Aptamers with fluorogenic ligands are emerging as useful tools to quantify and track RNA molecules. The RNA Mango family of aptamers have a useful combination of tight ligand binding, bright fluorescence, and small size. However, the simple structure of these aptamers, with a single base-paired stem capped by a G-quadruplex, can limit the sequence and structural modifications needed for many use-inspired designs. Here we report new structural variants of RNA Mango that have two base-paired stems attached to the quadruplex. Fluorescence saturation analysis of one of the double-stemmed constructs showed a maximum fluorescence that is ∼75% brighter than the original single-stemmed Mango I. A small number of mutations to nucleotides in the tetraloop-like linker of the second stem were subsequently analyzed. The effect of these mutations on the affinity and fluorescence suggested that the nucleobases of the second linker do not directly interact with the fluorogenic ligand (TO1-biotin), but may instead induce higher fluorescence by indirectly altering the ligand properties in the bound state. The effects of the mutations in this second tetraloop-like linker indicate the potential of this second stem for rational design and reselection experiments. Additionally, we demonstrated that a bimolecular mango designed by splitting the double-stemmed Mango can function when two RNA molecules are cotranscribed from different DNA templates in a single in vitro transcription. This bimolecular Mango has potential application in detecting RNA–RNA interactions. Together, these constructs expand the designability of the Mango aptamers to facilitate future applications of RNA imaging. Studying the dynamics and interactions of RNA molecules is central to understanding cellular regulatory processes. Recently, fluorogenic RNA aptamers have emerged as useful tools to detect and report intracellular RNA events due to their small size and genomic encodability . TypicalKD and have fluorescence enhancement of ∼1000-fold . To facilitate the design process we developed an abstract representation based on the known structure of Mango I  . Both stems had GAAA tetraloop-like linkers where the stems connected to the G-quadruplex . Fmax values for dsMango variants are reported as relative to the fluorescence observed for the single-stemmed Mango I , very similar to single-stemmed Mango I. The brightest double-stemmed construct was dsMango i-iv ii-i, which was ∼75% brighter than the single-stem Mango I (Fmax = 1.75 ± 0.08). In addition to curve fitting, the KD for dsMango i-iv ii-i was validated using a KinExA binding affinity assay . This technique measures the unbound Mango aptamer remaining on an affinity column after mixing with a series of TO1-b dilutions. The resulting data was fit using KinExA software which resulted in a measured KD of 4.35 nM (95% CI of 3.28–5.57 nM) . This demonstrates that the i-iv corner is not a requirement for double-stemmed designs , and showed reduced ligand affinity  E. Of not designs B. The thdsMango i-iv ii-i construct could achieve brighter maximum fluorescence than the single-stemmed Mango I. The presence of a second stem adds thermodynamic stability from additional base pairs, which could result in a greater fraction of correctly folded RNA molecules. However, because the concentration of RNA is saturating, this mechanism in isolation would require an ∼75% increase in the fraction folded. Alternatively, the second stem could create a new tertiary interaction that alters the quadruplex core. This has been previously observed in the structure of Mango III, where a pseudoknot-like tertiary interaction leads to structural rearrangements at the ligand binding site that alters the planarity of the TO1 in the bound state leading to an increased fluorescence enhancement relative to Mango I . Fmax was measured as relative to the unmutated dsMango i-iv ii-i, which was remeasured side by side with the mutational variants as a control. Most of the individual mutations had very little effect on Fmax or KD. We conclude that it is unlikely that nucleobases in the linker make direct hydrogen bonds or stacking interactions with the TO1-b ligand. Interestingly, some of the individual mutations appear to have slightly lower KD values in these experiments. We are hesitant to make strong conclusions about this enhanced binding affinity because the curve fitting in this experimental design is sensitive to experimental noise. In addition, some of the sequence variants appeared to have decreased binding affinity, including some individual mutations, a double mutant, and a variant where the GAAA tetraloop was completely replaced with a different tetraloop CUUG. It is likely that the decreased affinity in these cases was caused by misfolding events. Nevertheless, the results indicate the potential to modify ligand affinity by altering the linker sequence. These results suggest that further exploration of sequence variants may be warranted, which could be accomplished through randomizations, expansions, and reselections  was first investigated by separately transcribing and PAGE purifying each strand of the split sequence, and then mixing them back together in 1× Mango buffer with TO1-b . This primer-annealed template was added to a transcription reaction containing 1× T7 buffer, 2.5 mM ribonucleotide triphosphate mix (NEB: N0466S), and ∼800 units of T7 RNA polymerase (Thermo Fisher Scientific: EP0113). RNA was purified on a 12% polyacrylamide gel with 8 M urea. Bands were visualized with a brief exposure to UV light, and excised with sterile razor blades. Gel was crushed and eluted in 300 mM sodium acetate (RNase Free) at room temperature for 1 h. The RNA eluted from the crushed gel was filtered by centrifugation through a 0.2 µm sterile filter and ethanol precipitated with 3 volumes of 95% cold ethanol at −20°C for 5 min. RNA was pelleted by centrifugation at 4°C for 20 min and washed with 80% then 95% ethanol. Pellets were dried in a SpeedVac for ∼2 min, then rehydrated in RNase free water and quantified by A260 (NanoDrop). Purified RNA was run again on an analytical 12% denaturing polyacrylamide gel for quality control.Mango variants were made by in vitro transcription from synthetic oligonucleotide templates (IDT). The T7 promoter regions of each template were made double-stranded by annealing a second oligonucleotide with the sequence of the top strand of the promoter. DNA was heated to 95°C for 5 min and cooled to room temperature in 1× T7 buffer . Upon addition of TO1-3PEG-biotin , relative fluorescence was measured with a DeNovix QFX fluorometer. Best-fit curves were generated using the nonlinear regression function from the Scipy_Optimize package in python. The averages of the triplicate data were fit using a Trust Region Reflective least squares algorithm to Equation apt] and [TO1b] are the nanomolar concentrations of RNA and TO1-3PEG-biotin, respectively. F′ is the molar maximum fluorescence which is the Fmax/[TO1b]. The 68% confidence intervals were calculated using the Python uncertainties package.Serial dilutions of RNA were made using a 1× Mango buffer (10 mM NaHKD and Fmax for each mutation were determined by Student's unpaired t-test (n = 20).The variants with mutations in the tetraloop-like linker were evaluated very similar to above, but with the following differences. PAGE purified RNA was prepared as described above. For each double-stemmed construct, twofold serial dilutions were prepared and individually loaded onto 96 well plates. Dilutions ranged from 800 nM to 6.25 nM except for the A38 series of mutants which ranged from 500 nM to 3.9 nM. For each reaction, 1× Mango buffer containing TO1-b at a concentration of 40 nM was added prior to being briefly spun down. The samples were then incubated for 2 min at 65°C and cooled to room temperature for 2 min prior to measurement on a Tecan Spark Plate reader at 510 nm excitation, 535 nm emission. Triplicate data values were fit using the nonlinear regression procedure described above. Statistically significant changes in Mango RNA was prepared cotranscriptionally in triplicate from 1 µM synthetic oligonucleotide templates and 1 µM T7 promoter in the presence of T7 buffer, ∼400 units of T7 RNA polymerase, 1× Mango buffer, and 40 nM TO1-b. Two sets of identical plates were prepared simultaneously and individually ran on a preheated Eppendorf MasterCycler and Tecan Spark plate reader at 37°C for 3 h. Fluorescence measurements  were taken continuously in 1 min intervals from the plate reader. RNA concentration values were determined from 40 µL samples taken from the MasterCycler run plate which were collected at 0, 0.5, 1, 2, and 3 h timepoints and stored at −20°C. Band separation of the sample products was performed using analytical 12% Urea-PAGE gels. Gels were stained in Gel Red for 30 min and subsequently imaged. Densitometry was performed using GelAnalyzer using morphological background subtraction (10% width tolerance). Density values for each sample were compared to a purified RNA concentration reference to estimate sample RNA concentrations.dsMango were also prepared similarly. From this RNA, ½ serial dilutions were performed to obtain the specified concentration. For each concentration sample, purified RNA aptamer strands were individually loaded onto 96 well plates with 1× Mango buffer containing TO1-b at a concentration of 40 nM. The plate was then briefly spun down before incubation at 70°C for 3 min. After incubation, the plate was cooled to room temperature for 5 min prior to fluorescent measurement on a Tecan Spark plate reader at 510 nm excitation, 535 nm emission. Triplicate data values were fit using the nonlinear regression procedure described above.Split RNA aptamers were transcribed and purified separately as described above. Mango I and KD was calculated by the KinExA software using the equilibrium protocol.The dissociation constant was measured by an affinity exclusion assay with a KinExA model 3200 (Sapidyne). RNA (5 nM) in 1× Mango buffer was mixed with fivefold dilutions of TO1-3PEG-biotin beginning with 1.0 µM. Samples were also supplemented with 0.02% tween-20 to minimize nonspecific binding to the KinExA tubing system. Samples were allowed to equilibrate for 4 h at room temperature. The stationary phase of the system consisted of polymethylmethacrylate beads functionalized with TO1-3PEG-biotin according to Sapidyne protocols. Following introduction of the sample, buffer was flowed across the flow cell for 4 min at 0.25 mL/min. The amount of free aptamer from each equilibrium mixture was quantified by the fluorescent signal resulting from the binding of this free aptamer to the TO1-b functionalized beads in the stationary phase. Supplemental material is available for this article.


