dataesr/scientific-paragraphs-categorization huggingface dataset:
Illumination was then switched to TIRF mode to excite selectively molecules within ∼100 nm of the cover glass. Image acquisition was performed by illuminating with each wavelength in the TIRF mode for 6 s using a 300 ms integration time. Each set of images was then stacked using the average intensity and corrected for background using the rolling ball background subtraction algorithm  (29)  in ImageJ, with a radius of five pixels according to the size of spots. For colocalization analysis, the center positions of fluorescent spots were identified by their local maxima using a manually defined threshold in ImageJ. The colocalization of red-green spots was determined by calculating the distances between spots of one color to their mutual closest neighboring spot of the other color using R software (http://www.
Image stacks of the whole cell depth (10 m) were acquired with a Z-step size of 125 nm. Reconstruction and alignment of the 3D-SIM images was carried out with softWoRx v 5. 0 (General Electrics). 100 nm green fluorescent beads (Life Technologies) were used to measure the optical transfer function (otf) used for the 405 and 488 channels, and 100 nm red fluorescent beads (Life Technologies) were used to measure the otf used for the 561 channel. 170 nm TetraSpeck beads (Life Technologies) were employed to measure the offsets in the axial direction and the supplied target slide was used to calibrate X-Y image registration. All SIM data were checked to ensure they were of sufficient quality for analysis using the SIMCheck plugin  (30).
3D-SIM image analysis Segmentation of FISH spots and 3D colocalization analysis. 3D-SIM images were scaled to 16-bit prior to segmentation. Segmentation of FISH spots in the green and red channels was achieved using the 3D Spot Segmentation plugin in Im-ageJ  (31). The center positions of segmented spots in each channel were determined with subpixel precision using the 3D Objects Counter plugin and were then used to calculate the Euclidian 3D distances between spots of one color and their mutual nearest neighboring spot of the other color using R software (http://www. r-project. org). The spots of one color within a distance ≤160 nm to their mutual nearest neighboring spot of the other color were considered as colocalized, based on the distribution of nearest neighboring 3D distances (NNdist) obtained for an RNA that contained both tags on the same molecule (NV-lacZ-MS2 in Supplementary Figure  S2 ) and in accordance with the resolution limits of the microscope  (32).
Laser scanning and fluorescence detection were performed using an ALBA 5 system (ISS, Champain, IL, USA), a Zeiss Axiovert 200 microscope stage, and a 60× Plan-Apochromat 1. 4NA oil immersion objective. The emitted fluorescence was detected on two separate avalanche photodiode detectors with an appropriate dichroic 580LP (chroma, AHF analysentechnik, Tuebingen; DE)) and emission filters to separate the green (eGFP) (530/43, Chroma) and red (mCherry) (653/95, Chroma) emissions. The excitation volume was calibrated as diffraction-limited two-photon PSF using 60 nM Fluorescein in Tris pH9 with a w0 value of 340 nm and a z0 value of 1. 4 m. For Cross N&B data acquisition, we collected 50 frames of 20×20 m size (256 Pxl X 256 Pxl). The laser power was 16 mW and the laser dwell time 32 s. During the 50 images stack acquisition, photobleaching was always below 10%.
The data were analyzed using in house software written in IgorPro. Scanning cross number and brightness extends fluorescence cross-correlation spectroscopy (FCCS) to twodimensional cellular images by a covariance analysis of the raster-scanned fluorescence fluctuations in two channels  (33, 34)  (extended information is provided in Supplementary Information). In two-color mode (here using GFP and mCherry fluorescent protein fusions for each of two interacting partners), calculation of the covariance between the intensity fluctuations in the green and red detection channels (ch1 and ch2, respectively) allows evaluation of the cross-brightness (Bcc) or covariance of the intensities  (34). B cc = σ ch1 • σ ch2 F ch1 • F ch2 where σ and F are the variance and the average of the fluorescence intensity signal in each channel, respectively.
Sequencing and Bioinformatics Whole genome sequencing (WGS) was performed at the iGE3 Unige Genomics platform. Sanger sequencing was performed by Fasteris (Fasteris SA, Switzerland). Multiple alignments were performed using Clustal 2  , and boxshade 3. Ortholog analysis relied on the OMA database 4   (Altenhoff et al. , 2015).
