These, in turn, reveal previously unstudied properties and motifs not characteristic for small molecules with simple architectures. Examples include entanglements of structural elements in RNA molecules and knot-like folds discovered, among others, in the genomes of RNA viruses. Recently, we presented the first classification of entanglements, determined by their topology and the type of entangled structural elements. Here, we introduce RNAspider – a web server to automatically identify, classify, and visualize primary and higher-order entanglements in RNA tertiary structures. The program applies to evaluate RNA 3D models obtained experimentally or by computational prediction. It supports the analysis of uncommon topologies in the pseudoknotted RNA structures. RNAspider is implemented as a publicly available tool with a user-friendly interface and can be freely accessed at   Knowing the structure of RNA is key to understanding many biological processes like gene regulation and translation ,2. It isDetermination and prediction of RNA 3D structure is still a challenge. However, better experimental techniques and new computational methods are helping us to learn and analyze it with increasing accuracy, also for large molecules with composite structures. It is becoming possible to capture the specificity of complex structural motifs involving, i.a., large chain fragments or long-range tertiary contacts. Examples include knots and knot-like folds. Knots have been detected in protein structures ,13 and din silico modeling of RNA folds or programs to reconstruct the 3D structures from experimental data. Others may be functional motifs. Among the latter is the knot-like fold that – according to our classification – is assigned to higher-order entanglements.In our recent paper , we haveHere, we introduce RNAspider – a webserver to analyze entanglements of structure elements in RNA 3D structures. It identifies primary entanglements and the higher-order ones that involve pseudoknotted base pairs of various orders. It provides the classification of entanglements according to , visualiRNAspider begins its processing workflow with the validation and preparation of input data. It filters out any non-RNA chain, deletes surplus atoms in modified residues, and removes incomplete nucleotides. Then, it applies the incorporated RNApdbee algorithm  to extraThe system computes a polygonal chain for each open and closed structural element. The chain connects points corresponding to selected atoms and pseudoatoms, i.e. centers of mass of nucleobases involved in base pairing. By definition, a polygonal chain encircles each closed element, which gets covered with a polygon mesh via a recursive triangulation procedure. RNAspider uses Möller–Trumbore algorithm  to check - consists of elements, in which one is only punctured, while the other only punctures. In interlace, represented by &, they do both to each other. Entangled elements are marked in the system using capital letters—L stands for a loop, S for a single strand, and D for a dinucleotide step. Combining symbols from both groups allows us to describe entanglement classes, e.g. L&D denotes a loop interlaced with a dinucleotide step, L(S) is a loop lassoed over a single strand. The system additionally supports multiple punctures  is a loop lassoed over a single strand that punctures it thrice . Structural elements are constructed anew based on pseudoknotted base pairs of the current order. This time, nucleotides of the core structure might end up embedded inside. Such iterative procedure allows identifying higher-order entanglements defined upon long-distance, pseudoknotted base pairs. It runs by default unless switched off by the user selecting the Ignoring pseudoknots option in the advanced settings panel.RNAspider uses the concept of pseudoknot order ,34 to diRNAspider has a multi-layer architecture. The business logic layer integrates a computational module, message broker, and database, providing RESTful API employed by a user-friendly web interface. The computational module is developed using Java 11 with Apache Maven 3.5 and other third-party libraries. It provides efficient data processing, even for large, multi-model RNAs. RabbitMQ, an open-source message broker, ensures that no submitted task is lost. Python 3.6 and Gunicorn support the Flask web application. The database uses PostgreSQL, an open-source, object-relational database management system. Celery, a reliable distributed queuing system, performs cyclic operations like removing expired tasks. The RNAspider UI implemented in React follows the single-page application pattern. It supports all modern browsers and platforms, including mobile devices. Visualization of the RNA structure with entanglements is possible with suitably modified VARNA  and Mol*After accessing the RNAspider homepage , users uFile List tab). It is possible to download an archive with selected output data: cleaned 3D structure, secondary structure in extended dot-bracket notation, secondary structure diagram, or the result table (Download tab). The right panel presents the main findings divided into sections. The top one includes general information and a resubmission form to run data analysis with different parameter values. Below, users can see the RNA sequence and secondary structure in extended dot-bracket notation  entanglement – the inter-domain single-stranded fragment (U175–U177) punctures the center of the dinucleotide step   or L(S) with strand S that connects two helical regions and punctures a dinucleotide step or a loop between these regions. We suggest eliminating such RNA 3D models as unreliable.First, we focused on untranslated regions of the SARS-CoV-2 RNA genome. Structure motifs present in SARS-CoV-2 are crucial for developing RNA-targeted small-molecule drugs. Thus, many researchers have recently attempted to characterize the tertiary structures of selected fragments of this viral genome, both with experimental methods and computational modeling ,39. AlsoL(S) type – loop lassoing the single-stranded fragment is closed by pseudoknotted base pair(s). Thus, it can be identified if RNAspider runs with accepting pseudoknots option turned on (default settings). In the xrRNA structure, the loop – closed by base pair (G33-C49) of the first order – is punctured by the single strand (g1-G31) from the 5′-end, intersection point between C4’ in G2 and P in C3  from the flavivirus molecule. Its structure, determined by X-ray crystallography  , containair G33-C9 of the In , we discFrom the initial set of 5804 RNA structures, we have excluded 11 false positives, that is, molecules with overlapping atoms misrecognized as punctures. Among 5793 instances left, we found 1307 (22%) entangled cases. Fifty-three structures have only primary entanglements, 1232 – only higher-order entanglements, and 22 have both types. Analysis of the results indicates a strong correlation between pseudoknot order and the number of entangled structural elements (0.711 Pearson’s and 0.778 Spearman’s correlation coefficient). Also, the size of a molecule affects the formation of entanglements – the Person's correlation coefficient equals 0.812.L(S) type. Only 12% constitute interlaces, mainly from the L&L class. 3308 higher-order entanglements were found in 1254 structures from the input set. They consist almost entirely of the L(S) type arrangements. Detailed results of this analysis are available in A total of 120 primary entanglements were recognized in 72 RNAs (Table We hypothesize that interlaces and some lassos are incorrect geometries in molecule 3D models. However, PDB structures containing them should not be unreflectively considered wrong. We suggest their careful analysis to draw correct conclusions. For example, some entanglements in experimental structures may result from two molecules having alternative conformations.RNAspider is the first bioinformatics tool that automatically identifies, classifies, and visualizes entanglements in RNA 3D structures. Such uncommon arrangements of structural elements – taking on various topologies – arise when one of the elements punctures the area defined by the other. In , we showgkac218_Supplemental_FilesClick here for additional data file.


Background and purpose: Although immune checkpoint inhibitors (ICIs) have become the first-line treatment for metastatic non-small cell lung cancer (mNSCLC), their efficacy is limited. Meanwhile, recent reports suggest that radiotherapy (RT) can activate the systemic antitumor immune response by increasing the release of antigens from tumor tissues. Therefore, in patients with mNSCLC treated with ICIs, investigations were performed to determine whether the addition of RT improved the outcomes. Furthermore, the adverse events rate was evaluated.Methods and materials: Pubmed, Embase, and Cochrane Library were searched using the keywords “radiotherapy,” “immune checkpoint inhibitors,” and “non-small cell lung cancer” from the date of inception to 2 May 2022. Randomized controlled trials (RCTs) and nonRCTs (NRCTs) comparing the efficacy and safety of RT combined with ICIs versus ICIs alone in metastatic NSCLC were assessed. The primary outcomes were progression-free survival (PFS) and overall survival (OS), and the secondary outcomes were abscopal response rate (ARR), abscopal control rate (ACR), adverse events rate, and pneumonia rate. The analyses were conducted using the Mantel–Haenszel fixed-effects or random-effects model. The I2 statistic was used to determine heterogeneity, whereas funnel plots and Egger’s test were used to assess publication bias.Results: In 15 clinical studies, 713 patients received RT combined with ICIs and 1,275 patients received only ICIs. With regard to PFS and OS, the hazard ratios of RT combined with ICIs were 0.79  and 0.72 , respectively. In terms of ARR and ACR, the odds ratios (ORs) of RT combined with ICIs were 1.94  and 1.79 , respectively. Subgroup analyses based on study type (RCT/NRCT), RT target , number of RT sites (single site), previous ICI resistance (yes/no), and sequencing of RT and ICIs (concurrent/post-RT ICIs) revealed that the addition of RT significantly prolonged PFS and OS. However, subgroup analyses based on radiation dose/fractionation indicated that the addition of hypofractionated RT significantly prolonged OS but not PFS. When grouped according to the level of PD-L1 expression, the addition of RT prolonged PFS only in patients who were PD-L1-negative. Furthermore, subgroup analyses of ARR and ACR signified that the combination therapy resulted in better local control of lesions outside the irradiation field in the hypofractionated RT, extracranial RT, and ICI-naïve subgroups. In terms of adverse events, the addition of RT did not significantly increase the adverse events rate but was associated with a higher pneumonia rate .Conclusion: Meta-analysis of existing data suggests that the addition of RT can significantly prolong PFS and OS in patients with metastatic NSCLC receiving ICIs. In addition to lesions in the irradiation field, RT can improve the local control rate of lesions