dataesr/scientific-paragraphs-categorization huggingface dataset:
Proteins were blotted using a basic SDS-PAGE protocol. Primary antibody incubation was performed overnight at 4 °C (anti-UCP1, Calbiochem #662045, dilution 1:750; anti-perilipin, Interchim #BP5015, dilution 1:5000; anti-ERK1/2, Cell Signaling #9102, 1:2000; anti-CS, Abcam #ab96600, dilution 1:10000). Primary antibodies were detected with HRP-conjugated anti-rabbit or anti-mouse immunoglobulins (Promega) and anti-guinea pig immunoglobulins (Santa Cruz). Detection was performed using Immobilon Western Chemiluminescent HRP Substrate (Merk-Millipore). OD band intensities were evaluated using PCBas Software. Immunostaining analysis. Cells were fixed with PAF 4% for 10 min, permeabilized with 0. 1% Triton X-100 for 10 min, and then sequentially incubated with primary antibody overnight at 4 °C in a humidified chamber (anti-UCP1, Calbiochem, #662045, dilution 1:100) and with secondary antibody coupled with Alexa 488 (Invitrogen, dilution 1:500) for 30 min at room temperature.
A PBS wash was performed twice between all steps. Cells were finally mounted in Mowiol and visualized with an Axiovert microscope (Carl Zeiss, Le Pecq, France) under oil immersion and pictures were captured and treated with AxioVision software (Carl Zeiss). For mitochondria quantification, cells were incubated with MitoTracker ® Green FM (Invitrogen) following the manufacturer's protocol. Cells were washed and visualized with an Axiovert microscope and the fluorescence intensity evaluated using ImageJ. Measurement of metabolic parameters. For respiration analysis, hMADS cells were seeded in 24 multi-well plates and differentiated as previously described  34, 50. At day 14 of differentiation, hMADS cells were transfected with mimic Let-7i or mimic control and converted in brite adipocytes. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of 18 day-old differentiated cells was determined using an XF24 Extracellular Flux Analyzer (Seahorse Bioscience).
Acknowledgements The authors greatly acknowledge the IRCAN Animal core facilities, IRCAN  Cytomed  and IBV histopathology platforms. This work was supported by  CNRS ,  EU   FP7  project  DIABAT  ( HEALTH-F2-2011-278373 ),  French Agence Nationale de la Recherche  ( ANR-10-BLAN-1105  miRBAT). DL is a member of  Institut Universitaire de France.
Postoperative Endothelial Cell Density and Morphometry Vital staining was performed with trypan blue and alizarin red S (Sigma-Aldrich Corp. ). 51 Two nonoverlapping fields of the central cornea were photographed (SteREO Discovery V12; AxioVision Rel. 4. 8. 2. ; Carl Zeiss Canada), and a minimum of 100 cells per field were counted for endothelial cell density and morphometry analysis (KSS-409SP software, version 2. 10; Cellchek XL; KonanMedical USA, Torrance, CA, USA). The percentage of hexagonal cells was used to describe pleomorphism (differences in cell shape) and the cell area coefficient of variation to describe polymegethism (differences in cell size). 52, 53 stopathology Two samples per cornea, the iridocorneal angle, and the lens specimens were fixed in 10% neutral buffered formalin and processed for SEM (6360LV; JEOL, Tokyo, Japan) and light microscopy (Axio Imager.
Acknowledgments The authors thank  Myriam Bareille ,  Xinling Liu ,  Steve Breault , AndreDeveault,  Benjamin Goyer ,  Angèle Halley ,  Delphine Mathieu ,  Catherine Mauger-Labelle ,  Natalie Tessier ,  Nour Haydari ,  Marie-Claude Perron ,  Eugen Lungu ,  Caroline Audet ,  Jeanne d'Arc Uwamaliya , and  Élodie Samson , as well as the LOEX histology and the IBIS electron microscopy research assistants for their technical and/or intellectual support.