SA0657 has a Predicted Transmembrane Region and a Crucial CBS Pair in a Universally Conserved Layout SA0657 encodes a protein of 449 AA, predicted to be composed of a transmembrane region in the N-terminal part (DUF21: IPR002550), followed by a pair of CBS domains and a CorC_HlyC domain of unknown function in the C-terminal part (Figure  5 ). CBS domains (IPR000644) have poor sequence similarity but are defined by a conserved structure and are found in pairs that are referred to as a "Bateman module. " The binding of adenosine nucleotides (AMP, ATP, S-adenosylmethionine) or Mg 2+ triggers conformational changes of the protein leading to change in activity  (Baykov et al. , 2011; Ereño-Orbea et al. , 2013) , and a CBS pair is, for example, responsible for the regulation of the activity of the magnesium transporter MgtE  (Imai et al.
, 2012). The OMA orthology database describes 2043 proteins as orthologs of SA0657, among which is Salmonella CorB (StCorB: STM2679, see Supplementary Figure  S5 )  (Altenhoff et al. , 2015)   5. These two proteins share the same domains architecture but their overall similarity is low (22%; Figure  5  and Supplementary Figure  S7 ). StCorB was identified in a screen for S. Typhimurium mutants resistant to cobalt  (Gibson et al. , 1991) , where the authors proposed CorB to be an additional factor necessary for Mg 2+ efflux (along with two other proteins CorC and CorD), while influx was suggested to be solely mediated by CorA. Among the SA0657 orthologs, we identified a paralog of CorB in S. Typhimurium genome (STM4407), whose function is unknown and which was not identified in the cobalt resistance screen by  Gibson et al.
(1991). The S. aureus genome additionally encodes SA0780, a paralog to SA0657 that is 103 residues shorter (Figure  5  and Supplementary Figure  S7 ). To get a better grasp of the evolution and the distribution of SA0657 homologs in the genomes of the OMA database, we performed a phylogenetic analysis of the 3743 predicted orthologs of SA0657, SA0780, StCorB, StCorC, STM4407, human CNNM2 and murin CNNM2 (see Discussion) (Supplementary Figure  S4 ). This analysis reveals that SA0657 orthologs are widely distributed in the prokaryotic kingdom and that each phylum often possess several paralogs of SA0657, most often three, with up to nine in the Streptomyces coelicolor genome (Supplementary Figure  S6 ). This suggests that the SA0657 ancestor arose long before phyla separation. Proteobacterial proteins, StCorB (STM2479), StCorC (STM4407) and STM4407 are present in different clusters each counting several hundreds leaves, and SA0657 belong to a cluster of 273 Firmicute proteins.
Therefore, miRNAs can be used as a tool to uncover the pathways and targets that underlie changes in skeletal muscle in different pathophysiological conditions and after specific treatments. To identify the miRNA targets and pathways that are shared by more than one miRNA, a computer program named SID1. 0 (simple String IDentifier) was developed  [30]. This string identification program has proven to be a very useful tool to predict new genes, miRNAs and related targets and pathways involved in different pathophysiological processes  [30, 31]. In the present study, we performed the in silico analysis of the pathways shared by different miRNAs involved in the effects of n-3 PUFAs on myogenesis to support the experimental in vivo and in vitro observations.
Histological analysis Histological sections (4-μM) were cut from paraffin-embedded skeletal muscles, deparaffinized in xylene, rehydrated in ethanol and stained with H&E (Bio-Optica, Milan, Italy) according to standard procedures in order to quantify the morphological observation. The images were acquired by means of a Leica DMRB microscope coupled with a digital camera. To determine the percentage of myofibers with internalized nuclei, micrographs of H&E stained skeletal muscle sections were captured using a digital camera, and fibers with internalized nuclei were counted using NIH ImageJ software from five sections taken from each hamster (n=5 animals/group).
Principal Component Analysis on histological section The images of H&E stained skeletal muscle sections (from six sections taken from each hamster, n=5 animals/group) were processed by ImageJ software (http://imagej. nih. gov/ij/docs/intro. html). All the commands related to statistical measurements on image data, profile and histogram plotting and plugins related to image analysis can be found in http://imagej. nih. gov/ij/docs/guide/146-30. html#t oc-Subsection-30. 7. The following morphometric parameters were selected as being the most representative of fiber shape in the skeletal muscle: Area (area of selection in square pixels); Circ: (calculate to display circularity), AR (aspect ratio, major axis/minor axis); Round: (roundness); Solidity (area/convex area). Extracellular Area represents the extension of extracellular matrix.
The variables correlated with the two principal components can be identified by the highest score coefficients in absolute values. A multivariate analysis of variance (ANOVA) was performed to compare the groups with regard to the variables extracted from the image analysis. Moreover, a univariate ANOVA test was applied for each variable, followed by a multiple comparison between the three groups performed by means of the least significance difference (LSD) test. The significance level was set at alpha = 0. 05. Statistical analyses were performed with SPSS 18. 0. (Statistical Package for Social Sciences). For each PCA the variance explained by the model was evaluated  [37].
