Polyanions have been reported to induce tau aggregation in vitro, but the precise trigger to convert tau from an inert to a seed-competent form in disease states is unknown. RNA triggers tau fibril formation in vitro and has been observed to associate with neurofibrillary tangles in human brain. Here, we have tested whether RNA exerts sequence-specific effects on tau assembly and strain formation. We found that three RNA homopolymers, polyA, polyU, and polyC, all bound tau, but only polyA RNA triggered seed and fibril formation. In addition, polyA:tau seeds and fibrils were sensitive to RNase. We also observed that the origin of the RNA influenced the ability of tau to adopt a structure that would form stable strains. Human RNA potently induced tau seed formation and created tau conformations that preferentially formed stable strains in a HEK293T cell model, whereas RNA from other sources, or heparin, produced strains that were not stably maintained in cultured cells. Finally, we found that soluble, but not insoluble seeds from Alzheimer’s disease brain were also sensitive to RNase. We conclude that human RNA specifically induces formation of stable tau strains and may trigger the formation of dominant pathological assemblies that propagate in Alzheimer’s disease and possibly other tauopathies.Tau aggregation underlies neurodegenerative tauopathies, and transcellular propagation of tau assemblies of unique structure,  It is unknown why tau forms unique strains, but the observation that a seed-competent tau monomer encodes limited strain ensembles  and related tauopathies , which mnsembles  suggestsnsembles , but it nsembles .in vitro have been described , cytidine (C), and uracil (U), omitting guanidine because of its difficulty to synthesize. We purified recombinant, FL tau monomer (2N4R) according to our prior methods , termed Figs. 1in vitro. We incubated tau (8 μM) in the presence of polyA, polyU, and polyC RNA  for 24 h before measuring seeding with v2L biosensor cells that stably expressed the tau repeat domain with the P301S mutation fused to cerulean and mClover fluorescent proteins. After 48 h, we quantified intracellular aggregation by fluorescence resonance energy transfer (FRET) as per prior studies (B). A polyU/polyA hybrid also failed to elicit seeding. Two single-stranded DNA homopolymers  did not induce seeding (A). Two randomly generated single-stranded, complementary DNA sequences of the same length and similar molecular weight as the RNA both effectively seeded, while the annealed double stranded complex did not, nor did an RNA/DNA complex of polyA hybridized with polydT (B).We next tested the ability of RNA homopolymers to induce tau fibril formation  studies . PolyA g studies B. A poly seeding A. Two rah polydT B.A–C). We observed fibril formation via transmission electron microscopy (TEM) that correlated with seeding activity after 48 h incubation (C). Thus, despite universal binding of RNA to tau, only certain single-stranded nucleotide sequences induced seed-competent conformations.To rule out kinetic differences in seed induction, we incubated tau and RNA for up to 2 weeks. While polyA induced seed-competent forms, polyC and polyU never did , A–C. Wecubation C. Thus, D). We defined the minimal length of RNA necessary to induce seeding by incubating tau with increasing sizes of polyA ranging from 10 to 40 nucleotides (nt) for various time periods. At 48 h, the smallest length of RNA capable of inducing seed-competent tau to a significant degree was 40 nt  (E). After 7 days, we observed minimal increases in seed formation with 30 nt (4.1% FRET positive), which were still not significant compared to buffer control , while 40 nt seeding increased to 55% FRET positive.The optimal ratio of RNA:tau to induce seeding was 1:2 D. We defns test) E. After A). To exclude a direct effect of RNase on biosensor activity, we added RNase directly to the seeds without preincubation and detected no loss in seeding activity . We next used TEM to test fibril stability in the presence of nucleases. RNase eliminated all detectable fibrils, but not DNase or heparinase . Conversely, fibrils induced by heparin remained unchanged after DNase and RNase treatment but disassembled and lost 74% of seeding after incubation with heparinase (A and B). Incomplete loss of seeding after removal of heparin is consistent with our prior observations that it converts tau monomer to a highly stable, seed-competent conformation . Hence, despite starting with 100% inclusion-bearing cells derived from tau seeds, across multiple inducers, human RNA most efficiently induced conformations of tau that created stable strains.Tau was incubated in the presence of total human RNA, total yeast RNA, polyA RNA, or ssDNA before treating DS1 cells with induced tau seeds to initiate inclusion formation. After 72 h, we used fluorescence activated cell sorting to isolate 384 single aggregate-containing cells for each condition in the individual wells of 96-well plates , while remaining colonies were ordered (21%) (B and C). Colonies derived from polyA RNA were primarily disordered (65%), in addition to thread (17%), speckle (12%), and ordered (6%) morphologies (B and C). While relatively fewer ssDNA colonies stably maintained aggregates, we observed a distinct spiked morphology in 71% of colonies and additional colonies with speckle (14.5%) and ordered (14.5%) patterns (B and C). The sole remaining yeast RNA–derived colony produced speckle morphology (B and C). Although distinct inducers of intracellular aggregates generally displayed a heterogenous profile of intracellular morphologies, the predominant morphology for each inducer was unique.To further explore the effect of RNA origin on strain identity, we maintained remaining stable colonies  for characterization of inclusion morphology, a surrogate for strain identity . Colonieed (21%) , B and Chologies , B and Cpatterns , B and Crphology , B and Cin vitro and strains in cells, we tested its stabilization of seeds derived from the most common tauopathy, Alzheimer’s Disease (AD) (Given the ability of RNA to facilitate tau seeding ase (AD) . We homoase (AD)  and soluase (AD) . We thenase (AD) . By contin vitro and in AD. This preliminary work provides a conceptual framework for further tests of the origins of strains in humans. We initially found that tau bound multiple forms of single-stranded RNA with relatively high avidity, yet formation of seeds and fibrils depended on RNA sequence and size, with a maximally effective size >40 nt. RNA was critical to maintaining induced seed integrity in vitro. While RNA from a variety of sources induced seed-competent tau, when this was used to transduce a human biosensor cell system, strain stability was far more robust when seeds had been produced by induction with human RNA. Finally, soluble tau seeds from AD brain were highly sensitive to RNase but not DNase, indicating a critical role for RNA in the maintenance of AD-derived seeds. While more studies are required, these observations suggest that certain RNAs might specifically trigger the formation and maintenance of unique tau assemblies or strains, contributing to the diversity of tauopathies.This study has examined the role of RNA in the development and maintenance of tau seeds and strains Our finding that RNA modulates the conformation of tau assemblies builds on many prior reports that have linked tau and RNA-binding proteins (RBPs) in disease  seeded acutely into biosensor cells. However, only certain RNAs preferentially formed structures that propagated faithfully over multiple cell divisions, eviously , 6, 8, weviously .This work puts these findings in a new light. Specifically, we found that the source of inducer used to create tau seeds had an enormous impact on the stability of the strains that subsequently propagated in cultured HEK293T cells. For example, when we used heparin or yeast RNA, we readily formed fibrils and seeds that induced aggregation acutely in cultured cells, but inclusions did not persist. By contrast, induction of tau with human RNA efficiently triggered the formation of more stable strains. Coupled with data which indicated that RNA played a critical role in stabilizing induced fibrils and soluble AD seeds, these data suggested a role for RNA within the cell in the stabilization of strains. We hypothesize that strain stability in a cell will depend on the ability to bind specific classes or even sequences of RNA.in vitro creates a tau conformation competent to bind similar RNAs within the cell and thus more stably propagates strains. These findings are consistent with the observation that RNA is associated with neurofibrillary tau