It is known that an RNA’s structure determines its biological function, yet current RNA structure probing methods only capture partial structure information. The ability to measure intact  RNA structures will facilitate investigations of the functions and regulation mechanisms of small RNAs and identify short fragments of functional sites. Here, we present icSHAPE-MaP, an approach combining in vivo selective 2′-hydroxyl acylation and mutational profiling to probe intact RNA structures. We further showcase the RNA structural landscape of substrates bound by human Dicer based on the combination of RNA immunoprecipitation pull-down and icSHAPE-MaP small RNA structural profiling. We discover distinct structural categories of Dicer substrates in correlation to both their binding affinity and cleavage efficiency. And by tertiary structural modeling constrained by icSHAPE-MaP RNA structural data, we find the spatial distance measuring as an influential parameter for Dicer cleavage-site selection. Sequencing methods such as icSHAPE were developed to probe RNA structures transcriptome-wide in cells. To probe intact RNA structures, the authors develop icSHAPE-MaP and apply to Dicer-bound substrates showing that distance measuring is important for Dicer cleavage of pre-miRNAs. Dimethyl sulfate (DMS), 1-methyl-7-nitroisatoic anhydride (1M7), 2-methylnicotinic acid imidazolide-azide (NAI-N3), and Kethoxal are widely used reagents for RNA structure probing in vivo6. DMS modifies the N1 and N3 positions of adenine and cytosine bases within single-stranded regions in vivo3, whereas NAI-N3 acrylates the free 2′-hydroxyl groups for all four single-stranded bases, allowing for in vivo structure probing of the transcriptome by selective 2′-hydroxyl acylation followed by Primer Extension (icSHAPE)4. icSHAPE has been used to uncover structural variations of RNAs associated with different biological processes, such as translation, RNA-protein interactions, and N6-methyladenosine modification in living cells7.Genome-wide RNA structure  studies marry chemical probing with next-generation sequencing8, and SHAPE-MaP9 measure the rate of mutations generated during reverse transcription. However, DMS-MaPseq provides partial nucleotide coverage (only adenosine “A” and cytidine “C” nucleotides could be probed) and current SHAPE-MaP reagents  have only moderate cell membrane-penetration abilities, limiting their usage in vivo.Structure probing methods, including DMS-seq and icSHAPE, measure reverse transcription truncations arising at chemically induced nucleotide modifications to determine the probability that a nucleotide is in a single-stranded conformation. A limitation, however, is that structural information at the 3′ terminus of a probing target will be missing, due to the loss of mapping of short sequencing reads  and miRNA biogenesis. Studies have proposed that Dicer measures a certain number of nucleotides from either the 3′ overhang of dsRNA substrates (the 3′ counting rule)14 or from the phosphate group of the 5′ end for select pre-miRNAs and dsRNAs (the 5′ counting rule)15. In addition, our in vivo studies of short hairpin RNAs (shRNAs) and pre-miRNAs revealed that Dicer uses a single-stranded region  to precise anchor the cleavage site 2-nt downstream (the loop counting rule)16. However, questions remain, in regard to when and to what extent these mechanisms operate in pre-miRNA processing. In addition, Dicer also binds to a variety of substrates without apparent classical miRNA or siRNA processing activities17, suggesting that it has other roles in RNA metabolism. Whether and how Dicer differentiates between the cleavable and non-cleavable substrates is unknown.Dicer belongs to the RNase III family and cleaves double-stranded RNAs (dsRNAs) and pre-miRNAs into mature small interfering RNAs (siRNAs) or miRNAs, respectivelyIn this work, we present an approach to probe the structures of intact RNAs in living cells. Briefly, we harness the advantages of icSHAPE reagents and of mutational profiling in reverse transcription to develop a structure probing method, icSHAPE-MaP. To demonstrate its capabilities, we use icSHAPE-MaP to determine the complete structural information for cellular sRNAs. In addition, we combined icSHAPE-MaP with RNA immunoprecipitation (RIP) to determine the structural landscape for substrates of the RNA endonuclease Dicer. By combining structural information obtained by icSHAPE-MaP and tertiary structural modeling, we discover that spatial distance measuring is an influential parameter in Dicer-mediated pre-miRNA processing.3 to modify RNA, and subsequently maps mis-incorporation events generated by the reverse transcriptase Superscript II  at the nucleotides with NAI-N3-induced RNA modifications , that uses the icSHAPE reagent NAI-N3 was added directly to cells, which preferentially reacts with free 2′-hydroxyl groups of unstructured and flexible nucleotides, and then the sRNA fraction was purified. For in vitro probing, the sRNA fraction was first purified, refolded, and then treated with NAI-N3 in a tube. The remaining library construction steps were essentially the same for both, where sRNAs were ligated with two adapters at the 5′ and 3′ ends, and reverse transcribed with Superscript II reverse transcriptase. In principle, both reverse transcription mutations (RT-mut) and reverse transcription stops (RT-stop) indicate NAI-N3 modification and hence the structural flexibility of nucleotides. However, Superscript II reverse transcriptase usually adds a random number of non-template nucleotides at the 3′ end of cDNA18, which confounds accurate RT-stop identification. In icSHAPE-MaP, we thus only used RT-mut for RNA structural probing. To remove RT-stop fragments, we added adapters at both 5′ and 3′ end with polymerase chain reaction (PCR) primers prior to reverse transcription, thus only full-length sequences were amplified for subsequent analysis  in HEK293T cells. We performed both in vivo and in vitro icSHAPE-MaP structure probing (see “Methods”). For in vivo probing, NAI-NTo map RT-induced mutation sites, we performed deep sequencing and computational analyses, generating an icSHAPE-MaP reactivity/structure score for each nucleotide (see “Methods”). The score negatively correlates with the likelihood of the nucleotide being paired, providing a measure of its secondary structure information. The icSHAPE-MaP experiments were reproducible between independent biological replicates as the mutation rates of each transcript were highly correlated between two replicates  . We sequenced ~200 M reads and obtained structure scores for 186 transcripts with in vivo samples and 250 transcripts with in vitro samples  and 1-methyladenosine (m1A) modifications. This finding underscores the importance of analyzing the background DMSO libraries to determine NAI-N3-independent signals. Our analysis also revealed that the increase in mutational rates was more significant at A and U residues, consistent with previous observations that single-stranded regions are enriched for A/U compared to G/C4  between replicates of regions at different sequencing depths. We found that a cutoff of 2000× sequencing coverage yields very high-quality scores, and that 1000× or even 500× coverage is a reasonable cutoff when considering the trade-off between the cost and the reproducibility  was purified and structures were profiled by icSHAPE-MaP.We combined icSHAPE-MaP with RIP to profile the RNA structure landscape of Dicer substrates Fig. . Enriche17. Despite the different enrichment strategies, the two lists of RNAs agreed well with each other, with more than 50% common pre-miRNA coverage . Most of the AUCs are well above 0.5  RNAs   Fig. , top. Inops Fig. , bottom.ops Fig. .4. This is consistent with our observations of the more extended terminal loops of pre-miRNAs in our experimentally inferred models compared to the theoretical miRBase models. This analysis highlights the necessity of using experimental information to constrain computational modeling, which will otherwise generate more base-pairings as a result of energy minimization. These results show that the use of icSHAPE-MaP scores can more precisely model RNA secondary structures, providing a structural basis for RNA processing and functional studies.In general, most (78/100) pre-miRNAs have at least one, and almost half (47/100) of pre-miRNAs have at least five structurally different positions between the constrained experimentally inferred and theoretical models. Remarkably, the structurally different positions were often found around the terminal loop region (~±5 nt around the coordinate “0”) Fig. . In geneTo identify the structural determinants of Dicer substrates, we developed a computational method to de novo cluster the icSHAPE-MaP structure profiles (see “Methods”). We first aligned the profiles based on the central loop in predicted structures with constraints of icSHAPE-MaP scores and then performed principle component analysis (PCA) to reduce them into a two-dimensional space. The substrate structures obtained from Dicer RIP experiments established three distinct groups by the K-means clustering, based on the top two principal components Fig. . Interesp = 2.15e−27 and p = 1.05e−20, respectively). And substrates from Cluster II are slightly more enriched than those of Cluster III but with no statistical significance . Together, these results suggest that perfect hairpin-like structures, e.g., pre-miRNAs, are generally the preferential binding substrates for Dicer.Cluster I of Dicer substrates showed a relatively large terminal loop with a median size of about 9-nt, flanked by a near-perfect double-stranded stem, which resembles the characteristic hairpin structure of pre-miRNAs . Likewise, Cluster II had a higher cleavage score than Cluster III . These data suggest a direct correlation between Dicer binding affinity and cleavage activity.To directly measure the cleavage activities of Dicer on its substrates, we expressed wild-type (WT) or catalytic-dead Dicer in 293T Dicer-deficient cells, isolated the sRNA fraction of 40–200 nt, and performed RNA-Seq (see “Methods”). As a proxy for the cleavage activities of Dicer, we calculated a “cleavage score” as the log2 fold of change in target abundance in catalytic-dead Dicer vs. WT Dicer cells , an intron of the signal recognition particle 68 (SRP68), and an intron of ER lipid raft associated 1 (ERLIN1)  that belongs to a repeat family with sequence homology to pre-tRNA-Tyr. These findings suggest additional Dicer functions beyond miRNA biogenesis .We also detected Dicer substrates within mRNA introns and exons that display both high binding and cleavage activities  2-nt downstream a bulge or loop (the loop counting rule); (2) a physical distance from the C5′ atom of the 1st nucleotide of miRNA on 3p arm to the O3′ atom of the 3′ end of pre-miRNA . For each pre-miRNA hairpin, we searched for single-stranded regions on both the 5p and 3p arms. We found that 45% of the 1st nucleotides for 3p miRNAs . The distances of both D3p_miRNA and D5p_miRNA were in proximity to 59 Å for most pre-miRNAs, irrespective of the length of cleaved product  of longer RNAs. The examples