dataesr/scientific-paragraphs-categorization huggingface dataset:
A528R Interacts with NF-κB p65 to Suppress its Promoter Activity In order to further explore the relationship between A528R and co-immunoprecipitation (Co-IP) were performed. The results show that there is teraction between A528R and porcine p65 (Figure  4A ). Based on the data retrieve UniProt (https://www. uniprot. org/uploadlists/), porcine p65 is composed of N-te Rel homology domain (RHD) region and C-terminal non-RHD region. We cloned RHD region and non-RHD region into pEGFP-N1 vector, respectively. The Co-IP s that there is an interaction between A528R and p65 RHD region and not p65 no (Figure  4B ). According to the prediction of UniProt, there are five Ankyrin repeats existing in A528, including amino acids 54-83, 129-158, 261-290, 292-321, and 32 We made the five individual deletion mutants of A528R, and the Co-IP results s that all five deletion mutants had weakened interactions with p65 relative to full A528R (Figure  4C ).
A528R Interacts with NF-κB p65 to Suppress its Promoter Activity In order to further explore the relationship between A528R and p65, co-immunoprecipitation (Co-IP) were performed. The results show that there is an interaction between A528R and porcine p65 (Figure  4A ). Based on the data retrieved from UniProt (https://www. uniprot. org/uploadlists/, accessed on 16 September 2021), porcine p65 is composed of N-terminal Rel homology domain (RHD) region and C-terminal non-RHD region. We cloned the p65 RHD region and non-RHD region into pEGFP-N1 vector, respectively. The Co-IP showed that there is an interaction between A528R and p65 RHD region and not p65 non-RHD (Figure  4B ). According to the prediction of UniProt, there are five Ankyrin repeats (ANK) existing in A528, including amino acids 54-83, 129-158, 261-290, 292-321, and 322-352.
Funding: The work was partly supported by the  National Key Research and Development Program of China  ( 2017YFD0502301 ),  Jiangsu Provincial Key R&D plan  ( BE2020398 ), Jiangsu agricultural science and technology independent innovation fund project: CX(21)2035,  National Natural Science Foundation of China :  31672523  and a project funded by the  Priority Academic Program, Development of Jiangsu Higher Education Institutions (PAPD). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable.
, 2015). However, these studies do not use purified polysaccharides to confirm the absorption bands identified by comparison with the literature. Moreover, some studies have also performed polysaccharide analysis using spectral data but obtained from KBr pellets or aqueous solutions, the classical way before the development of the ATR method  (Kacurakova et al. , 2000). Fruits and vegetables are highly hydrated and susceptible to environmental conditions. Drying, not only prevents samples from oxidation and hydrolysis under the action of endogenous enzymes, but also concentrates samples by water elimination, so it significantly improves the reflectance spectra of some specific components present in lower content than water  (Lan et al. , 2020). Therefore, the systematic analysis of purified solid materials from cell wall polysaccharides by ATR-FTIR may improve their identification.
To track the characteristic peaks of each cell wall component, spectral data and conventional chemical methods are used to study the composition of cell walls and internal structures. In order to evaluate the available information based on extracted samples, powders of cellulose, hemicelluloses, and pectins were also scanned in ATR-FTIR. To identify the typology of the cell walls, both PCA and Hierarchical Cluster Analysis (HCA) were performed. This study provided new explanations and experimental ideas for studying complex natural polymer systems, and guidance for using ATR-FTIR data to clarify carbohydrate structures, physical properties and interactions. Switzerland). Arabinan (sugar beet), linear 1,5-α-L-arabinan (sugar beet), debranched arabinan, galactan (potato), rhamnogalacturonan I (from potato pectic fibre), and rhamnogalacturonan (from soybean pectic fibre), xylan, arabinoxylan, glucomannan, and xyloglucan were provided from Megazyme (Bray, Ireland).
ATR-FTIR spectra All cell wall polysaccharide samples, in the form of dry powder, were stored in P 2 O 5 atmosphere before analysis to remove residual water. ATR-FTIR spectra data (4000 to 600 cm -1 ) were acquired at room temperature in a Tensor 27 FTIR spectrometer (Bruker Optics®, Wissembourg, France), using a single-reflectance horizontal ATR cell (Golden Gate with a diamond crystal, Bruker Optics®) equipped with a system to press the dried homogenized samples on the crystal surface  (Bureau, Ścibisz, Le Bourvellec, & Renard, 2012). Each sample was analyzed three times (using after homogenization three different aliquots of the powders) to consider its heterogeneity, and each spectrum was the average of 16 scans. Spectral pre-processing and data treatment using multivariate analyses were performed with MATLAB 7.