deposits in human brain .All cells were grown in Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10% fetal bovine serum (HyClone) and 1% Glutamax (Gibco). Cells were maintained at 37 °C, 5% COWe prepared FL wildtype (WT) tau (2N4R) protein as previously described .Saccaromyces cerevisiae) was purchased from Sigma. All nucleic acid preparations were stored in diethylpyrocarbonate treated H2O at −80 °C. All ssRNA and ssDNA sequences were synthesized by Sigma.Total human RNA fractions were purified from HEK293T cells using Zymo Direct-zol RNA miniprep plus kits. Total yeast RNA . Filtered protein was collected, and protein concentration determined via DeNovix DS-11 Spectrophotometer. RNA or DNA was boiled for 7 min and snap-cooled on ice before addition to filtered monomer at determined mass ratio. All nucleic acid concentrations were quantified on a DeNovix DS-11 Spectrophotometer. Tau monomer was brought to a final concentration of 8 μM using tau buffer . DTT was added at a final concentration of 11% before adding inducer  or tau buffer. Fibril mixtures were agitated at 350 rpm using a Thermomixer (Eppendorf) set to 37 °C for determined time points.FL WT tau monomer was filtered through a 100 kDa molecular weight cut-off filter (Corning) as instructed by the manufacturer  and evaluated by flow cytometry  as described previously , 24. ThrStable cell lines expressing tau-repeat domain with P301L/V337M mutation fused to YFP (DS1)  were plaSorted monoclonal colonies grew in 96-well plates for 2 weeks before being scored and amplified. After 2 weeks, viable cell colonies containing aggregates were monitored for 2 months and scored as maintaining aggregates or as being sectored . Colonies were passaged into 24-well plates and then 12-well plates before storage in liquid nitrogen.via confocal microscopy . Each colony was imaged from 13 distributed points in each well, across 5 z-stacks. IN CELL software then compiled maximum intensity projections across z-stacks to create a single image from each point scanned. Maximum intensity projection files were coded and blindly scored for tau aggregate morphology before unblinding and quantification.Stably passaged monoclonal cell lines were plated onto a Corning Special Optics 96-well plate and grown to ∼40% confluency before fixing in 4% paraformaldehyde, permeabilizing in 0.1% Triton-X 100, and staining with DAPI. Cells were then imaged for GFP (488 nm) and DAPI (405 nm) Exemplar colonies were plated onto Ibidi 8 well μ-slides (cat no. 80826) and grown to ∼40% confluency before fixing in 4% paraformaldehyde, permeabilizing in 0.1% Triton-X 100, and staining with DAPI. The fixed cells were then imaged for GFP (488 nm) and DAPI (405 nm) on a confocal microscope (Nikon CSU-W1) at 100X objective using immersion oil. Images were acquired using an Orca-Fusion sCMOS camera (Hamamatsu) with the NIS Element Software.2 spirit transmission electron microscope (FEI), serial number: D1067, equipped with LaB6 source at 120 kV using a Gatan ultrascan CCD camera.Fibrils (4 μM) were deposited onto glow-discharged Formvar-coated 400-mesh copper grids for 30 s, washed with distilled water, and then negatively stained with 2% uranyl acetate for 1 min. Images were acquired on a Tecnai GSecond Generation Amine Reactive Biosensors  were incubated with EDC and Sulfo-NHS to activate carboxylic acids. FL WT tau was diluted in acetate (pH 6) to charge primary amines and allow immobilization on biosensors. After quenching biosensors with ethanolamine, tau-coated sensors were dipped into serial 3-fold dilutions (1/2 log) of RNA to determine association and disassociation kinetics. Data from wells containing no RNA and biosensors containing no tau were background subtracted. Data were analyzed using Octet Data Analysis software using y-axis alignment, Savitz-Golay filtering, and interstep correction.2O (Control) and were agitated at 750 RPM in a Thermomixer (Eppendorf) set to 37 °C for 24 h.Pre-formed tau fibrils were incubated with a mixture of RNase A  and RNase T1 , DNase I , Heparinase I (3797 U/ml), or Millipore Hg for 1 h at 4 °C to separate pellet from supernatant. Supernatant was flash frozen and stored at −80 °C for subsequent size fractionation. Pellets were washed with PBS for 30 min at 186,000g, 4 °C and resuspended in 50 mM Tris-HCL (pH 7.5) before flash freezing and storing at −80 °C.0.5 g sections of frontal lobe from AD brain were dounce homogenized in PHF buffer   and sarkE, we demonstrated through use of crosslinking with SDS-PAGE that the size-exclusion chromatography protocol used in this work reliably purifies monomer, dimer, and trimer.AD ultra-centrifuged supernatant (1 ml) was loaded onto a Superdex 200 Increase 10/300 Gl Column  and eluted in PHF buffer. Fractions were concentrated using 2000kD molecular weight cut-off filters (Vivacon), evaluated for protein concentration using A215 spectra, flash frozen in liquid nitrogen, and stored at −80 °C. Aliquots were thawed immediately before use in biochemical and seeding assays. The molecular weight/radius of proteins in each fraction was estimated by running gel filtration standards (Bio-Rad): Thyroglobulin (bovine) 670 kDa/8.5 nm; γ-globulin (bovine) 158 kDa/5.29 nm; ovalbumin (chicken) 44 kDa/3.05 nm; myoglobin (horse) 17 kDa/2.04 nm; and vitamin B121.35 kDa/0.85 nm. In a prior publication , Figure Figure 1All data generated and analyzed during this study are included in this article.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Here, we applied synthetic small RNA (sysRNA) to identify the RDT region in vivo by exploiting its complementary base-pairing ability to target mRNA. Through the combined analyses of rapid amplification of cDNA 3′ ends, primer extension, and capillary electrophoresis, we could precisely map and quantify mRNA 3′ ends. We found that complementary double-stranded RNA (dsRNA) formed between sysRNA and mRNA was efficiently cleaved by RNase III in the middle of the dsRNA region. The formation of dsRNA appeared to protect the cleaved RNA 3′ ends from rapid degradation by 3′-to-5′ exonuclease, thereby stabilizing the mRNA 3′ end. We further verified that the signal intensity at the 3′ end was positively correlated with the amount of mRNA. By constructing a series of sysRNAs with close target sites and comparing the difference in signal intensity at the 3′ end of wild-type and Rho-impaired strains, we finally identified a region of increased mRNA expression within the 21-bp range, which was determined as the RDT region. Our results demonstrated the ability to use sysRNA as a novel tool to identify RDT regions in vivo and expand the range of applications of sysRNA.Rho promotes Rho-dependent termination (RDT) at the Rho-dependent terminator, producing a variable-length region without secondary structure at the 3′ end of mRNA. Determining the exact RDT site IMPORTANCE sysRNA, which was formerly widely employed, has steadily lost popularity as more novel techniques for suppressing gene expression come into existence because of issues such as unstable inhibition effect and low inhibition efficiency. However, it remains an interesting topic as a regulatory tool due to its ease of design and low metabolic burden on cells. Here, for the first time, we discovered a new method to identify RDT regions in vivo using sysRNA. This new feature is important because since the discovery of the Rho protein in 1969, specific identification of RDT sites in vivo has been difficult due to the rapid processing of RNA 3′ ends by exonucleases, and sysRNA might provide a new approach to address this challenge. The Rho2–d tracts , 6.Rho is a ring-shaped homohexameric protein with RNA-dependent ATPase activity , which e8–in vivo, depending on whether the terminator is detected on the plasmid or the chromosome. The first method is to construct a series of plasmids, clone the terminator upstream of the reporter gene, and determine the strength of the terminator by comparing the expression level of the reporter gene. Using this method, Mott et al. and Hart and Roberts identified the Rho-dependent terminator trp t′ of the tryptophan operon and the Rho-dependent terminator tR1 of the λ phage, respectively  to impair Rho activity (E. coli RNAP distribution in response to BCM by chromatin immunoprecipitation and microarray (ChIP-chip) as well as RNA sequencing (E. coli by quantitative reverse transcription-PCR (RT-qPCR) analyses  play an important role in regulating gene expression in response to extracellular stress by base-pairing with mRNA or other sRNAs –30. Most–33–galM RDT region in vivo, we designed a sysRNA with an approximately 20-bp target-binding sequence that is perfectly base paired with