demonstrated in the present study showcase the application of icSHAPE-MaP to unveil the genome-wide structural landscape for Dicer substrate sRNAs. In the future, icSHAPE-MaP can be applied to reveal the structural features of binding by other RBPs.Our work presents a biotechnology “icSHAPE-MaP” to accurately probe RNA secondary structures in vivo while obtaining the complete information for their intact form. icSHAPE-MaP leverages mutational profiling of reverse transcriptase to detect NAI-N27. Our RIP-icSHAPE-MaP data suggests this structural feature for pre-miRNAs to be a general property of Dicer processing. In terms of Dicer cleavage-site selection on pre-miRNAs, our data suggest that distance counting in three-dimensional space is an important parameter. We hypothesize that Dicer cleaves pre-miRNAs at the phosphodiester bond of the nucleotide with the closest proximity to the physical distance D3p_miRNA (59 ± 1 Å), upon which mature miRNA lengths are determined 15. Together with the “loop counting rule”, they coordinately regulate Dicer cleavage-site selection on pre-miRNAs.In the analysis of the RNA structure of Dicer substrates, we observed that most pre-miRNAs bear a large terminal loop with a nearly perfect stem, structurally and statistically different from other Dicer substrates Fig. . This is29. Furthermore, we observed that pre-miRNAs with long 3p arms tend to display the “3′ counting rule”, with a lesser percentage featuring the “3′ loop counting rule” , and 10% fetal bovine serum in a humidified incubator at 37 °C with 5% CO2. All transfection assays were done by using polyethylenimine (PEI) (Sigma-Aldrich).The 293T cell line was purchased from ATCC (cat. #CRL-3216). The Dicer-deficient 293T cell line (NoDice 2-20) was a gift from Dr. Bryan R. Cullen at Duke University. Cells were maintained in DMEM/HIGH glucose with 6 cells were seeded on the first day and transfected with 20 µg plasmids 24 h later with 60 µl (1 µg/µl) PEI. In detail, the plasmids and PEI was first incubated with 1 mL Opti-MEM I Reduced Serum Medium (Gibco) separately. Then the two mixtures were combined and kept at room temperature for 15 min before adding to cells. Forty-eight hours later, cells were lysed in the lysis buffer , supplemented with the proteinase inhibitor cocktail (Roche) and RNase inhibitor RiboLock . The lysate was centrifuged at 15,000g for 10 min at 4 °C to remove the insoluble cell debris. The supernatant was incubated with anti-FLAG M2 magnetic beads (Sigma) for 3 h at room temperature.To analyze Dicer substrates at a large scale, NoDice 2–20 cells were transfected with a plasmid expressing human Dicer with two mutations in its RNase III domains . For one 15-cm plate, 9 × 102, 150 mM NaCl, 50 mM NAI-N3) on a Thermomixer at 37 °C for 12 min at 1,000 rpm (NAI-N3 group). For the DMSO group, NAI-N3 was replaced by DMSO in the modification buffer. RNA was isolated with Trizol, following the manufacturer’s instruction.After incubation, the beads were washed once with the high salt wash buffer , RiboLock ) and twice with the low salt wash buffer , RiboLock ). After the last wash, the beads were incubated with the modification buffer . Then the cells were resuspended in 100 mM NAI-N3 and incubated at 37 °C on a Thermomixer at 1,000 rpm for 5 min. The reaction was stopped by centrifugation at 2500g for 1 min at 4 °C, and the supernatant was subsequently removed. The cells were resuspended in 250 µl PBS, and then 750 µl TRIzol LS Reagent was added for RNA extraction. The extracted RNA was size-selected on a 6% Urea–PAGE gel for 25–200 nt. For the DMSO control group, the preparation of small RNA was the same as the in vivo modified group except that the HEK293T cells were treated with DMSO instead. For the in vitro group, the RNA extracted from the DMSO group was first heated in metal-free water at 95 °C for 2 min, then was chilled on ice. The 3.3 × SHAPE folding buffer  was added to RNA followed by incubation at 37 °C for 10 min. Then 1 M NAI-N3 was added to a final concentration of 100 mM, followed by incubation at 37 °C for 10 min. Finally, the reaction was stopped by adding 2 × Binding buffer of an RNA concentration kit (Zymo), and the RNA was purified, following the manufacturer’s instruction. The details of library construction are described in the following section “Construction of icSHAPE-MaP library”.For in vivo small RNA (<200 nt) structure probing, HEK293T cells were treated with NAI-NThe RIP pulled-down and “input” RNA was size-selected on a 6% Urea–PAGE gel for 25–200 nt. Gel purification was performed by crushing the gel and incubating in the gel crush buffer  with rotation at 4 °C overnight. The eluate was collected by centrifuging through 0.45 µm Spin-X columns (Thermo Fisher), concentrated, and purified by an RNA concentration kit (Zymo). The 8 µL purified RNA was ligated with a 3′ linker by incubation with the 3′ ligation mix . Forty-eight hours later, sRNA (<~200 nt) was extracted using the mirVana kit (Thermo Fisher). One microgram sRNA was then incubated with the end-repairing mix , 1 µL Fast AP (Thermo Fisher), 1 µL RiboLock, 4 µL nuclease-free water) at 37 °C for 1 h. The 3′ ligation mix  were transfected into the NoDice 2–20 cells (5 × 102), 1 µL RiboLock, and 1 µL SuperScript II (Thermo Fisher) were added. The reaction mix was incubated at 25 °C for 3 min, and then 42 °C for 3 h. cDNA products were purified by running on 6% Urea–PAGE gel. The following steps, including circularization, PCR amplification and PCR products purification, were done as previously described4. Briefly, circularization was conducted as follows, 16 µL cDNA products were mixed with 2 µL 10 × Circligase buffer, 1 µL Circligase II, 1 µL 50 mM MnCl2 and incubated at 60 °C for 2 hr. The circularization reaction was purified with a DNA concentration kit (Zymo). PCR was set up with 20 µL eluted circularized cDNA and PCR reaction mix . PCR primers were designed to amply the genomic regions flanking pre-miRNAs (~200-nt upstream and downstream). Mouse mir124-1 was cloned downstream of the CMV promoter between Hind III and BamH I to serve as a control for transfection and expression experiments. Pre-miR-217-WT was cloned downstream of the EF-1α promoter between Not I and Kpn I. All other variants of pre-miR-217 were made using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent). All oligo sequences for cloning can be found in Supplementary Data 16, with the exception that luciferase activities were measured 48 h after cotransfection of 100 ng pre-miRNA plasmids and 100 ng corresponding reporter plasmids.Cloning of luciferase reporters for miRNA binding sites was done as previously described16. Briefly, total RNA was extracted with the mirVana miRNA Isolation Kit (Thermo Fisher). The 3′ linker ligation was set up in 20 µL for 16 °C overnight, using 5 µg total RNA and the ligation mix , 100 mM DTT, 1 × RNA ligation buffer, 1 µL T4 RNA Ligase 2 with truncated KQ (NEB), 0.4 U/µL RiboLock (Thermo Fisher)). The ligation product was size-selected on a 15% Urea–PAGE gel (Thermo Fisher) for ~17–35 nt long sRNAs. Gel purification was performed by crushing the gel and incubating in the RNA elution buffer  with vigorous shaking at 37 °C overnight. The eluate was collected by centrifuging through 0.45 µm Spin-X columns (Thermo Fisher), followed by ethanol precipitation. The 5′ adapter ligation was set up in 20 µL for 16 °C overnight, using the 3′ ligation products and the ligation mix  was used to transfect HEK293T cells. To reduce the viscosity, cell lysate was subject to sonication on ice . The supernatant was incubated with 40 μL of anti-Flag M2 affinity gel (Sigma) with constant rotation for 2 h at 4 °C. The reactions were performed in a total volume of 30 μL in 2 mM MgCl2, 1 mM DTT, 5′/3′-end-labeled pre-miRNA of 1 × 104 to 1 × 105 c.p.m. and 15 μL of the immunopurified Dicer in the reaction buffer . The reaction mixture was incubated at 37 °C for 60 min, followed by the addition of 2 × loading buffer (Thermo Fisher) and separation on 18% polyacrylamide gel with 8M urea.Immunoprecipitation of Flag-Dicer and in vitro processing for pre-miRNAs were conducted as previously describedImmunoblotting was used to verify the enrichment of FLAG-tagged Dicer in RIP. The experiment was performed with primary antibodies for GAPDH , DICER , FLAG tag , and secondary antibodies: goat anti-mouse IgG (H + L)-HRP Conjugate ; Goat anti-rabbit IgG (H + L)-HRP conjugate . 1% of the sample volume of input or pull-down samples was used for immunoblotting.32-labeled DNA probes . Totally, 10–30 μg RNA was run on an 18% (w/v) polyacrylamide/8 M urea gel for electrophoresis, transferred onto an Amersham Hybond-XL membrane , and blotted with P3 (5%) or treated with 1 µL DMSO (unmodified control) in presence of 3 × SHAPE buffer  for 15 min at 37 °C. RNA was extracted in phenol:chloroform and resuspended in 5 µL RNase-free water or 4 µL for sequencing lanes. A DNA primer complementary to the Universal miRNA Cloning Linker (NEB) ligated to the 3′ end of RNA was 5′ labeled with T4 polynucleotide kinase (NEB) and γ-P32-ATP. 1 µL of the 5′ labeled primer (~250 nM) was annealed to the resuspended RNA samples at 95 °C and slowly cooled down to ~40 °C. Finally, primer extension was carried out in presence of Superscript III (Thermo Fisher), first-strand buffer, DTT, RiboLock, and 0.5 µL of 2 mM dNTP (plus 1 µL of 10 mM ddNTPs for sequencing lanes) at 55 °C for 15 min in a 10 µL final volume reaction. The reaction was terminated with 1 µL of 4 M NaOH and incubated at 95 °C for 5 min, followed by ethanol precipitation on dry ice. DNA pellet was resuspended in 10 µL of 2 × Gel Loading Buffer II (Thermo Fisher) and separated on 15% Urea-PAGE gel. All (−) lanes were those from DMSO-treated samples.RNA structure analysis was performed by selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE). 