dataesr/scientific-paragraphs-categorization huggingface dataset:
2. 5  [34]. The sequences produced in this study (OR229954-OR229985; Table  1 ) were compared to 48 reference genomes retrieved from GenBank database for DENV-1, to 57 reference genomes for DENV-2, and to 45 reference genomes to DENV-3 (Table  S1 ). In Guadeloupe (IPG and CHUG), 682 cases were confirmed positive for DENV by real-time RT-PCR, and 522 of these were tested for serotype (Figure  1A ). This revealed the predominance of DENV-2 (81. 8%, N = 427), followed by DENV-1 (11. 3%, N = 59) and DENV-3 (6. 9%, N = 36). During the 2019-2021 epidemic, the number of cases increased drastically on two occasions: between October 2019 and March 2020 (N = 148), and between August and December 2020 (N = 297) (Figure  1A ). Throughout the epidemic, DENV-1 was recorded at a low level, and DENV-3 appeared in the second half of the epidemic in August 2020, with very low circulation until the beginning of 2021 (Figure  1A ).
Two Different DENV-3 Genotype III Introductions in Guadeloupe and Martinique The complete genome of a panel of nine DENV-3 sequences (two from Guadeloupe and seven from Martinique), with a length of 10,649 bp, was obtained, and a multiple alignment was performed with GenBank sequences (Tables  1  and S1 ). No recombination events were found between the sequences from Guadeloupe and Martinique, or with those from GenBank, used to construct this phylogenetic tree. All the DENV-3 sequences of the study belonged to genotype III (Figure  4 ). However, they grouped into two different.
Two Different DENV-3 Genotype III Introductions in Guadeloupe and Martinique The complete genome of a panel of nine DENV-3 sequences (two from Guadeloupe and seven from Martinique), with a length of 10,649 bp, was obtained, and a multiple alignment was performed with GenBank sequences (Table  1  and Table  S1 ). No recombination events were found between the sequences from Guadeloupe and Martinique, or with those from GenBank, used to construct this phylogenetic tree. All the DENV-3 sequences of the study belonged to genotype III (Figure  4 ). However, they grouped into two different clades (Clade 1 and Clade 2), with between 2. 9% and 3. 1% divergence, and grouped under a node with a bootstrap of 100. The CHUM 11, CHUM 21, and CHUM 22 sequences belonged to Clade 1 (99. 7% of similarity) and grouped with sequences originating from Africa (Burkina Faso MT261978 and Ethiopia ON890788; 98.
Clade 1 grouped with samples with Caribbean/Central American origins from Saint Martin and Florida collected in 2020 and 2021 and emphasize an active circulation of the clade in this region. Clade 2, corresponding to an isolate from Guadeloupe collected in 2020 (IPG 8), grouped with strains from South America. This strain is linked to other sequences which circulated in Peru, Venezuela, and Colombia between 2015 and 2021, sharing 98. 1% identity. DENV-1 genotype V is known as the predominant DENV-1 genotype, which has circulated in South America for over 50 years  [40]. During the latest epidemics in Colombia and Venezuela, which occurred in 2015-2016 and 2018-2019, the DENV-1 strains isolated also belonged to genotype V  [44]. Although the DENV-1 serotype was recorded at a low level in both Guadeloupe and Martinique islands, all these phylogenetic data revealed the active circulation of this serotype and especially genotype V in Americas.
DENV-2 was the predominant serotype circulating in Guadeloupe, whereas it was detected at a low level in Martinique. In both islands, genetic data revealed the presence of the cosmopolitan genotype only. All the strains from the two territories were, therefore, genetically homogeneous and belonged to the same clade. The 19 strains sequenced showed a high homology with sequences from the Indian Ocean (Reunion Island and the Seychelles) and India, which circulated between 2016 and 2018 (98. 9%). Unfortunately, as there were no recent sequences published on GenBank, we could not determine the importation origin of these strains into the two French territories. This may be an indirect consequence of the COVID-19 pandemic, as, since the end of 2019, all sequencing efforts appeared to be mainly focused on this virus  [45].
