dataesr/scientific-paragraphs-categorization huggingface dataset:
We note that slicer-inactive mutant MIWI protein that fails to cleave target transcripts also shows such a dominant-negative effect in heterozygous mice carrying both the wild-type and knockin alleles  (Reuter et al. , 2011). We propose that the dominantnegative effect of the catalytic-dead mutation in MVH is likely due to a combination of effects, including the failure to eliminate/process slicer cleavage fragments and creation of stalled inactive complexes containing the mutant MVH, PIWI proteins, and piRNAs, as we demonstrated (Figures  4E  and 4F ). The TDRD9 ortholog in insects, Spn-E, is described as a piRNA biogenesis factor, and depends on its ATPase activity for ensuring piRNA biogenesis and fertility  (Malone et al. , 2009; Nishida et al. , 2015). However, study of the mouse ortholog already indicated that piRNA biogenesis is not affected by loss of TDRD9  (Shoji et al.
, 2016)  to bring the artificial piRNA reporter into the different Mvh genetic backgrounds. Briefly, the artificial piRNA reporter had a DsRed coding sequence with a 3 0 -UTR based on noncoding (all ATGs were mutated) LacZ sequence. The 3 0 -UTR contains 35 perfectly complementary binding sites for different MILI-bound piRNAs that are abundantly expressed in the embryonic mouse male germline. (Yang et al. , 2016). We previously demonstrated that this reporter is a target for MILI slicing for generation of piRNAs. New born pups (P0) and adults were collected for piRNA analysis. Mutant mice (Mvh E446Q ) are deposited with the European Mouse Mutant Archive (EMMA) under the accession number EM:09169. Genotyping Primers to detect bands (Figure  S2D ) corresponding to the wild-type (859 bp), knockin (1102 bp) and knockout (444 bp) alleles were ZY530 (TAGCAGGAATTTGGAGGCCA) and ZY533 (ACCTTCGTTTCTGAGACAGG).
Heterozygous males were crossed with Del-FLPeR (flipper)  (Farley et al. , 2000)  female mice to remove the PGK-Keo cassette to generate the Tdrd9 E257Q knockin allele (hereafter referred to as Tdrd9 KI ). Heterozygous Tdrd9 null mice (hereafter referred to as Tdrd9 +/À ) were generated by crossing the Tdrd9 KI females with male transgenic mice expressing the X-linked CMV-Cre recombinase  (Schwenk et al. , 1995). This leads to deletion of exons 3 to 5, potentially leading to a frame shift and a stop codon in the exon 6. Intercrosses generated the following experimental mice: Tdrd9 +/À , Tdrd9 +/KI , Tdrd9 KI/KI and Tdrd9 À/À. Only males were used for all experiments and the age of the donor animals is indicated in the text and relevant figures. Mutant mice (Tdrd9 E257Q ) are deposited with the European Mouse Mutant Archive (EMMA) under the accession number EM:08489.
Clones and Constructs The full-length complementary DNA (cDNA) for Mouse Vasa Homolog (MVH; 1-728 aa; GenBank Accession no. NM_001145885) was isolated by reverse-transcription PCR (RT-PCR) from mouse testis total RNA. The cDNA for mouse Tdrd9 (1383 aa; GenBank Accession no. AB362563) was a kind gift of Dr. Shinichiro Chuma  (Shoji et al. , 2009). For the production of full-length TDRD9 protein we used eukaryotic expression systems based on insect ovary-derived cells: Spodoptera frugiperda 21 (Sf21) or the Trichoplusia ni High Five cells. Full-length coding sequence for mouse TDRD9 (1-1383 aa) and TDRD9 E257Q ATPase mutant, which has a single amino acid mutation E257Q in the catalytic motif (DEVH/DQVH), were cloned into the NheI and KpnI restriction sites of the vector pACEBac2 to express the recombinant proteins as 6xHis-Sumo-StrepIII-fusions.
28-9564-75) and scanned (GE Healthcare; Typhoon FLA 9500 IP, cat no. 29-1885-90). The signal of 23S and 18S RNA visualized by ethidium bromide staining was used as a loading control. The LINE-1 (L1) probe was amplified with primers RP469 (gaagttcccaacatagagtcc) and RP470 (agtgggcagagtattctctgc), on the template of cloned L1 fragment (kindly provided by Donal O'Carroll). The sequence corresponds to 513-1,628 bp of Mouse L1Md-A2 repetitive element (GenBank accession No. M13002. 1). IAP probe was similarly prepared using primers JW7 (GGGAATAC TAATGTCCCTCG) and JW8 (CAACCAGAATCTTCTACGGC). The sequence corresponds to 3758-5171 bp of Mus musculus retrotransposon IAP (GenBank accession No. EU183301. 1).