Statistical analysis All biochemical analyses were presented as mean values of analytical triplicates and the reproducibility of the results was expressed as pooled standard deviations (Pooled SD). Pooled SD was calculated per series of replicates using the sum of individual variances weighted by the individual degrees of freedom  (Box, Hunter, & Hunter, 1978). A PCA was applied on the ATR-FTIR spectra in the range between 1800 and 800 cm -1 in order to study the repartition of the cellulose, hemicelluloses and pectins in a space according to their composition and absorption bands. Spectral data pre-processing and PCA were performed using MATLAB 7. 5 (Mathworks Inc. Natick, MA) software using the SAISIR package  (Cordella & Bertrand, 2014). HCA was performed using R software using FactoMineR (for computing) and factoextra (for visualizing the results) (R Core Team, 2014).
, 2021) ? As shown on the PCA, the kiwifruit cell walls were at the upper left in the middle of the Fig. 2C , while the apple and beet cell walls were at the bottom right in the middle of the Fig. 2C. This was probably linked to the high cellulose and relatively low galacturonic acid for kiwifruit, and the high galacturonic acid for both apple and beet (140-206 mg/g for apple, 141-225 mg/g for beet)  (Liu et al. , 2021). However, the cell walls of apple and beet were not distinguished, in spite of marked chemical and structural differences  (Liu et al. , 2021). Even a PCA performed on the ATR-FTIR spectra of the cell walls of apple and beet alone did not separate them well (data not shown). Beet cell walls are rich in arabinan (111-189 mg/g), contain ferulic acid, and have only minor amounts of rhamnose, fucose, xylose and mannose.
Therefore, ATR-FTIR spectroscopy coupled with chemometrics allowed a good discrimination of cell walls related to their compositions and structures, giving some classes according to the different kinds of pectins, hemicelluloses and cellulose. The cell walls represented a polymer system in a complex mixture with a diversity of both compositions and structures. PCA and HCA performed on the spectral data allowed to discriminate samples according to their cell wall polysaccharides. ATR-FTIR could be considered as a fast and easy way to distinguish different types of cell walls. Due to their complexity and the numerous spectral bands for each component, with the addition of overlapping, it was therefore difficult to assign each band to a compound chemical structure. However, the observed changes in intensity or presence/absence of some bands reflected the differences in composition between the cell walls.
Mechanistically, linkage of cysteine, tyrosine or histidine to the 8α-methyl group or the C6 atom of the isoalloxazine ring (Figure  S1 ) has been reported and suggested to occur auto-catalytically  [16]. Alternatively, in prokaryotes flavin transferases of the ApbE (alternative pyrimidine biosynthesis; also Ftp: flavin-trafficking protein) family (PFAM accession: PF02424) attach FMN covalently to threonine residues by phosphoester bond formation  [17, 18]. ApbE recognises the degenerated consensus sequence Dxx(s/t)gAT and targets threonine 7 in this motif as the FMN acceptor residue  [19, 20]. A well described target of ApbE-mediated post-translational modification is Na + -translocating NADH:quinone oxidoreductase, a component of the respiratory chain  [17, 21]. Fumarate reductases (FRDs), which catalyse the reduction of fumarate to succinate during anaerobic metabolism  [22, 23] , predominantly occur as multi-subunit membranebound complexes.
Transgenic cell lines To express FRDg(1-350), a C-terminal truncation of FRDg (TriTrypDB accession: Tb927. 5. 930) with a length of 350 amino acids, followed by two Ty1 epitopes  [37] , linked and interspaced with (PS) x1 linkers (DNA: CCAAGT), was inserted between the HindIII and BamHI restriction sites of the tetracycline-inducible pLew82  [38]  expression vector. The respective fragment was amplified from genomic DNA of strain AnTat1. 1 Munich  [34]. NotI was used for plasmid linearisation before transfection of the AnTat1. 1 90-13 cell line with the Nucleofector II device (Amaxa Biosystems)  [39]. Continuous selection was performed with phleomycin (BSF: 2. 0 µg ml -1 ; PCF: 2. 5 µg ml -1 ). The same approach was used to express FRDg(1-363), FRDg(1-363)-S9N that is identical to FRDg(1-363) except for a substitution of serine 9 (TCA) with asparagine (AAT) and FRDg(1-363)-D316A that is identical to FRDg(1-363) except for a substitution of aspartic acid 316 (GAC) with alanine (GCA).