Genetic diversity, whole genome sequencing and bioinformatics analysis A 500 bp library was constructed and whole genome sequencing was performed using Illumina Hiseq 2000 platform (Illumina, Inc. , San Diego, CA, USA) to obtain a 100 bp paired-end read, with 1,463,765,173 bps in average for each sample (Accession number: SRP051490, Additional file 1: Table  S2 )  [17]. The multi-alignment sequences from read data and references (ST1/027: R20291, accession number: NC_013316; ST37: M68, accession number: NC_017175) were obtained by the method that combines alignments from mappings to multiple reference sequences via REALPHY  [18, 19] , and then the phylogenetic tree was reconstructed via Bayesian evolutionary analysis by BEAST  [20]. The genomic single nucleotide polymorphisms (SNPs) between NAP1/BI/027 strains were detected by mapping the C.
difficile strains in China and identification of the different PCR ribotypes requires the collection of reference strains. Therefore, distribution of C. difficile circulating clones including the NAP/BI/027 is not known in China. PCR based sequencing method can detect the pathogen without reference strain by comparing with the public database. Thus, it becomes one of replacement to other method in lots of bacteria. However, not like other hospital acquired infection (HAI) bacteria, such as Mycobacterium tuberculosis and Klebsiella pneumonia  [28] , C. difficile is characterized by large genome diversity and instability increasing the difficulties to select proper primers for detection. This characteristic will increase the difficulty to extend the existed molecular typing methods used in US or UK to develop country, and easy to cause clinic false-negative results.
So, the fact may be that: NAP1/BI/027 has existed in China before, yet it could not be identified immediately. Whole-genome sequencing can easily distinguish minor differences, providing valuable information for molecular typing, and pathogen or transmission route tracing  [12, 29]. Thus, the genome complication would boost the application of WGS in C. difficile research. With the help of WGS, our research will enrich HAI pathogen database, and HAI outbreak tracing clues. Also, our research revealed the potential NAP/BI/027 threat for China will improve the completion of C. difficile NAP/BI/027 global epidemiology distribution  [6, 30, 31]. In this study, we investigated CDI outbreaks using medical personnel interview, medical record review and WGS for strain typing. From the spatial analysis of the cases, we assumed that CDI patients are mainly concentrated near to the toilets, bathroom, and janitor's equipment room (Fig.
The colonies were obtained by spreading samples on a solid medium. They were then purified by subculture and identified by MALDI-TOF MS  [28, 29]. Colonies were deposited in duplicate on a MTP 96 MALDI-TOF MS target plate (Bruker Daltonics, Leipzig, Germany), which was analysed with a Microflex spectrometer (Bruker). The 12 spectra obtained were matched against the references of the 7567 bacteria contained in the database by standard pattern matching (with default parameter settings), with MALDI BioTyper database software 2. 0 (Bruker). An identification score over 1. 9 with a validated species allows identification at the species level, and a score under 1. 7 does not enable any identification. When identification by MALDI-TOF MS failed, the 16S rRNA was sequenced  [30]. Stackebrandt and Ebers  [31]  suggest similarity levels of 98.
Cellular fatty acid methyl ester (FAME) analysis was performed by gas chromatography/mass spectrometry (GC/ MS). Two samples were prepared with approximately 60 mg of bacterial biomass per tube collected from several culture plates. FAMEs were prepared as previously described (http://www. midi-inc. com/pdf/MIS_Technote_101. pdf), and GC/MS analyses were carried out as previously described  [32]. Briefly, FAMEs were separated using an Elite 5-MS column and monitored by mass spectrometry (Clarus 500-SQ 8 S; Perkin Elmer, Courtaboeuf, France). A spectral database search was performed using MS Search 2. 0 operated with the Standard Reference Database 1A (NIST, Gaithersburg, MD, USA) and the FAMEs mass spectral database (Wiley, Chichester, UK). Antibiotic susceptibility testing was performed using the disk diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST)  2015    blotted paper were observed with a Tecnai G20 transmission electron microscope (FEI Company, Limeil-Brevannes, France).
