dataesr/scientific-paragraphs-categorization huggingface dataset:
S2 ). The p. Arg92Trp variant was not identified in NR5A1 in more than 800 known fertile individuals and was absent from >70,000 individuals present in public databases. Moreover, our families were unrelated and came from geographically distinct regions, excluding a founder effect for a rare variant. All 46,XX children were SRY-negative and exome or genome sequencing in family 1, subject 2. 1 and subject 3. 1 did not show any other likely pathogenic causes. Furthermore in family 1, a hidden Y chromosome mosacism was excluded by both FISH analysis and the absence of SRY amplification by PCR using DNA from gonadal tissue. In silico analysis predicts a disruptive effect of p. Arg92Trp on DNA binding The p. Arg92Trp mutation affects a highly conserved amino acid in the RGGR motif of the "A-box" of NR5A1 (Figure  1D ).
Materials and Methods Complete methods are described in detail in Supplementary Material, Materials and Methods. Ethics committee approval and written informed consent was obtained (Supplementary Material, Methods). Exome sequencing and direct sequencing of NR5A1 was performed as described in the Supplemental Methods. Control data for NR5A1 was obtained from direct sequencing of healthy fertile controls and public variant databases (Supplementary Material, Methods). Models of wild-type, p. Arg92Gln NR5A1 and p. Arg92Trp NR5A1 (hSF-1, Uniprot Q13285) in complex with DNA were generated with MODELLER software  (29)  and the crystal structure of mouse Nr5a1 bound to the inhibin a-subunit promoter (PDBid 2FF0) as a template (Supplementary Material, Methods). Human embryonic and fetal tissues were obtained from the Human Developmental Biology Resource (www.
hdbr. org) following ethical approval and with informed consent (see Supplementary Material, Methods). Binding of wild-type and mutant NR5A1 to a consensus NR5A1 DNA binding site (CCAAGGTCA) was assessed using electromobility shift assays. Transient gene activation studies of NR5A1 were performed for wild-type and mutant cDNA using a range of different putative NR5A1 targets (Amh, Cyp11a1, Tesco) in different cell lines (HEK, KGN, NT2D1, mouse embryonic stem cells; 23). Synergistic activation of the TOP-Flash reporter system by NR5A1 and b-catenin binding was studied as reported previously (24 and see Supplementary Methods). dbSNP138, http://www. ncbi. nlm. nih. gov/snp/ ExAC database, http://exac. broadinstitute. org/ Exome Variant Server, http://evs. gs. washington. edu/EVS/ 1000 Genomes Project database, http://browser.
Funding This work was supported in part by  Actions Concertees InterPasteuriennes (ACIP  to A. B. ), the  EU  call  FP7-INCO-2011-6  (A. B. and K. M. ) and an  ESPE   Collaborative Project Grant   40000767  (A. B. ). The human embryonic and fetal material was provided by the  Joint MRC/Wellcome Trust  [grant #  099175/Z/12/Z ]  Human Developmental Biology Resource  (www. hdbr. org). E. V. and H. B. are partially funded by the  DSD-TRN  ( Disorders of Sex Development-Translational Research Network ), grant #  R01 HD068138  from the  Eunice Kennedy Shriver National Institute of Child and Human Development (NICHD). J. C. A. is a  Wellcome Trust Senior Research Fellow in Clinical Science [grant   098513/Z/ 12/Z ] with support from the  National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London.
With all samples, the data were corrected for the contribution of the solution (buffer ϩ denaturant). Unfolding of holoBcII was also monitored by measuring enzyme activity, as described above. Denaturant concentrations in the samples were determined from refractive index measurements  (94)  Folding of BcII Metallo-␤-lactamase  JULY 29, 2016  • VOLUME 291 • NUMBER 31 JOURNAL OF BIOLOGICAL CHEMISTRY 16133 using a R5000 hand-held refractometer from Atago (Japan). Protein concentrations of ϳ0. 15 mg⅐ml Ϫ1 (6. 0 M) were used throughout. Quenching of Tryptophan Fluorescence by Acrylamide-All fluorescence experiments were performed at 25 °C using a protein concentration of 0. 10 mg⅐ml Ϫ1 (4 M). The enzyme was diluted with acrylamide concentrations ranging from 0 to 0. 2 M and tryptophan fluorescence emission spectra were recorded from 305 to 450 nm, following excitation at 295 nm.