Funding: This work has been notably supported by  Programme Opérationnel FEDER-Guadeloupe-Conseil Regional "Une Santé" 2021-2024  and the  INTERREG CARAIBES "CARES" program  (no. 5004). M. G. V. S. and E. C. are partially funded by  Calmette & Yersin doctoral  and postdoctoral fellowships from the  Institut Pasteur Department of International Affairs. The funders played no role in the decision to publish or in the preparation of the manuscript.
366 ± 0. 002 (stat. ). For each R T bin the intervals under the N T column are inclusive meaning that for 0 ≤ R T < 0. 5, N T is equal to 0, 1, 2 or 3. Figure  3  shows the unfolded N T and R T probability distributions in the transverse region integrated over all the events with the leading particle along with different model predictions. For each model, the ⟨N T ⟩ corresponds to the mean value of the corresponding N T spectrum. It is observed that PYTHIA8 Monash and PYTHIA8 ropes give the best qualitative description of the N T distribution, while EPOS LHC (HERWIG7) overestimates (underestimates) the data for N T > 10. However, when R T is computed, all the models underestimate the data for R T ≳ 2. This is because the models poorly describe the low-N T region, so they predict larger ⟨N T ⟩ values than the measured ones.
We sought to identify additional splicing-derived, pre-miRNAlike hairpins, using deep sequencing data of intermediate-length RNA species. Currently, library construction of intermediatelength RNAs (which should contain pre-miRNA hairpins) is known to be inefficient and highly biased, due to challenges in linker ligation to structured RNAs. Consequently, very few such datasets are extant, in contrast to 10,000s of published small-RNA sequencing datasets. We focused on three such datasets of $50-100 nt species from human samples: (1) HEK293T Ago2-IP samples from the Brian Gregory lab,  49  (2) total normalized HeLa samples from the Burroughs group,  50  and (3) a newly generated dataset from K562 cell Ago2-IP material (see STAR Methods). Inspection of the vicinity of intron boundaries revealed numerous loci with intermediate-sized reads, but few or no corresponding small-RNA reads.
Accordingly, these escaped our previous annotations of mirtrons from hundreds of small-RNA datasets. 23 For example, the 27th intron of the recently recognized miRNA pathway factor Ankrd52 51 yielded >10,000 hairpin reads, but no mature miRNAs, in the Gregory lab paired Ago2-IP datasets (Figure  2B ). The Ankrd52 hairpin resides precisely at the 3 0 end of the intron, with an inferred 5 0 tail of several hundred nucleotides. Although corresponding small RNAs were not present in the paired HEK293T Ago2-IP dataset, a low level of Ankrd52 small RNAs from the hairpin arms was detected (Figure  2B ) using other aggregated small-RNA datasets (Table  S1 ). Thus, it is subject to a very modest level of dicing, but the predominant species seem to be hairpins. Loci such as these encouraged systematic analysis of mammalian intermediate-sized reads in the vicinity of splice sites.
Applying stringent criteria, we identified 273 newly recognized human intron termini that generate >30 intermediate-sized RNAs with pre-miRNA-like structure (Table  S3 ). We provide additional compelling examples of such loci in Figure  S1 , emphasizing that these loci generally yielded numerous intermediate RNAs but few or no small RNAs in the paired Ago2-IP datasets. These mirrored the subcategories of known mammalian mirtrons,  23  in that most were 5 0 -tailed hairpins followed by whole-intron hairpins, with a small subset of 3 0 -tailed hairpins.
e. , mature miRNAs). In this study, we now recognize a substantial population of structured, splicing-derived RNAs (ssdRNAs). These lack compelling small RNA evidence, but exhibit other hallmarks of mirtron hairpins. We document ssdRNAs in association with Dicer and/or Ago2, but they may also potentially exist in a free hairpin state. We emphasize that, beyond these confident annotations, additional intermediate-sized loci were plausible candidates but fell below read cutoffs, had overly heterogeneous termini, or had suboptimal structures. As these current annotations derived from only three cell-line datasets, additional loci may prove confident with broader cell/tissue profiling. The Gregory HEK293T intermediate RNA library was especially useful for these annotations, and many loci were detected only in this dataset (Table  S3 ).