1 pmol of RNA was modified with 1 µL NAI-NmirVana miRNA Isolation Kit (Thermo Fisher). Complementary DNA (cDNA) synthesis was performed after DNase treatment  using Superscript IV with the random oligo. qPCR analysis was performed with iTaq Universal SYBR Green Supermix and Bio-Rad CFX384 cycler as described in the manufacturers’ instructions.Cells were harvested and total RNA was isolated using TRIzol (Thermo Fisher). Isolation of small RNAs from total RNA Samples was done with the HEK293T cells were transfected with DICER1  or control siRNA  by Lipofectamine 3000 (Thermo Fisher). Totally, 72 h after transfection, RNA extraction and RT-qPCR were conducted as described above.34, filtering high-quality reads with Trimmomatic (Version 0.33)35, and duplication removal with an in-house Perl script (available upon request). Clean reads were mapped to the reference sequences  with STAR36 by default settings. The read count was calculated using an in-house Perl script (available upon request). The enrich score and cleavage score calculation is described in the part “Calculation of the RIP enrichment scores and cleavage scores for Dicer”. The transcripts with enrichment scores > 0 were defined as RIP-enriched ones. sRNA-Seq data analysis was done by following our published protocol, except that only reads with the perfect match to pre-miRNAs were used16.The sequencing data were processed by removing adapters with Cutadapt (Version 1.16)34, filtering high-quality reads with Trimmomatic (v0.33)35, and duplication removal with an in-house Perl script (available upon request).Pre-process. The sequencing data was processed by removing adapters with Cutadapt (v1.16)23, snoRNAs (from Gencode v26)37, snRNAs (from Gencode v26), tRNAs (from GtRNAdb v2.0)19, vault RNAs (from RefSeq v109)38, Y RNAs (from RefSeq v109), and 5S rRNAs. The processed reads as above were mapped to them with STAR (v2.7.1a)36 with parameters --outFilterMismatchNmax 3 --outFilterMultimapNmax 10 --alignEndsType Local --scoreGap -1000 --outSAMmultNmax 1. To find out other sRNA fragments with limited annotation on the human genome, the unmapped reads were mapped to human genome (version GRCh38.p12) for the reiteration of data analysis mentioned above.Mapping. Human sRNAs sequences less than ~200-nt in length were collected, such as miRNAs (from miRbase v22)39. Shapemapper2 (v2.1.4)40 was used to calculate final scores as follows:Mutations on each read were parsed with shapemapper_mutation_parser. The script counts 8 mutation types: mismatch, insertion, deletion, multi-mismatch, multi-insertion, multi-deletion, complex-insertion, and complex deletion.Number of mutations for each nucleotide was counted with shapemapper_mutation_counter.icSHAPE-MaP reactivity scores were calculated with make_reactivity_profiles.py.Raw scores were normalized with normalize_profiles.py.Calculate icSHAPE-MaP scores. Replicate samples were combined (with samtools merge)The calculation process for each base can be briefly summarized by the following formula:The icSHAPE-MaP score for base The total read counts from two replicates were balanced by down-sampling. All bases were sorted by coverage. The bases with coverage greater than 500, 1000, 2000, 3000, 4000, or 5000 were selected to calculate the replicate correlation of mutation rate with sliding window . Finally, the data under each cutoff was used to generate a cumulative distribution curve.22 was used to predict the secondary structures of RNAs. The icSHAPE-Map scores were used as constraints with parameters: -si -0.6 -sm 1.8 -SHAPE icSHAPE-Map.shape -mfe.The Fold program in the RNAstructure package (v5.6)41 command line. Colors of bases were applied with parameter “-basesStyle1 on and -applyBasesStyle1on”.RNA secondary structures were visualized with VARNAv3-93The secondary structures of RNAs were predicted with icSHAPE-MaP scores as constraints. They were aligned to the center of their central loop and the icSHAPE-MaP scores of flanking 30-nt were used for PCA analysis in the Sklearn (v0.20.3) package. The raw 60-dimentional space was reduced to a 2-dimentional space. K-means clustering (K = 3) was carried out with 2-dimentional vector in the Sklearn package.i was calculated asReads were pre-processed as described above, followed by mapping to reference sequences with STAR. The RIP enrichment score for RNA i was denoted as The number of reads mapped to RNA 42 from secondary structures with constraints from icSHAPE-MaP data. First, RNA helices were built using a custom Python script adopted from Rosetta (available upon request). These helices were fixed throughout the following modeling steps. Second, de novo modeling was done through an established algorithm, Fragment Assembly of RNA with Full Atom Refinement (FARFAR)42. A Python script (FARFAR_setup.py) was used to call the Rosetta-built pipeline for FARFAR. 500 tertiary structures for each pre-miRNA per modeling run were produced. The top 50 structures with the lowest Rosetta energy were used for the distance measurement of the Dicer cleavage site (see below).de novo tertiary structure models of pre-miRNAs were produced by Rosetta5p_arm) or for that between C5′ on the first nucleotide next to the terminal loop on 3p arm and O3′ on the last nucleotide of each pre-miRNA (D3p_arm).The Dicer cleavage sites on pre-miRNAs were inferred from miRBase, based upon the annotated 3′ end of 5p or the 5′’ end of 3p miRNA sequences. The distance of Dicer cleavage was calculated using the physical distance between the fifth carbon atom (C5′) on the first nucleotide and the third oxygen atom (O3′) on the last nucleotide for each miRNA. The median distance was presented from the measurement of top 50 predicted tertiary structures for each miRNA. To calculate the arm length, it was measured for the physical distance between C5′ on the first nucleotide and O3′ on the last nucleotide next to the terminal loop on 5p arm (DFurther information on experimental design is available in the Supplementary InformationPeer Review FileSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Description of additional supplementary filesReporting summarySource Data files


Immunotherapy with nivolumab  has become the standard treatment for patients with metastatic renal cell carcinoma (mRCC) after progression to single-agent tyrosine kinase inhibitors. However, the optimal duration of immunotherapy in this setting has not yet been established.We retrospectively reviewed all patients treated with nivolumab at our institution from January 2014 to December 2021 and identified those who discontinued treatment for reasons other than disease progression (PD). We then associated progression-free survival (PFS) and overall survival following treatment cessation with baseline clinical data.Fourteen patients were found to have discontinued treatment. Four patients (28.6%) ceased treatment due to G3/G4 toxicities, whereas the remaining ten (71.4%) opted to discontinue treatment in agreement with their referring clinicians. The median duration of the initial treatment with nivolumab was 21.7 months (7.5-37.3); during treatment, two patients (14.3%) achieved stable disease as the best response, and the remaining twelve (85.7%) a partial response. At a median follow-up time of 24.2 months after treatment discontinuation, 7 patients (50%) were still progression-free. The median PFS from the date of discontinuation was 19.8 months (15.2 - not reached); a radiological objective response according to RECIST and treatment duration of more than 12 months were associated with a longer PFS. Three patients were re-treated with Nivolumab after disease progression, all of whom achieved subsequent radiological stability.In our experience, the majority of patients who discontinued treatment in the absence of PD were still progression-free more than 18 months after discontinuation. Patients whose initial treatment duration was less than 12 months or who did not achieve a radiological objective response had a greater risk of progression. Immunotherapy rechallenge is safe and seems capable of achieving disease control. Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults and accounts for 3-5% of new cancer diagnoses each year , 2. NowaIn recent years, immunotherapy in the form of immune checkpoint inhibitors (ICIs) has revolutionised the treatment of metastatic RCC.Nivolumab, an ICI that targets the programmed cell-death protein 1 (PD1), has become the standard treatment for patients with mRCC following progression to single-agent tyrosine kinase inhibitors (TKI) . In combHowever, the maximum duration of treatment differed in those trials. In the 2015 Checkmate 025 trial (nivolumab vs. everolimus for pre-treated mRCC), the first trial that paved the way for nivolumab in the management of RCC, treatment continued until disease progression or the development of treatment-limiting toxicities . In the The reason for limiting the maximum duration of immunotherapy treatment is the growing body of evidence indicating that the disease’s clinical control is often long-lasting and may be maintained even after therapy is discontinued. In fact, due to their unique mechanism of action, ICIs are capable of achieving long-term disease control in many solid malignancies, even after treatment discontinuation or interruption –12. TherData from retrospective analyses indicated that treatment interruption after a certain number of cycles could be safe for selected patients –13. MoreA patient-tailored “stop and go” approach could be an alternative option for selected patients in order to reduce overtreatment, limit the occurrence of treatment-related toxicities, and improve the possible financial toxicity of those therapies without compromising the treatment’s oncological results .This paper presents a retrospective analysis of patients treated with nivolumab at our institution, who opted to discontinue treatment in the absence of disease progression.We retrospectively reviewed all patients treated with nivolumab at our institution from January 2014 to December 2021 and identified those who discontinued treatment for reasons other than disease progression. Clinical data were extracted from electronic patient records.Inclusion criteria included a histological diagnosis of RCC, previous treatment with nivolumab interrupted in the absence of PD, and the availability of all necessary data.From electronic patient charts, we collected baseline clinical data, the reason for treatment discontinuation, the treatment’s oncological outcome , and data about subsequent treatments administered after disease progression. Adverse events were graded in accordance with the Common Terminology Criteria for Adverse Events (CTCAE) v5.0; radiological response was defined using Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 criteria.Treatment duration was defined as the time between the first and last dose of nivolumab. Progression-free survival (PFS) was calculated using the Kaplan-Meier method from the date of treatment interruption to the date of disease progression or death (whichever occurred first); progression-free survival was censored at the last patient follow-up visit without progression. Overall survival was calculated from the date of drug interruption to the date of death from any cause. For patients re-treated with nivolumab after disease progression, PFS for the second course of immunotherapy was calculated from the beginning of the second course until the occurrence of new disease progression.Key metrics were summarised by means of descriptive statistics. Patient PFS and OS were compared using the log-rank test and Cox’s proportional hazards method (when applicable). We performed univariate and multivariate analyses to determine the association between baseline characteristics and PFS from the time of treatment discontinuation; the covariates that showed any association with the oncological outcome with a p value of at least less than 0.1 in the univariate analyses were included in the multivariate analysis. Results were classified as statistically significant if their p-values were < 0.05. All statistical analyses were performed with “R” v4.0.5 and the “survival” package v2.44-1.1.At the time of their first visit to our institution, all patients gave their written consent for the use of their clinical data for scientific purposes. The study was conducted in accordance with the Declaration of Helsinki. Data collection was approved by the local Ethical Committee.Fourteen patients were found to