Apoptosis quantification TUNEL assay was performed on paraffin hamster muscular sections using the terminal deoxynucleotidyl transferase (TdT)-mediated in situ fluorescein-conjugated, dUTP nick end-labeling technique (In Situ Cell Death Detection Kit, Fluorescein), according to the manufacturer's protocol (Roche Diagnostic Corp. ). Briefly, muscle sections (biceps femoris) were deparaffinized in xylene, rehydrated and then treated with proteinase-K before proceeding with the assay. Sections were stained with a mouse monoclonal anti-α-sarcomeric-actin (Sigma-Aldrich) and then with anti-mouse Alexa 546 secondary antibodies (Molecular Probes). Nuclei were stained with DAPI. To determine the percentage of apoptotic cells, micrographs of skeletal muscle sections were captured using a Leica microscope (Leica Microsystems DMRB), and positive and negative TUNEL nuclei were counted using NIH ImageJ software from five sections taken from each hamster (n=5-6 animals/group).
After nucleus staining with DAPI (Sigma-Aldrich), skeletal muscle sections were analyzed by means of a fluorescence microscope (Leica Microsystems DMRB). All quantifications were performed using ImageJ software (http://rsb. info. nih. gov/ij/). To determine the percentage of positive CD45 cells and positive Pax7 and myogenin nuclei, micrographs of skeletal muscle sections were captured and cells counted from six sec-tions taken from each hamster, n=5 animals/group. To determine the fluorescence of α-MHC and caveolin-3 levels in skeletal muscle sections, the total cell fluorescence (CTCF) was calculated from digital images using the following formula  [38] : CTCF = Integrated Density -(Area of selected cell X Mean fluorescence of background readings) Images were from six sections, taken from each hamster ( n=6 animals/group).
Principal Component Analysis on cell culture The morphometric parameters derived from light microscope images of H&E stained C2C12 cells were elaborated with the image processing software (ImageJ), as previously described for in vivo studies. The following morphometric parameters were considered: Area; StdDev (standard deviation of the gray values used to generate the mean gray value); X and Y Centroid (the average of the x and y coordinates of all of the pixels in the image or selection which uses the X and Y headings); XM and YM Center of Mass (the brightness-weighted average of the x and y coordinates all pixels in the image or selection which uses the XM and YM headings); Perimeter (perimeter, the length of the outside boundary of the selection); the shape descriptors: AR; Round; Solidity; Circ; Num Nuclei (the number of nuclei in each cell).
Apoptosis quantification To analyze apoptotic nuclei by TUNEL assay, C2C12 cells were fixed in 4% paraformaldehyde in PBS, pH 7. 4, and permeabilized with 0. 1% Triton X-100 in 0. 1% Na citrate. TUNEL assay was performed using the terminal deoxynucleotidyl transferase (TdT)-mediated in situ fluorescein-conjugated, dUTP nick end-labeling technique (In Situ Cell Death Detection Kit, Fluorescein), according to the manufacturer's protocol (Roche Diagnostic Corp. ). To assess cell apoptosis, nuclei were analyzed from 10 random fields selected from 5 cell wells for each treatment using NIH ImageJ software. Apoptotic cell counts were expressed as the percentage of the total number of nuclei counted.
Immunofluorescence Primary antibodies used for immunofluorescence were: monoclonal anti-myogenin mouse Mab (BD Transduction), MF20 monoclonal antibody (MAb) (Developmental Studies Hybridoma Bank, University of Iowa) and mouse anti-caveolin-3 (BD Transduction Laboratories™). Cells were fixed with 4% paraformaldehyde and permeabilized with 0. 1% Triton X-100 for 2 min and then incubated with primary antibody for 1 h and subsequently with an appropriate secondary antibody (488 Alexa Fluor, Molecular Probes) for 1 h. After nucleus staining with 1 µg/ml DAPI (Sigma-Aldrich), cells were analyzed using a fluorescence microscope (Leica Microsystems, Mod. DMRB) equipped with a digital camera. All quantifications were performed using ImageJ software. (http://rsb. info. nih. gov/ij/). To determine the percentage of positive myogenin nuclei above the total number of nuclei, and MHC and caveolin-3 positive cells above the total number of cells, micrographs were captured and cells counted from a minimum of six random fields selected from three slides.