Hepatic alveolar echinococcosis (HAE) of the metastasis-like pattern, according to the Echinococcus Ulm classification, is usually discovered as an incidental finding, and the diagnostic differentiation from “true metastases” is difficult. The aim of this study was to investigate whether lesions of the “metastasis-like pattern” in HAE show a typical contrast behavior that can be used for differentiation from metastasis in malignancies.This prospective clinical study included 11 patients with histologically confirmed HAE of the metastasis-like pattern , who had been examined by B-scan sonography and CEUS, from the National Echinococcosis Registry Germany.On contrast-enhanced sonography, 11/11 reference lesions showed annular rim enhancement in the arterial and portal venous phases. Throughout the entire 4-min study period, none of the reference lesions showed central contrast enhancement—i.e., all exhibited a complete “black hole sign”. A small central scar was seen in 81.8% of cases.In clinically unremarkable patients with incidentally detected metastasis-like lesions of the liver, contrast-enhanced sonographic detection of rim enhancement without central contrast uptake (black hole sign) should be considered evidence supporting a diagnosis of hepatic alveolar echinococcosis with a rare metastasis-like pattern. This can help to differentiate HAE from metastases, especially in high-endemic areas. Alveolar and cystic echinococcosis are two completely different diseases . Cystic Echinococcus multilocularis Ulm Classification Ultrasound (EMUC-US) is the first method enabling classification into five B-scan sonographic patterns: hailstorm, pseudocystic, ossification, hemangioma-like, and metastasis-like , the parasitic lesions show highly diverse sonomorphological presentations, and are often difficult to diagnose by B-scan sonography , 6. In pike Fig.  [6]. Howike Fig. . TherefoIn addition to US examination for the detection of HAE, imaging diagnosis is usually complemented by CT scan which best shows common HAE characteristic calcifications. Furthermore, HAE-related morphologic changes are comprehensively visualized by CT .Furthermore, positron emission computed tomography (PET-CT) is of crucial importance. It is the only imaging technique that allows an assessment of inflammatory activity of the disease based on metabolic activity. FDG accumulation, usually in the marginal area of a lesion, is a parameter for the metabolic activity of the parasite. This is particularly helpful in the assessment of progression under benzimidazole (BMZ) therapy and in the detection of recurrences, e.g., in the postoperative course .Since the introduction of second-generation ultrasound contrast enhancers, contrast-enhanced ultrasound (CEUS) has become the standard method of liver metastases diagnosis, due to improved imaging in the portal venous and late phases . MeanwhiIn recent years, CEUS has become increasingly important in the diagnosis of HAE –23. ImpoThe aim of this study was to investigate whether lesions of the “metastasis-like pattern type” in HAE show a typical contrast behavior that can be used for differentiation from metastasis in malignancies.A total of 11 patients with histologically confirmed HAE and proven metastasis-like pattern, according to the EMUC-US classification, identified from the National Echinococcosis Registry Germany, were contacted and consented for prospective participation in the study , 24. AllContrast-enhanced ultrasound was performed following the guidelines and good clinical practice of the EFSUMB . All exaThe period of 7–30 s post-injection (p.i.) was defined as the arterial phase, 31–120 s p.i. as the portal phase, and 121–360 s p.i. as the late phase. Video recordings were obtained during inflation of the contrast agent  and the arterial phase (7–30 s p.i.). A targeted punctate scanning of the liver (sweep) was performed in the portal venous phase (60 s p.i.), and additional sweeps were performed in the venous phase starting at 120 and 180 s, respectively. The total observation time was 4 min.U test. The tests were two-sided. A p value of < 0.05 (α = 0.05) was considered statistically significant with a 5% probability of error.Statistical analysis was performed using SAS version 9.4. We calculated frequencies, mean, and location and dispersion measures. Differences were determined using the nonparametric Mann-Whitney p = 0.7048), age (p = 0.5074), disease duration (p = 0.2193), or duration of benzimidazole therapy (p = 0.1564).In our cohort of 11 patients with alveolar echinococcosis and sonomorphological metastasis-like EMUC-US pattern, the gender distribution was 63.6% female (7/11) to 36.4% male (4/11). The mean patient age at the time of the study was 57.1 ± 15.3 years. In 7/11 (63.6%) of the patients, the BMI was 25–30. In 2/11 (18.2%) patients, echinococcosis was initially suspected, in 8/11 (72.7%), suspicion of malignancy of unclear genesis, and in one patient, other suspected diagnoses were assumed. The mean disease duration in patients with alveolar echinococcosis was 59.5 ± 46.2 months. A total of 9/11 (81.8%) patients received a benzimidazole therapy, one patient did not receive BMZ therapy and one patient received an outlet attempt. The average BMZ therapy duration was 38.5 ± 48.4 months. Benzimidazole toxicity occurred in 4/11  during BMZ therapy , AE was discovered as an incidental finding during the investigation of other non-hepatic complaints or during screening examinations. A total of 10 of the lesions were biopsied and one patient underwent surgery. In each case, the entire liver showed a metastasis-like pattern of AE, with a mean of 3.6 ± 2.4 lesions (range: 1–10). The majority of the reference lesions (9/11) were located in the left lobe of the liver  showed a signal with any of the conventional Doppler techniques. All lesions  exhibited annular rim enhancement in the arterial and portal venous phases Fig. b–f. The This is the first prospective study to characterize the contrast pattern of metastatic liver lesions according to the EMUC-US classification in HAE . Prior tThe current literature confirms that HAE is commonly misdiagnosed as liver metastases , 28, 29.On contrast-enhanced ultrasound, all 11 reference lesions exhibited annular rim enhancement in the arterial phase. The onset of this phenomenon coincided with the arrival time of the contrast enhancer at 11 s post-injection. The rim enhancement disappeared towards the end of the portal venous phase (> 60 s after injection). Tao et al. first described rim enhancement as a band-like enhancement in the arterial phase around the irregular rim of the lesion. This report also described the phenomenon of the “black hole”, a complete non-enhancement of the interior of the AE lesion . This obIn conclusion, our results showed that when contrast-enhanced sonography is performed in cases of HAE of the metastasis-like type, the typical findings are echo-poor small metastasis-like lesions with annular rim enhancement in the arterial phase, a “black hole sign” and a small central echoic scar. This information may help in making the difficult diagnosis of HAE in asymptomatic patients, especially in high-endemic areas.


The lymph node posterior to the right recurrent laryngeal nerve (LN-prRLN) is a crucial component of the central lymph nodes (LNs). We aimed to evaluate multimodal predictive factors of LN-prRLN metastasis in patients with papillary thyroid carcinomas (PTCs), including the clinical data, pathologic data, and preoperative sonographic characteristics of PTCs.A total of 403 diagnosed PTC patients who underwent unilateral, sub-total, or total thyroidectomy with central neck dissection were enrolled in this retrospective study. The clinical data, pathologic data, conventional ultrasound (US) and contrast-enhanced ultrasound (CEUS) characteristics of PTCs were collected and evaluated for predicting LN-prRLN metastasis.In this study, 96 PTC patients with LN-prRLN metastasis and 307 PTC patients without LN-prRLN metastasis were included. Univariate analysis demonstrated that PTC patients with LN-prRLN metastasis more often had younger age, larger size, multifocal cancers, A/T < 1, well-margins, microcalcification, petal-like calcification, internal vascularity, centripetal perfusion pattern and surrounding ring enhancement. Multivariate logistic regression analysis revealed that the CEUS centripetal perfusion pattern, central LN detected by ultrasound and LN-arRLN metastasis were independent characteristics for predicting LN-prRLN metastasis in PTC patients.According to our research, it is essential for clinicians to thoroughly dissect central LNs, particularly LN-prRLNs. PTCs are one of the most prevalent thyroid cancer histological subtypes, with rising incidence and substantial lymph node metastatic rates , 2. AccoDue to the difficult surgical technique and the high rate of complications as a result of the deep position with confined exposure, some researchers contend that routine LN-prRLN removal is not necessary. Furthermore, it can be difficult to assess metastases before surgery, and even experienced surgeons are likely to ignore this location. However, the LN-prRLN metastasis rate in PTC patients has been reported to be as high as 2.7–30.4% , 7. The Some studies have reported the clinicopathologic risk factors for LN-prRLN metastasis for PTCs. Zhang et al. reported that tumor size larger than 1 cm, multifocality, and extrathyroidal extension were independent predictors of LN-prRLN metastasis in right-sided PTC . Zhou etCEUS, which provides more information about vascularity than conventional US, could reveal the thyroid nodule’s microvasculature and increase the diagnostic accuracy of thyroid nodules , 13. OurBy analyzing the preoperative US characteristics of PTCs and the postoperative pathological data in 403 PTC patients, this study seeks to identify potential predictors for the preoperative clinical evaluation of LN-prRLN metastases.The study was approved by the Ethical Committee of the Second Xiangya Hospital of Central South University in China and performed in accordance with the Declaration of Helsinki for human study. The requirement of informed consent from human subjects was waived by IRBs due to the retrospective nature of the review of images acquired for clinical diagnostic purposes. From May 2021 to August 2022, 1253 consecutive patients with thyroid nodules who received conventional US and CEUS examinations were prospectively enrolled in this single-center study. The inclusion criteria were as follows: (i) patients with thyroid nodules who underwent conventional US and CEUS examinations