outside the irradiation field via immune activation. Combination therapy does not increase the overall risk of adverse reactions, except for pneumonia. According to the global epidemiological survey of malignant tumors, lung cancer has the highest mortality rate and the second-highest incidence rate among 36 malignant tumors . Of thesImmune checkpoint inhibitors, including anti-PD-1/anti-PD-L1, significantly improve the rate of survival in cases with advanced NSCLC . The KeyAs an important treatment modality for mNSCLC, radiotherapy (RT) can enhance the systemic release of antigens from the tumor tissues. These antigens are recognized by antigen-presenting cells and are subsequently presented to T lymphocytes, especially CD8 cytotoxic T cells. The priming and activation of these cells trigger a systemic immune response against the tumor tissue, both in irradiated lesions within the irradiated field and in unirradiated lesions outside the irradiation field. In multiple murine solid tumor models, including glioma, NSCLC, and melanoma, RT combined with ICIs has demonstrated stronger antitumor effects than monotherapy . On the We conducted a systematic review and meta-analysis following the preferred reporting items of the guidelines for systematic reviews and meta-analyses . The proWe searched PubMed, the Cochrane Library, and Embase databases using the keywords “radiotherapy”, “immune checkpoint inhibitors”, and “non-small cell lung cancer” according to the PICOS principles from the date of inception to 2 May 2022 in order to identify the published randomized controlled trials (RCTs) and non-RCTs (NRCTs). P: mNSCLC; I: RT combined with ICIs; C: ICIs alone; O: progression-free survival (PFS), OS, abscopal response rate (ARR), abscopal control rate (ACR), adverse events rate, and pneumonia rate; S: Studies directly comparing RT plus ICIs with ICIs alone. No language restriction was placed. We also searched the references of articles that met our inclusion criteria. Detailed search strategies are reported in • NSCLC confirmed by histopathology.• Disease being metastatic or stage IV disease.• Studies directly comparing RT combined with ICIs and ICIs alone, either RCTs or NRCTs. In addition, we confirmed that RT received by the mNSCLC patients included in this study was the first RT after the diagnosis of mNSCLC.• Quantitative meta-analysis data reported for at least one outcome measure .We included published studies that met the following criteria:• Studies on malignancies other than NSCLC.• Studies including Abstracts only (full version not available), which we believe may lead to bias.• Ongoing research.Studies that did not meet these criteria were excluded, as were the studies that met the following criteria:After searching for studies, two researchers discarded all duplicate studies obtained from various databases and independently screened the titles and abstracts of all remaining articles to exclude the manuscripts that did not meet the inclusion criteria. In addition, the researchers carefully reviewed the full text of all shortlisted articles for inclusion in this study. Finally, the third investigator discussed the controversial study with the two investigators who conducted the screening. Any disagreements regarding study eligibility were resolved through consulting with the fourth investigator.The methodological quality of the included RCTs was independently assessed by two researchers (Liu Zijing and Xu Tiankai) according to the Cochrane risk of bias criteria. The 7 items used to assess bias in each trial included random sequence generation, allocation concealment, participants, and personnel, blinding of outcome assessments, complete outcome data, selective reporting, and other biases. We defined other bias as the difference in the baseline characteristics between the intervention groups. The Newcastle–Ottawa scale was used in cohort or case-control studies. The included trials were classified as either low-quality, high-quality, or moderate-quality based on the following criteria: 1) the trials were considered low-quality if randomization or allocation concealment was assessed as a high risk of bias, irrespective of the risk of other items; 2) trials were considered to be of high quality when randomization and allocation concealment were assessed as a low risk of bias and all other items were assessed as low risk of bias or unclear risk of bias in trials; 3) trials were considered to be of moderate quality if they did not meet the criteria for high risk of bias or low risk.Two researchers independently extracted the following information from each study: major author; publication year; publication country; type of studies; the number of patients; RT target; RT technique; RT dose (Gy)/fraction; timing of RT and ICIs intervention; type of ICIs; ICIs dose. The differences were resolved by consensus. Primary outcomes were PFS and OS, while the secondary outcomes were ARR, ACR, adverse events rate, and pneumonia rate. Furthermore, as some of the data were not explicitly reported, we contacted experimental institutions to obtain as much comprehensive information as possible. If the HR values for OS and PFS were not provided in the original text, we extracted data from the survival curves using digitizing software.2 statistic to assess heterogeneity between trials. If there was no significant heterogeneity, we pooled the data using a Mantel–Haenszel fixed-effects model (I2 < 50%); however, if there was significant heterogeneity (I2 ≥ 50%), a random-effects model was employed. Dichotomous outcomes  were expressed as ORs with 95% confidence intervals (CIs). Survival data  were expressed as HR with 95% CI. According to the Cochrane Handbook’s recommendations for practice, trials with zero events in both the intervention and control groups were not included in the meta-analyses when calculating HR and OR. We performed sensitivity analyses as per the “leave-one-out” method, which was used to determine the effect of each study on the overall outcome by deleting each study. Each estimate and its upper and lower CIs represent the HR or OR after removing a single study. The critical value for both OR and HR was 1. The results were considered robust if the new HR or OR value was consistent with the original HR or OR value on the same side of the cut-off after excluding any single study. All tests were 2-tailed, and p < 0.05 was considered to indicate statistical significance.We used STATA 15  for meta-analysis and the assessment of publication bias, generation of funnel plots, and assessment of heterogeneity. We also judged the publication bias of the included studies by using a funnel plot, followed by a quantitative assessment by Egger’s test. We used the IOur search strategy identified 1,434 records. After eliminating the duplicates, 1,295 unique records remained. Of these, 1,140 records were ineligible for inclusion, and 155 full texts were assessed for eligibility. After excluding 139 studies because the article type was not suitable for inclusion , 3 RCTs and 12 NRCTs remained. A total of 15 clinical studies were reported in 16 publications  Kataoka.The RT targets for three RCTs and five NRCTs were extracranial nonbrain metastases . Three np < 0.001). Ten studies that compared the OS of RT plus ICIs with ICIs alone were included (p < 0.001). The heterogeneity trend was low in PFS (I2 was 19.7%) and absent in OS (I2 was 0.0%)  .p = 0.008) and 1.79 , respectively. Furthermore, there was no heterogeneity in the results (I2 was 0.0% for both)  .p = 0.035) and 0.80 , respectively. The HRs of OS for RCT and NRCT were 0.67  and 0.76 , respectively. Heterogeneity was lacking in PFS and OS in RCTs and in OS in non-RCTs (I2 was 0.0% for both), unless RCTs had moderate heterogeneity in PFS (I2 = 33.3%)  , 3.p = 0.657) and 0.66 . There was no heterogeneity in the results (I2 was 0.0% for both). The OR of ARR was 2.92 . There was mild heterogeneity (I2 was 19.6%).We classify the type of RT in which the RT dose is greater than the conventional fractionated dose of 2 Gy/fraction as hypofractionated RT. according to this criterion, When subgroup analysis based on RT dose and fraction was performed, the HRs for PFS and OS of hypofractionated RT plus ICIs were 0.95  and 0.72 , respectively. The OR value of ARR was 2.31 . There was mild heterogeneity in PFS and ARR . For intracranial metastases, the HRs of PFS and OS for the RT plus ICIs were 0.55  and 0.61 , respectively. There was mild heterogeneity in PFS and OS .When subgroup analysis was performed according to RT target, for extracranial lesions, the HRs of PFS and OS of RT plus ICIs were 0.76  and 0.74 , respectively. Mild heterogeneity was seen in PFS (9.2% and 0.0% for I2).Subgroup analysis was conducted according to the number of RT sites. The HRs of PFS and OS of single site RT plus ICIs were 0.82  and 0.70 . The ORs of ARR and ACR were 2.15  and 2.22 , respectively. Mild heterogeneity was observed in PFS . For patients with immunotherapy rechallenge after ICI resistance, the HRs for PFS and OS of combination therapy were 0.80  and 0.78 , respectively. Heterogeneity was not noted in the results (I2 was 0.0% for both).Subgroup analysis was done based on resistance to ICIs. In ICI-naïve patients, the HRs for PFS and OS of RT plus ICIs were 0.78  and 0.73 , respectively. There was moderate heterogeneity in PFS . In the subgroup of ICIs after RT, the HRs of PFS and OS were 0.67  and 0.67 , respectively. There was no heterogeneity in the results (I2 was 0.0% for both), regardless of whether RT was concurrent or added prior to ICIs.Subgroup analyses were performed according to the sequencing of RT and ICIs. In the concurrent group, the HRs of PFS and OS were 0.83  and 0.47 , respectively. There was no heterogeneity in the results (I2 was 0.0% for both).Subgroup analysis was conducted according to the expression level of PD-L1. The HRs of PFS in the population with PD-L1 expression ≥50% and <1% were 0.74 . Four studies that compared the pneumonia rate between RT plus ICIs and ICIs alone were included (p = 0.014). Mild heterogeneity was seen in the pneumonia rate   .Sensitivity analysis performed by systematically excluding each specific study from the total counts showed that the new HRs for PFS and OS and the new ORs for ARR, ACR, adverse events rate, and pneumonia rate were similar to the previously mentioned raw HRs/ORs . This fiFurthermore, statistical analysis of publication bias indicated no evidence of statistical significance .In recent years, ICIs have gained attention for the treatment of advanced solid tumors, but their efficacy is limited. Although RT can activate the tumor immune microenvironment, its immune activation and abscopal effect are obviously insufficient. However, when added to ICIs, RT can improve the long-term efficacy of the treatment and cause a considerable abscopal effect, which has been confirmed in a variety of solid tumors . This meOn the basis of previous studies, this meta-analysis specifically focused on mNSCLC. The effect of adding RT on long-term survival, abscopal effects, and adverse events