FRDg  was N-terminally fused to T. brucei aconitase (ACO) (TriTrypDB accession: Tb927. 10. 14000)  [40] , replacing the mitochondrial targeting signal (FRDg(1-37)-ACO). Using XbaI, two fragments were inserted simultaneously between the HindIII and BamHI restriction sites of pLew82 with a puromycin resistance cassette; (1) a fragment containing the first 111 bp of the FRDg gene, followed by a flexible GSAGSAAGSGEF linker  [41]  (DNA: GGGTCTGCGGGGTCTGCAGCTGGGAGTGGTGAGTTT), (2) a 2664 bp fragment of ACO position 31-2694. AnTat1. 1 90-13 and the derived FRDg  cell line were transfected with the plasmid. Continuous selection was performed with puromycin (BSF: 0. 1 µg ml -1 ; PCF: 1. 0 µg ml -1 ). FRDg  was N-terminally fused to glycosomal isocitrate dehydrogenase (TriT-rypDB accession: Tb927. 11.
900), resulting in the fusion protein FRDg(1-363)-IDHg. A fragment containing the first 1089 bp of the FRDg gene, followed by a GSAGSAAGSGEF linker, was inserted between the HindIII and AgeI restriction sites of an IDHg open reading frame (TriTrypDB accession: Tb927. 11. 900) containing plasmid  [42]  based on the constitutive expression vector pTSARib  [43]. BglII was used for plasmid linearisation before transfection of the AnTat1. 1 90-13 cell line. Continuous selection was performed with puromycin (BSF: 0. 1 µg ml -1 ; PCF: 1. 0 µg ml -1 ). The same approach was used to express three additional FRDg-IDHg fusion proteins: (1) FRDg(1-9)-IDHg (FRD(1-9) N-terminally fused to IDHg), (  2 ) FRDg(1-37)-IDHg (FRD(1-37) N-terminally fused to IDHg) and (  3 ) FRDg(1-363)-D316A-IDHg (FRD(1-363)-D316A N-terminally fused to IDHg).
Subcellular digitonin fractionation 2. 0 * 10 9 procyclic T. brucei cells were harvested and washed twice in PBS. The cells were resuspended to 6. 5 * 10 8 ml -1 in STEN buffer (250 mM sucrose; 150 mM NaCl; 25 mM Tris-HCl (pH 7. 4); 1 mM EDTA) supplemented with 3. 5 % (V/V) PMSF (57 mM; solvent: isopropanol). 200 µl aliquots of the cell suspension (corresponding to 660 µg protein  [50] ) were incubated for 4 min at 25℃ in 14 different digitonin concentrations. Immediately after incubation, the samples were centrifuged at 14,000 g for 2 min. The supernatant was analysed by SDS-PAGE and western blotting. Pyruvate phosphate dikinase (PPDK), heat shock protein 60 (HSP60) and enolase (ENO) were used as glycosomal, mitochondrial and cytoplasmic marker proteins, respectively. The maximum signal intensity obtained for each analysed protein was set to 1 and the data points were fitted to the logistic function f (x) = A/(1 + exp((x -m)/s)) by application of the Levenberg-Marquardt algorithm for non-linear least square analysis.
15 µl were subjected to SDS-PAGE. The gel was stained with Colloidal Coomassie Brilliant Blue G-250  [52, 53]. The partially purified protein band was identified by in-gel fluorescence, excised (Figure  S2A ) and destained, followed by in-gel trypsin digestion. Isolated peptides were purified using C18 stage tips and analysed by LC-MS/MS. Proteomic analysis was performed at the Protein Analysis Unit (ZfP) of the Ludwig Maximilian University of Munich, a registered research infrastructure of the Deutsche Forschungsgemeinschaft (DFG, RI-00089). MaxQuant  [54]  software was used for peptide identification and quantification.