Genome annotation and comparison Genome annotation and comparison open reading frames (ORFs) were predicted using Prodigal  [33]  with default parameters, but the predicted ORFs were excluded if they spanned a sequencing gap region (contains N). The predicted bacterial protein sequences were searched against the Clusters of Orthologous Groups (COGs) database using BLASTP (E value 1e-03, coverage 70%, identity percentage 30%). If no hit was found, it searched against the NR database using BLASTP with an E value of 1e-03 coverage 70% and identity percentage of 30%. If sequence lengths were smaller than 80 amino acids, we used an E value of 1e-05. The tRNAScanSE tool  [34]  was used to find tRNA genes, whereas rRNAs were found using RNAmmer  [35]. Lipoprotein signal peptides and the number of transmembrane helices were predicted using Phobius  [36].
ORFans were identified if all the performed BLASTP procedures did not give positive results (E value smaller than 1e-03 for ORFs with sequence size higher than 80 aa or E value smaller than 1e-05 for ORFs with a sequence length smaller than 80 aa). Such parameter thresholds have already been used in previous work to define ORFans. Genomes were automatically retrieved from the 16s RNA tree using Xegen's PhyloPattern software  [37]. For each selected genome, complete genome sequence, proteome and   ORFeome genome sequence were retrieved from the National Center for Biotechnology Information FTP site. All proteomes were analysed with proteinOrtho  [38]. Then for each couple of genomes a similarity score was computed. This score is the mean value of nucleotide similarity between all orthologue couples between the two genomes studied (AGIOS)  [26].
Results Strain identification and phylogenetic analyses Strain MC1 (Table  1 ) was first isolated in February 2015 by spreading a urine sample on 5% sheep's blood-enriched Colombia agar (bioMérieux) in an aerobic atmosphere at 37°C after 48 hours of incubation. Using MALDI-TOF MS for identification, no significant score was obtained for strain MC1, suggesting that this isolate's spectrum did not match any spectra in our MALDI-TOF MS database. The nucleotide sequences of the 16S rRNA and the rpoB genes of strain MC1 (GenBank accession nos. LN849777 and LN849778 respectively) showed 96. 3% similarity with Corynebacterium simulans, the phylogenetically closest species with a validly published name (Fig. 1 ), therefore defining it as a new species within the  genus Corynebacterium. C. phoceense spectra (Fig.
Genome properties The genome is 2 793 868 bp long with 63. 23% GC content (Table  4 , Fig. 6 ). It is composed of 14 scaffolds (composed of 71 contigs). Of the 2575 predicted genes, 2508 were proteincoding genes and 67 were RNAs (four genes are 5S rRNA, three genes are 16S rRNA, three genes are 23S rRNA and 57 genes are tRNA genes). A total of 1804 genes (71. 93%) were assigned as putative function (by COGs or by NR BLAST). One hundred fifty-one genes were identified as ORFans (6. 02%). The remaining 475 genes were annotated as hypothetical proteins (18. 94%). The National Center for Biotechnology Information ID is PRJEB14666, and the genome is deposited under accession number FLTI01000001. The distribution of genes into COGs functional categories is presented in Table  5. 7 ). Among species with standing in nomenclature, AGIOS values ranged from 54.
D-Ribose, D-galactose, D-glucose, D-fruc- tose, D-mannose, arbutin, D-maltose and D-saccharose were metabolized. Cells were susceptible to vancomycin, rifampicin, doxycycline, erythromycin, amoxicillin, nitrofurantoin, gentamicin, ciprofloxacin, ceftriaxone, amoxicillin + clavulanic acid, penicillin G, gentamicin, trimethoprim + sulfamethoxazole, oxacillin, imipenem, tobramycin, metronidazole, amikacin, linezolid, clindamycin, daptomycin and chloramphenicol. The G+C content of the genome is 63. 23%. The 16S rRNA gene sequence and whole-genome shotgun sequence of C. phoceense strain MC1 T are deposited in GenBank under accession numbers LN849777 and FLTI01000001, respectively. The type strain MC1 T (= CSUR P1905 = DSM 100570) was isolated from the urine of a kidney transplant recipient.