have discontinued treatment for reasons other than disease progression. The median age was 77.7 years (range: 42.3-82.1 years). Eleven patients had been diagnosed with clear cell RCC (78.6%), one with papillary RCC, one with chromophobe RCC and one with RCC not otherwise specified. Twelve patients were treated with nivolumab in the second-line setting, while two patients were treated in the third-line. All but one patient had received nephrectomy prior to treatment. All patients were in good clinical condition at the start of Nivolumab treatment (ECOG PS of 0 or 1); 5 patients were classified as belonging to the good risk class according to IMDC criteria, while the remaining 9 patients were classified in the intermediate risk class; none of the patients were considered to be at poor risk. Patient clinical characteristics are summarised in The median duration of initial treatment with nivolumab was 21.7 months (7.5-37.3). During treatment, two patients (14.3%) achieved stable disease as the best radiological response, while the remaining twelve patients (85.7%) achieved a partial response. Twelve patients (85.7%) developed immune-related adverse events of any grade during therapy, requiring at least a brief interruption of nivolumab or treatment with systemic corticosteroids; four patients reported the onset of grade 3/4 toxicities . Data on treatment outcomes are reported in Ten patients (71.4%) opted to discontinue treatment in agreement with their referring clinicians; however, for 5 of these patients (50%), the previous occurrence of low-grade (G1-G2) adverse events was an important factor in their decision. The other four patients (28.6%) discontinued treatment after developing G3/G4 toxicities.At a median follow-up time of 24.2 months after treatment discontinuation, 7 patients (50%) were still progression-free. For 5 of the 7 patients who progressed, radiological progression was defined by the enlargement of known pre-existing lesions, and for the other 2, by the emergence of metastases at new sites .The median PFS from the date of discontinuation until disease progression was 19.8 months (15.2 - not reached); the median overall survival was not reached, with just one patient having died by the time of data cut off. Data on the post-interruption outcomes are reported in At univariate analysis, stable disease as the best radiological response and a treatment duration of less than 12 months were associated with a worse PFS Table 3;After disease progression, two patients were considered ineligible for other oncological treatments due to their poor clinical condition and were, therefore, only treated with best supportive care. One patient, whose CT scan revealed an oligoprogressive disease, was successfully treated twice in succession with stereotactic ablative radiotherapy and has not yet begun additional systemic therapy.Systemic therapy was initiated for the other four patients: due to the previous occurrence of immune-related colitis, one patient started third-line treatment with cabozantinib; the other three patients were re-treated with nivolumab. For two of these three patients, the cause for initial discontinuation was the emergence of an irAE (two grade 3 hypertransaminasemia) At the time of data cut off, the patients re-treated with nivolumab had been treated for 4, 5 and 12 months and are all progression-free; to date, no immune-related adverse event of any grade has been reported for either of them. Data on the treatments administered after disease progression and outcomes are reported in Immunotherapy has drastically improved the prognosis and natural history of patients with advanced renal cell carcinoma. The role of immunotherapy has been enhanced with the publication of recent trials, and a combination treatment with an immune checkpoint inhibitor is currently considered to be the standard of care in the first-line setting . NeverthDespite the fact that in the first and older trials, treatment with immune checkpoint inhibitors was continued until disease progression or the development of severe toxicities, ICIs have been shown to achieve long-term disease control even in the event of interruption  , 12. TheLong-term follow-up analysis of clinical trials using ICIs in melanoma and NSCLC demonstrated that many patients maintain the therapeutic benefit long after the end of treatment , 12.In the case of RCC, 27 patients with a response to nivolumab discontinued treatment in the Checkmate 025 trial and never received additional subsequent systemic therapy ; 13 of these patients were still alive and free from disease progression at the last follow-up .Many clinical trials are currently set for a maximum 2-year period of ICI treatment for all patients enrolled , 16. HowIn metastatic melanoma, a retrospective analysis of patients treated with anti-PD1 (pembrolizumab or nivolumab) for a median initial treatment duration of 12 months showed that the risk of relapse after treatment discontinuation was low, particularly in patients who achieved complete radiological response during treatment .Conversely, in non-small cell lung cancer (NSCLC), a randomised trial revealed that a fixed duration of one year seems to be inferior, in terms of PFS and OS, to continuous treatment with nivolumab in the whole population .Several authors have investigated the optimal duration and management of ICI treatment for RCC. In a recent phase II trial, 5 out of 12 patients (42%) who opted to discontinue nivolumab after achieving a radiological response within the first 6 months of treatment were progression-free one year after the discontinuation of treatment .Ornstein et al. conducted a phase II trial to evaluate the outcomes of intermittent treatment with nivolumab in a similar setting; of five patients who opted to discontinue nivolumab after obtaining a radiological reduction of 10% in tumor size, only one patient had to restart treatment at a median follow-up of 48 weeks .These small trials demonstrate that, for some patients, treatment interruption could be a viable option, but additional and larger studies are needed to increase the level of evidence and refine patient selection.However, following the decision to discontinue treatment, another important unanswered question concerns the immunotherapy rechallenge’s efficacy. Retrospective analysis in patients with other solid malignancies revealed an interesting response rate and a clinical benefit in patients re-treated with immunotherapy after disease progression (with the same ICI after a therapeutic pause or with a different ICI in the event of PD during treatment) , 20.In a retrospective, multicentric analysis of renal cell carcinoma, Ravi et al. found a response rate of 23% with low incidence of severe adverse events in a cohort of 69 patients  who underwent anti-PD1/anti-PDL1 rechallenge treatment . The occThe rechallenge strategy must be evaluated differently depending on whether the decision to discontinue therapy was due to the occurrence of toxicities or due to the patients’ or physicians’ preferences, as opposed to the progression of disease during treatment. Unfortunately, many studies, such as the abovementioned ones, did not distinguish between patients whose disease was under control or progressing when they discontinued treatment. These clinical situations are clearly distinct, and the results of re-treatment in one setting may not be applicable in another.In fact, recent trials specifically designed for patients after progression or a lack of response to treatment with a single ICI are evaluating the intensification strategy using combination treatment (TKI plus anti-PD1 or anti-PD1 plus another ICI) rather than a single ICI , 22.The final important question concerns the rechallenge’s toxicity profile. Many retrospective analyses demonstrated that, for patients who previously discontinued immunotherapy due to the occurrence of irAEs, these irAEs do not typically recur after the immunotherapy rechallenge’s commencement. Moreover, irAEs are usually milder and more manageable during rechallenge , 23–25. The majority of patients in our population who opted to discontinue treatment were safe and progression-free after more than one year from the start of the therapeutic break. As reported by other authors, the risk of progression was lower in patients who had been treated for more than 12 months and in patients who had previously achieved an objective radiological response. Re-treatment appeared to be safe for patients who had progressed; it is interesting to note that, despite the limitations of a short follow-up, no treatment-related adverse events were reported, in spite of the fact that two of the patients had initially discontinued treatment due to grade 3 toxicities (hypertransaminasemia). Accordingly, we decided not to re-treat the patient who had previously reported grade 3 colitis.Our analysis has several limitations. Due to the retrospective design, there was a selection bias in the population, which consisted of patients with a very good clinical condition and good prognostic characteristics at baseline. The small sample size limited the possibility of finding prognostic and predictive indicators for a prolonged drug holiday period; this could explain why many well-established prognostic factors, such as the IMDC class and performance status, did not seem to be associated with this PFS. Finally, radiological evaluation was performed as per the clinician’s decision, and radiological images were not re-examined.In our experience, the discontinuation of nivolumab treatment in a cohort of highly selected patients seems to be safe and capable of sustaining the disease’s long-term clinical control. Treatment duration of more than one year and the achievement of a radiological objective response were prognostic of longer progression-free survival from the date of treatment discontinuation. Rechallenge with nivolumab after the occurrence of progression seemed to be safe for the selected patients, including those patients who had previously reported the occurrence of certain toxicities.More studies are urgently needed to determine the optimal duration and management of treatment with ICIs, especially given the ever increasing importance of immunotherapy. An improvement in the selection of patients who can safely discontinue treatment with ICIs could result in a dramatic improvement in treatment customisation and individualisation.marco.maruzzo@iov.veneto.it.The data analyzed in this study is subject to the following licenses/restrictions: Available upon request. Requests to access these datasets should be directed to The studies involving human participants were reviewed and approved by Comitato Etico IOV IRCCS. The patients/participants provided their written informed consent to participate in this study.MM, DB, and VZ study design. EL, NC and AM data collection. MD, DB and FP data analysis. MD, DB and MM data interpretation and review. UB, DB and MM supervision. All authors: final review and approval of the final paper.This research received “Ricerca Corrente” funding from the Italian Ministry of Health to cover publication costs.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.