The synthesized cDNA libraries were resolved on 3% high-resolution MethaPhor agarose (Lonza, cat. No. 50180) gels in TAE buffer (Figure  1B ). Fragments in the size-range of $160 bp (short libraries) and $200-300 bp (long libraries) were gel-extracted with the use of MinElute Gel Extraction Kit  (Qiagen, cat No. 28604 ). Multiple libraries with different barcodes (at 3 0 end) were mixed in equimolar ratios and sequenced with the Illumina HiSeq 2000 platform (EMBL GeneCore facility, Heidelberg). The maximum sequencing length was 50 or 51 nt. Our library preparation strategy identifies the 5 0 end of these sequences, irrespective of how long such sequences might be. All the generated datasets are listed in Table  S1.
QUANTIFICATION AND STATISTICAL ANALYSIS Small RNA Data Analysis Reads were sorted into individual libraries based on the barcodes, the 3 0 adapter sequences were removed and mapped to the mouse genome (mm9). The software used for processing the data (genomic coordinates etc) from the raw data files are in-house tools developed by the Sachidanandam lab  (Olson et al. , 2008). Only reads perfectly matching the genome were kept for further analysis. Analysis of Libraries from Adult Mvh Animals After 3 0 adapter sequence removal, the read length profile of short and long libraries was similar (data not shown). Only short libraries were used for further analysis. The read lengths of MILI-, MIWI-and MVH-associated sequences were plotted  (Figures 4G and S5A). Whereas MILI piRNAs were preferentially 26-27 nt long, the MIWI piRNAs were mostly of 29-30 nt length (Figure  4G ).
First, we analysed the 5 0 end distances of targeting and produced reads by mapping the reads to L1_MM transposon consensus sequence (3 mismatches were allowed). The ping-pong signature was observed between MILI and MIWI piRNAs in both the wild-type and Mvh +/KI mutant indicating the cleavage of L1 transcripts and production of secondary piRNAs (Figure  S5F ). It was also present between MILI (and MIWI) piRNAs and MVH long RNAs of the wild-type mouse demonstrating that MVH associates with the 5 0 cleavage fragments resulting from slicer activity of MILI and MIWI. To find out whether the pachytene piRNAs might guide also the cleavage of into groups based on the nucleotide at their 1 st and 10 th position. No obvious difference was found between the wild-type (Tdrd9 +/KI ) and the mutants (Tdrd9 KI/KI and Tdrd9 À/À ).
DATA AND SOFTWARE AVAILABILITY Deep sequencing data generated in this study are deposited with Gene Expression Omnibus under the accession number GEO: GSE95580. Mouse mutants generated in this study are deposited with the European Mouse Mutant Archive (EMMA) under the accession numbers: Tdrd9 E257Q (EM:08489) and Mvh E446Q (EM:09169). Both mouse lines can be crossed with Cre mice to obtain the respective knockout lines.
ACKNOWLEDGMENTS We thank the following  EMBL core facilities: Genomics, Protein Expression, Histology ,  Gene Editing , and  Embryology. Support  from the  Imaging Facility, University of Geneva  is acknowledged. This work was supported by grants to R. S. P. from the  Swiss National Science Foundation :  ERC transfer grant  ( GermMethylation , no. 166923 ), project grant (Origin-of-pi, no. 166575 ), and funding from the  NCCR RNA & Disease. Funding from  Fondation Recherche Medicale  ( DEP20131128529 ) and  Agence National de la Recherche  ( GuidedMethylation ,  ANR-14-CE10-0011-02 ) to R. S. P. is also acknowledged. Work in the R. S. P. laboratory is supported by the  Republic and Canton of Geneva.
Background: Duchenne muscular dystrophy (DMD) is the most common disease in children caused by mutations in the DMD gene, and DMD and Becker muscular dystrophy (BMD) are collectively called dystrophinopathies. Dystrophinopathies show a complex mutation spectrum. The importance of mutation databases, with clinical phenotypes and protein studies of patients, is increasingly recognized as a reference for genetic diagnosis and for the development of gene therapy. Methods: We used the data from the Japanese Registry of Muscular Dystrophy (Remudy) compiled during from July 2009 to March 2017, and reviewed 1497 patients with dystrophinopathies. Results: The spectrum of identified mutations contained exon deletions (61%), exon duplications (13%), nonsense mutations (13%), small deletions (5%), small insertions (3%), splice-site mutations (4%), and missense mutations (1%).