85 Amino acid transport and metabolism F 74 2. 95 Nucleotide transport and metabolism H 105 4. 18 Coenzyme transport and metabolism I 73 2. 91 Lipid transport and metabolism P 123 4. 90 Inorganic ion transport and metabolism Q 44 1. 75 Secondary metabolites biosynthesis, transport and catabolism R 131 5. 22 General function prediction only S 78 3. 11 Function unknown - 997 39. 75 Not in COGs COGs, Clusters of Orthologous Groups database. a Total is based on total number of protein-coding genes in annotated genome.
That PARP inhibition prejudices efficacy of break repair has been exploited as anticancer strategies to potentiate the cytotoxicity of anticancer drugs  [14, 15]. Numerous phase I to III clinical trials based on this approach are in progress  [14]  (https://www. clinicaltrials. gov/). PARP inhibition has also proved to be exquisitely efficient to kill tumour cells deficient in double strand break repair by homologous recombination (HR), such as cells mutated for the breast cancer early onset (BRCA) genes BRCA1 or BRCA2  [16, 17]. The proposed explanation for this synthetic lethality between PARP and BRCA is that single strand breaks that arise spontaneously in cells are not efficiently repaired upon PARP inhibition, are thus converted during replication to double strand breaks that cannot be repaired in BRCA deficient cells, leading to cell death.
4a, b ). In agreement with our previous observations  [11] , siPARG alone slightly reduced HR compared to scramble siCTL (0. 89 ± 0. 06 %). In contrast, BRCA1 depletion dramatically reduced HR efficiency (0. 23 ± 0. 05 %), as expected. In the context of simultaneous PARG and BRCA1 To further examine the consequence of PARG and/ or BRCA1 siRNA-mediated depletion on HR efficiency, we examined by immunofluorescence the appearance of RAD51 foci in U2OS cells treated with the DSB inducer, topoisomerase II inhibitor etoposide. Cells were treated with 10 µM etoposide for 1 h and released in complete fresh medium for additional 2 h (Fig. 4c, d ), time chosen from kinetic experiment as the best moment to detect RAD51 foci in these conditions of treatment (data not shown). The presence of DSB was revealed by co-staining for γH2AX (Fig.
Statistical analyses Statistical analyses were carried out using R statistical packages. For clonogenic assays, short-term MTS viability assays and RAD51 foci analyses, data are represented as box plot graph where the lower and upper hinges represent the 25th and 75th percentile respectively. The middle horizontal line represents the median or 50th percentile. Whiskers are drawn to the lower and upper adjacent values. Far out values are represented by small "o". Significance tests, such as Anova and TukeyHSD (honest significant difference, for multiple comparison) were performed in R using the dataset used to draw the box plot. For cell counting at 72 h post-transfection and HR-assays, data are represented as bar plot graphs with standard deviation (SD) and significance evaluated using Student t test.
HR-assay HR was performed as described in Illuzzi et al. [11] , using U2OS cells containing the HR reporter DR-GFP and the inducible mCherry-I-SceI-GR (U2OS-DR-GFP-mCherry-I-SceI-GR). Cells were transfected with the respective siRNA twice, with interval of 48 h, treated for 2 days with 100 ng/ml of triamcinolone acetonide (TA, Sigma) to induce nuclear translocation of the mCherry-I-SceI-GR before evaluation of the GFP-positive cells out of the mCherry-positive cells by flow cytrometry (FACS-Calibur and Cell Quest software, Becton-Dickinson).