and (ii) pathological examination confirming PTCs after surgery. Patients with the following criteria were excluded: (i) a history of neck surgery or neck radiotherapy; (ii) central cervical lymph node dissection not performed; and (iii) LN-prRLN without clear pathological diagnosis. The final diagnosis of malignant thyroid nodules and metastatic lymph nodes was confirmed by histopathology after surgery. For patients with multifocal PTCs, the largest one was selected for CEUS examination. Finally, 403 PTC patients  were included in this study. In addition, TSH, thyroid peroxidase antibody (A-TPO), and thyroglobulin antibody (A-TG) levels were evaluated in all patients within one week before surgery.A GE E11 US scanner with a 3-12L (3-12 MHz) linear array transducer or Siemens Acuson S3000 US scanner with a 9L4 (4–9 MHz) linear array transducer was used for conventional US and CEUS examination. All examinations were performed by operators with more than ten years of experience in thyroid ultrasound diagnosis and five years of experience in performing CEUS examinations. Two other investigators with 5 years of experience in thyroid ultrasound diagnosis analyzed the US images independently. Disagreements were solved by consensus. Two-milliliters of SonoVue  or 0.4 mL of Sonozoid  was injected intravenously, followed by a saline flush of 5 mL. Thyroid nodule imaging lasted at least 30 seconds.Conventional US features of the thyroid nodules were classified as follows : shape  and inteThe following categories describe the thyroid nodules’ CEUS characteristics : enhanceThree surgical options  are available, according to the Chinese Thyroid Association’s guidelines. The procedure was chosen based on the surgeon’s desire and a comprehensive analysis of the patient’s condition. Prelaryngeal LN, pretracheal LN, left paratracheal LN, LN-arRLN, and LN-prRLN were the five subgroups of the central LNs and sent for individual pathological evaluation. If the lateral cervical LNs were diagnosed as metastatic LNs from PTCs, lateral cervical lymph node dissection was performed.The histopathological results after surgery were used as the only reference standard for the final diagnosis of malignant thyroid nodules and metastatic lymph nodes.The statistical analysis was carried out using SPSS version 21.0. . The mean and standard deviation (SD) of continuous data were displayed, and they were compared using an independent t test. Categorical data were evaluated using the chi-square test and are shown as percentages. To evaluate multimodal factors and their independent associations with LN-prRLN metastasis, binary logistic regression was performed. P < 0.05 was used to evaluate whether differences were statistically significant.This study comprised a total of 403 PTC patients, including 307 individuals without LN-prRLN metastasis and 96 patients with LN-prRLN metastasis. The univariate analysis of predictors for LN-prRLN is reported in Compared with PTCs without LN-prRLN metastasis, PTCs with LN-prRLN metastasis had more A/T < 1 and well-defined margins, microcalcification, petal-like calcification and internal vascularity on conventional US images. For CEUS images, the PTCs with LN-prRLN metastasis showed a more centrifugal perfusion pattern and the presence of surrounding ring enhancement than the PTCs without LN-prRLN metastasis 2.Level VI lymph nodes were detected in 74 (77.1%) patients in the PTC with LN-prRLN metastasis group compared with 102 (33.2%) patients in the PTC without LN-prRLN metastasis group on preoperative conventional US examination. In addition, all metastatic rates of left paratracheal LNs, prelaryngeal LNs, pretracheal LNs, LN-arRLNs, LN-prRLNs and lateral LNs were higher than those of the PTCs without LN-prRLN metastasis group on histopathological examination.All of the statistically significant factors (p < 0.05) underwent multivariate logistic regression analysis. The outcomes showed that the CEUS centrifugal perfusion pattern , central LN detected by ultrasound , and LN-arRLN metastasis  were independent characteristics for predicting LN-prRLN metastasis Table 3.Currently, there is still controversy about whether to dissect the LN-prRLN in the absence of certain LNs that are positive. Dissection of the LN-prRLN requires patience and experience and could lead to nerve damage and parathyroid injury, affecting the patient’s quality of life . AccordiAlthough PTCs have a favorable prognosis, other scholars contend that the central LNs are the most frequent sites for lymph node metastasis with a high incidence of 13.7%-72.2% , 16, 21.Univariate analysis in this study demonstrated that age < 45 years was a clinical risk factor related to LN-prRLN metastasis in PTC patients, demonstrating that patients with LN-prRLN metastasis were more likely to be young people, which was consistent with other previous studies , 26. In Recent studies have shown that US characteristics could help predict cervical central LN metastasis in PTC patients , 10, 27.Compared to color Doppler technology, the accuracy of thyroid nodule diagnosis may improve with the use of CEUS, which can detect more tumor microvessels. However, to the best of our knowledge, these CEUS parameters for predicting LN-prRLN metastasis in PTC patients have not been explored to date. In our study, we found that the centrifugal perfusion pattern and presence of surrounding ring enhancement were significantly associated with LN-prRLN metastasis in PTC patients. Multivariate analysis demonstrated that the centrifugal perfusion pattern was one of the independent characteristics for the presence of LN-prRLN metastasis.Although central LNs have a low display rate on preoperative conventional US examination, the present study found central LNs in 176 (43.7%) patients. Moreover, the display rate of central LNs in the PTC with LN-prRLN metastasis group was as high as 77.1%, which was significantly higher than that of 33.2% in PTC without LN-prRLN metastasis group. Multivariate analysis demonstrated that central LN detected by US was another independent characteristic for the prediction of LN-prRLN metastasis, indicating that the centrifugal perfusion pattern on CEUS and central LN detected by US could serve as preoperative factors for predicting LN-prRLN metastasis. In addition, all histopathological metastatic rates of left paratracheal LNs, prelaryngeal LNs, pretracheal LNs, LN-arRLNs, LN-prRLNs and lateral LNs were significantly higher than those of PTCs without LN-prRLN metastasis. In this study, individuals who had LN-arRLN metastases had a nearly fourfold increased probability of LN-prRLN metastasis than patients who did not. According to the multivariate analysis, LN-arRLN metastasis in PTC patients was a significant risk factor for LN-prRLN metastasis, suggesting that the LN-arRLN compartment may be used intraoperatively to predict LN-prRLN metastasis.According to the findings of univariate and multivariate analyses, the centrifugal perfusion pattern on CEUS, central LN detected by US, and LN-arRLN metastasis were independent predictors of LN-prRLN metastasis in these patients. In that case, suggesting that the LN-prRLN should be removed with thorough neck dissection to prevent metastasis.There are several limitations to our study. First, it was a single institutional retrospective analysis, hence the findings may not have been applicable to other PTC patient populations. Second, selection bias was also present. To further explain these findings, a large-scale prospective multicenter investigation is needed.In summary, complete central LN dissection should be taken into consideration given the rate of LN-prRLN metastasis and the challenges and complications of reoperation. Centrifugal perfusion patterns on CEUS, central LN detected by US and LN-arRLN metastasis are helpful in predicting metastasis to LN-prRLNs. Thus, preoperative conventional US and CEUS may serve as useful tools in predicting LN-prRLN metastasis in PTC patients.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by the Ethics Committee of Second Xiangya Hospital, Central South University, China. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.CN contributed to the conception and design of the work. YG participated to data analysis and manuscript writing. ZZ, KT, YX, RZ and QP participated to data collection and patients’ follow-up. All authors contributed to the article and approved the submitted version.


Average protein content in the OM was 0.33 mg of protein g−1 CaCO3 (±0.10 SD) and a 0.03% of OM relative to the sample weight (n =100). The average of N g−1 CaCO3 was 0.002% (± 0.001 SD). The results showed an increase (p < 0.001) in δ15N over the time, positively correlated with population growth derived from touristic development. These findings emphasize the need to generate urban-planning remediation strategies that consider the impact on natural environments, reduce sewage pollution, and mitigate local stressors that threaten the status of coral-reef communities in the Caribbean region.Coral-reef ecosystems provide essentials services to human societies, representing the most important source of income  for many coastal developing countries. In the Caribbean region, most touristic and coastal developments are in the vicinity of coral reefs where they may contribute to reef degradation. Here we evaluated the influence of sewage inputs in the coral reef lagoon of Puerto Morelos during a period of 40 years (1970–2012). Annual δ In the last century, sewage inputs in tropical coastal ecosystems have become an ever-increasing problem that compromise the future of coral reef communities  precipitated in their skeletons. The coral skeletons consist of a two-phase structure comprising fiber-like crystals of aragonitic calcium carbonate closely associated with organic molecules (organic matter = OM) trapped during crystal growth  ratio contained within the skeleton structure  atmospheric fixation and the discharge of DIN from terrestrial runoff, (2) nitrogen fixation resulting from nutrient depletion in the system, and (3) proportional changes in autotrophic and heterotrophic nutrition due to coral metabolism  using the following expression:Hermatypic corals build reefs through the accumulation of calcium carbonate  of recently fixed N oscillated from −3 to 0‰, while the DIN from sewage discharges exhibits δ15N values between +6 and + 22‰  input, which has increased over the past decade associated with the development of the tourist industry  at the Mesoamerican Barrier Reef (Mexican Caribbean). We measure δ15N using 40 years of historical composite record (1970–2012) in the coral