Exon deletions were found most frequently in the central hot spot region between exons 45-52 (42%), and most duplications were detected in the proximal hot spot region between exons 3-25 (47%). In the 371 patients harboring a small mutation, 194 mutations were reported and 187 mutations were unreported. Conclusions: We report the largest dystrophinopathies mutation dataset in Japan from a national patient registry, "Remudy". This dataset provides a useful reference to support the genetic diagnosis and treatment of dystrophinopathy.
Nonsense mutations are present in approximately 15% of all DMD patients  [7] [8] [9]. However, the precise analysis of the DMD mutation is required for the application of these molecular therapies to patients. In Japan, a hospital based dataset of 127 patients harboring small mutations by Takeshima et al. in 2010  [10] , and a registry based dataset in Japanese Registry of Muscular Dystrophy (Remudy) of exon deletions and duplications by Nakamura et al. [11]  have been compiled. Here, we extend these previous reports not only by increasing the number of the patients (1497 patients), but also by comprehensive genetic analysis of exonic and small nucleotide mutations and immunostaining of dystrophin on muscle biopsies to deduce genotype-phenotype correlations in patients with dystrophinopathies.
Registry-based datasets Remudy was developed in 2009 in collaboration with the Translational Research in Europe-Assessment and Treatment of Neuromuscular Diseases (TREAT-NMD) Network of Excellence  [11] [12] [13]. The Remudy database for male patients with dystrophinopathies includes clinical and molecular genetic data, as well as all mandatory and highly encouraged items for the TREAT-NMD global patient registry. To classify into DMD, BMD or IMD, the attending physicians reviewed their clinical information. Then, our clinical and genetic curators independently evaluated physicians' classification by reviewing the clinical information, and pathological data (including dystrophin immune-staining, if applicable), and report of genetic analysis for DMD. In the present study, we used the registry data compiled during from July 2009 to March 2017.
Small mutations The spectrum of identified mutations included nonsense mutations (186/371, 50%); small deletions (77/371, 21%); small insertions (39/371, 11%); splice-site mutations (61/371, 16%); and missense mutations (8/371, 2%) (Fig. 3a ). The location of small mutations in patients with DMD and BMD patients is shown in Fig. 3b. Detailed results of the small mutations in the 371 patients, with 312 kinds of mutations, are shown in Additional file 1: Table  S1. Among the 312 mutations, 149 were reported and 163 were unreported by HGMD (Human Gene Mutation Database)  [18].
Ethical considerations The study respects the ethical standards established in the Declaration of Helsinki. 40 Investigators undertake to respect law no. 2004-806 of 9 August 2004 relative to public health policy on clinical trials, made effective by implementing Decree 2006-477 of 26 April 2006. 41 pproval of the study protocol was obtained from the human ethical research committee (Comité de Protection des Personnes EST II, process number 15/494) and the French National Agency for Medicines and Health Products Safety (process number 2015-A01575-44). The study is registered on clinicaltrial. gov with the following number: NCT02870608. All participants will sign a statement of informed consent and will be allowed to abandon the study at any time, without negative consequences. All procedures of the study will be confidential.
The study will involve the collection and storage of ultrasound images, and study participants will therefore be thoroughly informed. All variables (patient parameters, data items, data elements) will be aggregated into electronic case report forms. As explained in the background, the FDA has so far not yet approved the use of SWE for obstetric clinical applications because of the paucity of data in the literature. In this study, fetuses will be included from 24 to 34 WG, over the period of organogenesis. In the patient information sheet, it is specified that previous studies explored the liver or brain of premature infants with SWE. Alison et al studied liver stiffness in preterm neonates with IUGR and gestational age at birth between 26 and 31 WG. 31.
Technical validation Feasibility will be evaluated by assessing the number of examinations performed and the number of examinations with interpretable results. All the variables and data that will be stored for each SWE examination are summarised in table  1. Intra-and inter-observer reproducibility will be evaluated by the intra-class correlation coefficient (ICC) with 95% CI. Intra-observer reproducibility will be calculated using the two cycles of measure, inter observer reproducibility between the operators will be calculated by means of the two cycles of measures if the intra-observer is high. 46 nly repeatable and reproducible values of each ROI will be considered to modellise the LLE ratio.
Clinical evaluation Evolution of fetal lung SWE, fetal liver SWE and lung-toliver-SWE ratio will be assessed by group and overall. Lungto-liver-SWE ratio will be initially defined as the value of the lung elasticity divided by the value of the liver elasticity. SWE values before corticosteroids administration will be compared between cases and control groups. If there is a statistically significant difference, a confusional bias will have to be sought between fetal lung elasticity values and threatened preterm labour. All results will be presented as 'delta' variation between two measurements in order to limit 'non-comparability bias' between both groups. If corticosteroids affect fetal lung elasticity, 'delta before/ after' will be more important in cases than controls. Analysis of mean differences between groups will be carried out using a Student's test or Wilcoxon test according to the distribution of data for quantitative variables.