Acknowledgements We are grateful to  Michal Goldberg  ( Hebrew University, Israel ) and  Evi Soutoglou  ( Illkirch, France ) for providing the U2OS-DR-GFP-mCherry-I-SceI-GR cell line. This work was supported by the  Centre National de la Recherche Scientifique ,  Université de Strasbourg, Ligue contre le Cancer ,  Agence Nationale de la Recherche  ( ANR-13-BSV8-0003-01 ) and  Fondation pour la Recherche Médicale. This work has been published within the LABEX  ANR-10-LABX-0034_  Medalis and received a financial support from  French government managed by ANR  under " Programme d'investissement d'avenir ". VS/FD team was  Equipe Labellisée Ligue contre le Cancer  ( 2011-2015 ).
) denitrificans were prepared in 20 mM sodium phosphate buffer (pH 7. 2) containing 0. 05% dodecyl maltoside. For each purified complex extinction coefficients were calculated from a knowledge of the stoichiometry, thanks to the known structures of the complexes, and BChl a extraction from purified complexes as described previously  12. Briefly, the UV-visible absorption spectrum was measured, the BChla concentration of the sample was determined from the absorption of an acetone methanol (7:2) extract using the extinction coefficient of ϵ 770nm 76 mM -1 cm -113. The stoichiometry of the complex was calculated as 4 BChl per reaction center plus 2 BChl per LH1 type αβ pair for core complexes and 3 BChl per LH2 type αβ pair for LH2 complexes. The complex extinction coefficient was then calculated as ϵ(cm -1 mM -1 ) = Absorbance * stoichiometry/(1cm * [BChl]), see Dataset 1.
Conclusions We have developed a novel protocol based on copper-free click chemistry that allows the formation and purification of specific dimers between highly oligomeric proteins. This protocol can be used to prepare biologically relevant dimers of certain LH2s with very asymmetrical lysine distribution, for example the LH2 of Roseobacter denitrificans or Rhodobacter sphaeroides. Such dimers will be of considerable interest for studying energy migration in light-harvesting arrays. F1000Research The authors described an approach to hetero-coupling of bacterial photosynthetic apparatuses, LH2 and core complex. I think their approach using copper-free Huisgen cyclization is reasonable. Although reaction yields are unfortunately very low, careful and patient chemistry could overcome these problems that they described.
Controlling degree of labeling paragraph, remove "this" in the second sentence. d) Same paragraph as part b) refer to Figure1when referring to "extinction coefficients calculated as described above, and then check spelling of fluorescein immediately following. e) same paragraph, when talking about threshold, could you mention how many lysines do you expect to have available? It could be at least a measure of expected threshold. f) The second and third sentences under "Purifying low yield labeled proteins" I would paraphrase F1000Research f) The second and third sentences under "Purifying low yield labeled proteins" I would paraphrase into "We chose to use succinimidyl-2-(biotinamido)ethyl-1,3,dithiopropionate, allowing strong and specific binding of labeled protein to streptaidin beads. Pure protein was eluted either with biotin or by reducing the disulfide bond" g) Next sentence I would take out "as expected", and add "non-specific binding" when referring to contamination of unlabeled protein without biotin.
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. No competing interests were disclosed. Competing Interests: 06 June 2016 Referee Report doi:10. 5256/f1000research. 9335. r14129 Tatas Brotosudarmo Ma Chung Research Center for Photosynthetic Pigments (MRCPP), Ma Chung University, Malang, Indonesia.