galM mRNA. Our results demonstrated that dsRNA formed by sysRNA-mRNA hybridization recruited the endonuclease RNase III to form a new mRNA 3′ end in the middle of the dsRNA region, thereby protecting the cleaved upstream mRNA fragment from rapid 3′-to-5′ exonuclease degradation and thus stabilizing the 3′ end of mRNA. We found that the intensity of the signal at the 3′ end of the RNA was proportional to the amount of mRNA expressed. Based on the above results, we designed and constructed four sysRNAs that bind to different regions upstream and downstream of the predicted galM RDT region. By measuring the signal intensities of these four newly generated 3′ ends, we verified a region of increased mRNA expression in the Rho-impaired strain, which was identified as the galM RDT region located within this 21-bp region. To our knowledge, this is the most accurate localization of the RDT region in vivo. We believe that this approach could be extended to identify other Rho-dependent terminators and is important for further understanding of RDTs.In this study, to identify the in vivo, we used the Rho-dependent terminator downstream of galM as an example. In a previous study, we reported the presence of two tandem terminators, a Rho-independent terminator and a Rho-dependent terminator, located at +7 to +30 and +31 to +108 downstream of galM, respectively  , a 3′ fragment of the pre-galETKM  . Based o at +124 . In vivotructure . To dete-galETKM . Furtherplasmids . If the  in vivo .galETKM, we extracted total RNA from wild-type E. coli MG1655 strains harboring MicC-galM expression plasmids (WT-Ms) and amplified mRNA 3′ ends using a rapid amplification of cDNA 3′ ends (3′ RACE) assay. Subsequently, we applied primer extension reactions using mixtures containing specific primers labeled with 32P to identify the sequences of interest in the amplified fragments. Finally, we performed capillary electrophoresis to visualize the fragments , the temperature-sensitive RNase E ams1Ts mutant , the temperature-sensitive RNase P rnpA49 mutant , and the RNase III rnc mutant     , 38. Altaterial) , whereasaterial) . MeanwhiIII-con) . These rd-1-thiogalactopyranoside (IPTG). We then detected the expression of MicC-galM2 by dot blot assay. The results showed that there was no MicC-galM2 expression in the WT-con strain, while the expression of MicC-galM2 increased over time in the WT-M2 strain  strain for subsequent testing. We named these two derivative strains Δgal-pGal-gpmA and Δgal-pRDTo, respectively. Δgal-pRDTo has been reported to be incapable of galM RDT  high specificity, (ii) ease of design and implementation, and (iii) minimal impact on cell growth. Therefore, our method might open up space for further investigations of RDTs in vivo.Our previous results showed that the in vitro ; howeverofactors . Severalofactors –52. Therion rate . Our unp process –56. SincNatural sRNAs serve a variety of physiological purposes, but the use of sysRNAs is still limited to inhibiting gene expression. Here, we used sysRNAs for the first time to detect RDT loci, extending their applications. In eukaryotes, RNA editing is an area of great interest. Compared to DNA editing, RNA editing does not require permanent changes to the genome, and this reversible and easily regulated editing method may have advantages in terms of safety. A major problem with editing technologies that rely on a CRISPR/Cas system is their dependence on the expression of exogenous editing enzymes or effector proteins. This can lead to issues such as cytotoxicity or delivery difficulties , 58. WeiE. coli strains MG1655 and HME60 and the Δgal and Δhfq mutants , SDF205 , GW10 , GW11 , GW20 , NHY312 , and NHY322  were generous gifts from Y. H. Lee    to generate sysRNA expression plasmids. Plasmids used are listed in Table S2.The pGal plasmid is a derivative of pCC1BAC  containing a coding region from guanines . To consplasmid)  to obtaiE. coli strains as previously described .Total RNA was extracted from 2 mL of escribed . For theTaq polymerase , a 0.2 mM concentration of a dNTP mixture, 0.3 μL of the 32P-labeled gal-specific primer M4-F (Table S3), and 2 μL of PCR product. Reaction products were separated on 8% polyacrylamide/urea sequencing gel. Radioactive bands were visualized by exposure of the gel to X-ray film, and the intensities of the radioactive signals were quantified using a PhosphorImager . Data were subjected to one-way analysis of variance (ANOVA) using the Bonferroni test (n = 3). GraphPad Prism 9.0 was used for one-way ANOVA and graph plotting.To visualize the mRNA 3′ ends, the PCR products were purified and used as templates for primer extension reactions, which were performed in a volume of 20 μL using 1 U of 32P, mixed with ULTRAhyb ultrasensitive hybridization buffer , and hybridized with RNA extract on a nylon membrane overnight at 42°C according to the manufacturer’s instructions (n = 3). GraphPad Prism 9.0 was used for one-way ANOVA and graph plotting.For RNA dot blotting, total RNA was extracted, and 10 μg of this was treated with DNase I  and purified with a G-50 column . One microgram of RNA was then mixed with 2× RNA loading buffer and incubated at 80°C for 5 min to remove the RNA secondary structure and inactivate DNase I. RNA was spotted onto a positively charged nylon membrane . The membrane was adhered to Whatman filter paper and baked at 80°C for 1 h. The MicC probe (Table S3) was labeled with ructions , 38. AftE. coli strains were collected. Total RNA was extracted, and RT-qPCR was conducted essentially as previously described . GraphPad Prism 9.0 was used for one-way ANOVA and graph plotting.For RT-qPCR experiments, 2-mL samples from escribed . Results


Molecular testing of indeterminate thyroid nodules informs about the presence of point mutations, insertions/deletions, copy number variants, RNA fusions, transcript alterations and miRNA expression. American Thyroid Association (ATA) guidelines suggest molecular testing of indeterminate thyroid nodules may be considered to supplement risk of malignancy (ROM). Although these recommendations have been incorporated in clinical practices in the United States, molecular testing of indeterminate thyroid nodules is not common practice in Asia. Here, we performed molecular testing of 140 indeterminate nodules from Chinese patients using a novel molecular platform composed of RNA and DNA‐RNA classifiers, which is similar to Afirma GEC and ThyroSeq v3. Compared with reports from North America, the new RNA and DNA‐RNA classifiers had a higher positive predictive value (p1 = 0.000 and p2 = 0.020) but a lower negative predictive value (p1 = 0.004 and p2 = 0.098), with no significant differences in sensitivity (p1 = 0.625 and p2 = 0.179) or specificity (p1 = 0.391 and p2 = 0.264). Out of 58 resected nodules, 10 were borderline and 33 malignant, indicating a 74.1% ROM, which was higher than reports in North America (10–40% ROM). Our findings emphasize molecular testing with the newly reported RNA and DNA‐RNA classifiers can be used as a ‘rule‐in’ test when ROM is high. About 20% of thyroid cytology findings are indeterminate, and molecular testing can be used for risk stratification. Here, we developed a novel molecular platform and performed molecular testing of indeterminate thyroid nodules. Our findings emphasize that molecular testing with the newly reported RNA and DNA‐RNA classifiers has a high positive predictive value and can be used as a ‘rule‐in’ test in Asia. ATAAmerican Thyroid AssociationAUS/FLUSatypia of undetermined significance/follicular lesion of undetermined significanceFN/SFNfollicular neoplasm/suspicious for a follicular neoplasmFNAfine‐needle aspirationNIFTPnon‐invasive follicular thyroid neoplasm with papillary‐like nuclear featuresNPVnegative predictive valuePPVpositive predictive valueROMrisk of malignancyTBSRTCThe Bethesda System for Reporting Thyroid CytopathologyWT‐UMPwell‐differentiated tumour of uncertain malignancy1In the last three decades, the number of cases of thyroid cancer has increased, becoming the most common type of cancer worldwide . In the Molecular alterations in indeterminate thyroid nodules can be identified via various molecular detection platforms, including point mutations, insertions/deletions, copy number variants, RNA fusions, transcript alterations and miRNA expression. The commercial Afirma platform quantifies RNA expression alterations and applies an expression classifier; ThyroSeq v3 determines both DNA and RNA alterations. In addition, ThyraMIR uses a microRNA risk classifier , 7, 8, 9There is increasing evidence concerning differences in the clinical practice of thyroid nodule management between Eastern and Western