skeleton-bound organic matter and their relationship with the human population increase to understand the influence of anthropogenic sewage discharge in the coral reefs of the Caribbean Sea.The Mexican Caribbean coast (MCC) has a particular nutrient-load input, due the karstic geomorphology and permeable limestone that interconnect terrestrial infiltration to groundwater systems, and in turn into coastal lagoons and reef-lagoon systems  is in the Riviera Maya between the cities of Cancun and Playa del Carmen; these locations represent the primary hubs of tourist development in the Mexican Caribbean. The basement of the Riviera Maya encompasses a karstic system with extensive aquifers that facilitate communication among them and discharge groundwater into the reef lagoon at a rate of 2,297,000 L dayOrbicella faveolata colonies (n= 3) were obtained with a pneumatic drill at 5-m depth in La Bocana reef of PMNMP. The core samples were dried at 60°C using a conventional oven during 7 h. Then, coral cores were X-radiographed using General Electric X-ray machine  with exposure settings at 73 kV for 20 mAs. The resulting digitalized images were dated retrospectively using the pattern of high- and low-density bands that represent an annual period  were made where the average standard deviation of repeated measurements of USGS41 standards was 0.09‰.Coral cores of iod Fig. . The OM iod Fig. . The reshttps://www.inegi.org.mx), and hotel occupancy and wastewater treatment data from the Secretary of Tourism of the Quintana Roo State  and the National Water Commission  respectively. Statistical analyses were performed using R software (R Team core 2018).Historical record of population growth (habitants per year) was obtained from the National Institute of Statistics and Geography , these differences are likely attributed to variations in the number of years examined, with a maximum of 15 years. Average protein amount in OM per sample was 0.33 mg of protein g−1 CaCO3  and a 0.03% of OM relative to the sample weight .Temporal changes of δA 1975 – 012, B [1ht Table  0.8–1% aht Table  who repo15N values in the OM of O. faveolata were 4.16‰ which coincided with that reported by Muscatine et al.  for many different coral species (n= 17). The nitrogen (μg) located in the coral OM of this study was positively related  with δ15N  with a significant increment between 1993 and 2012 of 4.40 ± 0.60‰ ; these values are closer to levels proper from sewage records . Thus, the formulation of localized complementary environmental policies and mitigation strategies, tailored to the hydrologic region in the Yucatán Peninsula, becomes imperative. The establishment of marine protected areas has emerged as the primary conservation strategy aimed at preserving the integrity of coral reef ecosystems. However, the efficacy of this approach remains incomplete without the comprehensive inclusion of vital components, such as a well-structured wastewater system, within urban design frameworks. Furthermore, ensuring the preservation of interconnected habitats, including mangroves and groundwater systems, which demonstrate functional linkages, assumes significant importance (Michener and Kaufman


Most contemporary theories for the chemical origins of life include the prebiotic synthesis of informational polymers, including strong interpretations of the RNA World hypothesis. Existing challenges to the prebiotic emergence of RNA have encouraged exploration of the possibility that RNA was preceded by an ancestral informational polymer, or proto-RNA, that formed more easily on the early Earth. We have proposed that the proto-nucleobases of proto-RNA would have readily formed glycosides with ribose and that these proto-nucleosides would have formed base pairs as monomers in aqueous solution, two properties not exhibited by the extant nucleosides or nucleotides. Here we demonstrate that putative proto-nucleotides of the model proto-nucleobases barbituric acid and melamine can be formed in the same one-pot reaction with ribose-5-phosphate. Additionally, the proto-nucleotides formed in these reactions spontaneously form assemblies that are consistent with the presence of Watson–Crick-like base pairs. Together, these results provide further support for the possibility that heterocycles closely related to the extant bases of RNA facilitated the prebiotic emergence of RNA-like molecules, which were eventually replaced by RNA over the course of chemical and biological evolution. The concept of proto-RNA and pre-RNAs (genetic polymers intermediate between proto-RNA and RNA) has been the subject of experimental investigation during the past decade, including efforts to understand which alternative backbones, linker moieties, and recognition units can exhibit RNA-like properties  [The ability of RNA to function as an informational polymer and to catalyze reactions is seen by many as support for the hypothesis that RNA was the primary polymer of early life before the emergence of DNA and coded protein synthesis (the RNA World hypothesis) ,2. A “stant life . In a reant life . However Pairing ). This f Pairing . In thisctivity) . Modificctivity) . In contctivity) .s-triazine heterocycles with H, NH2, and O as exocyclic groups—the class of molecules that potentially existed on the prebiotic Earth along with the canonical nucleobases—reveals 91 molecules from which life could have selected the first base-pairing compounds for a proto-RNA [2) drives their stacking in water  were purchased from Sigma-Aldrich and used as received.Barbituric acid (BA), melamine (Mel), and Mixtures of R5P with BA and Mel were prepared by combining stock solutions of R5P, BA, and Mel in deionized water to create two reaction mixtures: a dilute mixture containing 20 mM R5P, 10 mM BA, and 10 mM Mel, and a concentrated mixture containing 100 mM R5P, 50 mM BA, and 50 mM Mel. These mixtures were subjected to the reaction conditions described below.2O containing a quantitatively known small amount of sodium trimethylsilylpropanesulfonate, DSS . These samples were analyzed using a Bruker Avance IIIHD 800 MHz NMR using standard 1D 1H methods. CD analysis was performed by taking a 20 µL aliquot of the intermediate (suspension) phase of the thawed crude reaction mixture and adding 5 µL of 5 M NaCl before placing between the two plates of a strain-free 0.01 mm quartz demountable cell , which were analyzed using a JASCO 810 CD spectropolarimeter with 3 spectra collected and averaged per temperature, and with sample temperature increased in increments of 4 °C. AFM images were obtained with a Nanoscope IIIa  in tapping mode using Si tips  on freshly cleaved mica. A 2 μL sample of the assembly solution, prepared the same as for CD, was spread over the mica using N2 flow and dried with N2 gas. Excess sample was washed with cold water, and the mica surface was dried by N2 flow.At the conclusion of each reaction, the crude product solution was frozen to stop further changes. Each product mixture was subsequently analyzed by NMR and CD spectroscopies and by AFM. Samples for NMR spectroscopy were prepared by freeze-drying portions of the crude reaction mixture, followed by dissolution in DKa of 3.9–4.0 [An initial survey of the reactivity of BA and Mel under various conditions indicated that a pH range of 3–7 and a temperature range of 35–65 °C could be satisfactory for the simultaneous reaction of both bases with ribose-5-phosphate (R5P). The importance of the pH to this reaction was previously recognized and confirmed by the formation of these nucleotides when carried out in separate reactions . The pH  3.9–4.0  due to t1H-NMR spectroscopy . For example, the reaction carried out at 33 °C produced the α and β-C anomers of BMP in yields  of 4 and 12%, respectively, while simultaneously generating the α and β-C anomers of MMP in yields of 1.2 and 1.5%, respectively (see The reaction mixtures (at 20 °C and at 33 °C) containing R5P, BA, and Mel were prepared as slurries at pH 5. The solids in the stirred slurries dissolved slowly over time and became pale yellow solutions, presumably because the nucleotides being formed are more soluble than their precursor bases, an observation made previously for nucleoside-forming reactions involving the base 2,4,6-triaminopyrimidine  . After 5troscopy . These r spectra . Specifionformer . The nucNext, we observed that thawing the frozen reaction mixture gives three apparent phases: a solid phase likely to be the unreacted bases; a solution phase; and a third transitory phase suspended between the solid and the solution that was suspected to be a hydrogel a. We preA 20 µL aliquot of the suspected gel phase was withdrawn from the thawed reaction mixture, diluted with 5 µL of 2 M NaCl, and adjusted to pH 5 to stabilize the hydrogel at the lower nucleotide concentrations that are more suitable for analysis by CD and AFM. CD spectroscopic analysis b of this2 flow. Excess sample was removed by rinsing with cold water, followed by drying again under N2 flow. The resulting images show structures with lengths ranging from 100 to 600 nm and heights of ca. 1.6 nm (The gel phase was also analyzed by AFM. We previously reported that supramolecular assemblies composed of BMP and MMP form linear structures . Samples. 1.6 nm , which aThe increased reactivity of the noncanonical nucleobases BA and Mel toward sugars and sugar phosphates positions them as feasible alternatives—and potential precursors—to RNA nucleobases. Their spontaneous reaction with R5P and their pairing properties both suggest that their potential as proto-nucleobases is superior to that of the canonical bases ,13. We rIt is feasible that the prebiotic formation of proto-RNA provided the starting point for an evolutionary process that ultimately led to the emergence of RNA and DNA. Our demonstration that melamine and barbituric acid readily form glycosidic bonds with ribose provides a possible resolution to a major challenge to the prebiotic synthesis of RNA, that of nucleoside formation. As noted above, the propensity of melamine and barbituric acid nucleotides to form supramolecular assemblies may also provide a means to address the challenge of having a sufficiently high local nucleotide concentration to support covalent polymer formation. Despite these apparent advances, even if a one-pot synthesis can be demonstrated that gives rise to such RNA-like polymers, new questions will arise that need to be addressed, such as: How and why did a polymeric system that originated as a six-stranded assembly with three sets of two complementary sequences make the transition to a duplex system like that which is used in life today?