9 M, respectively. NMR spectra were collected using a homebuilt 750 MHz NMR spectrometer, controlled with GE/Omega software and equipped with an Oxford Instruments Co. magnet and a home-built triple-resonance pulse-field-gradient probe head. Spectral widths of 10204. 08 and 2659. 57 Hz in F2 ( 1 H) and F1 ( 15 N) respectively, were used for two-dimensional 1 H-15 N HSQC spectra. All spectra were collected at 25 °C, processed with NMRPipe (96), and analyzed with CcpNmr Analysis  (97). For chemical-induced unfolding under equilibrium conditions, the concomitant population of folded and unfolded species in the transition region was accompanied by changes in the NMR spectrum, both in terms of peak intensity and chemical shifts. The combined CSP was calculated as shown in Equation 2  (98) , CSP ϭ ͱ͓͑⌬␦ 1 H͒ 2 ϩ ͑0.
All authors reviewed the results and approved the final version of the manuscript. Acknowledgments-We acknowledge Prof. Jean-Marie Frere for critical reading of the manuscript and Prof. Roger H. Pain for his suggestion of a "punchy" title. Also, we are grateful to Dr. Catherine Goodman (acting on behalf of the Journal of Biological Chemistry) for valuable suggestions to improve the manuscript. Some NMR experiments in Oxford, United Kingdom, were collected with access provided by the European Commission's Framework Program 7 (FP7) East-NMR (Contract 228461) and BioNMR projects (Contract 261863).
DNA microarray Total RNA from control and Rosi-or GW-treated cells (n ¼ 4 for each condition) was extracted using a TRI-Reagent kit (Euromedex). RNA quality was checked by capillary electrophoresis (Experion, Bio-Rad). Microarray experiments were performed using Agilent 4 Â 44k v2 oligonucleotide arrays as previously described  [39]. Microarray data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE71293. Significant microarray analysis (SAM) was performed to determine differentially expressed genes in Rosi-or GW-treated cells compared to control cells at day 18. Biological pathways and processes were analyzed using DAVID database v6. 7 with Benjamini correction of p values  [40].
Western blot Cells were lysed in RIPA buffer (Sigma) containing cocktail of protease and phosphatase inhibitors (Sigma). Equal amounts of solubilized proteins were loaded on 4e20% gradient SDS-PAGE gels (Bio-Rad), blotted onto nitrocellulose membranes and incubated with the following primary antibodies: HSL (#4107), ATGL (#2138) and GAPDH (#2118) were purchased from CST Ozyme; total PDHE1a (#Ab110334), phospho-Ser293 PDHE1a (#Ab177461), UCP1 (#Ab10983) and GLUT1 (#Ab40084) from Abcam; PLIN5 (#GP31) and PLIN 1 (#GP29) from PROGEN; PDK4 (#H00005166-A02) from Abnova and PCK1 (#AP8093b) from Abgent. Anti-rabbit or anti-mouse IgG coupled to horseradish peroxidase were used as secondary antibodies. Immunoreactive proteins were determined by chemiluminescence (Clarity, Bio-Rad) with a ChemiDoc MP System (Bio-Rad) and quantification was performed using Image Lab software (Bio-Rad).
Statistical analyses Statistical analyses were performed using GraphPad Prism (GraphPad Software v. 5. 0). Paired or unpaired one-way ANOVA were performed and followed by Tukey's post hoc test to determine differences between groups. Two-way ANOVA, followed by Bonferroni's post hoc test, was applied when appropriate. Data are presented as percent of control cells or percent of WT untreated-mice, AESEM, unless otherwise described in legend. Statistical significance was set at p < 0. 05.