countries. Unlike Western countries, where diagnostic surgery is the long‐established standard of care for all FN/SFN nodules , strict However, neither of the two commercial molecular tests approved by the US Food and Drug Administration (FDA) is to date approved or available in China. To stratify indeterminate thyroid nodules, we designed a panel of multigene tests and successfully established a novel molecular platform based on data from a public database and 293 Chinese thyroid carcinoma samples. Our special panel involves a DNA‐ and RNA‐based next generation sequencing assay analysing 48 genes with a variety of genetic alterations, including point mutations, insertions/deletions and fusions, and expression profiles of 156 genes. Two classifiers were established based on the results of molecular testing: an RNA classifier similar to Afirma GEC and a DNA‐RNA classifier similar to ThyroSeq v3.In the current study, we performed novel molecular testing of 140 indeterminate thyroid nodules from Chinese patients to enrich clinical practice data for molecular testing in regions outside North America.22.1®  solution remaining after routine ThinPrep® (Hologic Inc.) preparation. All patients signed informed consent before FNA.All patients who underwent thyroid FNA in the National Cancer Center between October 2018 and August 2019 were eligible for enrolment in this study. The study protocol was reviewed and approved by the Ethics Committee of the National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital (Reference Number: NCC2019C‐018), and written informed consent was obtained from each individual enrolled in this study. The study methodologies conformed to the standards set by the Declaration of Helsinki. Patients were selected on the basis of the following criteria: (a) cytological diagnosis of Bethesda III or Bethesda IV according to The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC); and (b) at least 10 mL of PreservCyt® 2000 (Hologic Inc.). Residual cells of cytologically indeterminate nodules (Bethesda category III or IV) were transferred to RNA Later within 48 h of ThinPrep® preparation and sent for molecular testing, which was performed blindly without any patient information.Fine‐needle aspiration specimens were prepared as slides with ThinPrepCases of indeterminate cytology were managed based on ultrasound characteristics, clinical features and patient wishes, regardless of the results of molecular testing. The histopathology of surgical specimens was reviewed by pathologists who were blinded to the molecular testing results. All cases with a borderline diagnosis were re‐evaluated by a thyroid pathologist.2.2To distinguish malignant from benign thyroid nodules, we developed a machine learning model named Thyroeva™. As illustrated in Fig. 2.3qubit 3.0 .Genomic DNA was extracted using the Mag‐Bind Blood & Tissue DNA HDQ 96 kit . RNA was extracted using the Mag‐Bind Total RNA 96 kit (Omega Bioservices), and reverse transcription was performed with RevertAid First Strand cDNA Synthesis Kit . DNA/RNA purity was detected by a UV spectrophotometer , and quantification was performed using 2.4A library was prepared using the Thyroeva™  panel, with three separate multiplex PCR amplification reactions: DNA (140 amplicons), RNA fusion (36 amplicons) and mRNA (169 amplicons) , 22. DNA2.5burrows–wheeler aligner 0.7.10 . Local realignment and variant calling were performed with gatk v3.2‐2 . To avoid false positive mutation calls arising due to DNA damage, calling thresholds of different mutations were applied to DNA samples of different quality. Variants with population frequencies > 0.1% in the ExAC, 1000 Genomes, dbSNP and ESP6500SI‐V2 databases were grouped as common SNPs and removed. Integrative Genomics Viewer  was used to visualize variants aligned against the reference genome to confirm the accuracy of the variant calls by evaluating possible strand bias and sequencing errors. The RNA sequencing data were aligned to target amplicons with burrows–wheeler aligner 0.7.10 , and in‐house‐developed software was used for subsequent data processing.The DNA sequencing data were aligned to the human genome (hg19) with 2.6The results of molecular testing were divided into three parts: DNA mutations, fusions and expression. A gene expression classifier was built based on RNA expression results and applied to assign a percentage value to each genetic alteration depending on the strength of the association with malignancy ≤ 10% (no association with cancer or low cancer probability) or > 10% (high cancer probability). In addition to the gene expression classifier, we trained a genomic classifier (GC) for all combined DNA and RNA alterations. The sum of the individual values for all detected alterations was calculated for the GC score, whereby ≤ 70% accepted was negative and > 70% was positive.2.7Histopathology was taken as the gold standard for malignancy. Histopathological borderline lesions, including non‐invasive follicular thyroid neoplasms with papillary‐like nuclear features (NIFTPs) and well‐differentiated tumours of uncertain malignancy (WT‐UMP), were considered malignant because they represent a pre‐malignant entity that should be managed surgically. An analysis considering borderline lesions as benign is provided in Table medcalc version 18.2.1 . Expected PPV and NPV curves were based on calculated sensitivities and specificities over the range of the possible prevalence of malignancy (cancer or borderline lesion). All statistical analyses were performed with spss 17.0 , and P < 0.05 was considered statistically significant.Cytological and molecular testing results were compared with histopathological results, and the sensitivity, specificity, PPV and NPV of each test were calculated with 95% Wilson confidence intervals. Performance comparisons between the RNA classifier or DNA‐RNA classifier and their similar classifiers, namely, Afirma GEC and ThyroSeq v3, were carried out using the chi‐squared test. A chi‐squared test was also used to compare the diagnostic performance of the RNA classifier and DNA‐RNA classifier between AUS/FLUS nodules and FN/SFN nodules  were reported as unsatisfactory samples, 377 (14.5%) were diagnosed as benign, 263 (10.1%) were classified as indeterminate, 354 (13.6%) were suspicious for malignancy and 1447 (55.7%) were malignant.Of the 263 thyroid nodules with indeterminate results, 193 (73.4%) were classified as Bethesda III and 70 (26.6%) as Bethesda IV. A total of 119 indeterminate nodules were not included in subsequent molecular analysis due to insufficient samples  or missed collection by the cytotechnologist . Among 144 cases subjected to molecular detection, four cases were not processed molecular tests, one suspicious for parathyroid and three  insufficient for DNA or RNA. Ultimately, 140 samples from 136 patients were included in the ensuing statistical analysis  indeterminate nodules were categorized as negative and 114 (81.4%) as positive. For nodules with negative RNA test results, nine (34.6%) were surgically resected; for nodules with positive RNA test results, 49 (43.0%) were resected. In the DNA‐RNA molecular testing cohort, 34 (24.3%) indeterminate nodules were categorized as negative and 106 (75.7%) as positive. For nodules with negative and positive DNA‐RNA test results, 35.3% (12/34) and 43.4% (46/106) were resected respectively. In both the RNA and DNA‐RNA test cohorts, the resection rates in the molecular positive group were slightly higher than those in the molecular negative group, but with no significant difference (p1 = 0.511 and p2 = 0.431). According to a retrospective study, the clinical management of cases with indeterminate cytology in our institute was based on ultrasound characteristics, clinical features and patient willingness rather than on molecular testing results. Our molecular results suggest that there is a considerable number of patients with indeterminate cytology and malignancy who do not choose surgery.3.3Among 140 indeterminate thyroid nodules, 58 (41.4%) were managed with surgical resection and subsequent histopathological analysis, of which 15 were benign , 10 borderline (three NIFTP and seven WT‐UMP) and 33 malignant (29 papillary thyroid carcinoma (PTC), one follicular carcinoma, two Hürthle cell carcinoma and one anaplastic carcinoma, Table 3.4P = 0.264)  curve. The AUC was 0.81 for the RNA classifier and 0.77 for the DNA‐RNA classifier, without a significant difference between them (64) Fig. . Hence, P = 0.458) for the DNA‐RNA classifier. The specificities were 40.0% (95% CI: 11.9–68.1%) for the RNA classifier and 53.3% (95% CI: 24.7–81.9%) (P = 0.464) for the DNA‐RNA classifier. For