Immune checkpoint inhibitors (ICI) have become the cornerstone of treatment of certain malignancies. However, they can result in systemic toxicities including hepatitis. Societal guidelines recommend initial management with high dose steroids, then a slow taper as hepatitis resolves. However, there is significant variation in steroid response with some patients experiencing a relapse of hepatitis as steroid doses are tapered (“steroid relapse”).Identify clinical features that predict relapse, and to explore variations in steroid management, in patients with ICI hepatotoxicity.Patients receiving ICI in early phase clinical trials at Princess Margaret Cancer Centre, or treated at the Toronto Centre for Liver Disease for ICI hepatotoxicity, were included. Patients with CTCAE Grade (G)3 ICI hepatotoxicity (ALT >5 x ULN) were identified and clinical records reviewed for management and outcomes. Patients with an alternate cause for ALT elevation; who did not receive corticosteroids; or with HCC or viral hepatitis, were excluded.rd line therapy with Tacrolimus. Ultimately, hepatitis resolved in all patients.Between August 2012 and December 2021, 36 patients with G3 ICI hepatotoxicity were identified. Most  had metastatic melanoma. Thirteen received anti-CTLA-4/PD-1; 18 anti-PD-1 or anti-PD-L1, and 5 anti-CTLA-4 monotherapy. All patients initially received corticosteroids . Thirteen patients (36%) were steroid relapsers. Consistent steroid response was seen in 18 (50%). Age, sex, liver metastases, prior ICI exposure, peak ALT or starting dose of steroids  did not predict relapse, although relapsers were more likely to have been treated with combination anti-CTLA-4/PD-1 (7 (54%) relapsers, vs 3 (16%) responders, p = 0.02). Relapse occurred after a median of 14.5 days (range 8-111), and after taper to median 54% (5-100) of initial steroid dose. In responders, ALT normalisation occurred after median 14 days (range 3-56). In 27 patients where sufficient data were available, societal guidelines on ALT thresholds to initiate steroid taper were followed in 13 . However, initiation of steroid taper was delayed in responders compared to relapsers (after median 7 days (2-15) in responders vs 4 days (range 2-9) in relapsers, p = 0.04). Overall, 5 relapsers responded to re-escalation of steroids. Eight required additional treatment with MMF, and 4 required 3In patients with ICI hepatotoxicity, combination ICI therapy confers a higher risk of steroid relapse than monotherapy. There is significant heterogeneity in management of steroid dosing in patients with ICI hepatotoxicity. Delayed initiation of steroid taper may be associated with a reduced risk of relapse and warrants prospective evaluation as part of a standardised management algorithm.NoneNone Declared


This study aimed to investigate the possible factors affecting dentists’ behavior relating to performing oral cancer examinations as part of routine clinical examination. A total of 95 direct clinical observation sessions—utilizing an instrument consisting of 19 evidence-based observational criteria for oral cancer examinations—were observed by four calibrated dentists. Thirty-two final-year students, 32 interns, and 31 faculty members of Jazan Dental School were examined between April 9 and May 4, 2017. A descriptive analysis was conducted to investigate the frequencies/percentages of the performed observing criteria by all examiners. ANOVA and Tukey tests were carried out to investigate the difference between the examiner groups. A total number of 32 patients participated in the study, whereby each patient was examined by three different examiners from each group, as well as by the attending observer/s. Fewer than 50% of the examiners performed the clinical steps necessary for an oral cancer examination—for example, taking into account past medical history, as well as extra and intra-oral examinations. More than 90% of the examiners examined hard tissue, whereas fewer than 30% of them educated their patients about possible risk factors. A significant difference between examiner groups was found in favor of faculty members. A gap between knowledge and actual practice of oral cancer examinations was evident: majority of participants failed to perform the necessary steps for an oral cancer examination. Previous experience and confidence in performing oral cancer examination are possible explanations for the dentist’s behavior toward oral cancer examination. Global incidences of oral cancer are still rising, with South Asian countries having the highest incidence rates . In SaudMost oral cancer cases are detected at a late stage when the tumor has already metastasized to another location in the body . SeveralThe present study was performed according to the ethical standards of the institutional research committee, as well as the 1964 Helsinki Declaration. It received ethical approval from Jazan University (registry no. [CDREC-06]), dated 21 December 2016. All participants have consented prior to their participation, including with regard to the publication of the findings. The reporting of this present study followed the STROBE guidelines for reporting cross-sectional studies .The present study utilized a descriptive cross-sectional study design, in which direct clinical observation was carried out among final-year dental students, interns, and faculty members between April 9, 2017, and May 4, 2017. The main targets of this study were the dental interns who are the first line of treatment in JDS clinics and who oversee the completion of the primary dental charts of all new patients. The total number of interns was 40 at the time of planning this study. A personal invitation was sent to all the interns and an explanation of the study process was delivered to them in the form of two discussion sessions by the principal investigator (PI). To minimize effects on the behavior of clinicians under observation in dental examination sessions, the study and its process were explained to the participants without mentioning that the study would focus on the dental examinations and, in particular, oral cancer screening performance and the required related steps as a follow-up. We aimed to reduce the Hawthorne effect   were required. Items 1–10 related to reporting the patient’s general health, family history of cancer, habits, and diet. Items 11–16 related to the performance by the examiner of different steps for the head, neck, and oral examination. Item 17 was for obtaining a plain radiograph, and items 18 and 19 were for conditions when the further evaluation was necessary to diagnose potential positive oral cancer cases. Each item was given a weight of one, two, or three, based on the degree of significance and relevance as supported by current evidence [An instrument for the observation of oral cancer screening practice in form of checklist was developed and was based on the current available evidence relating to the recommended practice of oral cancer screening. The observation instrument included the most appropriate clinical steps to be taken during comprehensive clinical examinations of dental patients , 36, as evidence –53, withp < .000). Prior to the present study’s dental examination sessions, each participating patient was first examined by the observer/s who then observed the clinicians’ dental examination sessions. All 95 dental examination sessions were observed by the main observer and mentored by the PI. In addition, 18 of these sessions were observed by all the observers, while others were overseen by two or three observers  with Tukey’s HDS comparison between each occupation category. Additionally, Pearson’s correlations were run to investigate any correlation between patient age and total score by the main observer and mean total score by all observers. A qualitative approach was utilized to perform post-exam investigations in the form of two focus group discussions (FGDs) with the participants of the current observation study. The FGDs aimed to explore the possible factors explaining the observed findings. The 95 participants in the cross-sectional study were invited to participate in the follow-up discussions by direct contact.Descriptive analyses were run to investigate the participants’ demographics, as well as the frequency of the individual performance items in the observational checklist by the main observer. Furthermore, paired The total number of participating examiners was 95, with an almost equal distribution of gender and occupation . The total number of participating patients was 32, with ages ranging between 19 and 70 years old and with 62.5% males (see Table Table r = .807) between the main observer and the second observer (see Table p = .571 and fourth observer p = .062), except for the third observer (p = .018); see Table p = .007 and p = .031, respectively)  as they assumed it has no clear relationship with their patients’ oral health. Participants linked the cultural and religious unacceptability of alcohol use to the observed low score in item 8 . For items 6, 7, and 19 , female students and interns had higher scores than their counterpart. The participants mentioned that female students/interns are more vigilant to the oral changes associated with tobacco as they are used to examine mainly female patients who are usually non-smokers. Moreover, female participants mentioned that the tobacco advice they had given to the patients was based on their personal beliefs, as they did not receive formal training on tobacco counseling. In relation to item 17 (obtaining radiograph), participants denoted the reason for obtaining radiograph is related to the chief complaint only. Dental interns revealed that two factors could be related to their general low score in comparison to students and faculty members. The first one was because they rely on the other dentists whom the patient will be referred to do the next oral care/treatment. While the second factor was because they cannot perform full oral screening on each patient as they have a busy clinical schedule with large number of patients.The present study investigated possible explanations for dentists’ behavior by means of direct observations of routine dental clinical examination sessions. The interns in this study were recruited from the same group that were evaluated in two recent studies that included students, interns, and faculty members for their knowledge, attitude, self-efficacy, and opinions regarding oral cancer practice , 54, in The knowledge of oral cancer among dentists has been investigated thoroughly in previous studies and