The influence of structural modification on electrochemical, electronic, optical, and biosensing properties was systematically investigated. The results showed that the cyclic voltammograms of EDOT-containing materials displayed a high charge capacity over a wide range of scan rates representing a quick charge propagation, making them appropriate materials for high-performance supercapacitor devices. UV-Vis studies revealed that EDOT-based materials presented wide-range absorptions, and thus low optical band gaps. These two EDOT-modified materials also exhibited superior optical contrasts and fast switching times, and further displayed multi-color properties in their neutral and fully oxidized states, enabling them to be promising materials for constructing advanced electrochromic devices. In the context of biosensing applications, a selenophene-containing polymer showed markedly lower performance, specifically in signal intensity and stability, which was attributed to the improper localization of biomolecules on the polymer surface. Overall, we demonstrated that relatively small changes in the structure had a significant impact on both optoelectronic and biosensing properties for TPD-based donor–acceptor polymers.The molecular engineering of conjugated systems has proven to be an effective method for understanding structure–property relationships toward the advancement of optoelectronic properties and biosensing characteristics. Herein, a series of three thieno[3,4- Semiconducting π-conjugated materials with well-defined structures have drawn considerable attention owing to their low fabrication cost and ease of processability and tunability of their electrochemical and optical properties through the modification of chemical structures ,5,6,7,8.Electrochromism is a distinctive characteristic of certain materials that experience an electrochemical-induced change in coloration . During In addition to the use of electrochromism itself in biosensing, the utilization of conjugated polymers in electrochemical biosensing platforms has shown strong potential in the last two decades ,30. Sevec]pyrrole-4,6-dione (TPD) scaffold, one of the promising electron-accepting units, has several advantages, mainly providing facile synthesis, great coplanarity, a robust π-π stacking feature, and strong electron transporting, owing to the ease of switching to its quinoid form pyrrole-4,6-dione (TPD) acceptor core, an n-octyl-based linear alkyl chain for solubility, and electron-rich heterocycles as the donors were synthesized via Pd-catalyzed Stille cross-coupling reactions. Pristine selenophene (molecule 2) and EDOT (molecule 3) moieties were initially mono-stannylated from their two positions and used for the next step without further purification. Then, these building blocks (molecules 4-5) were separately coupled with molecule 1, and target selenophene-containing (Se-TPD) and EDOT-containing (EDOT-TPD) novel monomers were achieved with high yields. To synthesize the EDOT extended derivative of Se-TPD (EDOT-Se-TPD), further facile two-step reactions were performed. In the first step, Se-TPD was bis-brominated with the treatment of NBS. In the final step, the previously synthesized mono-stannylated EDOT moiety (molecule 5) was coupled with molecule 6 through Stille cross-coupling. The synthetic route of target monomers is given in Three novel conjugated small organic materials with a thieno3−/4− solution containing 0.1 M KCl at a scan rate of 50 mV/s. Clearly, the electrochemical response of GE/poly(EDOT-TPD) was greatly improved when the poly(EDOT-TPD) structures were grown on the GE surface. The high surface area and good conductivity of poly(EDOT-TPD) should be credited for this improved cyclic voltammetric response. The effective surface areas of the modified electrodes were estimated using the Randles–Sevcik equation 3−/4− solution, including 0.1 M KCl. poly(EDOT-TPD), and GE/poly(EDOT-TPD)/GOx. The introduction of poly(EDOT-TPD) served an outstanding film-forming ability and encouraged the electron transport of [Fe(CN)6]3−/4− to the surface of the electrode, which may be responsible for the improvement in the interfacial conductivity. After the GOx was combined with poly(EDOT-TPD) to modify GE, the electron transfer resistance changed, indicating that the GOx had been successfully immobilized on the electrode surface. The CV and EIS results proved the success of the electrode modification and were in good agreement with each other.Another effective method for analyzing the interface characteristics of GE with polymer and GOx film modification is electrochemical impedance spectroscopy (EIS). EIS experiments were performed in a 5.0 mM pyrrole-4,6-dione (TPD)-centered polymer-based biosensors have the potential to be reliable and promising electrochemical biosensing platforms. In addition, the apparent Michaelis–Menten constant (Kmapp) and Imax values were estimated as being 0.608 mM and 1.17 μA, respectively, using the Lineweaver–Burk equation. The lower Km value signifies an enzymatic function and an increased binding affinity of the immobilized GOx to the GE/poly(EDOT-TPD).Specific steady-state current as a function of glucose concentration was demonstrated to have a linear relationship in the 0.1–0.5 mM range (y = 5.65x − 0.3837 (R 0.995)) . The bioreported  in the lpoly(EDOT-TPD)/GOx for ten successive amperometric signals toward 0.5 mM glucose. A low-percentage relative standard deviation (% RSD) was determined as being 4.4. To analyze the reproducibility, five new GE/poly(EDOT-TPD)/GOx electrodes were prepared in the same conditions but on different days, and their amperometric responses were recorded. As shown in poly(EDOT-TPD)/GOx biosensor. For the long-term stability of the proposed biosensor, the amperometric responses of the newly prepared GE/poly(EDOT-TPD)/GOx biosensor were tested for 30 days. According to the results, it maintained 54% of its initial current even after 30 days of storage at 4 °C, and a negligible decrease was observed later, which demonstrates remarkable long-term stability , acetaminophen (APAP), citric acid (CA), and urea. The amperometric responses followed for all interfering chemicals (0.25 mM) in a 50 mM pH 7.0 phosphate buffer at −700 mV were insignificant compared to the clear and fast current signal for glucose d. These ® Zero sugar sample, the recovery test was performed in order to assess the effectiveness of the GE/poly(EDOT-TPD)/GOx biosensor for real sample analysis. When the Coke® Zero sugar sample was tested with the biosensor, no amperometric response was recorded, as expected. Once the drink sample was spiked with different concentrations of glucose (0.3 and 0.5 mM), significant amperometric responses were observed , based on electron-deficient thienopyrrole-4,6-dione (TPD) as the acceptor and two common electron-rich building blocks (selenophene and EDOT) as the donor, was synthesized via Pd-catalyzed Stille cross-coupling and electrochemically polymerized. Among the three materials, poly(EDOT-TPD) demonstrated the best characteristics for electrochromic device applications, considering optical contrast and switching time values. Additionally, poly(EDOT-TPD) was also shown to be the best candidate for the fabrication of glucose biosensing platforms. The sensitivity and LOD value of the biosensor are remarkably higher than those of other glucose biosensors reported in the literature, and the utilization of the biosensor electrodes in real-life samples was also successfully demonstrated. While other selenophene-containing donor–acceptor polymers showed high potential for electrochromic device applications and biosensors, with the TPD acceptor, selenophene was shown to have detrimental effects compared to its EDOT counterpart. This study clearly demonstrates that the performance of donor–acceptor polymers in electrochromic and biosensing applications heavily depends on the unique combination of the donor and acceptor moieties, and should shine light on further design efforts for improved performance in corresponding applications.In this study, a family of three conjugated monomers (


Fusion of RNA-binding proteins (RBPs) to RNA base-editing enzymes (such as APOBEC1 or ADAR) has emerged as a powerful tool for the discovery of RBP binding sites. However, current methods that analyze sequencing data from RNA-base editing experiments are vulnerable to false positives due to off-target editing, genetic variation and sequencing errors.We present FLagging Areas of RNA-editing Enrichment (FLARE), a Snakemake-based pipeline that builds on the outputs of the SAILOR edit site discovery tool to identify regions statistically enriched for RNA editing. FLARE can be configured to analyze any type of RNA editing, including C to U and A to I. We applied FLARE to C-to-U editing data from a RBFOX2-APOBEC1 STAMP experiment, to show that our approach attains high specificity for detecting RBFOX2 binding sites. We also applied FLARE to detect regions of exogenously introduced as well as endogenous A-to-I editing.https://github.com/YeoLab/FLARE.FLARE is a fast and flexible workflow that identifies significantly edited regions from RNA-seq data. The FLARE codebase is available at The online version contains supplementary material available at 10.1186/s12859-023-05452-4. Transcriptomics assays that leverage RNA base editing, such as DART-seq , TRIBE  and STAMTo address this need to identify regions exhibiting significant enrichment for various types of RNA edits, we present the FLARE (FLagging Areas of RNA-editing Enrichment) analysis pipeline. FLARE allows for analyses of all types of RNA base changes, including A-to-I, C-to-U, and U-to-C , with onWe demonstrate FLARE’s application to analyzing C-to-U RNA-editing data in the context of STAMP using the RNA-binding protein RBFOX2, in A-to-I editing in the context of TRIBE, and in the context of endogenous ADAR-deposited A-to-I edits. We also include a Snakemake  implemenFLARE takes as inputs the edited positions, which are the sites that contain at least one edit conversion at that position within any read as determined by the SAILOR algorithm . The corGiven that FLARE only considers genomic regions known to have at least one edited site, we employ a zero-truncated Poisson model, which enforces a 0% probability of a given window having zero edits. Simulating edit count data for one replicate using this model, based on the number of editable bases in a window, lines up well with empirical data  peaks, and to contain the canonical GCAUG RBFOX2 binding motif, irrespective of window read depth. However, the correlation of edit fraction with these two “ground truth” metrics only approaches steady-state value for windows with at least 50 reads in our example  are then merged to form clusters. Window-merging distance, which defaults to 15 base pairs, can be increased by users desiring coarser clusters  value of a given cluster edit fraction within a negative binomial distribution, using mean editing fraction among clusters as the The FLARE pipeline is command-line based, and uses the Python-based pipelining framework Snakemake . All proWe initially tested FLARE on RBFOX2-APOBEC1 STAMP data (C-to-U edits) in HEK293T cells, which were transiently transfected with RBFOX2-APOBEC1 fusion construct (doxycycline-induced expression for 72 h) before being prepared for RNA sequencing at a depth of 30 million reads. Based on our experience, we recommend that any library being used for RNA editing analyses with SAILOR and FLARE be sequenced at least this deeply.log2(fold-change) > 1 and p-value < 0.05. By our overlap metric, it is clear that applying more stringent adjusted p-value filters results in better window filtering and higher quality clusters, regardless of window read depth. More specifically, 28%, 32% and 35% of windows with adjusted p-values of below 0.1, 0.01, and 0.001 overlapped with eCLIP peaks or the canonical motif, respectively, with these proportions increasing to 36%, 42% and 47% for windows with a read depth exceeding 50  overlap, under the assumption that true binding sites should