in patients treated with ICIs was evaluated. Moreover, the effects of RT dose and fraction, RT target, the number of RT sites, previous ICI resistance, RT and ICI sequencing, and PD-L1 level on the efficacy of RT combined with ICIs were determined. Such results have rarely been reported in previous meta-analyses .Subgroup analysis of RT dose and fraction revealed that hypofractionated RT plus ICIs can significantly improve long-term survival as well as abscopal local control rates. Notably, the addition of hypofractionated RT did not offer a significant benefit in terms of PFS. No heterogeneity was observed in PFS only when grouped based on PD-L1 levels. Therefore, the reason for hypofractionated RT not significantly improving PFS could be that ICIs demonstrate good efficacy in mNSCLC with high PD-L1 levels. Hence, the improved efficacy of RT was overshadowed by the favorable efficacy of ICIs. The hyperfractionated RT group could not be evaluated in this study because of data limitations Hyperfractionated RT tends to alter the immune microenvironment and activate the immune cells . ExtensiSubgroup analysis of the RT target showed that the addition of RT, whether extracranial or intracranial, significantly improved long-term disease control and offered survival benefits. In the case of intracranial metastases, the blood–brain barrier prevents cytotoxic T cells from entering the brain, thereby inducing tumor evasion . The gooWith regard to the number of RT sites, only single-site RT combined with ICIs showed obvious benefits in terms of PFS and OS. There is insufficient data to judge whether multi-site RT is beneficial. Owing to the tumor’s specificity and site-specific immunogenicity, multi-site RT can elicit a more pronounced systemic immune response than single-site RT and is now considered to be the new paradigm . ClinicaWith regard to previous immune resistance, the addition of RT can improve PFS and OS regardless of the presence of immune resistance. However, RT cannot improve the abscopal effect of patients with immune resistance. In addition to the immune-resistant microenvironment, limited RT sites and insufficient doses of RT limit the benefits of abscopal effects .Regarding RT and ICI sequencing, the results suggest that combined therapy can improve the efficacy of ICIs when RT is used concurrently with ICIs or when ICIs are followed by RT. RT can act both as an inducer of immune activation and a beneficiary of immune activation . IdeallySubgroup analysis based on PD-L1 expression levels showed that in patients who were PD-L1-negative, the addition of RT to ICIs significantly improved the PFS compared with those who had a high PD-L1 expression. However, the survival benefit of RT based on PD-L1 expression levels could not be assessed owing to data limitations. Moreover, due to the large variety of immune checkpoint inhibitors used within each study, we were unable to obtain practically meaningful results through statistical analysis. PD-L1 is currently a biomarker that effectively predicts the efficacy of ICI. However, after RT, tumor cells release tumor-associated antigens and cytokines. Whether these can be used as predictive biomarkers to assess the degree of improvement in the efficacy of immunotherapy with the addition of RT remains to be assessed.In terms of adverse events, previous studies have established that combined therapy does not increase the incidence of adverse events compared with ICIs alone, regardless of irradiation time and target . HoweverThis study conducted a meta-analysis on whether the addition of RT can improve the efficacy of ICIs in the treatment of mNSCLC. The content of the discussion is rare in related studies in this field. For patients with mNSCLC who experience limited efficacy after receiving ICIs, this study has reference value regarding intervention with local RT (whether to intervene and how) as well as the screening of patients benefiting from RT. However, there are some deficiencies in this study. First, under objective conditions, the number of published RCT studies is limited. Therefore, this study may have a certain degree of bias due to the small number of RCT studies included. Controversy exists regarding radiation dose, target, number of irradiations, and intervention time. On the basis of confirming that hypofractionated RT can significantly improve the curative effect, the optimal single RT dose and the number of irradiations should be further determined. On the one hand, it is necessary to consider fully activating the immune microenvironment. On the other hand, it is imperative to avoid the occurrence of obvious adverse reactions. These reactions correlate with the pathological types of tumors, tumor size, tumor location, metastatic status, intrinsic radiosensitivity, and host characteristics. Compared with hypofractionated RT, hyperfractionation and extensive irradiation may alleviate the adverse reactions while fully activating systemic immunity. Relevant clinical studies are required. Moreover, owing to the differences in the immune microenvironment of liver, bone, and brain metastases, RT exhibits varying degrees of efficacy improvement for different metastases. Whether RT for brain metastases can obviously improve the efficacy of ICIs by damaging the blood–brain barrier needs to be supported by adequate medical evidence. For different patients, the size of the radiotherapy target volume varies with the size of the patient’s lesion. Therefore, quantitative analysis cannot be performed based on the size of the target area. The volume size of the irradiated target area also needs to be further explored to improve the efficacy of immunotherapy.There is mutual induced sensitization between RT and immunotherapy, and the period for the increase in immune cells after hypofractionated RT varies . Therefovia its immune-activating effect. Furthermore, the findings imply that the addition of RT does not increase the overall risk of adverse events, except for pneumonia.Meta-analysis of the existing data suggests that the addition of RT can significantly improve PFS and OS in patients with mNSCLC treated using ICIs. In addition, RT can significantly improve the abscopal local control of lesions outside the irradiation field


The transient nature of RNA has rendered it one of the more difficult biological targets for imaging. This difficulty stems both from the physical properties of RNA as well as the temporal constraints associated therewith. These concerns are further complicated by the difficulty in imaging endogenous RNA within a cell that has been transfected with a target sequence. These concerns, combined with traditional concerns associated with super-resolution light microscopy has made the imaging of this critical target difficult. Recent advances have provided researchers the tools to image endogenous RNA in live cells at both the cellular and single-molecule level. Here, we review techniques used for labeling and imaging RNA with special emphases on various labeling methods and a virtual 3D super-resolution imaging technique. The flow of genetic information is a multi-stage process centered around RNA metabolism. Since Francis Crick proposed the central dogma of molecular biology , RNA hasRNA world hypothesis has drawn more attention recently in the scientific world with emerging evidence [RNA world hypothesis as a model for the origins of life. In addition to enhancing our knowledge of cellular activities and revealing the possible origins of life, RNA also has been demonstrated with promising disease diagnostic and therapeutic potentials. RNA vaccines against SARS-CoV-2 have exhibited high prophylactic efficiency against the disease during the two years since their development [Interestingly, the evidence ,9 suggesevidence . An excievidence . Recentlelopment ,13. Furtelopment . Lastly,elopment . TogetheDrosophila melanogaster gene bicoid functions as an anterior body pattern organizer. The bicoid mRNA is localized only in the anterior pole region of the egg via random dynein-mediated transport and anchoring [bicoid spreads posteriorly, generating a concentration gradient for arterial/posterior patterning.Controlling the localization of RNA is a widespread, evolutionarily conserved, and efficient way to target gene products to a specific region of a cell or embryo . Using mnchoring . After fThe examples above show that mRNA function is closely tied to its subcellular localization and transportation, and thus methods and tools are needed to examine this aspect of RNA research. Unfortunately, conventional biochemistry and genetic tools primarily focus on identifying the properties of the RNA molecule, such as length, sequence, structure, and functions, whereas pulldown assays provide insight into the protein-RNA interactions. In order to obtain a complete spatial-temporal profile of RNA throughout its entire lifespan, from transcription to degradation, methods to visualize RNA within cells are required. Such methods are critically important to enhance our understanding of RNA and thus offer unparalleled opportunities for advancement in cellular and molecular biology, therapeutic discovery, and medical diagnostic.Generally speaking, the size of mRNA is far smaller than the resolution limit of conventional light microscopy. Thus, only two approaches are feasible regarding mRNA imaging: electron microscopy (EM) and fluorescent light microscopy. Among various EM techniques, two techniques have been used in mRNA imaging: first, an EM-level adaptation of in situ hybridization (ISH) technique that combines antisense probes and gold-coupled antibodies for detection ,20; and Another challenge in the fluorescent light microscopy approach of mRNA imaging is how to label the RNA. Fluorescent light microscopy relies mainly on fluorophores, including fluorescent proteins and dyes. At the time of this writing, no naturally fluorescent RNA has been discovered ; as a reAn appropriate fluorophore used to label RNA needs to have three outstanding characteristics: quantum yield, extinction coefficient , and phoLastly, choosing an imaging method that matches the experimental approach can be challenging but essential. The experimental design must take into consideration the speed of image acquisition, temporal resolution, signal-to-noise ratio, and effects on cell viability. Wide-field fluorescent microscopy with deconvolution could provide sufficient speed and sensitivity to study mRNA dynamics . Howeversuper-resolution microscopy (SRM) has been developed that bypass the diffraction limit. The diffraction limit is a barrier in optical microscopy caused by the physical property of light, restricting the optical resolution to roughly 250 nm [In the past two decades, a series of revolutionary techniques termed y 250 nm . There ay 250 nm , the firy 250 nm  and photy 250 nm  and the y 250 nm  and struy 250 nm . STED any 250 nm . Thus, ty 250 nm ,39,40. Oy 250 nm ,42,43.SMLM methods typically utilize conventional wide-field excitation and achieve super-resolution by fitting and localizing individual molecules, which are subsequently