Statistical analysis Results are expressed as the mean ± SD. The analysis of variance was performed (ANOVA) for comparisons between more than two groups, whereas the two-tailed unpaired Student's t-test was used for comparisons of the mean differences between two groups. A suitable post-hoc test was used in combination with ANOVA to test for significant differences between groups. Differences were considered statistically significant when P< 0. 05. (SPSS for Windows, version 11. 5; SPSS, Inc. , Chicago, IL).
In silico A literature search was performed using Pub-Med or ISI Web of knowledge Databases to identify miRNAs with experimental evidence of involvement in murine muscle differentiation and modulated by n-3 PUFAs or TNF (last accessed December 21 2014). The following key words were used: miRNAs and differentiation and C2C12 or TNF and miRNAs or n-3 PUFAs and miRNAs. Because a lack of data was found , regarding the influence of ALA on microRNA we extended PubMed searches to n-3 PUFAs. Moreover, we extended PubMed searches to humans miRNAs, because, (at the best of our knowledge) no literature on n-3 PUFAs modulation of miRNAs in murine cells was found as yet. Thus, the murine miRNAs, which are equivalent to humans mi-croRNAs, were considered in the present research. To obtain the common pathways of specific miRNAs, the list IDs of KEGGs pathway and database DIANA-mirPath  [39]  were indexed using SID1.
0. Since a visual inspection of the IDs would be unpractical due to their large number (thousands IDs), they have been automatically indexed using a simple Fortran written program (SID1. 0; String IDentifier), developed by us  [30] , that looks for Refseq IDs shared by the predicted pathways of the different datasets. The database also allows to report the -ln(p-value) (the negative natural logarithm of the enrichment p-value calculated for the specific pathway). The p-value is a measure of the association between a selected gene from the list of pathways. In pathway analysis, generally p-values less than 0. 05 indicate that the association is not statistically significant and the pathway could be rejected.
Six morphometric parameters were opportunely selected by ImageJ software, as reported in the Materials and Methods section (Fig. 1C ). The measurements of these parameters were used as variables to construct two Principal component functions. The diagram of the values (Fig. 1D ) obtained from the morphometric parameters for each group (Healthy, Dystr/P and Dystr/FS) showed that the Principal component 1 (PC1) was strongly correlated with the Area, AR and solidity parameters; the Principal component 2 (PC2) was strongly correlated with the Circ, Round and Extracellular Area parameters identified by the highest score coefficients in absolute values (Fig. 1C  in the red box). Dystr/P (yellow dots) revealed a growing PC2 (y-axis value) compared with the Healthy group (blue dots) (the total variance explained by the PC1 and PC2 was 53.
Flaxseed diet reduces inflammation and cell death In order to determine whether the altered morphology in dystrophic skeletal muscles was associated with an inflammatory status, the level of plasma TNF in 150-days-old hamsters was investigated by ELISA analysis. TNF levels were markedly increased in plasma of Dystr/P (3. 2 fold) vs. healthy controls (Fig. 2A ). The FS diet induced a slight, non-significant decrease in the cytokine levels. Inflammation of hamster muscle tissue was also monitored by pan-leukocyte marker CD45 immunofluorescence, which demonstrated a marked increase in the number of inflammatory cells in the Dystr/P compared with the healthy muscle sections (Fig. 2B ). By contrast, in the skeletal muscles of Dystr/FS, the number of CD45 positive cells significantly decreased. Quantitative data are shown in Figure  2C.
ALA preserves the morphology of differentiating myoblast C2C12 by attenuating the deleterious effects of TNF To test the effects of ALA on the inhibition induced by TNF on myogenesis, murine C2C12 myoblasts were used as an in vitro cell model to recapitulate myogenic differentiation. After 5 days in DM, TNF inhibited differentiation and myotube formation in C2C12 cells, as shown in the H&E stained image (Fig. 4A ), thus confirming previous observations  [40]. The addition of ALA to the DM prevented the inhibitory effect of TNF on myogenesis, thereby allowing the formation of myotubes in C2C12 cells. (Fig. 4 A ). To quantify any morphological changes, the PCA analysis was elaborated from twelve morphometric parameters opportunely selected and measured by ImageJ software. The resulting diagram (Fig.
e. miR-1, miR-133a, miR-133b and miR-206, involved in C2C12 cell differentiation are modulated by TNF in the same cells  [54]. It is noteworthy that miR-206 is modulated by both n-3 PUFAs and TNF. Therefore, the search in the literature yielded the following seven miRNAs for the computational analysis: miR-206, miR-181a, miR-29a, miR-26a, miR-1, miR-133a and miR-133b. The web-based computational database DIANA-MicroT 3. 0 was used to extract the KEGG pathway IDs potentially altered by the expression of these seven miRNAs. These pathways were then filtered using the SID1. 0 program to detect the common pathways. The database selected 149 common pathways (p-value range between 0. 05 and 32. 36). Table  1  shows all the common pathways with p values above 10 (13 common pathways). The common pathways selected included those related to the cell-cell and cell-ECM interaction, such as the FAK pathway and pathways related to communication between the external microenvironment and the interior of the cells (DNA), such as MAPK.