postulated to be related to dentists’ practice of oral cancer examinations –29, 55. Dentists have actual control over their practice of oral cancer examinations when they demonstrate their ability to perform the following essential sub-behaviors: extra- and intra-oral screening skills, obtaining radiographs, taking a biopsy, writing referral reports, specialist consultations, communicating with or counseling patients , specifying an oral cancer provisional treatment plan , and referring suspicious cases to specialized centers Table . AdditioDirect clinical observation methods strengthen this study by capturing the clinical steps that dentists may or may not have performed, which leads to a better understanding of the behavior’s potential causes. Due to the cross-sectional nature of this study, the findings need to be tested through experimental study design, in order to measure the effect of experience, skills, and self-efficacy on dentists’ behavior. Furthermore, having a main observer present, who observed all dental clinical examination sessions, added to the reliability of the comparison between different examiners. Similarly, having four observers to compare findings with added to the internal validity of this study. Moreover, the observational instrument that was developed for the study had not been tested in previous independent work. However, the findings of this study indicated that the developed instrument had the capacity to investigate oral cancer examinations as part of routine dental clinical examinations. All observers had strong statistical correlations, with no statistical difference being found between the three of them.To conclude, the present study has shed light on the gap that existed between knowledge and actual practice of oral cancer examinations by dentists. The practice of oral cancer examinations is a complex behavior that is influenced by multiple factors: oral cancer knowledge, perceptions, experience, self-efficacy, actual control, and other external factors such as the afforded clinic time per patient. Furthermore, experience and confidence are essential determinants for performing oral cancer examinations. Therefore, dental schools and decision-makers should be aware about the influence of these determinants on oral cancer examinations and should be stressed in the future interventions that intend to improve the practice of oral cancer examinations as part of routine dental clinical examination sessions.


People from the general population often tend to believe that psychiatric patients may be incurable, dangerous, and unpredictable. Stigma represents a critical issue which should be defeated. In spite of the interest of research, little is known about the relationship between personality traits and level of stigma toward people with mental illness.To evaluate whether certain personality traits can influence the level of stigma towards mental illness in a population of university students.A web-survey was spread on social networks between March and June 2020 through Google Forms. Eligibility criteria for inclusion were:1) Being 18 years of age or older; 2) Attending a degree course in an Italian University; 3) Provide informed consent. Socio-demographic characteristics of the participants were collected. Stigma was measured using the Attribution Questionnaire (AQ-27), personality traits were evaluated through the Big Five Inventory (BFI) and the Mental Health Knowledge Schedule (MAKS-i) investigated the knowledge about mental illness. Statistical analyses were performed using SPSS 24.0.We computed a multiple linear regression to calculate potential predictors of stigma, adjusted on the basis of the knowledge of mental illness. Results showed that age and faculty class were not related to stigma. Agreeableness (A) and Openness to experience (O) were associated with less stigmatizing attitudes. Conversely, Neuroticism (N) and Conscientiousness (C) seemed to predict higher levels of stigma.Our results suggest an interesting relationship between personality traits and stigmatizing attitudes, which deserves to be further studied. They also confirm the importance of implementing appropriate strategies against the stigma of mental illness.No significant relationships.


The alterations of gut microbiota have been associated with multiple diseases. However, the relationship between gut microbiota and adverse outcomes of hyperlipidemic stroke patients remains unclear. Here we determined the gut microbial signature to predict the poor outcome of acute ischemic stroke (AIS) with hyperlipidemia (POAH).Fecal samples from hyperlipidemic stroke patients were collected, which further analyzed by 16s rRNA gene sequencing. The diversity, community composition and differential gut microbiota were evaluated. The adverse outcomes were determined by modified Rankin Scale (mRS) scores at 3 months after admission. The diagnostic performance of microbial characteristics in predicting adverse outcomes was assessed by receiver operating characteristic (ROC) curves.Enterococcaceae and Enterococcus were increased, while the relative abundance of Lachnospiraceae, Faecalibacterium, Rothia and Butyricicoccus were decreased. Moreover, the characteristic gut microbiota were correlated with many clinical parameters, such as National Institutes of Health Stroke Scale (NIHSS) score, mean arterial pressure, and history of cerebrovascular disease. Moreover, the ROC models based on the characteristic microbiota or the combination of characteristic microbiota with independent risk factors could distinguish POAH patients and GOAH patients (area under curve is 0.694 and 0.971 respectively).Our results showed that the composition and structure of gut microbiota between POAH patients and good outcome of AIS with hyperlipidemia (GOAH) patients were different. The characteristic gut microbiota of POAH patients was that the relative abundance of These findings revealed the microbial characteristics of POAH, which highlighted the predictive capability of characteristic microbiota in POAH patients. Acute ischemic stroke (AIS) was a leading cause of death and chronic disability worldwide. Stroke survivors frequently had various complications, such as cognitive impairment and physical disability, which had a great impact on the quality of life . Recent Recent studies have emphasized that the characteristic gut microbiota (GM) are associated with AIS. It was reported that stroke patients showed significant dysbiosis of bacteria with enriched short-chain fatty acids (SCFAs) . Our preAlcaligenaceae and Acinetobacter could remarkably distinguish autism spectrum disorders from the healthy group  > 2.28 mmol/L or total cholesterol (TC) > 6.2 mmol/L or high-density lipoprotein (HDL) < 0.91 mmol/L or low-density lipoprotein (LDL) > 3.4 mmol/L. Exclusion criteria: application of antibiotics or probiotics within three months, restriction of diet, concurrent pregnancy, schizophrenia, bipolar disorder, or other serious life-threatening illnesses . The modified Rankin Scale (mRS) was applied to assess the post-stroke functional outcome of each patient in a 90-day follow-up after the stroke onset. The included AIS with hyperlipidemia were divided into the good functional outcome group (mRS score < 3) and the poor functional outcome group (mRS score ≥ 3).All hyperlipidemic stroke patients were collected basic information at enrollment, including sex, age, years of education, history of smoking and drinking, presence of hypertension and diabetes, and history of cerebrovascular disease. Hypertension was considered as blood pressure ≥ 140/90 mmHg. Diabetes was defined as fasting blood glucose ≥ 7.0 mmol/L or 2 h blood glucose ≥ 11.1 mmol/L in an oral glucose tolerance test. The blood samples were extracted on an empty stomach after fasting overnight and centrifuged at 1300xg for 10 minutes. The biochemical indicators analyzed included TG, TC, HDL, LDL, creatinine, vitamin B12, folic acid (FOA), uric acid (UA), homocysteine (Hcy), C-reactive protein (CRP), hypersensitive C-reactive protein (hs-CRP), fasting blood glucose (FPG), glycosylated hemoglobin, thyrotropin, free triiodothyronine (FT3), free tetraiodothyronine (FT4), mean arterial pressure (MAP), D-dimer, alanine transaminase (ALT), aspartate transaminase (AST) and troponin. Moreover, computed tomography (CT) and magnetic resonance imaging (MRI) were used to identify new lesions of patient. Stroke severity was evaluated based on the National Institutes of Health Stroke Scale (NIHSS) by professional physicians within 24 hours of admission. Sleep condition was also quantified through Pittsburgh Sleep Quality Index (PSQI) during hospitalization.® Manual of soil Kit , and the concentration and purity of which were detected with NanoDrop2000 UV-vis spectrophotometer . The hypervariable regions of the 16s rRNA gene were amplified using PCR with primers 338F: ACTCCTACGGGAGGCAGCAG and 806R:GGACTACHVGGGTWTCTAAT. Next, PCR products were recycled by 2% agarose gel, and paired-end sequenced (2 × 300) on an Illumina MiSeq platform . Alpha diversity was analyzed through Shannon and ACE. Principal coordinates analysis (PCoA) on the Bray-Curtis dissimilarity index was used for beta diversity analysis. The intestinal typing analysis was performed at the genus level by clustering samples with similar dominant microbiota structures into a class. Moreover, we identified the significant differences in relative abundance at levels of phylum, class, order, family, genus, and species by Wilcoxon rank sum tests based on the obtained community abundance data. Linear discriminant analysis (LDA) effect size (LEfSe) was applied to find significantly enriched taxa and their influence between the two groups using nonparametric Kruskal Wallis (KW) sum rank test, with thresholds of LDA score > 2.Fresh stool samples (200 mg) were obtained, and fed into a labeled 2 ml sterile centrifuge tube and quickly stored in a -80°C freezer. The bacterial DNA was isolated by E.Z.N.A. P value < 0.05 was considered to be of significance.Statistical analysis was carried out by SPSS V.22.0 . Chi-square test and multivariate logistic analysis were used to analyze the categorical variable data. Odds ratio (OR) and 95% confidence interval (95% CI) were figured out. The values of continuous variables were represented as median with quartile or mean with standard deviation (SD) based on the fact whether they were normally distributed, and compared by rank sum test or t-test respectively. The P < 0.001), and decreased MAP  were the independent risk factors of POAH.According to the follow-up