The Shapiro-Wilk test will be used to determine if the data set is well modelled by a normal distribution. The relationship of quantitative variables to each other will be tested using Pearson's or Spearman's correlation as appropriate. Qualitative variables will be expressed as frequencies and quantitative variables will be displayed as the mean ±SD if normally distributed or as the median ±IQR if asymmetrically distributed. Qualitative variables will be analysed using a χ 2 test or Fisher test. For statistical analyses, the level of statistical significance will be set at 5% (P<0. 05). Statistical analysis will be performed with statistical software SAS for Windows, version 9. 4 and MedCalc software, version 15. dIscussIon 2D-SWE could be considered as a new noninvasive tool for monitoring fetal lung development based on the evaluation of mechanical properties during pregnancy.
However, standardisation for measuring SWE in the human fetal lung is necessary to demonstrate applicability of the technique, and to understand limiting factors. This pilot study will focus on technical performances before considering clinical applications. In this protocol, SWE will be used after the fetal period of organogenesis, and pharmacovigilance data will be collected at birth and 3 months' later in order to assess the safety of the device. With the study design, we shall not attempt to define the reference value of fetal lung elasticity during pregnancy or to correlate SWE value with respiratory function at birth. The main objective is to evaluate the feasibility and reproducibility of 2D-SWE in the human fetal lung between 24 and 34 WG and to modellise a new LLE ratio. Indeed, each target organ will be explored through three measurements to define the most relevant and reproducible regions with the lowest intra-and inter-observer variability.
This platform will accelerate the development of chemical immunomodulatory agents as subpopulation-specific targeted therapies for immune-related disorders. ■ EXPERIMENTAL SECTION General Materials. Commercially available reagents were used without further purification. Thin-layer chromatography was conducted on Merck silica gel 60 F254 sheets and visualized by UV (254 and 365 nm). Silica gel (particle size 35-70 μm) was used for column chromatography. 1 H and  13  C spectra were recorded in a Bruker Avance 500 spectrometer (at 500 and 125 MHz, respectively). Data for 1 H NMR spectra are reported as chemical shift δ (ppm), multiplicity, coupling constant (Hz), and integration. Data for  13  C NMR spectra are reported as chemical shifts relative to the solvent peak. HPLC-MS analysis was performed on a Waters Alliance 2695 separation module connected to a Waters PDA2996 photodiode array detector and a ZQ Micromass mass spectrometer (ESI-MS) with a Phenomenex column (C 18 , 5 μm, 4.
HPLC (H 2 O-ACN with 0. 1% HCOOH): t R 5. 58 min. 1   S3 ) (7 mg, 0. 015 mmol) in DMF (0. 5 mL) was added DIPEA (8 μL, 0. 045 mmol). The solution was stirred for 15 min, and COMU (9 mg, 0. 022 mmol) was added predissolved in DMF (0. 2 mL). After 6 h stirring at rt, the crude mixture was extracted with CH 2 Cl 2 , and the organic extracts were dried over MgSO 4 , filtered, and evaporated under reduced pressure. The mixture was purified by semipreparative HPLC to obtain 8 as a red solid, synthetic yield 26%. HPLC (H 2 O-ACN with 0. 1% HCOOH): t R 6. 6 min. 1   Spectral Characterization of BODIPY-Prodrug Conjugates. Spectroscopic and quantum yield data were recorded on a Synergy HT spectrophotometer (Biotek). Compounds were dissolved at the indicated concentrations, and spectra were recorded at rt.
Briefly, RAW264. 7 macrophages were plated on 96-well plates and stimulated as described above when appropriate, reaching 90-95% confluence on the day of the experiment. Compounds were added to the cells at indicated concentrations and incubated at 37 °C overnight. Then cells were washed and treated according to the manufacturer's instructions, and their absorbance values (570 nm) were measured in a Synergy HT spectrophotometer (Biotek). Data analysis was performed using GraphPrism 5. 0. Cell viability data was normalized to the proliferation of the cells in cell culture medium. Cytokine Immunochemical Profiling. Cytokine levels were determined using a Mouse Cytokine Array Panel A (Proteome Profiler, R&D System) following the manufacturer's instructions. Briefly, RAW264. 7 macrophages were plated on 12-well plates on the day before the experiment, reaching 70-80% confluence on the day of the experiment.