However, we caution against inferring this to reflect cell specificity, as the efficiency of pre-miRNA cloning strategies and their individual biases likely vary widely across datasets. For example, the Burroughs HeLa data are from total RNA and not Ago2-IP, our K562 data may have less efficient hairpin cloning, or perhaps the Gregory data may exhibit PCR skew. Nevertheless, many of these newly recognized intronic hairpin loci were co-detected by long reads in two or more of the datasets, indicating their breadth. For example, of loci confidently annotated from HEK293T, HeLa data share 64 loci with R2 hairpin reads and 21 with R10 hairpin reads, while K562 expresses 139 loci with R2 hairpin reads and 56 with R10 hairpin reads (Table  S3 ). In addition, six known mirtrons were present in HeLa and/or K562 data, but not HEK293, and four ssdRNA loci were annotatable only from the HeLa data (none were exclusive to K562).
A B Figure  2. Exemplar mirtron and ssdRNA loci (A) A previously annotated mirtron (mir-1226) that is a definitive Dicer substrate, as evidenced by phased miRNA-5p, miRNA-3p, and terminal loop reads. However, paired intermediate-length and small RNA Ago2-IP data from HEK293T cells show that mir-1226 is predominantly uncleaved. (B) Example of a newly annotated ssdRNA derived from an intron of the miRNA factor Ankrd52. This locus generated abundant hairpin reads, but no small RNAs were detected in matched small and intermediate-sized Ago2-IP data from HEK293T cells (bottom), although inspection of 756 aggregated small-RNA datasets reveals modest small-RNA production from its hairpin arms (UCSC genome browser tracks).
Mammalian mirtrons belong to a larger class of ssdRNAs To provide a basis for understanding the properties of these newly recognized loci, we first examined 480 previously annotated mirtrons across a range of informative datasets. 23 Using the paired datasets of 50-100 and 20-25 nt RNAs from HEK293T Ago2-IP samples,  49  we observed that previously annotated mirtrons at 3 0 intron boundaries (i. e. , conventional and 5 0 -tailed mirtrons) were supported by substantial intermediatesized-and small-RNA reads. Because we previously annotated mirtrons from numerous small-RNA datasets, we also evaluated their coverage across aggregated data (Table  S1 ). As expected, the vast majority yielded small RNAs at a stringent cutoff (>10 reads per million mapped reads [RPM]) and exhibited paired read pileups that reflect 5p/3p duplexes (Figures  3A  and 3B ).
4] [55]  Nevertheless, as endogenous mirtrons typically generate only modest amounts of small RNA (Figures  3A  and 3B ), and are mostly not conserved,  23, 56  this raises questions as to their relevance to gene regulation. In fact, miRNAs must typically achieve substantial abundance to exert meaningful regulatory impacts, in part due to a plethora of low-affinity fortuitous sites throughout the transcriptome. 2  With this perspective, we assessed the relationship of known mirtrons and our newly recognized ssdRNAs with miRNA factors. We first analyzed two Dicer photoactivatable ribonucleosideenhanced crosslinking and immunoprecipitation (PAR-CLIP) datasets from HEK293T. 57, 58 Although Dicer and Ago proteins form complexes, the stringency of CLIP protocols is presumed to reduce indirect interactions.