have a higher overlap with such features. The publicly available eCLIP dataset we used for this calculation  represenThis increase in cluster quality corresponding to increasing p-value filtering stringency indicates that our model is successfully preferentially filtering out false positive sites. Importantly, using FLARE’s window-based approach to cluster detection attains higher sensitivity and specificity  than merely filtering individual SAILOR-called sites based on SAILOR confidence scores , as it appears to strike a balance between precision and statistical significance among the 10, 20, 30, 40, and 50 base pair size options we tested  to editing in U87 cells with higher levels of ADAR3 exogenously introduced, Kurup et al. determined that transcripts exhibiting reduced A-to-I editing in glioblastoma are more likely to be bound to ADAR3. Within such transcripts, Kurup et al. identified several dozen genomic sites consistently exhibiting reduced editing across three biological replicates, but note that apart from these specific instances, “there was substantial variability in the specific sites with altered editing.” Indeed, among the replicates, about ten times more differentially edited sites were only specific to one replicate than were shared. Examining editing changes at wider regions representative of clustered editing, using the approach offered by FLARE, enables the aggregation of RNA editing signals to elucidate interesting changes that might be missed when analyzing changes at a site-specific level.Running SAILOR followed by FLARE on the three wild-type (WT) replicates and ADAR3 + replicates yielded several thousand clusters across all six samples Fig. A. To ensAluJo- element towards the 3’ end of the non-coding RNA NEAT1, whose expression has been linked to tumor malignancy in glioma . In. In8]. IAlu elements in the ADAR1 knockdown condition compared to the non-targeting and baseline conditions  condition, which had the most RNAEditor editing clusters, replicable FLARE clusters coincided with 484 of 627 (77.2%) replicable RNAEditor clusters Fig. D. Howeveons Fig. F. Given Whether applied in the context of exogenous RNA-editing using technologies such as STAMP or TRIBE, or endogenous ADAR-based A-to-I editing, the FLARE pipeline is an effective tool to efficiently locate and prioritize loci significantly enriched for editing. We demonstrate that FLARE’s Poisson-based filtering approach preferentially filters out false positive editing sites to yield high confidence clusters, and that the scores FLARE subsequently assigns to these clusters enable further stratification and increased stringency.We emphasize that FLARE is capable of detecting clusters of any type of transition or transversion—to our knowledge, there are no existing RNA editing site detection and clustering pipelines currently available that can process all edit types.FLARE leverages the Snakemake python framework  and SingAdditional file 1: Figure S1. Validation of the zero-truncated Poisson model. a Simulated edit count distribution matches actual edit count distribution well. b Residuals at lower coverage windows exhibit little correlation to coverage. It is worth noting that although the model loses fidelity in regions at extremely highly covered windows (c), the resulting overestimation of expected edit counts in such areas will tend to lead to under-calling of regions rather than false positives, erring on the conservative side to increase precision at the expense of recallAdditional file 2: Figure S2. FLARE cluster properties and comparison to site-based approach. a The correlation of window edit fraction with window precision, which is expected to be both positive and uncorrelated to window read depth, only stabilizes above a read depth of approximately 50 reads. b Generating 50 base pair windows surrounding individual SAILOR sites, filtered by individual SAILOR site score exclusively, results in lower specificity than the approach employed by FLARE. c A site-based approach also yields lower sensitivity than the cluster-based approach. d Expanding RBOX2 FLARE clusters leads to higher rates of eCLIP or canonical RBFOX2 motif overlap. e FLARE clusters overlapping eCLIP are slightly likelier than ones not overlapping eCLIP to contain secondary RBFOX2 motifs. f A higher fraction of eCLIP overlapping clusters contain secondary motifs than control motifs. g A higher fraction of non-eCLIP overlapping clusters contain secondary motifs than control motifs. h Between approximately 25% and 40% of edit sites can be found within peaks depending on score threshold. i The cluster length distribution with 30 bp windows has a long tail but is centered around approximately 100 bpAdditional file 3: Figure S3. TRIBE and HyperTRIBE datasets are amenable to FLARE analysis. a There is an enrichment for overlap with Hrp48 CLIP peaks of Hrp48-TRIBE and Hrp48-HyperTRIBE, when normalized to WT overlapAdditional file 4: Figure S4. Window size affects sensitivity and statistical significance—graphs produced using a single replicate. a Larger windows yield more precise but slightly less statistically significant RBFOX2 clusters based on eCLIP overlap. b Larger windows yield more precise but far less statistically significant RBFOX2 clusters based on eCLIP overlap. c Marginal gains in precision from larger window sizes decrease for each size increase. d As window size increases, neighboring small clusters merge to form larger clusters, but this merging and the resultant decrease in total clusters is less dramatic at higher values. e The “efficiency score” metric can be serve as a guide to tune window sizes for optimal clustering


Structurally reconfigurable RNA structures enable dynamic transitions of the functional states in response to diverse molecular stimuli, which are fundamental in genetic and epigenetic regulations. Inspired by nature, rationally designed RNA structures with responsively reconfigurable motifs have been developed to serve as switchable components for building engineered nanomachines, which hold promise in synthetic biological applications. In this review, we summarize recent progress in the design, synthesis, and integration of engineered reconfigurable RNA structures for nanomachines. We highlight recent examples of their targeted applications such as biocomputing and smart theranostics. We also discuss their advantages, challenges as well as possible solutions. We further provide an outlook of their potential in future synthetic biology. They often cooperate with metal cofactors and perform specific phosphodiester bond cleavage and formation at the translational level  technology, a variety of artificially selected RNA aptamers and ribozymes have emerged, which can bind the molecules of interest, or show certain catalytic activities when incorporated with given molecules . Like their protein counterparts, these RNA aptamers can be fused to other RNA molecules of interest, thus are suitable for RNA imaging and detection in live cells. These fluorescence aptamers can be further integrated with other structurally reconfigurable aptamers, enabling switchable fluorescence in response to the molecules of interest  have attracted much interest. These RNA aptamers light up when binding to corresponding fluorophores  of mRNAs , allowing switches of translation initiation or transcription termination   have been constructed to disrupt the formation of an intrinsic transcription terminator placed upstream of a gene in et al. et al. A variety of riboswitches have been found involved in regulating post-transcriptional processing  that can bind and reconfigure the regulatory sites  of other mRNAs, leading to post-transcriptional/translational regulations of the latter  or repressed (by the off-antiswitch) in the presence of the ligands, which demonstrates the capability of these riboregulators for bi-directional modulation.For example, Bayer witches"    RNA motifs can interact with K-turn binding protein L7Ae and conduct structural reconfigurations, which can be utilized to build RNP nanomachines that can transform in response to L7Ae -CRISPR associated protein (Cas) system can be regarded as a natural ribonucleoprotein machine for adaptive immunity in microorganisms  responsive to small molecules, Tang et al. designed an inactive sgRNA precursor (pre-sgRNA)  repressors    and therapeutic RNAs such as small interfering RNAs (siRNAs) and anti-miRNAs, enabling cancer-targeted therapeutics   may help construct more complicated RNA systems with optimized performances. However, the efficiency of traditional evolution techniques is poor. It often takes years to find a new RNA structure with the desired functionality. Therefore, there is a need to develop high-throughput yet high-speed selection strategies for evolving input-output responses and dynamic functions.The combination with artificial evolution strategies (Taken together, we envision that in near future, the development of cutting-edge technologies in diverse fields would largely enrich the catalog of designer RNA structures, which enable novel nanomachines with functions that are robust, scalable, and applicable to a wide range of living organisms.Kai Jiao, Yaya Hao, Fei Wang, Lihua Wang, Chunhai Fan and Jiang Li declare that they have no conflict of interest.


In the era of immunotherapy, the optimal combination of immune checkpoint inhibitors (ICIs) and chemoradiotherapy (CRT) for stage III non-small cell lung cancer (NSCLC) is not defined. The current study investigated the efficacy and safety of definitive CRT(dCRT) plus consolidation ICIs with or without induction ICIs in stage III NSCLC.123 consecutive patients treated with dCRT followed by consolidation ICIs at our institution from 2018 to 2022 were retrospectively reviewed. Failure patterns, survival outcomes, and toxicity profiles were analyzed.The 1- and 2- year PFS rates were 75.3% and 56.9%, respectively, and median PFS was 30.83 months from the start of treatment. In-field failure (18.7%) was the most common failure pattern. The most common adverse event (AE) was pneumonitis caused by ICIs or RT. The incidence of Grade 3-4 and Grade 5 pneumonitis was 5.7% and 1.6%, respectively. Further analysis showed that the induction plus consolidation ICIs group has significantly lower cumulative incidence of distant metastasis rates  and higher incidence of pneumonitis (p=0.039) compared with patients in the consolidation ICIs group.Combined CRT and consolidation ICIs achieved encouraging efficacy and manageable toxicity in patients with stage III NSCLC in China. Induction plus consolidation ICIs might reduce distant metastasis and deserve further investigation. Approximately one third of patients with non–small-cell lung cancer (NSCLC) are initially diagnosed at stage III, locally advanced disease , 2. The Novel treatment strategies used in a variety of ongoing trials are under evaluation to improve outcomes in this setting, including different anti-programmed cell death protein 1/programmed death-ligand 1(anti-PD-1/anti-PD-L1) antibodies after CRT, induction ICIs with or without chemotherapy before CRT, etc . The maiTherefore, we assessed the benefit of the adding induction ICIs plus chemotherapy before dCRT in stage III NSCLC and exploring the optimal combination of ICIs and CRT.th AJCC staging system, undergone dCRT and consolidation anti-PD-1/anti-PD-L1 ICIs, ≥18 years of age, >6 months follow-up. The exclusion criteria were patients who received surgery, or palliative treatment, non-completion of radiotherapy, with incomplete clinical information and so on.This study was approved by our institutional review board of Cancer Hospital, Chinese Academy of Medical Sciences. Consecutive patients treated with dCRT followed by consolidation ICIs with or without induction ICIs plus chemotherapy at our institution from September 2018 to June 2022 now were retrospectively reviewed. The inclusion criteria for the study were patients who had a pathologically confirmed unresectable stage III NSCLC according to the 8All patients received intensity modulated radiation therapy or