Acknowledgments This work was funded by  Fondazione San Raffaele, Ceglie Messapica Italy Research Project MERIT-RBNE08HM7T -Italian Ministry of Health  and partially by  ANR  (no. 13-BSV1-0005 ),  NIH  (no. 5R01CA180057-02 ),  AFM  (no. 2012-0773 ),  UPMC Emergence 2011 , and IBPS 2015. The authors would like to thank  Dott. Mauro Sansò , Chief Operating Officer at  Fondazione San Raffaele  for his excellent management of the MERIT # RBNE08HM7T  funds.
[26]. Here, we present new scanning transmission X-ray microscopy, 2-D electron microscopy and 3-D electron tomography data acquired on both Candidatus Gloeomargarita lithophora C7 and Candidatus Synechococcus calcipolaris G9. Based on these new observations and a review of the literature, we discuss with unprecedented detail the molecular pathways possibly involved in the formation of intracellular carbonates in both cyanobacterial groups.
Transmission Electron Microscopy Analyses Conventional TEM analyses were carried out on a JEOL-2100F (JEOL Ltd. , Tokyo, Japan) microscope operating at 200 kV. This microscope was equipped with a field emission gun, an ultra-high resolution (UHR) pole piece, a Gatan energy filter (GIF200) system (Gatan, Inc. , Pleasanton, CA, USA), a JEOL detector with an ultrathin window allowing detection of light elements, and a scanning TEM (STEM) device, which allowed Z-contrast imaging in high angle annular dark field (HAADF) mode. Chemical analyses were acquired by performing energy dispersive X-ray spectrometry (EDXS) mapping in the HAADF STEM mode. STEM EDX spectrum analysis was made using the JEOL Analysis Station software, and the semi-quantitative chemical microanalyses of individual carbonate and polyphosphate inclusions relied on the use of K-factors.
8. 5. Tomograms were reconstructed using the IMOD software package (University of Colorado, Boulder, CO, USA) or DIGIECT software  [37]. The particle sizes (diameters) of carbonate inclusions were calculated from more than 200 measurements performed on HAADF-STEM images using Image J software (v1. 50b). The frequency distribution of particle sizes was analyzed by the IBM SPSS Statistics software (v. 22) (IBM, New York, NY, USA), and the skewness was calculated using the equation (µ ´ν)/σ, where µ is the mean, ν is the median, and σ is the standard deviation. A skewness value of zero means that the distribution is symmetric (Gaussian distribution), while a positive skewness indicates an asymmetrical distribution with a long tail towards higher values, and a negative skewness indicates an asymmetrical distribution with a long tail towards lower values.
Scanning Transmission X-Ray Microscopy Analyses STXM experiments were carried out at the P L 2,3 -edges. STXM and X-ray absorption near-edge structure (XANES) spectroscopy analyses were performed on beamline 11. 0. 2. 2 at the Advanced Light Source (Lawrence Berkeley National Laboratory, Berkeley, CA, USA). The ALS storage ring was operated at 1. 9 GeV and 500 mA current in a top-up mode. Energy calibration was achieved using the well-resolved 3p Rydberg peak of gaseous CO 2 at 294. 96 eV for the C K-edge. A 25-nm zone plate was used. Alignment of images of stacks and extraction of XANES spectra were done using the aXis2000 Software  [39]. Carbonate inclusions were discriminated based on their different spectral signature at the C K-edge and the Ca L 2,3 -edges following the approach detailed by Cosmidis et al.
Acknowledgments: The research leading to these results has received funding from the  European Research Council  under the European Community's  Seventh Framework Programme  (FP7/2007-2013 Grant Agreement No. 307110-ERC CALCYAN ). The TEM facility at IMPMC was purchased owing to a support by  Region Ile-de-France  grant  SESAME 2000 E 1435. Advanced Light Source (ALS) Molecular Environmental Science beamline 11. 0. 2 is supported by the  Office of Science, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences , and  Biosciences and Materials Sciences Division, U. S. Department of Energy , at the  Lawrence Berkeley National Laboratory.