Although mirtrons were not previously reported to associate with Dicer, metagene plots show positional enrichment that matches well to the annotated hairpins (Figure  3E ). Strikingly, even though ssdRNAs are by definition poorly diced, we observe a comparable local spike of Dicer occupancy precisely on ssdRNA regions (Figure  3G ). For both classes (B) Meta-analysis of known mirtrons across hundreds of small-RNA datasets shows a characteristic ''double peak'' reflecting 5p/3p duplexes. (C) Positional properties of 270 newly recognized ssdRNAs in HEK293T Ago2-IP library show they generate specific 3 0 -intron terminal intermediate-sized species with few small RNAs or adjacent exonic RNAs (i. e. , conventional ssdRNA or 5 0 -tailed ssdRNA). (D) Meta-analysis of ssdRNAs across hundreds of small-RNA datasets shows a signature of Dicer cleavage.
In addition, the rarer classes of mirtrons and ssdRNAs that reside at 5 0 intron termini were similarly detectable in both Dicer and Ago2 CLIP-seq data (Figures  S2A-S2G ). Overall, about half of known mirtrons and newly recognized ssdRNAs bound Dicer, and 80% of mirtrons/50% of ssdRNAs were associated with Ago, at levels >1 read/10 M. Although this is a modest level, no such positional enrichment on ssdRNAs was observed in CLIP-seq datasets of other core miRNA factors such as Drosha  62  or DGCR8  63  or a distinct RBP regulator, Pum2  64  (Figures  3I-3L ). We note that DGCR8 was substantially enriched precisely at canonical pre-miRNAs, but Drosha was not (Figure  S2H ). This may reflect that DGCR8 is technically easier to capture in association with pri-miRNA transcripts than the nuclease Drosha.
73  Thus, different TENTs have overlapping activities on certain miRNAs, but opposite activities on different miRNA substrates; they also have overall minor effects on accumulation of most miRNAs. 74  Our initial inspection of a then-limited set of mammalian mirtrons (11 in human and 20 in mouse) revealed that all mirtron-3p species were heavily tailed, similar to invertebrate mirtron-3p species. 75  Now, meta-analysis of the tailing properties of our highly expanded catalog of mammalian mirtrons,  23  across numerous public small-RNA datasets (Table  S1 ), broadly reaffirms this perspective. In particular, mirtron-5p reads exhibit similar modest U/A tailing, comparable to aggregate canonical miRNA-5p and miRNA-3p reads, while mirtron-3p species accumulate high amounts of untemplated adenylation and particularly of untemplated uridylation (Figure  S4 ).
The highly preferential modification patterns of mirtron-3p species implied that their hairpin precursors are the tailing substrate. For example, this was seen in the cases of pre-mir-1226 and mirtron-Ankrd52 (Figure  2 ). Of note, the dominant hairpin reads of mirtron-Ankrd52 have one or two nucleotides trimmed from the 3 0 -SA, and thus, tailed reads are a minority of the overall hairpin reads. However, for pre-mir-1226, >99. 9% of hairpin reads carry 3 0 untemplated modifications, mostly U tails and a smaller fraction of A tails. To evaluate this more systematically and uniformly, we compared canonical miRNAs and ssdRNAs/mirtrons using matched datasets of Ago-bound intermediate-length and small RNAs from HEK293T cells. 49 As expected, the bulk of canonical pre-miRNAs and mature miRNAs were unmodified (Figures  3N  and 3O ).
For these reasons, and unlike with canonical pre-miRNAs,  31, 32  it is difficult to assess mirtron hairpin termini systematically. However, tailing of ssdRNA/mirtron hairpins with 1-nt 3 0 overhangs or 5 0 overhangs may yield structures that are more favorable for dicing (Figure  S7 ). Thus, akin to group II let-7 members, abundant mirtron hairpin tailing by multiple TENT enzymes may paradoxically aid the dicing of a population of ssdRNAs/mirtrons. The newly recognized class of ssdRNA loci in this study intrinsically does not generate much small RNA, precluding their analysis. Only 12 ssdRNAs had even one small RNA in any of our newly made datasets, and we did not observe directional increase in TUT4/7 ± TENT2 mutant cells, except for three paralogs of the known mir-6511 mirtron family. The fact that tailing loss was insufficient to induce ectopic ssdRNA-derived small RNAs may reflect that they are also largely suboptimal Dicer substrates.