volumetric modulated arc radiation therapy with the prescribed dose of 60 Gy, concurrently or sequentially combined with platinum-based doublet chemotherapy. Radiation therapy simulation was performed using 4-dimensional computed tomography (4D-CT) scans for all patients. The gross target volume (GTV) of the primary tumor (GTVp) was defined as the primary tumor delineated on simulation CT images, and the GTV of the lymph nodes (GTVn) was defined as any regionally involved lymph nodes with short axis >1 cm on pretreatment CT or high fluorodeoxyglucose uptake on positron emission tomography scans. For patients received induction chemotherapy with or without immunotherapy, GTVp and GTVn included the post-induction volume on the 4D-CT. The clinical target volume (CTV) comprised a margin of 0.5 cm beyond the GTV (GTVp plus GTVn) and the pre-treatment involved hilar and mediastinal nodal regions, even when the enlarged lymph nodes disappeared after induction therapy. The PTV included a margin of 0.5 cm beyond the CTV. Details of simulation, target volume definition, prescription, planning were published previously , 15. PatBaseline demographic and therapeutic data, including age, ECOG status, sex, smoking history, NSCLC pathology, cancer staging, epidermal growth factor receptor (EGFR) and PD-L1 expression, radiotherapy dose and technology, ICIs cycle and sequence, and so on, were extracted from electronic medical records. The toxicities were graded according to the Common Terminology Criteria for Adverse Events, version 5.0. During the follow-up, radiographic imaging was performed every 12 weeks for 2 years, every 6 months to 5 years, and then every year until tumor progression. The tumor response evaluation was according to Response Evaluation Criteria in Solid Tumors version 1.1. Locoregional failure (LR) was defined as clinical and/or biopsy-proven recurrence in the primary tumor or the ipsilateral hilum, mediastinum, or supraclavicular, irrespective of distant metastasis. Distant metastasis (DM) was defined as any evidence of metastatic disease beyond locoregional regions previously mentioned. Locoregional failure was further classified as in-field failure or out-field failure based on the component of disease progression occurring within or without the 95% isodose line, respectively.All data are expressed as median  for continuous variables and as numbers (percentages) for categorical variables. Intergroup comparisons were performed using Mann-Whitney U test for continuous variables and the chi-square test or Fisher’s exact test when appropriate for categorical variables. One to one propensity score matching (PSM) was conducted to adjust for prespecified baseline characteristics that were potentially confounding variables. PFS was defined as the time from first treatment to the first documented event of tumor progression or death in the absence of disease progression. OS was defined as the time from first treatment to death from any cause. In the exploratory analysis for subgroup patients received consolidation ICIs, OS and PFS was further calculated from 6 weeks after RT, to better compare with the PACIFIC trial. Kaplan-Meier survival analysis was used to evaluate the PFS and OS. To properly evaluate the patterns of failure, the first site of recurrence  was analyzed by considering death as a competing risk, respectively. Univariate Cox regression model was used to calculate hazard ratio (HR) and 95% confidence interval (CI). All tests were two sided, and statistical significance was set at a p-value <0.05. Statistical analysis was performed with SPSS (version 26.0), data visualization was performed using R software (version 4.1.1).A total of 123 patients were identified. The baseline demographic and therapeutic characteristics of patients are presented in With a median follow-up of 25.57 months (IQR 18.8-32.4), 52 patients (42.3%) had experienced disease progression and 23 patients (18.7%) had died. The 1-, 2- and 3- year OS rates were 94.2%, 82.5% and 77.9%, respectively, and median OS was not reached  patients have completed 12 months or more consolidation ICIs, 5 patients were continuing the consolidation ICIs, and 67 patients had discontinued consolidation ICIs due to various reasons. The most frequent cause leading to discontinuation of consolidation ICIs was pneumonitis caused by ICIs or RT, which accounted for 22.8% of the total cases, the other reasons included disease progression (19.5%), other ICIs related AEs (2.4%), pneumonia (2.4%) and patient decision (6.5%) (31 patients (25.2%) experienced locoregional progression as the site of first failure, and 21 patients (17.1%) experienced distant metastasis as the site of first failure in the entire cohort Figure 2The patterns of first failure were different between these two groups. Specifically, locoregional progression and distant metastasis were observed in 12 (29.3%) and 2 (4.9%) patients, respectively in the induction plus consolidation ICIs group and 19 (23.2%) and 19 (23.2%) patients, respectively in the consolidation ICIs group. And the rate of distant metastasis was significantly lower in the induction plus consolidation ICIs group than consolidation ICIs group (p=0.011) Figure 2The incidence of new lesions is listed in All adverse events in these patients are summarized in After adjusting for confounding variables via PSM, all clinical features were balanced between the induction plus consolidation ICIs group and consolidation ICIs group  completed 12 months or more consolidation ICIs in our cohort, which is close to those reported in Pacific-R trial (47.1%) and Pacific trial (48.7%) . The inc2.3% compThe survival rates in this study were numerically superior to those in the PACIFIC trial  and PaciIn terms of toxicity, the most common AEs was pneumonitis caused by ICIs or RT. In some cases, the differentiation between radiation pneumonitis and immune-relate pneumonitis was difficult. The overall pneumonitis incidence was markedly higher (65.9%) than it reported in the PACIFIC trial 33.9%) (3.9% (3).There are several limitations in this study. Firstly, this is a single institution retrospective analysis. Secondly, not long follow-up time and significantly improved treatment effect in this study gave rise to limited events of tumor progression and death, a longer follow-up may be more informative. However, under the circumstance that there is no study to investigate the optimal combination of ICIs and CRT for LA-NSCLC, our analysis can provide a rationale for the design RCTs, as well as clinical practice. Future RCTs are expected to validate and update these results.This is the first study to evaluate the dCRT followed by consolidation ICIs with or without induction ICIs plus chemotherapy in one cohort of stage III NSCLC patients. This study identified encouraging efficacy and manageable toxicity of the combined ICIs and CRT for patients with stage III NSCLC in China, and demonstrated that induction plus consolidation ICIs could reduce distant metastasis and gain similar survival benefit compared with consolidation ICIs.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving humans were approved by National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College. The studies were conducted in accordance with the local legislation and institutional requirements. The ethics committee/institutional review board waived the requirement of written informed consent for participation from the participants or the participants’ legal guardians/next of kin because of the retrospective nature of this research.YY: Data curation, Formal analysis, Methodology, Writing – original draft. JYW: Investigation, Methodology, Resources, Writing – original draft. TZ: Data curation, Resources, Writing – review & editing. ZZ: Data curation, Resources, Writing – review & editing. YW: Data curation, Methodology, Writing – review & editing. YJ: Data curation, Methodology, Writing – review & editing. WL: Investigation, Resources, Writing – review & editing. ZX: Investigation, Resources, Writing – review & editing. LD: Investigation, Resources, Writing – review & editing. QF: Investigation, Resources, Writing – review & editing. XW: Investigation, Resources, Writing – review & editing. JL: Investigation, Resources, Writing – review & editing. WW: Investigation, Resources, Writing – review & editing. QX: Conceptualization, Resources, Writing – review & editing. JW: Conceptualization, Resources, Writing – review & editing. Y-XL: Conceptualization, Project administration, Supervision, Writing – review & editing. NB: Conceptualization, Funding acquisition, Supervision, Writing – review & editing.


Carbon ion radiotherapy offers ballistic and radiobiological advantages over conventional photon-based radiotherapy, making it an effective option in case of rare, radioresistant, and difficult-to-treat tumours. The current narrative review aims to critically report the state-of-the-art application of carbon ion radiotherapy in oncological settings, highlighting the clinical activity on carbon ion radiotherapy at the National Center for Oncological Hadrontherapy (CNAO). CNAO is the only Italian facility, and one of four located in Europe using both protons and carbon ions for oncological treatments. Proton and CIRT became fully operational at CNAO starting in 2011 and November 2012, respectively. After an initial ramp-up period, CNAO has progressively honed its clinical, technical, and dosimetric skills and contributed to increasing knowledge on the efficacy, feasibility, and safety of CIRT in selected tumour types, demonstrating the mild rate of toxicities also in case of re-irradiation and tolerance in case of association with systemic treatments.Background: Currently, 13 Asian and European facilities deliver carbon ion radiotherapy (CIRT) for preclinical and clinical activity, and, to date, 55 clinical studies including CIRT for adult and paediatric solid neoplasms have been registered. The National Center for Oncological Hadrontherapy (CNAO) is the only Italian facility able to accelerate both protons and carbon ions for oncological treatment and research. Methods: To summarise and critically evaluate state-of-the-art knowledge on the application of carbon ion radiotherapy in oncological settings, the authors conducted a literature search till December 2022 in the following electronic databases: PubMed, Web of Science, MEDLINE, Google Scholar, and Cochrane. The results of 68 studies are reported using a narrative approach, highlighting CNAO’s clinical activity over the last 10 years of CIRT. Results: The ballistic and radiobiological hallmarks of CIRT make it an effective option in several rare, radioresistant, and difficult-to-treat tumours. CNAO has made a significant contribution to the advancement of knowledge on CIRT delivery in selected tumour types. Conclusions: After an initial ramp-up period, CNAO has progressively honed its clinical, technical, and dosimetric skills. Growing engagement with national and international networks and research groups for complex cancers has led to increasingly targeted patient selection for CIRT and lowered barriers to facility access. The role of charged particle therapy in cancer treatments continues to grow with protons as the leading modality . Indeed,https://www.ptcog.ch/; accessed on 30 August 2023). Insurance reimbursement differs across the Nations in which CIRT is available. Due to the results obtained by several clinical trials, the Japanese National Health System (NHS) covers the cost for many indications [Currently, 13 of the world’s ion beam facilities provide CIRT for preclinical and clinical activity in Asia and Europe . Clinical data have mainly been derived from single-arm prospective studies, dose escalation trials, and small cohort case series reported by the few CIRT facilities operating worldwide.www.clinicaltrials.gov (accessed on 30 August 2023) website and three phase III trials are currently recruiting patients to test CIRT versus photon or PBT as standard treatment for unresectable or incompletely resected radioresistant tumours, such as axial