utilized to form a complete image in a pointillistic fashion . Since tBefore diving into various SMLM techniques used to image mRNA, the advantages and disadvantages of live cell imaging and fixed samples must first be addressed. Fluorescence microscopy of living or fixed cells is entirely dependent upon suitable labeling and detection strategies, as well as the overall scope of the study. In fixed cells, because all cellular activities and movements have been terminated during fixation, this imaging strategy allows repeat capture to maximize localization precisions, which contributes to the spatial resolution advantages of STORM and PALM. However, cell fixation also has significant limitations. For instance, fixed cells are no longer capable of providing dynamic information, and exhibit changes in cellular structures or molecules caused by the fixation process .Live cell imaging is a closer representation to the natural state of the cell. Whereas fixed cells are best described as in situ, live cells remain our best method of obtaining data regarding the behavior of cells in their native environments. For example, live cell imaging enables researchers to obtain real-time measurements at the temporal frequency necessary to sample the dynamics of most biological processes adequately . HoweverAnother consideration when comparing the two primary super-resolution approaches is the size of the dataset required to generate super-resolution localizations. Depending on the samples and applications involved, SMLM methodologies often require thousands of frames to reconstruct a high-quality image from an individual localization event, sometimes requiring minutes or hours to capture. As a result, many SMLM techniques are often performed on chemically fixed cells to prevent cell movement and facilitate the localization of single molecules. Despite this consideration, SMLM techniques have enabled significant progress in the arena of imaging mRNA dynamics and mRNA-protein interactions. For example, SRM and SMLM techniques have been utilized to great effect to localize a single RNA macromolecule with high spatial resolution, providing information regarding RNA binding protein (RBP) partners. However, it should be noted that not all SMLM techniques will achieve molecular resolution. In a study published in 2017 , an SMLMRNA has also been studied utilizing the single-particle tracking capacity of SMLM methodologies. The principles of SMLM can be expanded to track single-particles within live cells where a labeled single RNA macromolecule is localized with nanometer precision and observed over a period of time. Such fine-detailed and dynamic information, when paired with structural information of the cell, provides invaluable information pertaining to the dynamics and transport behaviors of RNA. A distinct advantage of tracking single particles is the ability to derive 3D information regarding the dynamic behaviors of RNA. One way in which this has been utilized is through the use of single-particle localization in two-dimensions to derive virtual three-dimensional (3D) information using a computational algorithm via single-point edge-excitation sub-diffraction (SPEED) microscopy. SPEED microscopy has been used to track messenger ribonucleoprotein (mRNP) movement through the nuclear pore complex (NPC) of eukaryotic cells ,48,49. TTaken together, super-resolution microscopy methodologies present an attractive option for obtaining both dynamic and static information regarding RNA localization, lifecycle, and interacting proteins. Detailed within this manuscript is a series of techniques developed to facilitate the imaging of RNA, with mRNA being given special consideration.3H- or 125I-labeled radioactive hybridization probes. By combining the hybridization approach in the previous autoradiography methods with observations made by Rudkin & Stollar [Fluorescence in situ hybridization (FISH) is a valuable method for imaging nucleic acids. First developed by Bauman and colleagues in 1980 , this me Stollar , wherein Stollar  , to label RNA . LikewisOne of the significant problems with mRNA FISH remains the photostability of the fluorophores used, particularly when attempting super-resolution, as the photon flux required in super-resolution environments results in the rapid deterioration of fluorophores during excitation ,58. One The second approach to improving upon ISH methodology, the addition of multiple fluorophores to a single probe, was first utilized in RNA FISH by attaching multiple CY3 fluorophores to a DNA probe. This approach resulted in significantly higher fluorescence and enabled researchers to identify discrete sequences of mRNA enabling the quantification of mRNA for the first time via mRNA FISH. Although this advance is often considered the first incarnation of single-molecule FISH (smFISH) , this apSingle-molecule FISH (smFISH) was developed by Raj and colleagues in 2008 to address limitations in FISH concerning low intensity of signal . The autAs described by Raj and colleagues, smFISH is quite sensitive because of the number of probes attached to each mRNA. In addition to providing more sensitivity, smFISH provides for a more robust system. The increased number of fluorophores over the single fluorophore found in traditional RNA FISH enables researchers to employ more photo intensive imaging techniques to investigate the 3D localization of the smFISH-labeled mRNA, and is considered by many to be the gold standard of mRNA localization at the organelle level. Further, the technique offers the option of labeling different segments of longer mRNAs with differently colored probes, which can serve to differentiate complete mRNAs from broken or partially degraded transcripts. Overall, smFISH serves as a useful technique in fixed cells for studies that require the detection and relative quantification of two to four mRNAs. This is accomplished by pairing smFISH with quantitative reverse transcription polymerase chain reaction (qRT-PCR). As this method allows a researcher to label the entirety of an mRNA transcript, this technique can be useful in studies where it is important to distinguish full transcripts from damaged or partial ones. This is done by calculating the fluorescent intensity of an individual fluorophore on an individual antisense targeting sequence, and then determining which spots have the appropriate fluorescent intensity commensurate to having a completely labeled and intact mRNA sequence.While smFISH has many technical strengths and has been invaluable in answering many questions regarding mRNA behavior within the cell, it suffers from many significant shortcomings. This technique is viable only in fixed cells. This presents four significant problems to imaging mRNA. First, static information derived from a fixed cells only provides researchers with a snapshot of their behavior. Second, the fixation of a cell often alters membrane structures, potentially leading investigators to draw potentially erroneous conclusions regarding interactions between mRNA and the nuclear envelope . Third, Using techniques such as smFISH to resolve and identify mRNAs is limited by their proximity, which is frequently below the optical diffraction limit. This potentially causes spectral overlap and the loss of discrete signal. Whereas temporal separation of detection is a key feature of other single-molecule approaches such as SPEED microscopy ,65,66,67S. cerevisiae cells by combining spatial and spectral coding using  order of probes  and combinations of colors [spatial barcoding [Lubeck and Cai in 2012 and Shah and colleagues in 2016 described sequential FISH (seqFISH) as a way to overcome these limitations to result in a technique that resolved transcripts from 32 stress-responsive genes in single f colors ,72. Theif colors .) The auarcoding  , ignoring spatial order. Because transcripts are labeled multiple times throughout their length, partially degraded and nonlinear mRNAs as well as those entangled with proteins and other macromolecules can be detected. These features ease the resolution requirement for confident detection. The authors term this approach arcoding  -coated coverslip. Oligo(dT) consists of short, single-stranded sequences of deoxythymine. This surface provides a 2D space for mRNAs to adhere and creates some distance between individual transcripts so they can be identified more easily. Oligo(dT) ligands covalently bind to a solid support (in this case the treated coverslip) and hybridize to the mRNA via the poly-adenylated tail. Then, seqFISH is performed as described previously  see FiguRNA SPOTs shows its strength in the sheer number of mRNA species it can identify and differentiate, achieving this by extracting them from the cell and spreading them out on a surface suitable to examination via SLM. Thus, this strategy is well suited to studies in which the identification of a large number of mRNAs is a must (such as in comparing the transcriptomes of two different cell types), but in which subcellular localization or colocalization information is not needed and thus extracted tissue can be used in lieu of fixed cells. However, like other methods discussed here, neither the dynamics of the live cell environment nor the spatial or temporal relationships of mRNA expression in the cell can be studied with this technique.It is currently desirable to obtain information regarding the transcriptome localization during various cellular states. To this end, multiplexed error-robust FISH (MERFISH) was developed by Chen and colleagues in 2015 . MERFISHIn computer science, a bit is a binary unit that can be read as a 1 or a 0. Similarly, a qualitative off or on designation can also be applied in fluorescent imaging. With this rationale, Chen and colleagues use sequential FISH probes to generate a code word that is unique to each target mRNA, permitting up to 140 code words using the 16-bit MHD4 code or 1001 possible code words using the MHD2 code ,76. A siThis technique provides researchers with a robust, error-resistant methodology for quantifying and localizing specific RNA of interest within the cell. Similar to smFISH, this method can only be utilized in fixed cells as the immobility of the RNA is critical to the success of this method. This makes this an ideal technique for capturing whole transcriptome information at key points in the cell life cycle, but not well-suited for capturing details regarding RNA dynamics. Further, producing so many smFISH probes is costly and untenable; to address this, the authors have adapted an existing Oligopaint approach ,78. ThisSMLM is a broad discipline that covers a wide array of methods and techniques that have variously been utilized to image RNA. All of these techniques fundamentally rely upon the principles of spatial localization, meaning that sufficient temporal or physical space is required between individual fluorophores to permit the mathematical fitting of the point-spread function (PSF) without overlapping, thereby determining the 2D localization as well as the localization precision ,46,49. TIn addition to