the RNA and DNA‐RNA classifier, the PPV was 81.6% (95% CI: 70.4–92.9%) and 84.4% (95% CI: 73.4–95.5%) (P = 0.717), and the NPV was 66.7% (95% CI: 28.2–100.0%) and 61.5% (95% CI: 30.9–92.1%) (P = 0.806) respectively  for the RNA classifier and 88.4% (95% CI: 78.4–98.4%)  and a lower NPV (P = 0.004), but no significant differences was observed for sensitivity (P = 0.625) or specificity (P = 0.391). Similarly, the DNA‐RNA classifier showed a higher PPV (P = 0.020) and a lower NPV (P = 0.098) than ThyroSeq v3, with no significant difference in sensitivity (P = 0.179) or specificity (P = 0.264)  for indeterminate thyroid nodules, we generated an expected PPV curve as well as an expected NPV curve for our molecular tests Fig. . In Fig.3.6In the DNA‐RNA testing cohort, 26 DNA mutations and fusions were detected by molecular analysis, as shown in Table Nevertheless, RAS or RAS‐like mutations (e.g. BRAF K601E) are not specifically associated with malignancy and are usually detected in follicular‐patterned neoplasms, which commonly include follicular adenoma, NIFTP, follicular variant PTC and follicular carcinoma. Among our 10 nodules with RAS mutations, two were benign hyperplasia (one NRAS and one HRAS), two were NIFTP (two NRAS), one was WT‐UMP (NRAS) and five were PTC (three NRAS and two KRAS). When RAS mutations were identified alone, the probability of cancer, NIFTP or WT‐UMP was 80%. Two cases of BRAF K601E mutation were detected: one in a NIFTP nodule and the other in a WT‐UMP.The expanded gene mutation panel of the DNA‐RNA test contributed greatly to the diagnosis of thyroid cancer based on the specific molecular alteration detected (e.g. BRAF V600E), which is important for a ‘rule‐in’ test.4Fine‐needle aspiration is now the most successful diagnostic test in the management of thyroid nodules. Nonetheless, approximately 10–40% of FNAs are not conclusively diagnosed by cytology and are categorized as indeterminate , 27, 28.In the current study, a ROM of 74.1% was calculated for indeterminate nodules, which is much higher than that reported for indeterminate categories in North America, at 10–40% , 12. OneGiven that no commercial molecular test for indeterminate thyroid nodules approved by the FDA is available in China, we developed a novel molecular platform that included both RNA and DNA‐RNA sequencing tests, corresponding to the commercial Afirma GEC and ThyroSeq v3 tests respectively. With regard to reclassifying indeterminate thyroid nodules, the AUCs for the RNA and DNA‐RNA classifiers were 0.81 and 0.77, respectively, showing an acceptable ability to identify thyroid carcinoma among indeterminate thyroid nodules. To better evaluate the diagnostic ability of the novel platform, we performed statistical analysis between our results and those reported in North America , 13. As Nevertheless, in our cohort, the PPVs of the RNA and DNA‐RNA classifiers were higher  and the NPVs were lower  than those of Afirma GEC and ThyroSeq v3 respectively , 13. TheAccording to the expected PPV and NPV curves for our novel molecular platform, the application mode of ‘rule‐in’ for RNA and DNA‐RNA testing is determined by the high ROM of the indeterminate nodules we examined. When the ROM value decreased to 30.9% or less, both classifiers may be suggested as ‘rule‐out’ tests. If molecular testing is performed as ‘rule‐out’ test, RNA classifier is superior to DNA‐RNA classifier for the higher NPV and cheaper cost. In general, the application mode of ‘rule‐in’ or ‘rule‐out’ for molecular testing depends on the ROM in different regions and populations.Although no statistically significant differences in diagnostic capacity between the RNA and DNA‐RNA classifiers was observed, the expanded gene mutation revealed by DNA‐RNA testing may be valuable for diagnosing thyroid cancer based on specific molecular alterations. Particularly when molecular testing is used as a ‘rule‐in’ test, characteristic high‐risk mutations will directly lead to a decision of surgery for indeterminate nodules. Both our current and previous studies show that TERT and BRAF V600E mutations and gene fusions are associated with a very high probability of malignancy , 35, 36.RAS or RAS‐like mutation (e.g. BRAF K601E) is not specifically associated with malignant outcomes, with an approximately 40–80% probability of cancer or NIFTP , 32, 35.® material to perform molecular testing, which was different from a previous experimental design [® material, a single puncture is sufficient for both cytological diagnosis and molecular testing because the liquid‐based sample remaining requires no additional FNA, reducing the burden on patients and saving medical resources. In the current study, among the indeterminate FNA results, residual liquid‐based samples for further molecular testing were available for 54.8% of patients, 97.8% of them having sufficient RNA and DNA to carry out molecular testing. Therefore, we strongly recommend using residual liquid‐based samples for molecular testing when available.One advantage of the present study is that we used residual ThinPrepl design  in which® FNA samples, more nodules categorized as AUS/FLUS were excluded as there were fewer cells in the AUS/FLUS residues compared to those categorized as FN/SFN. Hence, the proportion of FN/SFN nodules among indeterminate nodules based on molecular testing was higher than that among all indeterminate nodules. Finally, because our current study is a retrospective analysis, the clinical management of patients was not based on molecular testing results. A large number of cases with positive molecular test results did not undergo surgery; as such, next‐step follow‐up and/or prospective studies should be carried out. Moreover, the application of molecular testing in our clinical practice should be strengthened.There are some limitations in our study. First, as no commercial Afirma GEC and ThyroSeq v3 tests are available in China, we could not directly compare the performance of these two tests between regions. Second, there were some histopathological borderline tumours in our study that were diagnosed with a high level of disagreement between pathologists , 42. Our5In regions with high ROM of indeterminate nodules, RNA and DNA‐RNA tests may serve as ‘rule‐in’ tests in our clinical practice, especially an expanded DNA‐RNA testing may be more valuable than RNA testing alone. Whereas, in regions with low ROM, RNA and DNA‐RNA tests may serve as ‘rule‐out’ tests and a single RNA test is enough. In one word, the performance mode of ‘rule‐in’ or ‘rule‐out’ for molecular testing correlates closely with the ROM value of indeterminate thyroid nodules. In addition to the difference in thyroid cancer practice between Western countries and Asia highlighted in our study, it should be noted that there is still a considerable amount of variability among countries on the same continent as well as among national institutions. It remains challenging to define a universally acceptable approach of molecular testing for indeterminate thyroid nodules.The authors declare no conflict of interest.CH and WJ contributed equally to this work. CH reviewed the histopathological sections of the resected thyroid samples and wrote the manuscript. WJ carried out the data acquisition and assisted in writing the original draft. QC performed the ultrasonic diagnosis. ZZ and JC performed the cytological diagnosis. LZ prepared the cytological sections. CW collected the cytological samples. YS and ZL carried out the RNA and DNA extractions. HZ performed the cytological diagnosis and data analysis. TX conducted the molecular detection and data analysis. GH obtained the financial support and designed and conducted this study. All authors approved the final version of the manuscript.https://publons.com/publon/10.1002/1878‐0261.13205.The peer review history for this article is available at Table S1. Diagnostic performance of molecular tests considering borderline lesions as benign. The ROM of indeterminate nodules and the diagnostic performance of molecular tests were analysed considering borderline lesions as benign. An additional comparations of the diagnostic performance of the RNA classifier and DNA‐RNA classifier between Bethesda III and Bethesda IV nodules were performed.Table S2. Diagnostic performance of molecular tests considering borderline lesions as malignant. The ROM of indeterminate nodules and the diagnostic performance of molecular tests were analysed considering borderline lesions as malignant. An additional comparations of the diagnostic performance of the RNA classifier and DNA‐RNA classifier between Bethesda III and Bethesda IV nodules were performed.Click here for additional data file.