Data analysis was performed with the software Flowjo. Live-Cell Fluorescence Confocal Microscopy. Cells were plated on glass chamber slides Lab-Tek II (Nunc), stimulated with Texas Red conjugated Zymosan A S. cerevisiae BioParticles (0. 05 mg mL -1 ), washed with PBS, and incubated with compound 5 (150 nM) at 37 °C. Cells were imaged under a Zeiss LSM510 META fluorescence confocal microscope equipped with a live cell imaging stage. Fluorescence and brightfield images were acquired using a 40× oil objective. Fluorophores were excited with 488 nm (BODIPY) or 543 nm (Texas Red) lasers. All images were analyzed and processed with ImageJ.
Research Article Transwell Assays in M1 and M2 Macrophages. RAW264. 7 macrophages were plated on 24-well plates equipped with Transwell insert membranes, presoaking the insert in cell growth medium prior to cell seeding overnight. Macrophages were polarized toward M1 (LPS, 100 ng mL -1 ) or M2 (IL-4, 100 ng mL -1 ) on separated wells at 37 °C for 24 h. Then, macrophages were washed with PBS and compound 5 (10 μM) was added and incubated in M1/M2 macrophages at 37 °C for 18 h. Macrophages were detached, resuspended in CaCl 2 buffer, and analyzed by flow cytometry using Annexin V-AF647. Data analysis was performed with Flowjo. Assays in ex Vivo Human Monocyte-Derived Macrophages. Ex vivo experiments with fresh human peripheral blood from healthy donors were approved by the Accredited Medical Regional Ethics Committee (AMREC, reference number 15-HV-013).
cDNA samples were analyzed using a SYBR green based quantitative fluorescence method (Invitrogen) and Kiqstart primers (Sigma). Data was analyzed with β-actin as the housekeeper gene and presented as ΔCT values. Zebrafish Tissue Regeneration Model. Transgenic macrophage reporter fish Tg(cfms::Gal4-UAS::mCherry) were used to monitor macrophage recruitment in tail fin r e g e n e r a t i o n a s s a y s. A d o u b l e t r a n s g e n i c T g -(tnfα::eGFP;mpeg1::mCherry) was used to image M1 macrophage polarization in vivo upon LPS treatment. For wholemount TUNEL staining experiments, the transgenic macrophage reporter zebrafish Tg(mfap4::tdTomato-CAAX) were used. Fish were fixed for 2 h in 4% p-formaldehyde (PFA) in PBS, permeabilized for 12 h in PBS containing 0. 1% Triton X-100 and 3% bovine serum albumin (BSA), and washed in PBS.
Preliminary search Before starting the development of a COS on medication review in older patients with multimorbidity, we checked that there was no existing or ongoing work on this subject. A systematic search was performed in the COMET (Core Outcome Measures in Effectiveness Trials) database which collects all details of ongoing and completed COS developments. 33 We found one ongoing study of COS development on prescribing in older patients living in the nursing home setting. The primary investigator of this COS study was contacted, and after a discussion, the conclusion was that the two studies would be complementary with a slight overlap. Furthermore, an ongoing project attempting to develop a COS for the assessment and management of frail older patients was retrieved. 34 The principal investigator was contacted for discussion and the reply was that the COS study had not yet started.
Overview of the work performed The development of the COS was achieved through a four-step approach represented in Figure  1. The scope was defined by the OPERAM research team in order to best fit with the OPERAM project and to be of the highest relevance for future RCTs in this research topic. We followed the guidelines published for the development of a COS  35, 36  and the project was registered on the COMET database (http:// www. comet-initiative. org/studies/details/806?result=true). The methodological aspects addressed in this study protocol are detailed in Table  S1 , following recommendations made by Sinha et al. 37  A steering committee was set up with the members of the OPERAM board and the local principal investigators of the participating centers.
data extraction All data extractions on outcomes and outcome measurement instruments were performed by two independent reviewers (ST and J-BB for RCT protocols; LP and J-BB for published studies). Any disagreement was resolved by discussion and consensus. AS was called upon to resolve any divided opinion toward consensus, if needed. The characteristics of the RCT protocols and the published studies were extracted by JBB and included the following: setting (hospital setting, primary care, and nursing home); number of patients intended to be recruited (RCT protocols) or actually included (published studies); and mean age (published studies only). Outcomes used to compare the two groups under investigation (in RCTs and prospective before/after studies) or to evaluate the medication review process were extracted: name of the outcome in free text (what was measured in published studies and what was planned to be measured in protocols); primary or secondary outcome.