25, 26 This seems to be accounted for by intrinsic recognition of intronderived hairpins bearing signature 3 0 splice sites by Tailor. 90, 91 ammalian mirtrons similarly evolve extremely rapidly, as only a few percent of ssdRNAs/mirtrons in humans are shared in mouse. 23 However, is there also evidence that the inferred intragenomic conflict between splicing-derived hairpins and tailing pathways has had collateral effects on the modification and/or evolution of mammalian canonical miRNAs? If so, that might be the clearest evidence that the role of ssdRNA/mirtron tailing in mammals is to suppress their incorporation into miRNA regulatory networks. We first addressed this by performing a meta-analysis of aggregated human small-RNA data (Table  S1 ). Even though the overall level of modifications to canonical miRNAs is very low, by combining numerous datasets, we gained sufficient power to resolve trends for preferred trends of untemplated additions to different individual 3 0 miRNA termini (Figure  7 ).
We then mapped small RNA-seq reads from these datasets to the hg38 genome assembly. Unmapped reads were iteratively trimmed one nucleotide each iteration retaining a read length of 15 nt, and then mapped to the genome using Bowtie with no mismatches, up to 30 iterations. The reads were required to match the micro-RNA mature sequences of both canonical miRNAs and mirtrons. 23 with at least 15 nt overlap and within 2 nt of the 5' end. The reads mapped to the spike-in sequences were counted. We normalized small RNA-seq by total spike-in reads in each library. We performed small RNA differential expression analysis between TUTase double and triple mutants against wild type using Bioconductor R package Limma.
ssdRNA identification and annotation The sequencing coverage bigwig files from intermediate-length RNA libraries were merged. We applied a custom method to call peaks genome-wide as follows. We first called peaks genomewide with different coverage cutoffs: R 2 RPM, R 5 RPM, and R 10 RPM. The peaks were combined from different cutoffs and merged to keep the unique peaks by filtering out the peaks with lower cutoffs if they overlapped with those with higher cutoffs. The called peaks were annotated using Ensembl gene annotation GRCh38 (v100), and the peaks with a distance of % 10 nt to the intron ends and R 30 intermediate-length RNA reads were retained. The known RNAs and mirtrons, as well as the reads from sRNA, intermediate-size, AGO CLIP-seq and DICER CLIP-seq datasets, were further used to annotate these peaks.
ACKNOWLEDGMENTS We thank  Fuqu Hu  for conducting some initial analyses of small-RNA responses to ectopic TENT enzymes,  Erik Ladewig  for initial studies of mirtron tailing, and  Jorgen Kjems  for sharing mirtron plasmids. S. L. was supported by a training award from the  NYSTEM , contract  C32559GG , and the  Center for Stem Cell Biology at MSKCC. Work in S. P. lab  was funded by the  European Research Council  ( ERC-CoG-647455 RegulRNA ) and was performed under the framework of the  LABEX :  ANR-10-LABX-0036_NETRNA  and  ANR-17-EURE-0023. J. W. was supported by an  Australian Research Council (ARC)   Future Fellowship  ( FT16010043 ) and an  Australian National University (ANU) Futures Scheme. Work in E. C. L. lab was supported by  NIH  grants  R01-GM083300  and  R01-HL135564  and by the  MSK  Core Grant  P30-CA008748.
METHOD DETAILS Processing and curation of human small RNA, intermediate-sized RNA, and CLIP-seq data We collected 756 small RNA, 8 intermediate-length RNA, and 255 CLIP-seq datasets from various tissues/cell lines from NCBI GEO/ SRA. The accession numbers of datasets analyzed in this study are summarized in Table  S1. Amongst these, 180 human small RNA datasets were added since our previous meta-analysis. 22 Of particular importance to the annotation of splicing-derived RNAs in this study were datasets of human intermediate-length RNAs, generated by sequencing of Ago2-associated RNAs from HEK293T cells  49  or from HeLa total RNAs depleted of abundant ncRNA species using antisense LNAs. 50 fter removing 3' adaptor sequences, we mapped small RNA reads to human (hg38) genome assembly. Unmapped reads were iteratively trimmed one nucleotide each iteration retaining a read length of R15 nt, and then mapped to the genome using Bowtie with no mismatches, up to 5 iterations.