As dairy cows are being housed for longer periods, with all-year-round housing growing in popularity, it is important to ensure housed environments are meeting the needs of cows. Dairy cows are motivated to access open lying areas, although previous motivation studies on this topic have confounded surface type and location (i.e. pasture outdoors vs cubicles indoors). This study measured cow motivation for lying down on an indoor open mattress  compared to indoor mattress-bedded cubicles, thus removing the confounding factor of surface type and location. This was repeated for an identically sized indoor deep-bedded straw yard (ST), to investigate whether surface type affected motivation for an open lying area. Thirty Holstein-Friesian dairy cows were housed in groups of 5 (n = 5 x 6) in an indoor robotic milking unit with access to six mattress-bedded cubicles. To assess motivation, cows were required to walk increasing distances via a one-way indoor raceway to access the open lying areas: Short (34.5 m), followed by Medium (80.5 m) and Long (126.5 m). Cows could choose to walk the raceway, leading to the MAT or ST, to lie down or they could lie down on the cubicles for ‘free’. Overall, cows lay down for longer on the open lying areas at each distance compared to the cubicles, with cows lying down slightly longer on ST than MAT, although lying times on the open lying areas did decrease at the Long distance. However, cows were still lying for >60% of their lying time on the open lying areas at the Long distance. This study demonstrates that cows had a high motivation for an open lying area, the provision of which could better cater for the behavioural needs of housed dairy cows and improve housed dairy cow welfare. The majority of dairy cows are housed at some point throughout their lives  versus the avoidance of another (suggesting preference is based on choosing the lesser of two evils) ,22, i.e.The aim of the current study was to measure cow motivation for lying down on an indoor open mattress lying area [MAT] when cows had free access to indoor mattress-bedded cubicles, thus removing the confounding factors of surface type and location. This was repeated for a deep-bedded straw yard (ST) of an identical size to the open mattress, to further investigate the value cows place on lying surface type and the relationship between this and the value cows place on lying space. Walking distance was used as a measure of cow motivation to access these open lying areas via an indoor raceway, set at three different increasing distances . Given that previous studies appear to show that cows are motivated to access open lying areas, and particularly at night, we predicted that (1) cows would spend more time lying in the open lying areas compared to the cubicles at all walking distances, (2) that total lying times on the open lying areas would decrease with walking distance imposed and (3) that lying times at night on the open lying areas would not be effected by walking distance. Finally, we predicted that (4) surface type would also influence lying times on the open lying surfaces, with cows being more motivated to access the deep-bedded straw than the mattress.Ethical approval for this study was given by the Harper Adams University Research Ethics Committee (0488-201905-PGMPHD).st 2019 to July 21st 2020  and clean water, respectively. PMR was provided daily at approximately 0730 h, with feed refusals being removed every day before fresh feed was provided. Cows were fed concentrates, based on milk yield, during milking in the milking robot.The trial area was located in an area of 453-min areas . The ‘CuThe ‘Experimental Area’  compriseThroughout the study cows had continuous access to cubicles, with each experimental period lasting a total of 31-d : 3-d of While on trial, cows continued to have free access to the milking robot, were fed concentrates during milking, and were segregated into the trial area after milking via an automatic segregation gate . At apprFour video cameras , were set up to continuously record cow behavior within the trial area see . Each exEach experimental period began with a 3-d familiarization period. During this time, cows only had access to the ‘Cubicle Area’ so that they could familiarize themselves with their new grouping, robot access and the cubicles. For the duration of the study, cows had ‘free’ access to the cubicles at all times and did not have to work to gain access to them.During training periods, each cow was encouraged to walk the raceway distance, with a researcher walking quietly behind them, to gain access to the open lying space they had access to at the time. Following the familiarization period, the first training period, which always occurred at the Short distance, was 3-d long, to allow for one-way gate training see , and cowFor experimental periods 1–3, the open lying area marked ‘Surface Area 1’ as per Throughout the study, milk yield was recorded automatically at each milking for individual cows via the robotic milking system and was used to calculate average yield per cow for the duration of their time on the study. Milking permission, in terms of milking frequency and latency between milkings, was managed by the herd manager depending on the stage of lactation for individual cows. The milking status of trial cows was monitored regularly by the herd manager and LSC, and any trial cows that had not successfully taken themselves to be milked for >12 h were encouraged to the robot for milking.LS was evaluated after the trial area was cleaned each morning, at approximately 1000 h, on the first day of training for Surface A and Surface B , raceway distance , and surface type for open areas .post hoc differences were made using the Sidak correction [Linear mixed effects models were used to analyse the (fixed) effects of chosen lying location, raceway distance, and surface type. Assumptions of Gaussian residual distribution and homoscedasticity were examined and met for our analyses. Because treatments were applied at the group level, to meet the assumption of independence of observations , group was treated as a random effect to identify it as the level of independent observation. All data were analysed using R version 4.1.0  using thrrection .1,5.0 = 11.53, P = 0.019; 1,5.0 = 19.25, P = 0.0071; 2,46.1 = 1.06, P = 0.35), but a significant interaction was detected between distance and location . As distance to access the open lying areas increased, lying time on the open lying areas decreased and increased on the cubicles. No overall effect of location on the frequency of lying bouts per 24 h was found , but there was a significant interaction between distance and location . A strong effect of location was also found on lying bout duration, with cows exhibiting longer lying bouts on the open lying areas than in the cubicles , nor was there a significant interaction between distance and location .Overall lying time, lying bout frequency and lying bout duration results are shown in 2,6.7 = 6.3, P = 0.029; 1,5.0 = 27.59, P = 0.003; 1,5.7 = 6.61, P = 0.044), and no significant interaction was found between distance and surface . Distance strongly influenced the frequency of lying bouts in the open lying areas, with the number of lying bouts decreasing with distance , and no significant interaction between distance and surface . The mean duration of a lying bout was not higher for ST versus MAT  or a significant interaction between distance and surface . We found that the time spent not lying was also influenced by distance, with less time spent not lying as distance increased  nor an interaction between distance and surface . Finally, the frequency of raceway completions was analysed and a strong decrease in the number of completions as distance increased was found . No interaction effect was found between distance and surface for the frequency of raceway completions .Lying time and other behaviors were also analysed with respect to choices made while in the open lying areas in order to evaluate the effect of surface type and distance . DistancThe main objective of the study was to establish the extent of dairy cow motivation for lying on an open lying area when the cows had free access to cubicles, both of which were indoors and of the same surface type, , so as to remove these as confounding factors. This was repeated for a different surface type  to investigate the influence surface type has on motivation for an open lying area. Overall, time spent lying on the open lying areas declined as distance to access the open lying areas increased to the Long distance. However, cows still chose to lie down for longer on the open lying areas at this distance (>60% of lying time), compared to the free access cubicles, showing they were motivated to access the open lying areas rather than lying in cubicles. With a longer raceway we have observed shorter lying times for the open lying surfaces, but a ceiling effect (when the cost is too much and never paid) was not reached in the current study.Surface type did influence motivation, with cows expressing a higher motivation and lying down longer on the open lying area when it was a deep-bedded straw yard (ST) compared to MAT. This could be due to a lack of previous experience the cows had with an open mattress lying area, whereas they did have previous experience of open straw yards during dry periods. An animal’s previous experience can affect the results of preference and motivation tests, altering an animal’s attraction or avoidance of either a familiar or unfamiliar resource ,22. PrevDespite cows lying down longer on the ST compared to the MAT, there was no interaction between surface type and distance to access the open lying areas, suggesting that surface type had a limited effect on motivation and that access to an open lying area was the main driving factor for motivation in the current study. This is in agreement with a previous study whereby cows were found to trade lying down on their preferred lying surface with a cubicle for lying on an open lying space with a less preferred lying surface, demonstrating the increased value cows place on an open lying area than the lying surface . An addi2 per cow on the open lying surfaces and had an 83% cubicle stocking density, compared to 3.1 m2 per cow on the open mattress area and 67% cubicle stocking density in the other study [2 and 9m2 lying area per cow, respectively) and high stocking densities  were found to have no difference in lying times. Therefore, it is possible that only the difference in cubicle stocking densities between these two studies accounts for the low preference in the previous study and high motivation in the current study for an open mattress lying area. These previous studies investigating the effects of stocking densities in straw yards on cow behaviour are relatively short term studies  and further research is needed to investigate the long term effects on behaviour. Alternatively, the difference in results between that study and the relatively high motivation found in the current study may be due to the physical differences between the open lying areas. The current study offered cows a flat open lying area without obstruction, whereas the previous study offered cows a sloped lying area, due to the area’s previous function as cubicles, and the lying area was obstructed with the brisket board, limiting where the cows could lie down [Unlike the relatively high motivation for an open lying area found in the current study, a previous study found that cows showed a relatively small and varied preference to lie down on a mattress open lying area  compared to cubicles of the same surface . Both ther study . Cows haer study ,39 and ver study . The lower study . Howeverer study  and lacter study  cows houlie down .The total time cows spent in the open lying areas and lying on the open lying surfaces in the current study are similar to motivational studies for access to other open lying areas in the form of pasture access that also used walking distance as an indicator for motivation ,29. A 20Cows in the current study had longer lying bouts on the open lying areas compared to the cubicles, which can account for longer overall lying times on the open lying surfaces compared to the cubicles . HoweverCows spent longer lying down on the open lying surfaces at night compared to in the day-time in the current study. Cows exhibit a clear diurnal lying pattern, with cows lying down primarily at night ,48, whicActivities such as eating, drinking and sleeping are said to be ‘resilient’ activities which animals tend to show inelastic demand to perform, meaning that an animal will continue to work to perform such activities despite an increasing cost ,50. LyinCows in the current study were motivated to access and lie down in the open lying areas compared to the cubicles, with cows having a higher motivation, in terms of time spent lying, for an open deep-bedded straw area than an open cow mattress. Surface type had a smaller effect on motivation than walking distance, demonstrating the value cows place on an open lying area regardless of the surface type in the current study. Given cows value access to open lying areas, the provision of such areas has the potential to improve cow welfare in commercial housing systems in the future.S1 TableMean (± SEM) and range of lactation number, days in milk, milk yield (kg/d), body condition score and lameness score for all cows in the study. * This study included 6 primiparous and 24 multiparous cows.(PDF)Click here for additional data file.S2 TableStart and end date for each experimental period (1–6) within the study.(PDF)Click here for additional data file.S3 TableSummary, averaged for all cows, of time spent lying (h/d), lying bout frequency per day, lying bout duration (hr), time spent lying during the day (h/d), and time spent lying during the night (h/d) on MAT, ST, Open Lying Surfaces and Cubicles at the Short, Medium and Long distances and time spent not lying (h/d) and raceway completion frequency per day for ST and MAT at the Short, Medium and Long distance (± SEM).(PDF)Click here for additional data file.S1 FileA detail description of the training protocol.(PDF)Click here for additional data file.S1 DatasetA collated dataset for the behavior of each cow during each choice period (3 d) and a table of content (ToC) for the dataset.(XLSX)Click here for additional data file.