Data availability F1000Research: Dataset 1. Extinction coefficients for purified complexes, 10. 5256/f1000research. 8676. d121349 21 F1000Research: Dataset 2. Degree of labeling for different complexes, 10. 5256/f1000research. 8676. d121350 22 F1000Research: Dataset 3. HPLC trace after cross-linking, 10. 5256/ f1000research. 8676. d121351 23 F1000Research: Dataset 4. Spectra of peaks in HPLC profile, 10. 5256/f1000research. 8676. d121352 24.
c) Controlling degree of labeling paragraph, remove "this" in the second sentence. d) Same paragraph as part b) refer to Figure1when referring to "extinction coefficients calculated as described above, and then check spelling of fluorescein immediately following. e) same paragraph, when talking about threshold, could you mention how many lysines do you expect to have available? It could be at least a measure of expected threshold. f) The second and third sentences under "Purifying low yield labeled proteins" I would paraphrase F1000Research f) The second and third sentences under "Purifying low yield labeled proteins" I would paraphrase into "We chose to use succinimidyl-2-(biotinamido)ethyl-1,3,dithiopropionate, allowing strong and specific binding of labeled protein to streptaidin beads.
l) Same paragraph, impressive statistical analysis of the distribution of modifications. Could you provide that as a supplementary material or show it somewhere? m) Last paragraph before discussion, about halfway: "the labeling positions" seems to be typed twice in a row. I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. No competing interests were disclosed. Competing Interests: 06 June 2016 Referee Report doi:10. 5256/f1000research. 9335. r14129 Tatas Brotosudarmo Ma Chung Research Center for Photosynthetic Pigments (MRCPP), Ma Chung University, Malang, Indonesia.
Dataset 3. HPLC trace after cross-linking http://dx. doi. org/10. 5256/f1000research. 8676. d121351 This table includes the data used to generate Figure 4A. Column 1, retention time; column 2 absorbance at 280 nm in OD. Dataset 4. Spectra of peaks in HPLC profile http://dx. doi. org/10. 5256/f1000research. 8676. d121352Thistableincludes the data used to generate Figure4B. Column 1, wavelength (in nm); column 2 to 4 absorbance at in order 5. 84, 6. 13 and 6. 60 min.
Proteins were denatured for 3 min at 95 °C, and cellular debris were pelleted by centrifugation. 10 l of supernatant were loaded on 10% SDS-PAGE. After transfer to nitrocellulose membrane with a semidry system, proteins were detected by hybridization with the appropriate antibodies. Mass Spectrometry Experiments and Data Analysis-For sample preparation from stacking gels, each lane was excised, cut in small cubes, and subjected to in-gel trypsin digestion after reduction and alkylation as described in Ref. 23. All steps from in-solution trypsin digestion, peptide sample analysis by LC-MS/MS on an Orbitrap Velos, protein identification, to comparative label-free quantification using MaxQuant suite were performed as previously described in Ref. 14. Fluorescence Microscopy-Fluorescence microscopy was performed with GFP-NonStop-expressing yeast cells grown in liquid synthetic medium containing galactose without uracil to a final A 600 of 4.
0 and resuspended in sterile water. DNA was stained with Hoechst 33352 (5 ng⅐l Ϫ1 ) for 5 min, and cells were washed twice in sterile water. Samples were imaged using a Leica DMRXA fluorescence microscope. Fluorescent signals were collected with single band pass filters for excitation of GFP (Leica) for 3500 ms, mCherry (Cy3; Leica) for 800 ms and Hoechst 33352 (A; Leica) for 40 ms. Images were acquired with a Hamamatsu ORCAII-ER cooled CCD camera controlled by the Openlab software (version 3. 5; Improvision) and processed using Adobe Photoshop CS3 software (version 10; Adobe).
Acknowledgments-We thank  Onn Brandman  for providing RQC2and RQC2 aaa -containing plasmids and  Véronique Albanèse  and  Jonathan Warner  for providing the anti-Sis1 and anti-Rpl3 antibodies, respectively. We thank  Antonia Doyen  for yeast strain construction,  Frank Feuerbach  for helpful advice in fluorescence microscopy and all the members of the lab for discussions and criticism on the manuscript. We are grateful to the proteomics platform of the  Pasteur Institute  for the availability of the  Orbitrap Velos.