To further support the benefits of NPs-PLGA on autophagic clearance, autophagic flux was assessed in the presence of Baf A1. Consistently, Baf A1-induced blockade of autophagic flux was mitigated by NPs-PLGA, as indicated by the smaller accumulation of GFP + RFP + vesicles (Figure  S6B ) and lower LC3II and p62 protein levels (Figure  S6C ). Altogether, these data support the beneficial role of NPs-PLGA in the improvement of Dox-impaired autophagic flux in cardiac cells.
6 mg for SMPs) was dissolved in DMF (15 mL for NPs, 80 mL for SMPs), and EDC (9. 3mg for NPs, 24. 8 mg for SMPs) and NHS (0. 7 mg for NPs, 1. 86 mg for SMPs) were added to the PLGA solution. Then, the PLGA solution was added to the particle suspension, which was agitated at 40°C for 2h. The particles were recovered by centrifugation (4000 g for 10 min), washed three times with distilled water, and dried under vacuum. Particle shape and size were examined via transmission electron microscopy (TEM), using a Philips Model CM20 microscope (Eindhoven, The Netherlands) or scanning electron microscopy (SEM, JEOL JSM 7800F). Size distribution was assayed by measuring the size of 100 particles using Image J software. Zeta potentials were measured in water by dynamic light scattering (DLS) using a Zetasizer Nano (Malvern Instruments, Malvern, UK).
Intracellular uptake rate (FACS) Particles were solubilized in water and sonicated for 15 cycles of 1 min to facilitate mixing and avoid aggregation. Then, after exposure to MPs-PLGA-FITC or NPs-PLGA-FITC, cells were trypsinized, thoroughly rinsed in PBS, and analyzed with a FACSVerse flow cytometer (BD Biosciences). A minimum of 30,000 cells were analyzed after the exclusion of cellular debris. The FACSuite software was used for data acquisition.
Immunofluorescence and histological studies Heart tissues were embedded in OCT (Sigma-Aldrich) using ice-cold 2-Methylbutane. Immunofluorescence experiments were performed as previously described  [46]. Briefly, frozen sections (6 µm) were fixed in 4% paraformaldehyde, followed by permeabilization and blocking in PBS with 0. 02% FBS, 1% BSA, and 0. 3% Triton X-100 at RT. Sections were immunostained O/N with the following antibodies: anti-alpha actinin (Abcam ab9465), anti-LC3 (Cell Signaling Technology #3868), anti-Vinculin (Sigma-Aldrich, V9131) followed by secondary Alexa Fluor antibodies (Molecular Probes). Nuclei were counterstained with DAPI. Images were acquired by confocal Microscope Zeiss LSM 780 and ZEN image analysis software (Zeiss) or digitized with a Hamamatsu NanoZoomer, and fluorescence intensity was quantified by ImageJ software.
Average cardiomyocyte area was measured manually after vinculin staining on two different regions (150-200 cells counted per heart). To assess fibrosis, the hearts were transversely sectioned at a thickness of 6 μm. These sections were then fixed using 4% paraformaldehyde and subsequently stained with Sirius Red. Slides were scanned with Hamamatsu NanoZoomer, and fibrosis was quantified using ImageJ software (RSB). The positively stained area with Sirius Red, indicating fibrosis, was expressed as a percentage of the total area in the scanned slides. Apoptosis was evaluated using the TUNEL staining method with the In Situ Cell Death Detection Kit from Roche, following the manufacturer's instructions. The proportion of apoptosis was quantified by calculating the percentage of apoptotic nuclei relative to the total number of nuclei observed.