It predicts that life evolved from increasingly complex self-replicating RNA molecules4. The question of how this RNA world then advanced to the next stage, in which proteins became the catalysts of life and RNA reduced its function predominantly to information storage, is one of the most mysterious chicken-and-egg conundrums in evolution5. Here we show that non-canonical RNA bases, which are found today in transfer and ribosomal RNAs7, and which are considered to be relics of the RNA world12, are able to establish peptide synthesis directly on RNA. The discovered chemistry creates complex peptide-decorated RNA chimeric molecules, which suggests the early existence of an RNA–peptide world13 from which ribosomal peptide synthesis14 may have emerged16. The ability to grow peptides on RNA with the help of non-canonical vestige nucleosides offers the possibility of an early co-evolution of covalently connected RNAs and peptides18, which then could have dissociated at a higher level of sophistication to create the dualistic nucleic acid–protein world that is the hallmark of all life on Earth.The RNA world concept Peptide synthesis can take place directly on RNA, which suggests how a nucleic acid–protein world might have originated on early Earth. Comparative genomics21 suggests that ribosomal translation is one of the oldest evolutionary processes23, which dates back to the hypothetical RNA world4. The questions of how and when RNA learned to instruct peptide synthesis is one of the grand unsolved challenges in prebiotic evolutionary research5.A central commonality of all cellular life is the translational process, in which ribosomal RNA (rRNA) catalyses peptide formation with the help of transfer RNAs (tRNA), which function as amino acid carrying adapter molecules14 demands a stepwise evolutionary process11. From the perspective of the RNA world, at some point RNA must have gained the ability to instruct and catalyse the synthesis of, initially, just small peptides. This initiated the transition from a pure RNA world1 into an RNA–peptide world13. In this RNA–peptide world, both molecular species could have co-evolved to gain increasing ‘translation’ and ‘replication’ efficiency17.The immense complexity of ribosomal translation13, we analysed the chemical properties of non-canonical nucleosides7, which can be traced back to the last universal common ancestor and, as such, are considered to be ‘living molecular fossils’ of an early RNA world12.To gain insight into the initial processes that may have enabled the emergence of an RNA–peptide world29, we used naturally occurring non-canonical vestige nucleosides and conditions compatible with aqueous wet–dry cycles31.This approach, which can be called ‘palaeochemistry’, enabled us to learn about the chemical possibilities that existed in the RNA world and, therefore, sets the chemical framework for the emergence of life. In contrast to earlier investigations of the origin of translation32. Some tRNAs, however, contain additional amino acids in the form of amino acid-modified nucleosides, for example, g6A (ref. 33), t6A (ref. 34) and m6t6A (ref. 35), which are found directly next to the anticodon loop at position 37. Other non-canonical vestige nucleosides often present in the wobble position 34 are nm5U and mnm5U (refs. 38).In modern tRNAs Fig. , the ami6)aa6A. Cleavage of the urea40 (step 3) would furnish RNA with a peptide connected to a (m)nm5U (step 4). Subsequently, strand displacement with a new (m6)aa6A strand may finally enable the next peptide elongation step.Close inspection of their chemical structures Fig.  suggests1a–1j and 2a–2c  as RNA donor strands. The complementary RNA acceptor strands were prepared with an (m)nm5U nucleotide at the 3′ terminus (2a–2c). Figure 1a and 2a. The analytical data are presented in Fig. 1a with 2a and activated the carboxylic acid of 1a using reagents such as EDC42/Sulfo-NHS43, DMTMM·Cl43 or methyl isonitrile44 . In all cases we observed high yielding product formation  couples to 2c with an apparent rate constant (kapp) of 0.1 h−1. For the amino acids L (in 1d), T (in 1e) and M (in 1h) a fourfold higher rate constant (≈0.4 h−1) was determined, and the highest rate was measured for F (in 1g) with kapp > 1 h−1. These differences establish a pronounced amino acid selectivity in the coupling reaction, probably as a result of distinct pre-organizations. We next reduced the length of the RNA donor strand to five, and finally to three, nucleotides (M NaCl and 0 °C). The interaction of three nucleotides on the donor with the corresponding triplet on the acceptor seems to be the lower limit for productive coupling. Interestingly, this is the size of the codon–anticodon interaction in contemporary translation18.A kinetic analysis shows that the nature of the amino acid affects the coupling rate Fig. . For exaleotides . We dete1a  with the different acceptors 2a–2c under the recently described prebiotically plausible thiol activation conditions45 . Here also, the coupling products were obtained within a few hours  of approximately 87 °C was determined, which in comparison to the starting duplex  in water at pH 6 Fig. . After 6peptide-mnm5U-RNA-5′ acceptor strands as starting materials  led to the preferential formation of the peptide product, 5c (approximately 7:1 5c:cyclic-5c ratio).During urea cleavage we detected competing formation of hydantoin side productsure Fig. . Under m6peptide6A). This is essential because an RNA–peptide world, with initially low chemical efficiency, might have been limited to the synthesis of smaller peptides. We found that the required adenosine nucleosides, containing a whole peptide attached to the N6-position, are available if the peptides that are produced by RNA degradation of the RNA–peptide chimeras, for example, can react with nitrosated N6-methylurea adenosine  and added the solution to triglycine (pH 9.5), we obtained the peptide-coupled adenosine nucleoside ggg6A in approximately 65% yield. Incorporation of (m6)ggg6A into RNA and hybridization of this donor strand with a 3′-ggvmnm5U-RNA-5′ acceptor strand furnished, after coupling and urea cleavage, the RNA–peptide chimera 3′-gggggvmnm5U-RNA-5′  to a single RNA acceptor strand (21-mer) with a central gmnm5U and a 3′ terminal nm5U , containing both a 3′-m6g6A and a 5′-m6v6A, to two different acceptor RNAs, containing a central vmnm5U (21-mer) and a 3′ terminal vmnm5U (11-mer)  Fig. , right. 6g6A-RNA-3′ and 11-mer: 5′-m6v6A-RNA-3′) to an acceptor strand with a vmnm5U at the 3′ end (11-mer: 2c) . The first contained a 5′-m6l6A and the second a 5′-m6g6A, together with two mismatches. We added this mixture to an RNA acceptor strand (11-mer: 2c) with a 3′-vmnm5U nucleotide  Fig. , left. O2c) Fig. , only foide Fig. , right. 2g, in which the contemporary canonical bases were replaced by the non-canonical 2′-OMe nucleotides: Am, Cm, Gm and Um. The strand 2g was equipped with an additional 3′-mnm5U nucleotide. For the experiment we used the same amount of donor strand for all coupling steps and performed filtration steps to remove remaining activator. After two couplings, two urea cleavages and two filtrations, we observed, by high-performance liquid chromatography (HPLC) analysis, the presence of the product 3′-ggmnm5U-RNA-5′ 7g  were annealed with NaCl (5 μl from a 1 M aqueous solution) by heating at 95 °C for 4 min, followed by cooling down slowly to room temperature. After that, MES buffer pH 6 (25 μl from a 400 mM aqueous solution) and NaCl (5 μl from a 1 M aqueous solution) were added to the oligonucleotide solution. Finally, carboxylic acid or nitrile activator/s (10 μl of each component from a 500 mM aqueous solution) and water  were added to the solution mixture. The peptide coupling reaction was incubated at 25 °C for 24 h. The crude reaction mixtures were analysed by HPLC and MALDI-TOF mass spectrometry.The hairpin-type intermediate (0.5 nmol) was diluted with MES buffer pH 6 or acetate buffer pH 4 (12.5 μl from a 400 mM aqueous solution), NaCl (5 μl from a 1 M aqueous solution) and water . The urea cleavage reaction was incubated at 60–90 °C at different time intervals. The crude reaction mixtures were analysed by HPLC and MALDI-TOF mass spectrometry.Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-022-04676-3.Supplementary InformationSupplementary text, compounds, figures and references.Peer Review File