PPARg and PPARa agonists drive similar transcriptional response and mitochondrial adaptation in white adipocytes DNA microarrays were used to investigate the whole transcriptional effects of PPARg and PPARa agonists on hMADS cells. SAM analysis of 12891 transcripts using false discovery rate of 5% revealed that, compared to untreated white adipocytes, 265 and 210 genes were significantly up-and down-regulated by Rosi, respectively, and 470 and 209 genes were significantly up-and down-regulated by GW, respectively (data not shown). Among the 215 genes upregulated by the two treatments, 96 and 57 were induced by more than 3-fold by Rosi and GW, respectively. On the contrary, genes upregulated specifically by Rosi or GW displayed weak fold induction, suggesting major overlap in the regulation of gene expression by PPARg and PPARa agonists in adipocytes.
PPARa deficiency disrupts WAT britening-induced PDK4 expression in vivo Data in human adipocytes indicated that PDK4 induction was responsible for major metabolic adaptations during britening. To confirm these in vitro results, we aimed at investigating PDK4 regulation in vivo. As PPARg but not PPARa deficiency in WAT provokes lipodystrophy, PPARa-null mice were used. Inguinal WAT was investigated as it is a fat depot prone to britening compared to other fat pads. Two cohorts of mice were investigated. WT and PPARa-null mice at 21 weeks of age acclimated at thermoneutrality were treated with the b 3adrenergic agonist CL316,243. Acclimation to thermoneutrality is essential to avoid the confounding effects due to activation of brown and brite fat depots at conventional housing temperature. Body weight and inguinal fat pad weight were higher in PPARa-null mice compared to age-matched WT mice (Figure  S6A ).
ACKNOWLEDGMENTS We gratefully acknowledge  Frédéric Martins  and  Jean-José Maoret  (Get-TQ transcriptomics facility) from the  Institute of Metabolic and Cardiovascular Diseases  ( I2MC , Toulouse). We also thank  Arnaud Polizzi  ( UMR1331 ToxAlim, Toulouse ) for excellent technical assistance and  Colette Bétoulières  ( UMR1331 ToxAlim, Toulouse ) for mouse breeding and care. The authors also acknowledge the  Cytomed Platform  from  IRCAN (Nice). D. L. is a member of  Institut Universitaire de France. This work was supported by  Inserm ,  CNRS ,  EU   FP7  project  DIABAT  ( HEALTH-F2-2011-278373 ),  French Agence Nationale de la Recherche  ( ANR-10-BLAN-1105  miRBAT and  ANR-12-BSV1-0025Obelip ) and  Région Midi-Pyrénées.
A previous study showed that Klf2 and Klf4 are induced by ERK5 signaling and contribute to muscle cell fusion  (Sunadome et al. , 2011). Microarray analyses in that study indicate that Klf5 expression is not affected by inhibition of ERK5 signaling (GSE25827). This is in agreement with our previous observations that Klf5 expression was unaffected by a MEK inhibitor in smooth muscle cells  (Oishi et al. , 2010). These findings indicate that members of Kru ¨ppel-like factor family differentially contribute to different processes during muscle differentiation and maturation. That said, it is possible that the loss of Klf5 is compensated by other Klf members, as we observed modest changes in the expression of other Klfs in Klf5-deficient C2C12 cells (Figure  3 -figure supplement 1). Future studies of possible cooperation and competition among Klf members will be important for elucidating their functional roles in muscle biology.
In addition, based on a published microarray analysis of effects of an individual Mef2 isoform (GSE63798)  (Estrella et al. , 2015)  and our results from smooth muscle cells  (Oishi et al. , 2010) , it appears Klf5 is regulated by Mef2A. This reciprocal regulation of Klf5 and other myogenic transcription factors support the notion that Klf5 is a component of an intricate transcription factor network that regulates muscle differentiation and regeneration. In addition to MyoD and Mef2 identified in the present study, Klf5 has been shown to interact with various other transcription factors and cofactors. For instance we showed that Klf5 interacts with C/EBBb and d to regulate adipocyte differentiation  (Oishi et al. , 2005) , interacts with PPARd to regulate lipid metabolic genes  (Oishi et al.