generating pointillistic images, SMLM techniques can be utilized to localize and track single particles. This can, in principle, be accomplished using any appropriate fluorophore or technique that enables sufficient temporal and spatial separation permitting the fitting of single particle PSFs at the single-molecule level . Our labIn addition to 2D localization information, some SMLM methodologies are capable of capturing 3D localization information of single particles. This is accomplished in a variety of ways, the most prominent being PSF engineering methodologies. The PSF of a standard fluorescence microscope changes relatively slowly as the Z-dimension changes above or below the focal plane. This can be altered by the addition of a variety of microscope modifications to generate an engineered PSF that will change in relation to the location of the fluorophore to the focal plane . These eEmesvirus zinderi also known as Bacteriophage MS2 (MS2), a virus that stands out for being the first genome ever fully sequenced [The MCP-MS2 loop system is the current gold standard for labeling mRNA in living cells and has been utilized for numerous studies in a variety of model systems over the past few decades ,95,96,97equenced ,99. MS2 equenced ,101. Folequenced .This observed process has provided researchers with a method to label RNA in living organisms. As stem-loop structures do not exist in mammalian cells, MCP does not interact with macromolecules endogenous to mammalian cells. This affords researchers the ability to develop a bipartite labeling methodology, where two plasmids would be introduced to a cell of interest. First, a plasmid containing a mammalian promoter, the sequence encoding for the RNA of interest, and the MS2 loop sequences in the 3′ UTR. Second, a plasmid containing a mammalian promoter, nuclear localization signals (NLS), and the sequence for MCP conjugated with a fluorophore on the 3′ end A. When bEarly versions of this approach suffered from significant signal-to-noise complications, as unbound fluorescently tagged MCP proteins are ubiquitous within the nucleus. If the chimeric RNA sequence contains a single MS2 sequence, it is impossible to differentiate between MCP associated with RNA and unbound MCP. This problem was addressed by Bertrand and colleagues by adding 24× MS2 loop sequences to the 3′ UTR of their target sequence . By addiUsing this system, Mor and colleagues were able to collect some of the first dynamic information regarding nuclear export at the single-mRNP level . Using tS. cerevisiae [In summation, the MCP-MS loop system provides several distinct advantages detailed here, most notably being the ease of use in live cells. However, there are also many distinct disadvantages to this system. Chief among the disadvantages is the high background noise present in the system, as unbound MCP-fluorophore conjugates aggregate in the nucleus. Next, the MS2 loop, as it is not present in mammalian cells, confounds cellular machinery. Chimeric mRNA transcripts are untranslatable, and the tight bonding between the MCP-MS2 loop impairs accessibility of mRNA decay enzymes to the MS2 array, leading to slow degradation in revisiae ,106,107.revisiae ,109.Several steps within the life cycle of an mRNA molecule may inhibit or prematurely end its progression to the ribosome. One of these inhibitory instances, occurring during transcription, is the formation of an RNA:DNA hybrid, R-Loop, from single-stranded RNA hybridization with the complementary DNA sequence. Numerous factors may cause RNA:DNA hybrid formation, relaxed upstream supercoiling, defective proteins related to stalled transcription and RNA:DNA hybrid resolution, G-rich mRNA regions, and non-template DNA strand nicks and secondary structures . TypicalThe complexity of the transcriptome limits the techniques used in microscopy-based assays of mRNA localization and quantification. Typically, localization of a specific mRNA occurs through in situ hybridization with either a fluorescently labeled complementary sequence or a tag recognized by a fluorescently labeled antibody . HoweverAdditionally, S9.6 antibody off-target binding may result from the labeling method used to fluorescent label S9.6 mAb. Commonly, to expose and label cysteines with a fluorophore by maleimide derivatization, the disulfide bonds within the hinge-region are reduced with a mild reducing agent. From this reduction, the antibody is halved and may exhibit a loss of affinity in the antigen binding regions ,118,119.Utilizing the same protein for binding and degrading hybridized RNA in RNA:DNA hybrids, RNase H, researchers have successfully inactivated the catalytic activity of the hybrid binding domain (HBD) . AdditioImaging endogenous mRNA is preferred as it provides a more accurate picture of biological processes unaffected by plasmid overexpression or other experimental artifacts arising from transfection. Further, endogenous mRNA also avoids restriction to cell types that can be efficiently transfected. Studying mRNA expression in live cells can provide dynamic information about how mRNA expression changes in response to varying conditions or over time. Santangelo and colleagues developed multiply labeled tetravalent RNA imaging probes (MTRIPs), a method of labeling native mRNA transcripts with multiple fluorophores . Synthethttp://crispr.mit.edu (accessed on 8 August 2022), “Benchling.” Available online: http://benchling.com (accessed on 8 August 2022). These tools are most effectively used in conjunction with Zhang’s protocol for assembling gRNA plasmids [Using endogenous tagging of mRNA such as the previously mentioned MS2 loop systems would be further improved by the ability to shorten the endogenous tag insertion steps and flexibility in insertion sites. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a hot topic field in genomic manipulation, first discovered in 1987  and pushplasmids . The CRISPR/Cas9 system is small enough to fit onto small format delivery mechanisms such as electroporation of mRNA nanoparticles ,132 and Several classes of Cas have been discovered that have an affinity to RNA binding, specifically Cas13 ,142,143.An RNA targeted molecular beacon functions along a similar premise as all ISH methodologies but is especially similar to smFISH. Specifically, a short, 15- to 20-nt, fluorescently tagged DNA oligo binds to a complimentary transcript of interest within the cell, thereby enabling researchers to visualize and localize that transcript. The primary difference is that this technology attempts to resolve the background noise problem inherent to so many of the mRNA labeling techniques discussed in this manuscript. Researchers have accomplished this by adding a fluorophore to one end of the sequence and a quencher to the other  Figure A. A quenThis probe can be used in vivo or in situ, making it a standout from other ISH techniques. The probe can be introduced into live cells through a variety of methods, including electroporation, gene guns, microinjection, and digitonin membrane degradation ,151,152.An aptamer is a single-stranded length of DNA or RNA that forms a secondary structure that selectively binds to a specific target . As therSaccharomyces cerevisiae [This system has been utilized in a variety of studies to evaluate the localization and behavior of telomerase-associated RNA in both mammalian cells and revisiae ,158,159;revisiae , the RNArevisiae . ProvidiIn summation, this system provides an attractive option for labeling RNA in fluorescent microscopy. In many ways, this approach can be combined with CRISPR-based strategies, as a degree of genomic engineering is required to ensure that the sequence of interest is labeled appropriately. This system also has a few significant shortcomings. Specifically, the early generation of spinach and broccoli aptamers suffered from poor folding in live cells, low quantum yields, and rapid photobleaching . HoweverThere are a wide variety of techniques for labeling mRNA for light microscopy, both for the cellular level and single-molecule level. Each of which have their own strengths and weaknesses . These tLiving-cell mRNA labeling presents a particularly difficult challenge when compared with protein labeling. The advent of genetic engineering made it possible not only to transfect cells with plasmids containing fluorescent proteins, but to modify endogenous proteins with a fluorescent protein. Because mRNA, by its very nature, is untranslated, there remains a significant challenge in imaging endogenous RNA to answer questions regarding native RNA behaviors. Some techniques have significantly contributed to resolving these questions; chief among them is the MCP-MS2 loop system. However, this system remains hampered by the two-part system requiring transfection with the MCP plasmid. This introduces two levels of uncertainty. First, MCP that is unbound is present and free floating through the nucleus. This free-floating property creates an unacceptably high background fluorescence, making it difficult to isolate nuclear neighborhoods, track single molecules, or observe behaviors related to splicing and packaging for export. However, this problem is not insurmountable as a super-folder GFP system has been utilized as a potential solution to this issue . Second,An intriguing development in the field of RNA study is the development of proximity labeling techniques. These techniques rely upon enzyme-catalyzed in vivo reactions that occur between a labeling enzyme and a protein of interest . A recenOf particular interest in recent years is the idea of an RNA-involved phase-separation. This can be seen during transcription where it is hypothesized that RBPs interact and synergize the phase separation of polymerase condensates to promote transcription . To inve