chordoma, adenoid cystic carcinoma (ACC), and sarcomas (NCT02838602 and NCT01182779), and for recurrent head and neck (H&N) cancers (NCT04185974).To date, 55 clinical studies including CIRT for adult and paediatric solid neoplasms have been registered on the https://www.ptcog.ch/; accessed on 30 August 2023), using both protons and carbon ions for oncological treatments. After an initial phase of activity (from 2001 to 2010) dedicated to design, construction, beam characterisation, and commissioning, CNAO’s treatment devices  received CE marking in December 2012 and the synchrotron was recognized as a medical device from the Italian NIH.The National Center for Oncological Hadrontherapy (CNAO) is the only Italian facility, and one of four located in Europe  compared with the CNAO’s literature data available so far. The endpoint was to describe the evolution of clinical activities across 20 years of experience at CNAO and in our 10 years with CIRT.After the first identification of 145 articles, 111 records underwent first-step screening. Of these, 20 records were excluded because they reported CIRT data regarding tumors that were currently not treated at CNAO or for which there were no studies  ongoing. Ten studies were excluded for being unfit according to the aim of the review or because they reported the same cohort of patients; in these cases, the most recent cohort was considered. An amount of 68 studies were finally analyzed.The most common H&N tumors treated at CNAO are primary sinonasal cancers (SNC), mucosal melanomas (MM), adenoid cystic carcinoma (ACC), and relapses of tumors at the edge or in previously irradiated fields.SNC forms a rare, heterogeneous group of histologies with substantially diverse biological behaviours. CNAO recently reported on only two phase II studies  to include CIRT for poor prognosis SNC , adopting a multidisciplinary approach and delivering chemotherapy, surgery, and (photon and/or PBT/CIRT) RT according to disease site and stage . Histolo-CIRT was one of the alternative therapeutic options for a mixed beam approach (boost with CIRT of 9–21 GyRBE followed by photons or protons up to 50–60 GyRBE to the large volumes) in the SINTART 1 study ;-CIRT was one of the approaches in the SINTART 2 study and can be delivered as a single modality , or in a mixed beam approach (CIRT boost of 21–24 GyRBE with 3 GyRBE per fraction in 7–8 fractions followed by 54 Gy or 54 GyRBE of photons or PBT) according to the volumes to be treated .Concomitant chemoradiation (CT/RT) was limited to good IC responders or primarily non-resectable tumours. Overall, this multimodality treatment proved to be safe, feasible, and effective, especially in IC-responsive patients. Notably, CT was not delivered concomitantly with CIRT. Observed survival rates were similar to those reported for other series, highlighting the need for further research efforts in this rare disease.p-value 0.007), confirming its safety and efficacy as a sole local treatment modality. Japanese data on unresected patients [Mucosal melanoma (MM) is an ultrarare, aggressive disease. In this scenario, Ramaekers and colleagues  reportedpatients ,13,14 supatients . To bettpatients  in a reaAdenoid cystic carcinoma (ACC), another rare tumour, represents a challenging scenario for radiation oncologists. Its radioresistance, anatomical complexity, and tendency to embrace or intersect radiosensitive structures and follow neural pathways often make it difficult to treat with conventional RT. In recent decades, Japanese centres deemed CIRT to be the sole modality for definitive RT of head and neck ACC (ACCHN), showing good results in terms of LC (5-year LC ranging from 51% to 92%) and acceptable toxicity rates ,17,18,19Few studies have described the role of CIRT in relapsed H&N cancers. A prospective randomised clinical trial (NCT04185974) is underway to analyse the safety and efficacy of re-intensity modulated RT (IMRT) compared to re-CIRT for recurrent H&N tumours . Jensen Held and coworkers studied 229 patients with recurrent H&N cancer retreated with CIRT, observing local PFS of 24.2 months , and a median OS of 26.1 months, proving better for ACC compared to SCC tumours. Moreover, the rate of late toxicity grade ≥ 3 was 14.5% .In this scenario, CNAO reported the outcomes of 51 patients with inoperable recurrent salivary gland tumours treated up to a median re-CIRT dose of 60 GyRBE, with 3.0–5.0 GyRBE per fraction . Our datSkull base (SB) chordomas pose a challenge for treatment given their proximity to radiosensitive normal tissues and their well-known radioresistance. Mizoe and colleagues  reportedThe CNAO series by Iannalfi and colleagues  represenAs in the above-reported experience with chordomas, CNAO reported treating SB chondrosarcomas with a tailored approach, delivering PBT or CIRT according to histological characteristics, patient toxicity risk, and GTV volume at baseline MRI. We recently described the outcome of 48 patients treated with PBT (67%) and CIRT (33%) up to a total dose of 70 GyRBE in 35 fractions, and 70.4 GyRBE in 16 fractions, respectively . After aIn advanced malignant pelvic bone sarcomas, often involving nerves and blood vessels, thus generally precluding wide resection, CIRT has proven to be effective. In unresectable chondrosarcomas, CIRT yielded 5-year LC, OS, and disease-free survival (DFS) rates of 53%, 53%, and 34%, respectively , with anCNAO recently published a retrospective experience of 54 consecutive axial bone and soft tissue sarcomas treated with CIRT at a total median dose of 73.6 GyRBE (range = 70.4–76.8), in 16 fractions . In our d in the pattern of failures in sacral chordomas. Our results showed that half of the recurrence volumes were in a well-covered high-dose region, but the median LETd values were significantly lower (27 vs. 30 keV/lm) [p = 0.003) [d maps showed potential as a risk factor for local recurrence in sacral chordoma, and dosiomics appeared to be the most promising approach against more conventional dose distribution parameters  [Furthermore, considering dose-averaged LET (LETd)—which steeply rises only in the distal part of the spread-out Bragg peak (SOBP)—we analysed the role of RBE and LET keV/lm) , suggest= 0.003) . Moreovemethods) .Currently, CNAO is involved in the randomised observational SACRO study NCT02986516), promoted by the Italian Sarcoma Group, comparing upfront surgery with CIRT in patients with resectable sacral chordoma, assessing relapse-free survival as the first endpoint. CIRT could represent an option in preoperative retroperitoneal sarcoma settings to limit toxicity in surrounding healthy tissue for further dose escalation in critical regions at risk for positive margins and in unresectable cases. In the former scenario, a phase II trial (NCT04219202) is ongoing , and, in6, promotα\β ratio of prostate adenocarcinoma, and its relative intrinsic radioresistance. The results of three recent phase III non-inferiority randomised trials showed that moderate hypofractionation (2.5 to 3.4 Gy per fraction) has the same efficacy as conventional fractionation photon RT, although long-term efficacy  and tolerability remain to be determined [By virtue of its ballistic characteristics, CIRT has recently been proposed for prostate cancer to minimise AE observed with conventional radiation. The focus is on the benefits of hypofractionation, delivered with CIRT, whose rationale resides in the low termined . At CNAOtermined ,54, therRadiation is a cornerstone in the treatment of rectal cancer across neoadjuvant, adjuvant, and salvage settings. The Japan Carbon-ion Radiation Oncology Study Group (J-CROS) multicentre study retrospectively analysed the data of 224 RT-naïve patients with local recurrent rectal cancers (LRRC), treated with CIRT at a total dose ranging from 70.4 GyRBE to 73.6 GyRBE , achieviConsidering its ballistic advantages over conventional RT, CIRT was applied also to LRRC reirradiation. The German experience  in 19 caAlthough in vitro studies provided a biological rationale for using CIRT for pancreatic cancer ,63, clinStudies on CIRT for gynaecological cancers have mainly focused on the most aggressive and poor prognosis tumours. The Japanese group assessed the role of radical CIRT in inoperable endometrial carcinoma (stage: I–III) , reporti2+ signaling [Murata and colleagues  performeignaling , paving CIRT proved to be safe in challenging presentations of cervical carcinomas  , showingIn view of its dosimetric hallmarks, CIRT was also tested on 16 cases of unresectable lymph node recurrences arising in a previous RT field, achieving 3-year OS, LC, and DFS rates of 74%, 94%, and 55%, respectively, with no grade ≥ 3 AE . ConsideThroughout CNAO’s 20-year history and after an unavoidable, intense, and successful learning curve , its clinical and dosimetric skills have progressively improved and matured technically.We dealt with a unique treatment planning process. CNAO beams are fixed, horizontal, and vertical, and they serve three treatment rooms. Robust plan optimization is used, accounting for set-up and range uncertainties and the patient set-up is verified and corrected, daily, through dedicated images .To manage the anatomical changes during the treatment we implemented a site-specific adaptive protocol and scheduled re-evaluation CT, based on the tumour location and uncertainties. The solutions to deal with the organ motion include immobilizing the patient with a solid thermoplastic mask, acquiring 4DCT, combining gated dose delivery with rescanning, and performing multiple pre-treatment 4DCTs with multiple breathing phases to assess the reproducibility of respiratory patterns and anatomy before CIRT.Moreover, CIRT (RBE)-weighted dose (DRBE) significantly depends on the RBE model implemented in the treatment planning system. Our group described a clinically oriented approach to translate LEM-based in mMKM-based prescription doses, reducing the dose distribution difference among CIRT plans .The retrospective dosimetric analysis, based on the dose conversion approach and the clinical outcomes of patients, has been crucial to finding new optimization aims, describing OAR constraints, and consequently ameliorating the treatment quality . To undeIn these 10 years of CIRT, we collected a large number of radiological images, the analysis of which hopefully allows us to find predictive and prognostic markers of clinical outcomes to validate prospectively.To better understand the radiobiological mechanisms of cell response to CIRT, the Radiobiological Unit is moving from in vitro 2D analysis towards the evaluation of 3D in vitro models and in vivo ones.Furthermore, CNAO centralizes rare tumours, and for many of them, no guidelines or consensus exists. To enhance the quality of care for this uncommon disease, the efforts in our CIRT decade were focused on creating national and international networking to share the treatment approaches on a case-by-case basis with the possibility of referring pathologic diagnosis to high-expertise centres, surgery to high-skilled groups, and enrol patients in clinical protocols .In our experience, consistent with the international hadrontherapy data, CIRT appears promising in several clinical settings, especially in the case of radioresistant tumors, in which the RBE has proved to be beneficial, and in tumours close to radiosensitive OARs, in which the peculiar ballistic characteristics are advantageous. CNAO has contributed and continues to contribute to increasing knowledge on the efficacy, feasibility, and safety of CIRT in selected tumour types , demonstHealthcare collaborations between CNAO and other national and international facilities have been established over the years, allowing for more targeted patient selection and management strategies. Clinical networking, along with collaboration with several scientific societies and research groups  has gradually led to a reduction in barriers to facility access. CNAO is fully committed to constantly reporting and updating data gathered from observational studies, and assessing oncological outcomes and toxicity. As summarized in