mRS results, 231 hyperlipidemic stroke patients were divided into two groups: 58 POAH patients and 173 good outcomes of AIS with hyperlipidemia (GOAH) patients. As showed in Alpha diversity was evaluated by the Ace index , Faecalibacterium (P < 0.01) and Butyricicoccus (P < 0.05) were negatively correlated with the mRS score, while Enterococcus was positively correlated with the mRS score (P < 0.05). Spearman correlation heatmap . Moreover, the predictive model combined with the five genera and the three independent risk factors could also distinguish POAH from GOAH .We screened out the five genera as biomarkers according to the LDA value, including Enterococcus increased while the abundance of bacteria producing SCFAs decreased, which was closely related to independent risk factors, such as cerebrovascular history, NIHSS score, and MAP. Moreover, the characteristic microbiota and microbiota plus with the three independent risk factors could establish a distinction for predicting POAH. These results indicated that GM might provide novel microbial biomarkers for predicting POAH.This study revealed that GM feature of POAH was that the abundance of Enterococcus in POAH was enriched, and positively related to the mRS score, indicating that the abundance of Enterococcus might be related to the risk of POAH. It was reported that Enterococcus was an opportunistic pathogen in the gastrointestinal tract, and the risen level of Enterococcus was relevant to many neurological and metabolic diseases, such as Parkinson’s disease, Alzheimer’s disease and diabetes  and post-stroke affective disorder  . The divdiabetes . Enterocdisorder , which w as IL-6 , and fur as IL-6 , which l as IL-6 . Evidenctabolism . Hu X etpatients , suggestLachnospiraceae, Faecalibacterium, Rothia and Butyricicoccus. Moreover, Lachnospiraceae, Faecalibacterium, and Butyricicoccus were associated with lower mRS score. Lachnospiraceae, a primary producer of butyrate, was related to the functional prognosis of diseases  can be found below: NCBI, PRJNA894329.The protocol of the study was reviewed and approved by The Ethics Committee of the Second Affiliated Hospital of Wenzhou Medical University (LCKY2020-207). 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. JL, JS and SC designed the experiments. JC, BC, JM, JZ, QG, HX, YK and SY performed the experiments and conducted the statistical analyses. All authors contributed to the article and approved the submitted version.This work was supported by Clinical Medical Research Project of Zhejiang Medical Association (2022ZYC-D10).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.


Understanding the factors that play a role in the initiation of alcohol use and the subsequent transition to later alcohol abuse adolescence is of paramount importance from the context of developing better-targeted types of secondary (“pro-active”) prevention interventions . Peer and family influences together with temperament traits have been suggested to be of cardinal importance regarding the initiation of alcohol use. In addition to these factors neurobiological and genetic factors play a major role in the risk of developing alcohol abuse upon initiation. The presentation will highlight the different psychological, neurobiological, and social factors underlying the risk of the transition to abuse and dependence in adolescence. In addition, examples of targeted prevention interventions will be highlighted.No significant relationships.


Therefore, this work aimed to investigate the overall nutritional quality of these new products. Regulated information [Regulation (EU) 1169/2011], the presence/absence of nutrition or health claim and organic declarations, the gluten-free indication, and the number of ingredients were collected from the food labels of 269 commercial meat analogues currently sold on the Italian market. Nutritional information of reference animal meat products was used to compare the nutrition profile. As an indicator of the nutritional quality, the Nutri-Score of meat analogues and counterparts was also determined. Plant-based steaks showed significantly higher protein, lower energy, fats and salt contents, and better Nutri-Scores than the other analogues. All the meat analogues showed a higher fibre content than meat products, while plant-based burgers and meatballs had lower protein contents than meat counterparts. Ready-sliced meat analogues showed a lower salt content than cured meats. Overall, all these plant-based products showed a longer list of ingredients than animal meat products. Results from this survey highlighted that plant-based steaks, cutlets, and cured meats have some favourable nutritional aspects compared to animal-based products. However, they cannot be considered a “ The high consumption of red meat and mostly processed meat is currently debated for many reasons related to human, planet, and animal health. First, diets high in red meat and processed meat have been considered risk factors for a high number of deaths and years lived with disability (DALYs) in the last Global Burden of Disease study . HoweverFor all these reasons, there is currently an urgent call to promote plant-based diets limiting the consumption of meat and animal foods. Plant-based diets foresee the inclusion of crops  and their traditional derivatives , but also of a large plethora of plant-based foods developed to mimic animal foods. These products include plant-based drinks, cheese substitutes, and even meat analogues, with the latter reaching a 4.6 USD billion global market in 2020 and expecting 6 USD billion within 4 years . It is aMeat analogues—also known as meat substitutes—are plant-derived food products usually processed to resemble meat flavour, texture, and appearance. They can be derived from various vegetable sources. Soy and textured vegetable proteins are the most common, but pulses , cereals , mushrooms, and seeds  are also used , 6. MoreMeat analogues can be produced with two different approaches aiming at obtaining a fibrous structure, which is essential for the texture . In the The interest in these innovative products and, consequently, their purchase in supermarkets is constantly growing. Moreover, these products are perceived by consumers as healthier than meat , even thThis work is part of the Food Labelling of Italian Products (FLIP) study that systematically investigates the overall quality of the pre-packed foods of the most important food groups and related categories sold on the Italian market.The selection of samples was performed, as previously described by Angelino et al. , in the The inclusion criteria taken into consideration for the selection were the item's availability in at least one online shop and all the data to be retrieved on the pack or in the retailer's online shop. Exclusion criteria were: incomplete images of all the sides of the pack, unclear images of nutrition declaration or list of ingredients, and products marked as “product currently unavailable” on all the selected online stores during the data collection period.Caprinae meat ; other animals  and the related energy and nutrient contents were retrieved as described for meat analogues.Data extraction was conducted as described by Angelino et al. . For eacThe following additional information was collected: fibre content (g/100 g), number of ingredients, presence/absence of a gluten-free indication, and plant they were made of .Collected data were organised in a dataset where commercial products were sub-grouped depending on: (1) category, (2) type, (3) presence/absence of an organic declaration, (4) presence/absence of nutrition claims, (5) presence/absence of health claims, (6) presence/absence of indications related to gluten. All the plant-based products investigated were divided into two major categories: plant-based meat analogues (PBMA) and plant-based ready-sliced meat analogues (PBSMA). Among the PBMA, we identified different types of products they intend to resemble, i.e., steaks, burgers, meatballs, and cutlets. As for the PBMA, we considered as control products: steaks, both white meat and red meat, burgers, meatballs, and cutlets.Santé publique—France , this value was estimated by subtracting the energy provided by each macronutrient from the total energy, divided by 2 kcal/g. The percentage of fruit, vegetables, pulses, nuts and rapeseed, walnut, and olive oil was retrieved from the list of ingredients. As the salt is added to steak during cooking, different amounts of salt (from 0.05 to 0.5 g) were considered to determine the score of this control product. In comparing the analogue products and the meat counterparts, 0.5 g of salt was considered. For meat products retrieved in the Food Composition Database for Epidemiological Studies, Nutri-score was calculated considering nutrition values reported in the database, even though this type of front-of-pack labelling is not applied to the not pre-packed products.For all the products, the Nutri-Score was also determined by using the Excel sheet provided by the e—France . Sodium p < 0.05 of the significance level. The normality of data distribution was rejected through the Kolmogorov–Smirnov test. Therefore, data related to energy, macronutrient, fibre, and salt were expressed as median and interquartile ranges. In order to investigate differences among categories, the Kruskal–Wallis non-parametric one-way ANOVA for independent samples with multiple pairwise comparisons test was used. Differences between products with or without organic declaration, nutrition and health claim declaration, and indication related to gluten were assessed by using the Mann–Whitney non-parametric test for two independent samples. In addition, nutritional values of meat replacer products were compared to those of control counterparts using the Mann–Whitney non-parametric test for two independent samples. Results were graphically shown using Origin software .The statistical analysis was carried out using the Statistical Package for Social Sciences software  and performed at Two hundred and ninety meat analogues were collected. After removing products not respecting inclusion criteria, a total of 269 products were considered in the final analysis, 229 PBMA and 40 PBSMA , 2. PBMAOverall, 59.3% of the PBMA products had pulses as the main ingredient, while 22.7 and 18.0% were mainly based on vegetables  and (pseudo)cereals , respectively. Among the PBSMA, 45.0% were based on cereals , 25.0% on pulses , 7.5% on oils , and the remaining 22.5% on other ingredients (water emulsions of different ingredients) (data not