A wide range of bacterial pathogens have been identified in ticks, yet the diversity of viruses in ticks is largely unexplored.Haemaphysalis concinna, Dermacentor silvarum, and Ixodes persulcatus, in North China.Here, we used metagenomic sequencing to characterize the diverse viromes in three principal tick species associated with pathogens, Flaviviridae, Picornaviridae, Luteoviridae, Solemoviridae, and Tetraviridae), negative-sense RNA viruses  and double-stranded RNA viruses (Totiviridae and Partitiviridae). Of these, Dermacentor pestivirus-likevirus, Chimay-like rhabdovirus, taiga tick nigecruvirus, and Mukawa virus are presented as novel viral species, while Nuomin virus, Scapularis ixovirus, Sara tick-borne phlebovirus, Tacheng uukuvirus, and Beiji orthonairovirus had been established as human pathogens with undetermined natural circulation and pathogenicity. Other viruses include Norway mononegavirus 1, Jilin partitivirus, tick-borne tetravirus, Pico-like virus, Luteo-like virus 2, Luteo-likevirus 3, Vovk virus, Levivirus, Toti-like virus, and Solemo-like virus as well as others with unknown pathogenicity to humans and wild animals.A total of 28 RNA viruses were identified and belonged to more than 12 viral families, including single-stranded positive-sense RNA viruses (Mononegavirales and Bunyavirales among the three tick species. Comparatively, I. persulcatus ticks had been demonstrated as such a kind of host with a significantly higher diversity of viral species than those of H. concinna and D. silvarum ticks. Our analysis supported that ticks are reservoirs for a wide range of viruses and suggested that the discovery and characterization of tick-borne viruses would have implications for viral taxonomy and provide insights into tick-transmitted viral zoonotic diseases.In conclusion, extensive virus diversity frequently occurs in  Argasidae (soft ticks), Ixodidae (hard ticks), Nuttalliae, and the newly proposed Deinotheriidae  are common hematophagous arthropods that have been implicated as vectors of human and animal diseases worldwide . Approxiheriidae , 3. Theiheriidae . Argasidheriidae . The incheriidae , 6. The heriidae . Attentiheriidae , 9, Alonheriidae , Bourbonheriidae , most reheriidae , Yezo viheriidae , Toyo viheriidae , and Sonheriidae , which hheriidae , Heartlaheriidae , tick-boheriidae , Powassaheriidae , and Afrheriidae , positivnown one . Howevernown one , 22. Sucnown one . TherefoIxodes persulcatus, Haemaphysalis concinna, and Dermacentor silvarum were collected by sweeping flags on the vegetation in Zhalantun county, Inner Mongolia Autonomous Region, and Mudanjiang city, Heilongjiang province, respectively . All tick specimens were thoroughly surface sterilized to remove the possible contaminants before DNA/RNA isolation. Surface sterilization included sequential 1 mL washes with a vortex for 1 min in 3% hydrogen peroxide (H2O2), two 30 s washes in 70% ethanol (w/v), 2 min in phosphate-buffered saline , and finally drying on clean tissue paper to avoid cross contaminations. Extraction of total DNA and RNA from ticks was performed using E.Z.N.A. DNA/RNA Isolation Kit (OMEGA) with modifications. In brief, ticks were homogenized in RLT solution under liquid nitrogen and then incubated at 55°C for 10 min with proteinase K (Qiagen) and centrifuged for 30 s at 15,000 × g. We took a proportion (~30 μL) of the homogenates for genomic DNA extraction with RNAase A treatment (15 min) for later DNA purification. Simultaneously, another 300 μL of tick homogenates was utilized to enrich viral particles and total RNA extraction. Before RNA extraction, the homogenates were filtered (0.45 μM) at room temperature and then treated with RNase A (15 min), followed by Turbo DNase and Benzonase  (30 min) (Questing ectively . Morpholectively  under st(30 min) . The enr(30 min) .To verify tick species identification, genomic DNA was extracted from each specimen pool using E.Z.N.A. DNA/RNA Isolation Kit (OMEGA). Two genes were used for tick identification: the partial 18S rRNA gene  which was amplified using primer pairs 18S-1 (5′-CTGGTGCCAGCGAGCCGCGGYAA-3′) and 18S-2 (5′-TCCGTCAATTYCTTTAAGTT-3′) and partial COI gene (~680 nt) using primer pairs LCO1490 (5′-GGTCAACAAATCATAAAGATA TTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′). PCRs were performed as previously described , 27. Forwww.genomics.cn).The enriched total nucleic acid (11 μL) from each tick pool was then subjected to first- and second-strand cDNA synthesis with Super Script IV reverse transcriptase  and exo-Klenow fragment , respectively. For all libraries, the Ribo-Zero Gold Kit (Illumina) was used to remove ribosomal RNA under the manufacturer's guidance. Subsequently, all rRNA-depleted RNA samples were resuspended to construct libraries using the KAPA Stranded RNA-Seq Kit  with TruSeq Index PCR Primer barcodes  following the manufacturer's instructions. Qubit high-sensitive RNA assays (Thermo Fisher Scientific) were performed to quantify cDNA levels before, during, and after library preparation, and the fragment sizes were simultaneously determined with an Agilent Bioanalyzer. Subsequently, equimolar amounts of nucleic acids were pooled and submitted for sequencing in each library. All libraries were sequenced on a single lane  on an Illumina HiSeq 2,500 platform at the BGI Sequencing Center   , 29. Cle program , 31.−5 or better collated. All potential virus assemblies were screened against the Conserved Doman Database (www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) with an expected value threshold of 1 × 10−3 to identify viral gene segments. Putative viral contigs were further merged by high-identity overlaps with a threshold value of 95% similarity using the SeqMan program of the Lasergene package v7.1 (I. persulcatus (GCA_013339685.1), D. silvarum (GCA_013339745.1), and whole-genome shotgun database of Ixodida , respectively. If aligned bases of the query contigs covered more than 50% and the nucleotide similarity exhibited higher than 85% from any comparison with the earlier databases, they were discarded from the downstream analysis. Transcript abundance containing RNA-Seq fragment counts for each transcript (or gene) across each sample was estimated using the alignment-based abundance estimation method RSEM  databases (available as early as May 2022) of reference RNA viruses as well as those recently published, with hits at an e-value of 1 × 10age v7.1 . To compage v7.1 , and theage v7.1 . A novelage v7.1 , namely hod RSEM . The triReverse transcription-PCR and nested PCR were performed to confirm the existence of virus on RNA samples from the tick pools which were originally subjected to RNA-seq with primer sets  targetinBunyavirales, Mononegavirales, Flaviviridae, Luteoviridae, Partitiviridae, Totiviridae, and Picornaviridae were retrieved from NCBI GenBank (www.ncbi.nlm.nih.gov/genbank) and aligned using MAFFT v.7.266, employing the E-INS-i algorithm (http://tree.bio.ed.ac.uk/software/figtree). All phylogenetic trees were mid-point rooted for purposes of clarity only. The flowchart of the study design diagram was illustrated with different types of treatment and analysis  RdRp-segments of the lgorithm . Ambiguolgorithm . Followilgorithm . Finallylgorithm . Phylogeanalysis .I. persulcatus  and 147 H. concinna  achieved alive from Mudanjiang city, Heilongjiang province, which were then randomly assigned into five groups of 30 individuals each species, sexes, and development stage. Among them, I. persulcatus groups included female, male, and nymphal ones, respectively, while only two groups  of H. concinna were arranged for inadequate nymphs. An average of 10.96 Gb data, including 7.1–7.6 × 107 or so 150-base paired-end reads, were generated from male, female, and nymph groups of I. persulcatus, while the male and female groups of H. concinna generated ~11.18 Gb of data on average, including 6.8 × 107 and 8.0 × 107 or so 150-base paired-end reads. While in Zhalantun city, Hulunbuir, Inner Mongolia, a total of 274 D. silvarum  and 40 I. persulcatus  were harvested alive. The D. silvarum ticks were also divided into three groups , each with 30 individuals, which generated ~12.30 Gb data on average. After quality filtration and host subtraction, a total of 614,213,814 reads remained which were assembled into 815,457 contigs. Assembled contigs were compared to the NCBI Viral Genomes database, resulting in 1,311, 1,033, and 709 viral contigs from I. persulcatus, D. silvarum, and H. concinna as viral origin through BLASTN and BLASTX, which were kept for a subsequent manual inspection , negative-stranded RNA viruses , and double-stranded RNA viruses (Totiviridae and Partitiviridae) were involved , were identified in all the three D. silvarum pools  and VP3 protein (QHW66956.1), respectively  with 75.65 and 39.62% identities on RdRp coverage over 2,620 amino acid sequences, respectively    Table 118640.1)  and Xian18640.1  18640.1) . FurtherI. persulcatus yields two viruses belonging to Chuviridae  sequences at 99.95% on the 100% query coverage of Nuomin virus (UKS70432.1) , G (AUW34383.1), NP (AUW34384.1), and VP4 (AUW34385.1)  . Nuomin 70432.1) , left, s70432.1) , and theuviridae . Fortuna34385.1) , right bin China .Chuviridae, we also identified several viral contigs belonging to Mononegavirales (Nucleorhabdovirus and two Alphanemrhavirus-like viruses (Tacheng tick virus 3 and Norway mononegavirus 1) were discovered from I. persulcatus with 88.44, 47.32, and 49.46% identities to those aligned RdRp sequences of Chimay-like rhabdovirus (62.52% AVM86063.1), Tacheng tick virus 3 (YP_009304331.1), and Norway mononegavirus 1 (ASY03261.1) (I. persulcatus and assigned as glycoprotein of Chimay rhabdovirus (AVM86062.1) and nucleoprotein of Tacheng tick virus 3  and Norway mononegavirus 1 , respectively , YP_009304331.1 , and UXX19014.1  in L segments, respectively  and American dog tick rhabdovirus (AUX13124.1) with 94.51% and 48.42% similarities over the 99.0% coverage, respectively    . Moreoveectively . As a caectively  , three Phlebovirus, two Ixovirus, and one Uukuvirus (Phenuiviridae), one Orthobunyavirus (Peribunyaviridae), and two unclassified Bunyaviruses from I. persulcatus and D. silvarum, whereas no Bunyavirales contigs detected in H. concinna  to Beiji orthonairovirus (BJNV)  and 77.61% in nucleocapsid protein (AII01798.1) over the 100% coverage (Orthonairovirus.Assembly of multiple contigs from s BJNV) , B, a noJNV , B, sulcatus  and humasulcatus . BJNV recoverage , B. HowePhenuiviridae are present in I. persulcatus and D. silvarum. The most diversity appeared in genus Phlebovirus, which is currently comprised of over 70 phleboviruses isolated from ticks, mosquitoes, and sand flies  and non-structural protein  and nucleoprotein  over the 98% coverage , was harvested from D. silvarum with 85.46% similarity on the 100% sequence coverage of RdRp gene , was also documented in I. persulcatus with 74.1% identity in L segments  with 92.61% similarity on the amino acids' sequences on polymerase gene   based on over 90% sequence coverage of RdRp gene  and ORF1  of Lonestar tick totivirus   . As a caRp genes .Leviviridae were also detected from I. persulcatus (Other more distantly related viruses including unclassified Vovk virus (44.24% RdRp aa identity with QKK82915.1) and Levivirus  and 35.34% coat (UJQ85258.1) in the family of sulcatus .D. silvarum, I. persulcatus, and H. concinna ticks from North China. The outcome from our sampling efforts supports the results throughout various Chinese regions, showing that I. persulcatus, D. silvarum, and H. concinna are three of the most abundant questing ticks within the regions  and Far-Eastern strain of TBEV  can be found in the article/YS contributed to the concept, design, and statistical analysis of the manuscript. TQ and MS were involved in the field survey. YS and HF were involved in the taxonomic identification of ticks. TQ and ZL were involved in the sequencing and molecular analysis of viruses. YS, TQ, MS, and MZ were involved in the analysis, interpretation, and drafting of the manuscript. All authors contributed to revising it critically for important intellectual content, final approval of the version to be published, and agreement to be accountable for all aspects of the manuscript in ensuring that questions related to the accuracy or integrity of any part of the manuscript are appropriately investigated and resolved.