To assess satellite cells obtained from Pax7 CE/+ ;Klf5 flox/flox mice, the cells were treated with 1 mM 4-hydroxytamoxifen (4OH-Tmx; Sigma-Aldrich, H7904) or vehicle 3 days after isolation, then grown in growth medium for another 2 days. C2C12 mouse myoblasts (RRID:CVCL_0188) were purchased from the ATCC (ATCC #CRL-1772, passage number 6-8, Rockville, MD). The identity was not authenticated by our hands. Cells were free from mycoplasma contamination confirmed by monthly tests for mycoplasma. Cells were cultured in DMEM (Nakarai) containing 10% FBS and 1% penicillin-streptomycin at sub-confluent densities. C2C12 cells were differentiated into myotubes by replacing growth medium with medium containing 2% horse serum with antibiotics (differentiation medium, DM). To knock down Klf5 in C2C12 cells, siKlf5 (Thermo scientific, Dharmacon, siRNA-SMARTpool, M-062477-01-0005) treatment was carried out using RNAiMAX (Life Technologies) according to the manufacturer's instructions.
After another 3 washes in PBS, the sections were counterstained with Hoechst 33342 solution at room temperature for 3 min. The slides were mounted with a coverslip and Fluoromount-G (SouthernBiotech, Birmingham, AL). Immunofluorescent signals from stained sections and cells were captured using a LSM710 confocal imaging system (Carl Zeiss, Inc. , Oberkochen, Germany) or OLYMPUS IX73 fluorescence inverted fluorescence microscope with an Olympus DP80 camera and Olympus cellSense Digital Imaging software (Tokyo, Japan).
Luciferase assay Murine Klf5 (Genbank accession number: NM_009769) was sub-cloned into p3xFlag-CMV-7. 1 vector. The Myog-Luciferase vector was kindly provided by Dr. T. Sato (Kyoto Prefectural University of Medicine, unpublished). The Myod1 expression vector was a gift of Dr. P. Maire (Institute Cochin)  (Santolini et al. , 2016). The Mef2d expression vector was kindly provided by Dr. E. Olson (University of Texas Southwestern Medical Center). The 293T cells were transfected with a CMV-Renilla luciferase control reporter plasmid along with Myog-Luciferase, Klf5, Myod1 and Mef2d expression plasmids. The total amount of plasmid DNA was kept constant using p3xFlag-CMV-7. 1 empty vector. Forty-eight hours after transfection, the cells were lysed in Passive Lysis Buffer (Promega) and shaken for 15 min.
Acknowledgement We are grateful to  Dr. A Aiba  ( The University of Tokyo ) and  Dr. Y Nakayama  for providing pSpCas9 plasmid,  Dr. P Maire  and  Dr. I Sakakibara  for providing Myod1 expression plasmid, and  Dr. T Sato  for providing Myog-Luciferase plasmid. We thank  Dr. M Kanagawa  and  Dr. S Fukada  for the technical advice for Pax7 CreERT2/+ mice and  Dr. S Takeda  and  Dr. N Ito  for the technical advice for SC culture. We thank  Mrs N Yamanaka  and  Mrs M Hayashi  for their excellent technical assistance (The University of Tokyo). We thank our colleagues in the  Oishi Lab  and members of the  Manabe Lab for discussion. This work was supported in part by  JSPS KAKENHI  Grant Number  26882020  ( Grant-in-Aid for Research Activity Start-up  to SH),  25H10  (to YO), and  15H01506  (to IM);  AMED-CREST  (to IM); and the grants from  Nakatomi Health Science Foundation  and the  Uehara Memorial Foundation  (to SH).