shown).As for controls, 25 out of the 225 items retrieved from the online stores were removed because not respect inclusion criteria. Besides these 200 commercial control products, 69 controls were obtained from the Food Composition Database for Epidemiological Studies in Italy . Controlp < 0.05). The median protein content was 14.0 (9.8–17.0) g/100 g, and the steaks had a significantly higher protein content than the other analogues. Overall, analogue steaks were also significantly lower in total fat, saturates, total carbohydrates, sugars, fibre, and salt than the other categories. The other categories  did not differ from each other for total fat, saturates, sugars, fibre, and protein contents (p > 0.05). Conversely, they differed from each other for salt, of which cutlets had the highest content (p < 0.05). In addition, cutlets also differ from burgers and meatballs for the total carbohydrates, having a significantly higher content.The median energy content of all plant-based meat analogues was 198 (155–230) kcal/100 g, with steaks being the analogues with the lowest content  1169/2011 , and theThe median energy content of all these products was 212 (198–247) kcal/100 g, with protein and fats as the main nutrients contributing to the energy content. Products bearing at least a nutrition claim were significantly lower in energy, total fat, saturates, and salt than products without a nutrition claim. The products that reported a protein claim, in addition to being, as expected, higher in protein, were significantly lower in total fat. Intriguingly, products with and without a claim on fibre did not significantly differ for their fibre content. Nutrition declaration of organic products was not different from conventional counterparts.p < 0.05), while plant-based cured meats showed a significantly lower energy content than controls . The salt content differed only between PBSMA and cured meats (p < 0.05).On the contrary, all the meat analogues showed a higher total carbohydrates, sugars, and fibre content than controls , higher in the absolute value than the median number [9 (6–10)] found in the commercial meat products. Instead, the plant-based meatballs showed a similar number of ingredients [13 (11–15)] as controls [13 12–15)] as well as the plant-based cutlets had a similar number of ingredients [14 (12–17)] as meat cutlets [13 (11–15)]. The PBSMA showed a number of ingredients around 13 (8–15). It was not possible to estimate the median number of ingredients for cured meats controls since this information is not provided in the database  had a D score. The plant-based cured meats had 32.5% of products with an A and B score, 65% of products with a C and D score. Only 2.5% of plant-based cured meats had an E score, while 69.2% of cured meats had that score. The remaining 30.8% of cured meats had a C and D score.Data shown in To the best of our knowledge, this is the first survey to evaluate the nutritional quality of several different types of PBMA and PBSMA sold on the Italian market. The critical evaluation of such products falls into a field in great expansion—the conception and development of meat analogue products—seen the consistent promotion of a transition towards plant-based and sustainable dietary models . Indeed,N = 50), which were lower in protein and higher in carbohydrate than meat burgers . The comparison with worldwide data is not easy. Despite the consumer attraction for these products, there are not so many published surveys on the presence of PBMA and PBSMA in the large-scale distribution. A very recent cross-sectional study considered the nutritional facts of 207 meat analogues  and 226 meat products available from 14 retailers in the UK . Althoug burgers , differeSafefood” has identified on the Irish market 354 analogue products, including also other categories not considered in the present survey, i.e., fish substitutes and pastry-based meat substitutes   , while irotein”)  this valrotein”) . These lrotein”) , the abstout court animal-derived protein substitutes due to: (i) a different PDCAAS, (ii) presence of trypsin inhibitors—usually inactivated by heat—which affects the protein digestibility, (iii) deficiency in some essential amino acids  availability of essential amino acids compared with unprocessed or minimally processed soy protein; (ii) protein digestibility-corrected amino acid score (PDCAAS) close to 1.00, referring value for animal-based proteins; (iii) improvement in colour, flavour and texture parameters of the products . Cerealsno acids . Besidesno acids , 31].Moreover, unlike meat which is a protein-rich food, cereal-based products have a higher content of carbohydrates and sugars in comparison with their meat counterparts. As for cereal-based analogues, pulses also lead to having carbohydrates and sugars in the final product. Therefore, these substitutes, which aim to replace meat, also add carbohydrates and sugars to the diet. On the contrary, meat products are not contributors to the intake of sugars, already widely introduced in our diet.In addition to this, carbohydrates are also added to the products because of their binding capacity. They are added as starches and flours to improve products' texture and gums to improve stability . Adding As for carbohydrates, other ingredients are added to the formulations to contribute to the final product. Salt is added as a seasoning but also for toughening the product. Even if in small quantities, the presence of salt involves a change in the structure of the proteins which allows obtaining the desired structure of the final product . Our surBesides comparing the nutrition facts, PBMA, PBSMA, and meats were also compared by the Nutri-Score. This is one of the most common front-of-package labels adopted in several countries. There is still an intense debate whether it is a good way to explore the nutritional quality of food items , 33. NutOur study has several strengths and limitations worth to be noted. Among strengths, to the best of our knowledge, this is the first survey investigating the nutritional quality and the Nutri-Score of PBMA and PBSMA sold in Italy, retrieving the large majority of products currently on the market. Moreover, focusing on information reported on the food labelling, we were able to focus not only on nutrition declaration but also on other aspects such as the presence of nutrition and health claims and the presence of gluten-free or organic declarations. However, this point can also be considered as a limitation since we cannot consider data on nutrients that are not mandatory based on the current European legislation . Moreover, we cannot exclude the presence on the marker of other items sold in other minor retailers or other channels .The first finding of the present survey on the nutritional profile of PBMA and PBSMA sold on the Italian market relates to the huge number of products present nowadays on the market. Moreover, wide variability in the nutritional values among products was observed. This variability was related to the heterogeneity of these products in terms of types and ingredients used. As for animal meats, plant-based steaks resulted nutritionally different from the other categories, showing a higher protein amount and a lower energy, other macronutrients, and salt content. The Nutri-Score values were more favourable in the meat analogues compared to the animal counterparts. Concerning nutritional and health claims and organic ones, as previously reported in other FLIP study papers, a nutritionally significant advantage of PBMA and PBSMA boasting such claims has not been observed compared to their counterparts.tout court alternative to animal meat. Thus, further studies focusing on evaluating the biological value of protein from plant-based sources and the bioaccessibility of micronutrients are needed. In addition, it is noteworthy the need to improve the formulation of meat analogues in terms of the number of added ingredients, processing, etc. At last, there is also a need for adequate nutritional education programs in order to increase consumers' knowledge and awareness about the differences between animal- and vegetal-based products. All these steps before and during the shopping time are the most important decisional times for the customers, as they drive their intention-to-buy and, finally, the consumption of products, which will directly impact their health.The similarity of the nutritional values of plant-based alternatives and animal meats retrieved by the nutrition facts, and more for steaks, should not induce the consumer to consider PBMA and PBSMA as animal meat equivalent or alternatives. In the present manuscript, as in a previous FLIP manuscript focusing on plant-based beverages vs. milk, it has been widely discussed that the difference in terms of the biological quality of proteins and the micronutrient amounts and bioavailability does not allow to consider PBMA and PBSMA a The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.Margherita Dall'Asta, Department of Animal Science, Food and Nutrition, Università Cattolica del Sacro Cuore, Piacenza, ItalyMarika Dello Russo, Institute of Food Sciences, National Research Council, Avellino, ItalyDaniele Nucci, Veneto Institute of Oncology IOV-IRCCS, Padova, ItalyStefania Moccia, Institute of Food Sciences, National Research Council, Avellino, ItalyGaetana Paolella, Department of Chemistry and Biology A. Zambelli, University of Salerno, Fisciano, ItalyVeronica Pignone, Freelance Nutritionist, San Giorgio del Sannio, ItalyAlice Rosi, Department of Food and Drug, University of Parma, Parma, ItalyEmilia Ruggiero, Department of Epidemiology and Prevention, IRCCS Neuromed, Pozzilli, ItalyCarmela Spagnuolo, Institute of Food Sciences, National Research Council, Avellino, ItalyGiorgia Vici, School of Biosciences and Veterinary Medicine, University of Camerino, Camerino, Italy.SC was involved in the protocol design, data analyses, and in the interpretation of results and drafted the manuscript. DA was involved in the protocol design and in the interpretation of the results and contributed to the drafting of the manuscript. TT was involved in the protocol design and critically reviewed the manuscript. NP participated in the conceptualisation of the study, in the interpretation of the results, and critically reviewed the manuscript. DM was involved in the interpretation of the results, conceived and designed the protocol of the study, critically reviewed the manuscript, and had primary responsibility for the final content. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.