To investigate human oral health and disease, models are required which represent the interactions between the oral mucosa and microbiome. Our aim was to develop an organotypic model which maintains viability of both host and microbes for an extended period of time.Streptococcus gordonii  or Aggregatibacter actinomycetemcomitans (pathogen) for 72 hours in agar. RHG histology, viability and cytokines (ELISA), and bacterial viability (colony forming units) and location (FISH) were assessed.Reconstructed Human Gingiva (RHG) were cultured air-lifted with or without penicillin-streptomycin (PS) and topically exposed to S. gordonii (with or without PS) did not influence low level of IL-6, IL-8, CCL2, CCL5, CCL20 or CXCL1 secretion. However, all cytokines increased (except CCL2) when RHG were co-cultured with A. actinomycetemcomitans. The effect was significantly more in the presence of living, rather than dead, A. actinomycetemcomitans. Both bacteria resulted in increased expression of RHG antimicrobial peptides (AMPs) Elafin and HBD-2, with S. gordonii exposure resulting in the most Elafin secretion.The low concentration of topically applied agar did not influence RHG viability. Topically applied bacteria in agar remained localized and viable for 72 hours and did not spill over to infect RHG culture medium. PS in RHG culture medium killed topically applied bacteria. Co-culture with living bacteria did not influence RHG viability  or histology . RHG exposed to S. gordonii and A. actinomycetemcomitans.This technical advance enables living human oral host–microbe interactions to be investigated during a 72-hour period and shows differences in innate immunology triggered by  Humans are host to a complex flora of microorganisms that colonize the oral mucosa and gingiva. More than 700 species of commensal and pathogenic, aerobic and anaerobic bacteria inhabit the oral cavity . The heaStreptococcus gordonii is a Gram-positive facultative anaerobic bacterium which has adapted to a commensal role during a long evolutionary relationship with the host. S. gordonii is one of the initial colonizers of dental plaque, and is also one of the streptococcal groups which dominates the oral mucosa of healthy individuals , leukotoxin, cytolethal-distending toxin, collagenase and outer membrane protein  consisting of a stratified and differentiated gingival epithelium on a fibroblast-populated collagen hydrogel which functions as the lamina propria . RHG is ituation . Using tituation , as well allergy . Even th allergy . Antibiovia the edges of the transwell, and b) the impact of the presence and absence of PS on the host-microbe co-culture over a period of 3 days. The second aim of this study was to investigate differences in innate cytokine immune responses when RHG is exposed to a commensal and a pathogenic bacterium for 3 days. In this study, S. gordonii represented the commensal and A. actinomycetemcomitans represented the pathogenic bacteria.The first aim of this study was to develop a model to investigate interactions between the RHG and living oral bacteria over an extended period of time of 3 days. The impact of two essential technical steps on RHG viability were investigated: a) the impact of topically applied agar as a vehicle to keep the bacteria localized on the surface of RHG thus preventing bacteria from spilling over into the culture medium 6 cells) was cultured exactly as previously described  (passage 10 to 20) and fibroblast  (passage 10 to 20) cell lines were used for constructing RHG. The RHG model, consisting of a fibroblast-populated collagen hydrogel (collagen I derived from rat tail) overlaid with keratinocytes  broth or on BHI containing 1.5% agar plates for determination of viable bacterial cell counts as described below. A. actinomycetemcomitans Y4 was routinely cultured in BHI containing 0.02M NaHCO3 and 1% glucose or on tryptic soy agar  plates containing 5% sheep blood (BA plate). Both strains were cultured anaerobically  at 37°C.S. gordonii of high and low densities, (4) exposed to live A. actinomycetemcomitans of high and low densities, (5) exposed to heat-killed A. actinomycetemcomitans of high density only. For conditions (1-4), the co-cultures were cultured in the presence or absence of PS in the culture medium. For condition (5) where no live A. actinomycetemcomitans were present, co-cultures were grown in the absence of PS.RHG underwent 5 exposure conditions as follows: (1) negative control: unexposed RHG, (2) vehicle control: RHG exposed to the vehicle agar , (3) exposed to live S. gordonii and A. actinomycetemcomitans were first diluted to 4 x 109 CFU/mL respectively. The dilutions were then mixed with 0.3% agar containing BHI either at the ratio of 1:1  or after 100 times dilution . A drop of the mixture (50µL) was then pipetted onto the center of RHG. The final bacterial cell counts on RHG were either 1 x 106 CFU (low) or 1 x 108 (high) CFU.For conditions (3) and (4) where living bacteria were used, prior to exposure to RHG, the stationary cultures of A. actinomycetemcomitans was first diluted in PBS to 4 x 109 CFU/mL. This dilution was incubated at 60°C for 1 h before mixing with 0.3% agar containing BHI and at the ratio of 1:1. A drop of the mixture (50µL) was then pipetted on top of RHG. The number of heat-killed A. actinomycetemcomitans was equivalent to the bacterial cell counts in the living high bacterial group. Heat-killed A. actinomycetemcomitans was plated on BA agar to confirm effectivity of heat-killing.For condition (5) with dead bacteria exposure, the stationary culture of 2 and 95% humidity representing the environment of the oral cavity. The culture medium of all conditions was refreshed and collected on day 1, day 2 and day 3 (every 24 hours). On day 3, each RHG was divided in half; one half was used to assess viable bacterial counts and the other half was used to assess RHG viability and for (immune-)histochemical analysis.During exposure, all RHG were cultured under aerobic conditions at 37°C, with 7.5% COTo determine the number of living bacteria, RHG was homogenized, collected, diluted and plated for colony-forming-unit (CFU) counting. All procedures were performed in a sterile environment. Cysteine peptone water  was used for processing the tissue homogenate as well as for serial dilutions before plating.S. gordonii) or BA agar plates (A. actinomycetemcomitans) and incubated anaerobically for 3 days at 37°C before CFU were counted.In detail, RHG in 1mL CPW was mechanically broken down to a homogenous suspension using a glass tissue homogeniser . Another 1 ml CPW was added to wash and collect the residual materials. All tissue homogenates were then filtered through a Falcon™ cell strainer  in order to remove tissue fragments, and serially diluted with CPW. The filter-through and the dilutions (100µL) were plated on BHI  assay as described previously . RHG bioFor each experimental condition, a strip of RHG tissue was fixed in 4% paraformaldehyde and processed for paraffin embedment. Tissue sections (5μm) were stained with hematoxylin and eosin (HE) for histological analysis or used for IHC of Ki67 , keratin10 , Elafin  and HBD-2 . The stained slides were visualized using a Nikon Eclipse 80i microscope with 20x/0.5 objective . Ki67 quantification was performed as previously described . The Ki6In Situ Hybridization (FISH) following the manufacturer’s protocol . The FISH probe EUB338 (5′-GCTGCCTCCCGTAGGAGT-3′) was used to visualize bacteria. After FISH staining, the tissue sections were mounted with a mounting medium containing DAPI . The images were taken under a fluorescence microscope with 40x/1.0 objective  at Ex/Em= 490/525nm (Probe) and Ex/Em= 375/460nm (DAPI) with NIS-Elements software  (Bacteria in RHG sections (5μm) were detected by Fluorescence erlands) ELISAs were performed as described by the suppliers and as described previously . In shorp < 0.05. Data are represented as mean ± SEM; *p<0.05; **p<0.01; ***p<0.001.Statistical analysis was performed with GraphPad Prism, version 9.1 . The statistical methods for analyzing the data were described in detail in the figure legends. All data represent at least three individual experiments, each with an intra-experiment duplicate. Differences were considered significant when via the edges of the transwell, and b) the impact of the absence of PS on RHG which would be necessary to maintain viable bacteria for 3 days. Clearly the topical presence of agar and the absence of PS in the culture medium did not influence RHG histology  the impact of topically applied agar which can be used as a vehicle to keep the bacteria localized on the surface of RHG thus preventing bacteria from spilling over into the culture medium istology Figure 1istology . The proFurthermore, metabolic activity, which correlates to cell viability remained unaltered by the presence of agar or the absence of PS  and A. actinomycetemcomitans (high) respectively. We further performed FISH to visualize the location of the bacterial cells  all cytokine levels increased when PS was absent (In order to determine whether the presence of bacteria influenced secretion of (pro-) inflammatory cytokines and chemokines, RHG culture supernatants were assessed by ELISA. Co-culture of RHG with e period Figure 3A. actinomycetemcomitans  were next compared ) and HBD-2 are antimicrobial peptides (AMPs) which are expressed at low levels in the upper epithelium layer of RHG  for 3 days an increased expression of both proteins was observed, in particular for Elafin inflammatory cytokine and chemokine secretion and expression of AMPs were observed. S. gordonii exposure resulted in a negligible inflammatory response whereas A. actinomycetemcomitans strongly stimulated cytokine and chemokine secretion indicating that the later had initiated TLR signaling and activation of the inflammazone, which, in vivo, would be expected to combat invasion of the pathogen into the host tissues in order to maintain homeostasis  is more than just a question of TLR activation by bacterial cell membrane components  since when the same number of heat killed bacteria were applied to RHG the inflammatory cytokine response was significantly lower.Moreover, the expression of AMPs (Elafin and HBD-2) increased in the upper regions of RHG epithelium after exposure to both bacteria, in line with our previous studies . Howeverinocytes . This woin vitro models our host-microbe model does have limitations and there is still room for further improvement. In this study, the RHG was constructed using TERT immortalized gingiva keratinocyte and fibroblast cell lines. In the past the cell line model was extensively compared with the RHG constructed from primary cells and was found to show excellent correlation with regards to histology, biomarker expression and cytokine secretion  for the research.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.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.


