrhoidectomy wound, and anastomotic suture line. Tumors at anastomosis could be classified into four groups: local recurrence, local manifestation of widespread metastasis, metachronous carcinogenesis, and implantation metastasis. However, all of the previous studies only reported the phenomena of implantation metastasis at anastomosis. No study had proved the origin of anastomotic metastasis by genomic analysis. In this study, a 43-year-old woman presented with persistent hematochezia was diagnosed as having severe mixed hemorrhoids. She was treated by procedure for prolapse and hemorrhoids (PPH), without receiving preoperative colonoscopy. Two months later, she was found to have sigmoid colon cancer by colonoscopy due to continuous hematochezia and received radical sigmoidectomy. Postoperative histological examination confirmed the lesion to be a moderately differentiated adenocarcinoma (pT3N1M0). Six months later, she presented with hematochezia again and colonoscopy revealed two tumors at the rectal anastomosis of PPH. Both tumors were confirmed to be moderately differentiated adenocarcinoma without lymph node and distant metastasis and were finally removed by transanal endoscopic microsurgery (TEM). Pathological examination, whole exome sequencing (WES), and Lineage Inference for Cancer Heterogeneity and Evolution (LICHeE) analysis demonstrated that the two tumors at the rectal anastomosis were probably implantation metastases arising from the previous sigmoid colon cancer. This is the first study to prove implantation metastasis from colon cancer to a distal anastomosis by WES and LICHeE analysis. Therefore, it is recommended to rule out colorectal cancer in proximal large bowel before performing surgery with a rectal anastomosis, such as PPH and anterior resection. For patients with a suspected implanted tumor, WES and LICHeE could be used to differentiate implantation metastasis from metachronous carcinogenesis. Colorectal cancer (CRC) is one of the most common malignancies worldwide. CRC can be spread by direct extension, lymphatic and blood vasculature, and intra-abdominal/transperitoneal spread . Implant2 IV day 1, capecitabine 10,00 mg/m2 twice daily PO for 14 days, repeat every 3 weeks) from 9 October 2018 to 23 February 2019. Furthermore, she was followed up regularly at our hospital. Six months after radical excision, she presented with hematochezia again. Flexible colonoscopy (9 March 2019) revealed two tumors at the rectal anastomosis of PPH . The patient was last followed up on 26 April 2022 by telephone interview, and she had remained disease-free  for 3 years after TEM. The timeline of important events, treatment, and follow up is shown in A 43-year-old woman presented with persistent hematochezia. She did not have special history of medical, psychosocial and hereditary disease, relevant past interventions, and family history of cancer. No obvious mass was palpated on digital rectal examination. Preoperative colonoscopy was recommended but refused by the patient. She was diagnosed as having severe mixed hemorrhoids and treated by procedure for prolapse and hemorrhoids (PPH) on 7 July 2018 at a local hospital. Unfortunately, hematochezia continued after PPH, and the patient visited our hospital approximately 2 months after PPH. Colonoscopy (1 September 2018) revealed an ulcerative mass in the sigmoid colon , which was confirmed to be adenocarcinoma on biopsy-based pathological examination. The rectal anastomosis performed during PPH had healed well, and no obvious abnormality was identified at the site of rectal anastomosis under colonoscopy. The patient underwent radical sigmoidectomy on 9 September 2018. Postoperative histological examination confirmed the lesion to be a moderately differentiated adenocarcinoma (pT3N1M0). She underwent 6 cycles of adjuvant chemotherapy  were implantation metastases originating from the sigmoid colon carcinoma (S tumor), peripheral blood of the patient and formalin-fixed paraffin-embedded specimens of the three tumors were obtained and analyzed by WES. LICHeE analysis is a well-accepted combinatorial algorithm designed to reconstruct multi-sample cell lineage trees and infer the subclonal composition of the given samples based on variant allele frequencies of single-nucleotide variants –31. TherImplantation metastasis of CRC to a distal anastomosis suture line is a rare situation, which has not yet been proved in the published literature by genomic analysis. To the best of our knowledge, this is the first study to prove the implantation metastasis of CRC to rectal anastomosis by comprehensive analysis, including pathological examination, WES, and LICHeE analysis. Differential diagnosis between implantation metastasis and metachronous carcinogenesis is very important, since treatment plan and prognosis vary greatly. In this case, the mucosa between the rectal anastomosis and sigmoid anastomosis was normal under colonoscopy; postoperative pathological examination showed that the sigmoid colon cancer and the two tumors at rectal anastomosis have similar histology; all of them were moderately differentiated adenocarcinoma. In addition, lymph node and distant metastasis were excluded. These above clinicopathological features indicated that the two anastomotic tumors probably originated from the sigmoid colon cancer. However, all of this evidence is only supportive, not conclusive. The best available method to prove the relationship between two tumors is WES and comparative study with LICHeE analysis, but it is time-consuming and very expensive. For these reasons, none of the published case reports of implantation metastasis had been proved by WES and LICHeE analysis.It has been demonstrated that viable exfoliated cancer cells presented within the bowel lumen both proximal and distal to the CRC mass . OperatiDue to the rarity of implantation metastasis at the rectal anastomosis, there is no well-established operation. Several operative procedures had been reported in the literature, including local excision, abdominoperineal resection, and anterior resection with anorectal anastomosis . In thisThis is the first study to prove the implantation metastasis of CRC to rectal anastomosis by WES and LICHeE analysis. This case highlights the catastrophic consequence of tumor implantation to a distal rectal anastomosis after radical excision of colon cancer. Therefore, it is recommended to rule out CRC in proximal large bowel before performing surgery with a rectal anastomosis, such as PPH for hemorrhoids and anterior resection for rectal cancer. Once the diagnosis of CRC is confirmed, hemorrhoidectomy  should not be performed until the removal of CRC. Similarly, for patients with a confirmed diagnosis of CRC, a complete colonoscopy is still required to rule out another synchronous CRC in the proximal colon. If the colonoscope cannot pass through the lumen of the tumor site, a careful intraoperative exploration of the unchecked proximal colon is suggested to rule out a second tumor. Otherwise, the exfoliated malignant cells of the proximal tumor may implant at distal anastomosis. For patients with a suspected implanted tumor, WES and LICHeE could be used to differentiate implantation metastasis from metachronous carcinogenesis. Furthermore, TEM could be a reasonable and curative operation for rectal implanted tumor limited within the intestine wall.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by the ethics committee of Shanghai Changhai Hospital. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the patient for the publication of any potentially identifiable images or data included in this article.GY and XG contributed equally to this article. GY and XG: material preparation, data collection, and draft preparation. LX and TL: data collection, critical revision, and final approval of the paper DS: data analysis, critical revision, and final approval of the paper. WZ: study conception, design, and final approval of the paper. All authors contributed to the article and approved the submitted version.This study was supported by the National Natural Science Foundation of China , Shanghai Pujiang Program (#2019PJD052), Natural Science Fund of Shanghai (20ZR1457200), and 234 Subject Climbing Plan of Changhai Hospital (#2020YXK022) Shanghai Sailing Program (21YF1459300), The 71st Batch of China Postdoctoral Science Foundation (48804).Author DS is employed by the Department of Medicine, Genecast Biotechnology Co. Ltd., Wuxi, China.The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.