Three-photon activation set-up The photoactivation of the DMNB cyclofen was performed using 280fs pulses at 1064 nm with a repetition rate of 20 MHz generated by a fiber laser (FemtoPower, Fianium Ltd). The pulses were sent by a set of galvo-scanners (Thorlabs, GVSM002/M, Newton, NJ, USA) through a telescope to fulfill the back aperture of the objective. The pulses then were sent to an inverted microscope (Olympus, Tokyo, Japan) equipped with a motorized xy-stage (Märzhäuser, SCAN IM 120 × 80, Wetzlar, Germany) and a motorized objective holder (Märzhäuser, MA42). Typically, a 40x objective with 1. 3 NA (Olympus, UPLFLN 40 XO) was used. Targeting the specific tissue was achieved using custom software (Labview, National Instruments, Austin, TX, USA) and a PCI board (National Instruments, NI PCI-6024E) that controlled the galvo-scanners and the motorized stage.
ACKNOWLEDGEMENTS We thank  Cristina García  for excellent technical assistance with zebrafish husbandry and  Angelique di Domenico  for critical reading of the manuscript. IT and DZ were partially supported by a pre-doctoral fellowship from  MINECO  and the  I3 program , respectively. Additional support was provided by grants from  MINECO  ( SAF2012-33526 ,  SAF2015-69706-R  and  BFU2012-38146 ),  ISCIII/FEDER (Red de Terapia Celular-TerCel   RD12/0019/0019 ),  AGAUR  ( 2014-SGR-1460 ),  Fundació La Marató  de  TV3  ( 201534-30 ) and  ERC  (Grant Agreement 242993).
Methods The present analysis includes gallbladder and other biliary cancer (C23-C24), as coded using the International Classification of Diseases for Oncology (ICD-O). The data sources and methods are described in detail in an earlier article in this issue. In brief, we obtained regional and national-level incidence data from 48 population-based cancer registries in 13 countries and nationwide cancer deaths from the World Health Organization (WHO) mortality database for 18 countries. To allow data comparisons across countries, we used standard methods to check the incidence data consistency and quality  [22]. All incidence data were converted to the latest version of ICD-O (ICD-O-3)  [23] ; the data were subsequently translated to the 10th edition of the International Classification of Diseases (ICD-10)  [24].
Nationwide mortality data from WHO systematically undergo data verification, and the data are coded in ICD-10 to avoid misclassification of cancer mortality over time  [25]. We estimated age-standardized incidence (ASIR) and mortality (ASMR) rates per 100,000 person-years using the direct method and the World standard population  [26, 27]. We estimated national ASIRs by aggregating the data from the available cancer registries using a weighted average of local rates. Trends in incidence were estimated only for 4 countries that provided written consent to use their data or submitted new data for $10-years and we matched mortality data to the same timeperiod (Table  1 ). To describe incidence and mortality time trends, we calculated the estimated annual percent change (EAPC) for the most recent 10-year period using the method proposed by Esteve et al.
[28]. All of the EAPCs were tested for equality to zero by using the corresponding standard errors. We considered EAPCs statistically significant if the p-value 0. 05. We conducted the data analysis in Stata version 12. 1 (StataCorp)  [29]. We also considered incidence information by anatomical subsite as coded in ICD-O-3, the most specific classification used by cancer registries, in order to differentiate between gallbladder site (C23), unspecified site (C24. 9) and other parts of the biliary tract (C24. 0-C24. 8).
Acknowledgements The authors would like to thank sincerely all of the cancer registry directors and their staff (listed in the Appendix to the Introduction of this Supplement) for their considerable efforts in collecting the data presented in this paper, together with members of the  IARC Section of Cancer Surveillance , especially  Sebastien Antoni ,  Murielle Colombet  and  Mathieu Laversanne  for their collaboration. The authors also wish to acknowledge  Drs Catterina Ferreccio  and  Luis Eduardo Bravo  for their valuable comments in reviewing earlier drafts of the manuscript.