Drosophila melanogaster, Argonaute 1 (Ago1) is the core RISC component in microRNA (miRNA) silencing, while Argonaute 2 (Ago2) is the core RISC component in small interfering RNA (siRNA) silencing. The expression of Ago1 and Ago2 is regulated by stress response transcription factor Forkhead box O (dFOXO) increasing siRNA silencing efficiency. dFOXO plays a role in multiple stress responses and regulates pathways important for longevity. Here we use a next-generation sequencing approach to determine the effects of aging on small RNA abundance and RISC loading in male and female Drosophila. In addition, we examine the impact of the loss of dFOXO on these processes. We find that the relative abundance of the majority of small RNAs does not change with age. Additionally, under normal growth conditions, the loss of dFOXO has little effect on the small RNA landscape. However, we observed that age affects loading into RISC for a small number of miRNAs.Aging can be defined as the progressive loss of physiological homeostasis that leads to a decline in cellular and organismal function. In recent years, it has become clear that small RNA pathways play a role in aging and aging related phenotypes. Small RNA pathways regulate many important processes including development, cellular physiology, and innate immunity. The pathways illicit a form of posttranscriptional gene regulation that relies on small RNAs bound by the protein components of the RNA-induced silencing complexes (RISCs), which inhibit the expression of complementary RNAs. In  Drosophila, each of which have distinct functions and small RNA content  following the manufacturer’s protocol. Libraries were sequenced on a NextSeq500 platform [Illumina].RNA was extracted from 40 whole mated female or male flies or from immunoprecipitated Ago1 or Ago2 using Trizol reagent [Invitrogen] following the manufacturer’s protocol. RNA extracted from whole flies was sized selected to 19 to 35 nt fragments using 17% 19:1 acrylamide:bisacrylamide urea gels [National Diagnostics]. Gels were sliced from 19–35 nt, crushed, and eluted overnight in 500 μL 0.3 M sodium acetate with rotation. Twenty micrograms of glycogen and small RNA spike ins  were addGATCGTCGGACTGTAGAACTCTGAACGTGTAGATCTCGGTGGTCGCCGTATCATT. The trimmed sequence files were processed through the Reverse-Complement application. These trimmed and reverse complemented files were then size selected using Filter FASTQ [Drosophila transposons, mature miRNAs, and miR* strands using sR_bowtie [et. al 2008 [Processing of sequence files was conducted using the Galaxy web platform  and the er FASTQ  to filteer FASTQ , known DR_bowtie  set to mR_bowtie . miR* stR_bowtie . Librari al 2008 . All seqS1 FigDAH) and dFOXO-null  flies were recorded 2–3 times weekly. (A) Mated wildtype males had a median lifespan of 90 days and a maximum survival of 105 days while mated dFOXO-null males had a median lifespan of 45 days and a maximum survival of 65 days. (B) Mated wildtype females had a median lifespan of 90 days and a maximum survival of 110 days while mated dFOXO-null females had a median lifespan of 55 days and a maximum survival of 70 days.Percent survival of wildtype (w(TIFF)Click here for additional data file.S2 FigDrosophila melanogaster Schneider 2 cells as input. Western blot of input, supernatant, and eluate was probed with a commercial rabbit (RB) anti-Ago1 antibody. Anti-Ago1 antisera immunoprecipitated Ago1 and was capable of depleting the lysate of Ago1. Preimmune sera and anti-Ago2 antisera did not immunoprecipitate Ago1. (B) A second blot was probed with a commercial rabbit anti-Ago2 antibody. Similarly, anti-Ago2 antisera was able to immunoprecipitate Ago2 and depleted the lysate of Ago2. Preimmune sera and anti-Ago1 antisera did not immunoprecipitate Ago2.(A) Guinea pig anti-Ago1 antisera, Guinea pig anti-Ago2 antisera, or preimmune antisera were used in an immunoprecipitation (IP) using lysate from (TIF)Click here for additional data file.S3 Figin silico, and mapped to known Drosophila miRNAs and transposons (n = 2). Transposon siRNAs are plotted as percent of mapped reads in Ago1 RISC against percent of mapped reads in Ago2 RISC. All transposon siRNAs that were detected in both RISC make up a greater percentage of Ago2 RISC.Small RNA immunoprecipitated with Ago1 RISC or Ago2 RISC from young (5 day old) or old (35 day old) whole flies were sequenced, size selected to 21 nt (TIF)Click here for additional data file.S1 TableDrosophila miRNAs and transposons, and normalized as a percent of total reads mapped (n = 2). miRNA reads were then normalized by total miRNA reads and shown here. (A) The 20 most abundant miRNAs in young wildtype males make up 87 to 94 percent of the total miRNA reads in the male libraries. (B) The 20 most abundant miRNAs in young wildtype females make up 79 to 87 percent of the total miRNA reads in the female libraries.Small RNAs from whole flies, Ago1 RISC immunoprecipitation (IP), or Ago2 RISC IP were sequenced, mapped to known (PDF)Click here for additional data file.S2 TableDrosophila miRNAs and transposons, and normalized as a percent of total reads mapped (n = 2). Transposon siRNA reads were then normalized by total transposon siRNA reads and shown here. (A) The 20 most abundant transposon siRNAs in young wildtype males make up 57 to 88 percent of the total transposon siRNA reads in male total small RNA and Ago2 RISC. (B) The 20 most abundant transposon siRNAs in young wildtype females make up 55 to 66 percent of the total transposon siRNA reads in the female total small RNA and Ago2 RISC.Small RNAs from whole flies, Ago1 RISC immunoprecipitation (IP), or Ago2 RISC IP were sequenced, size selected in silico, mapped to known (PDF)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


The availability of a simple web-based platform for DMS-MaPseq analysis will dramatically expand studies of RNA structure and aid in the design of structure-based therapeutics.RNA structures play critical roles in regulating gene expression across all domains of life and viruses. Chemical probing methods coupled with massively parallel sequencing have revolutionized the RNA structure field by enabling the assessment of many structures in their native, physiological context. Previously, we developed Dimethyl-Sulfate-based Mutational Profiling and Sequencing (DMS-MaPseq), which uses DMS to label the Watson-Crick face of open and accessible adenine and cytosine bases in the RNA. We used this approach to determine the genome-wide structures of HIV-1 and SARS-CoV-2 in infected cells, which permitted uncovering new biology and identifying therapeutic targets. Due to the simplicity and ease of the experimental procedure, DMS-MaPseq has been adopted by labs worldwide. However, bioinformatic analysis remains a substantial hurdle for labs that often lack the necessary infrastructure and computational expertise. Here we present a modern web-based interface that automates the analysis of chemical probing profiles from raw sequencing files (  RNA is an informational intermediate between DNA and proteins and drives a diverse set of biological processes . RNA’s aA wealth of techniques have been utilized to study RNA structures at different levels of resolution. Biophysical approaches such as NMR and X-ray crystallography have proven useful at resolving RNA structures at an atomic scale and can provide information on tertiary structure; however, these high-resolution approaches are time-intensive, requiring pure samples, making them not ideal for high-throughput data collection. In contrast, when combined with next-generation sequencing, chemical probing has allowed for the elucidation of the secondary structure of RNAs in a high throughput manner both in vivo and in vitro with single-nucleotide precision . One sucDMS-MaPseq has been used to determine the genome structure of both HIV-1  and SARSThe high-throughput nature of DMS-MaPseq produces orders of magnitude more data than previous chemical mapping procedures leading to a new analysis bottleneck. Furthermore, incorporating next-generation sequencing requires a host of new bioinformatics tools that may be unfamiliar to RNA structural biologists. These tools also require new computational infrastructure and know-how to run. These new challenges can lead to a significant barrier to analyzing high-throughput chemical mapping data. We have developed a MaP analysis webserver to alleviate the computational burden for extracting RNA structure information from mutational profiling chemical mapping data. This web-based platform will enable the scientific community to analyze high throughput chemical probing data without dedicated computational knowledge or infrastructure.The DMS-MaPseq protocol is extensively detailed in a dedicated paper (see ref: ). In shohttps://cherrypy.dev) framework based on Python3 (https://www.python.org/) for server web services and data management. The client-side utilizes HTML5 (http://www.w3.org/TR/html5/) styled with Bootstrap (http://getbootstrap.com/) coupled with jQuery (http://jquery.com) for interactive user-interface components. The MaP analysis server supports most web browsers, including Google Chrome, Mozilla Firefox, Apple Safari, and Microsoft Edge. The job submission page has been streamlined to be user-friendly  and support various functions, such as hovering for more information and zooming. Investigating the mutation fraction per nucleotide can reveal structural details for a reference RNA sequence. Generally, As and Cs with low reactivity (mutation fraction < 0.01) are in Watson-crick base pairs (e.g. AU or GC pairs). Conversely, nucleotides with high reactivity are unpaired or in noncanonical interactions. For a more in-depth discussion of DMS reactivity values, please see refs: , and if it's lower, this may be a sign of an incorrect reference sequence or too many similar reference sequences. For each reference sequence, an interactive bar plot displays the mutational fraction of each nucleotide. Coloring is static, with guanosine, cytidine, adenosine, and uridine as orange, blue, red, and green. These interactive plots were generated with Plotly  ,16. ThisIn the past decade, the coupling of next-generation sequencing to chemical mapping has seen the emergence of rapid and high-throughput probing experiments. We expect experiments such as DMS-MaPseq to increase in popularity, with more groups adopting and expanding chemical mapping to supplement their other molecular and structural biology experiments. With the ever-increasing usage of high-throughput chemical mapping techniques, there will be a corresponding demand for new computational resources to analyze the results of these experiments. The webserver presented herein brings the first free and easy-to-use computational tool for analyzing MaP chemical mapping experiments. The MaP analysis webserver will alleviate the existing computational burden allowing to dramatically expand studies of RNA structure and aid in the design of structure-based therapeutics.