Due to the implementation of global education and global citizenship education in the 21st century, more focus is given to developing teachers’ global competence in English language teaching. This study aims to examine the perceptions of English as a Second Language (ESL) teachers of (1) global competence integration in English teaching and (2) the professional development programs organized by the Ministry of Education (MOE) for their global competence development. A web-survey questionnaire was distributed to 172 Malaysian ESL teachers based on selected criteria. The data collected were analyzed descriptively. The main findings indicated that most ESL teachers showed positive perceptions regarding the importance of global competence and integration in English lessons. Although 83.1% of the 172 participants agreed to have attended 10 or more programs related to developing global competence, the descriptive analysis found the ESL teachers’ moderate knowledge and understanding of what constitutes global competence. In contrast, a high agreement was found regarding their perceptions of the importance of global competence in ESL lessons. Also, most respondents revealed the lack of support from the MOE through professional development programs specifically structured to develop ESL teachers’ global competence. The findings served as a template for a larger-scale study that focuses on implementing global competence in the local non-western context. For selecting respondents who corresponded to the fourth criterion, the ESL teachers made their judgments based on the descriptions of the criteria included with the Google Form Survey link. The requests for participation in the survey, the descriptions, and links were sent to the ESL teachers The data collection was conducted between April 5 and April 18, 2021. The teachers who fit the criteria voluntarily agreed to respond to the survey questionnaire during their free time within the period allocated by the researcher in a fortnight.This research utilized the quantitative research design to identify the perceptions of ESL teachers in Malaysia on global competence. The survey questionnaire was designed to extract information on the perceptions of ESL teachers. The construction referred to the study’s objectives and the research questions formulated. Through surveys, information from many respondents can be obtained and further analyzed.Before conducting the study, a pre-test was performed to confirm no ambiguity in each questionnaire item and that the respondents could clearly understand the questions . Five reCorrect wording for the items.Correct sequence of the items.Clearly understood items (by the respondents).Additional items or exclusion of items.Clear and adequate instructions.Necessary amendments were made to improve the items based on feedback from five respondents. Then, the web-based survey questionnaire was submitted for expert checking to further validate the survey items before distributing them for the actual data collection. It is summarized that all the items and the data collected from the survey were reliable. Besides, the web-based survey was appropriate for the safety guidelines of reducing physical contact and heightening physical distancing due to the recent Coronavirus or COVID-19 pandemic. It was also helpful to expedite and improve the process . In thisA web-based survey link or Google Form was given to ESL teachers and posted in English teachers’ groups through social media platforms and messaging applications such as Telegram, WhatsApp, Facebook, Twitter, and Instagram. It was the fastest and most efficient way to reach the teachers in different states.The online data gathered from the Google Form questionnaire were tabulated. The responses for each item were entered into an IBM SPSS Statistics database version 26 for the descriptive quantitative analysis procedure. The descriptive analysis described a particular situation through numerical data , and allThe data collected from the online survey questionnaire are presented and discussed in two sections, categorized below, with the first section on ESL teachers’ knowledge and perceptions of global competence in English lessons. Finally, the second section elaborated on the ESL teachers’ perceptions of professional development programs organized by the MOE to develop their global competence.Next, the respondents’ criteria in this study include the experience of attending professional development programs. In the questionnaire employed, one of the items is specific to gather responses on whether the ESL teachers have previously attended 10 or more programs on global competence. Regarding Item 6, 143 ESL teachers (83.1%) answered “Yes,” while the other 29 ESL teachers (16.9%) said “No.”In this section, 14 items measured the respondents’ knowledge and perceptions of global competence to answer this study’s first research question (RQ1). The results are presented in M = 3.27) and the perceptions that they have the characteristics of globally competent ESL teachers (M = 3.33). Similarly, a moderate level of agreement was found in the ESL teachers’ knowledge of the global competence definition (M = 3.39) and elements (M = 3.10). Another moderate agreement was found among some ESL teachers regarding their knowledge of the difference between intercultural and global competence (M = 3.38).As depicted in M = 3.71, the majority agreed. Interestingly, most of them understood the importance of global competence in teaching English. The ESL teachers mostly agreed (M = 4.27) with the notion that ESL teachers’ global competence is essential. Furthermore, the high level of agreement (M = 4.23) on the ESL teachers’ view of global competence integration in English lessons showed that most ESL teachers strongly agreed and agreed that they have a positive and favorable view of the integration. Most ESL teachers strongly felt that integrating global competence into lessons is crucial (M = 4.20). Most respondents also agreed that the integration creates meaningful lessons and assists students’ English learning (M = 4.17).The respondents were asked if they believed that global competence is closely related to English lessons. Based on the high mean score of M = 4.05) and interest (M = 4.11) in learning the target language when global competence was integrated into language lessons. From the findings, most ESL teachers agreed that English lessons should emphasize knowledge of local and global issues (M = 4.35); with 8.8% (15) ESL teachers quite agreed, 43.3% (74) agreed, and 46.8% (80) strongly agreed. The strong agreement among the respondents concerning such content to be imparted in ESL lessons showed their perceptions about current ESL teaching and learning. Most were also optimistic about the global competence integration in ESL lessons.Moreover, based on the high level of agreement for Items 12 and 13, most ESL teachers noticed that students displayed better engagement . According to the results, the high score implied that some ESL teachers were keen to learn and develop global competence through various programs. The vital role of programs and courses for ESL teachers’ professional development could not be denied. Based on M = 3.62). However, the data analysis also resulted in a moderate level of agreement on whether the ESL teachers have received knowledge on global issues from courses organized by MOE (M = 3.22). The disparity in ESL teachers’ perceptions about the courses organized by MOE (M = 3.22) showed the scarcity of suitable programs that cater to ESL teachers’ global competence needs.The quantitative data findings provided information regarding ESL teachers’ perceptions regarding the factors for the second research question (RQ2). The survey indicated how the programs and courses by the MOE might affect ESL teachers’ global competence development. A high mean score revealed some of the respondents’ high level of agreement that the CEFR-aligned syllabus and related courses offer platforms for teachers to integrate global competence (M = 3.16) proves the lack of help from MOE to train globally competent ESL teachers. The respondents had different opinions about it, and 34.9% quite agreed with the claim, 19.2% disagreed, and 7% strongly disagreed. The mixed reactions to the content of Item 5 may require the MOE’s attention on how to better assist ESL teachers in developing their global competence.Global competence knowledge must be embedded in offered courses so that teachers know the interdisciplinary connections of global competence development. The lowest mean score for whether the MOE has helped the ESL teacher develop global competence (M = 4.14) widen their content for the ESL lessons. Generally, it boosts their confidence in teaching global knowledge in language classrooms. The ESL teachers’ high level of agreement on Item 6 reflects their ability to find materials on global issues independently.In this study, some of the respondents believed that the availability of materials on global issues had helped them . She is an educational technology enthusiast and an e-learning practitioner. Her areas of concentration are ESL, mobile learning, Mobile-assisted Language Learning (MALL), technology acceptance as well as language pedagogy, and the use of technology in teaching English as a Second Language (ESL).The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.Funds received for open access publication fees, from my institution (grants), National University of Malaysia/ Universiti Kebangsaan Malaysia with grant no. GG-2020-024.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.