Silencing of PKCα reduced Ser and Thr phosphorylation of NS5 in vivo To investigate the functional interaction between host PKC and DENV NS5 in vivo, we depleted PKCα using PKCα-specific siRNAs and examined the level of NS5 phosphorylation in vivo. We first, tested the efficiency of siRNA-mediated PKC depletion. We found that at 24, 48, and 72 h after PKC-specific siRNA transfection, more than 70 % of the endogenous PKC levels were downregulated (Fig. 2a ), while host cell viability was unaffected (data not shown). We selected to examine NS5 phosphorylation at the 24 h after siRNA transfection. We expressed His-FLAG-tagged NS5 in host cells by infecting the cells with recombinant DENV expressing the His-FLAG-NS5 protein (etDENV), then pulled down the NS5 using anti-FLAG antibody. The phosphorylation on the His-FLAG-NS5 was analyzed by anti phospho- Ser and phospho-Thr immunoblottings.
Sequence and structure analysis To identify the conservation among flaviviruses, DENV1 (P27909), DENV2 (AHG23127), DENV3 (P27915), DENV4(P09816), West Nile virus (P06935), Japanese Encephalitis virus (P27395), yellow fever virus (AAC35930), St. Louis encephalitis virus (P09732), Omsk hemorrhagic fever virus (AAR98531), Zika virus (W8R1T1), Gadgets Gully virus (A0EKU2), Kama virus (W5VRZ2) and tickborne encephalitis virus (Q01299) NS5 sequences were compared by BioEdit 7. 2. 5. The phospho-conservative site was shown in crystal structure of NS5 (PDB ID: 4V0R).
Acknowledgements SJ is supported by  "Chalermphrakiat" Grant, Faculty of Medicine Siriraj Hospital, Mahidol University, The Thailand Research Fund  ( RSA5880038 ), and the  Advanced Research on Pharmacology Fund ,  Siriraj Foundation   D003421. TP is supported by a scholarship from  Science Achievement Scholarship of Thailand (SAST). SC is supported by  Grants-in-Aids for scientific research  from  Mahidol University (Thailand) , and by the  Mid-Career Grant  ( RSA5680054 ) from  Thailand Research Fund (TRF). This project was supported by  National Research University of Thailand (NRU)  and the  DENFREE  project (the  European Union Seventh Framework Programme  ( FP7/2007/2011 ) under Grant Agreement n°282 378).
They have been corrected for instrumental errors, environmental perturbations, the ocean sea state influence, the tide influence, atmospheric pressure, and multi-mission crosscalibration  (CLS, 2015). Two kinds of sea surface temperature (SST) data are used in this paper. One type is derived from the merged satellite's infrared sensors (AVHRR/NOAA) and microwave sensor (AMSR-E/AQUA), and in situ SST (buoy and ship) data global daily SST (MGDSST), with a 0. 25 • × 0. 25 • horizontal resolution, which are analyzed and published at the Office of Marine Prediction of the Japan Meteorological Agency (JMA). The data can be obtained from http://near-goos1. jodc. go. jp/. The other type is derived from the NOAA 0. 25 • × 0. 25 • daily optimum interpolation sea surface temperature (OISST), which is an analysis constructed by combining observations from different platforms, such as satellites, ships, and buoys, on a regular grid via optimum interpolation.
In situ data The in situ data employed in this paper for the comparison and validation of both systems are provided by the South China Sea Institute of Oceanology, Chinese Academy of Sciences. There was one mooring to measure the sea water velocity and five cruises conducted to measure the temperature and salinity (TS) in the SCS during 2012. The mooring station is located at Maoming (Fig. 1 ), where bottom-mounted upward-looking 75 kHz acoustic Doppler current profilers (ADCPs) were deployed to monitor the current profile (u component and v component) from the depth of 2 to 48 m with a 2 m vertical interval. The period of the monitoring is from 11 July to 8 October 2012, with a temporal interval of 10 min. Firstly, the outlier data are eliminated from the original measured data; secondly, a 25 h low-pass filter is applied to remove tidal currents; and daily mean currents are calculated using 25 h averaging.