Bioinformatic analysis and toolchain For statistical analysis and plotting R 3. 6. 2 [55]  was used with the integrated development environment RStudio 1. 1. 463 [56]. In silico cloning and sequence alignment was performed with CLC Main Workbench 8. 1 (https://www. qiagenbioinformatics. com). Sequence similarity was visualised using Sequence Manipulation Suite 2  [57]. Image post-processing was carried out with Fiji  [58] , quantification with Image Studio Lite (https://www. licor. com). Protein domains were annotated and predicted using InterPro  [59] , while general genomic information was retrieved from TriTrypDB  [60]  or GenBank  [61].
Only upon effective import, the tag on the fusion protein will be flavinylated in trans by FRDg, enabling analysis without prior cell fractionation. This N-terminal tagging approach is unlikely to interfere with the glycosomal import mechanism, which mostly relies on a C-terminal peroxisomal-targeting signal  [78]. The strategy can be used to map glycosomal import sequence requirements, study the import mechanism and its regulation, or screen for novel glycosomal proteins. Eukaryotic ApbE domains are rare and limited to few clades of the tree of life. The domain architecture of kinetoplastid FRDs (Figure  1A ) is also predicted for some algal FRDs, such as Symbiodinium microadriaticum (GenBank accession: OLP85325), Nannochloropsis gaditana (GenBank accession: EWM27529) or Nannochloropsis salina (GenBank accession: TFJ85565).
Samples were analyzed with a Leica SP2/SP8 confocal microscope (Leica Microsystems). For each FOV, a stack of two channels (one per staining) and 9-13 Z stacks with a step size of 0. 3 mm were acquired with a 63Â oil-immersion objective and processed with ImageJ software (National Institutes of Health). Representative images for each condition were prepared for figure presentation by applying color adjustments uniformly with Adobe Photoshop version CS6. For PLA imaging in brain sections, mice were deeply anesthetized and immediately perfused transcardially with PBS followed by 4% PFA/ PB. Brains were removed and postfixed overnight in the same solution, cryoprotected by immersion in 10%, 20%, 30% gradient sucrose (24 h for each sucrose gradient) at 4°C, and then frozen in dry ice-cooled methylbutane.
4 numerical aperture), and a 405 nm and a 561 nm laser lines. For each FOV, a stack of two channels (one per staining) and 9-13 Z stacks with a step size of 1 mm were acquired. Images were opened and processed with ImageJ software (National Institutes of Health). Quantification of cells containing one or more red dots versus total cells (blue nuclei) was determined by using the Fiji package (https://imagej. net/software/fiji/). Nuclei and red dots were counted on the maximum projections of each image stack. After getting the projection, each channel was processed individually. The blue nuclei and red dots were segmented by filtering with a median filter, subtracting the background, enhancing the contrast with the Contrast Limited Adaptive Histogram Equalization plug-in, and finally applying a threshold to obtain the binary image and the regions of interest.
To quantify receptor-Rluc expression, luminescence readings were also performed after 10 min of adding 5 mM DeepBlueC (Invitrogen). The net BRET is defined as [(long-wavelength emission)/(short-wavelength emission)] -Cf where Cf corresponds to [(long-wavelength emission)/(short-wavelength emission)] for the Rluc construct expressed alone in the same experiment. BRET is expressed as milli BRET units (mBU; net BRET Â 1000). In BRET curves, BRET was expressed as a function of the ratio between fluorescence and luminescence (GFP/Rluc). To calculate maximal BRET from saturation curves, data were fitted using a nonlinear regression equation and assuming a single phase with GraphPad Prism software version 8. 0. 1. Each representative curve shown is the mean of three internal replicates. Western blot and coimmunoprecipitation.
All antibodies were prepared in TBS-Tween-20 (0. 1%) with 5% BSA (w/v). Membranes were then washed 3 times with TBS-Tween-20 (0. 1%), and HRP-labeled secondary antibodies, selected according to the species of origin of the primary antibodies (Sigma-Aldrich, #NA-931-1 and #NA-934V), were added for 1 h at a 1:5000 dilution in TBS-Tween-20 (0. 1%) at room temperature. Finally, protein bands were detected by incubation with an enhanced chemiluminescence reagent (Bio-Rad), and densitometric analysis of the relative expression of the protein of interest versus the corresponding loading control was performed with ImageJ software. Western blot images were cropped for clarity. Electrophoretic migration of molecular weight markers is depicted on the left-hand side of each blot. For coimmunoprecipitation experiments, 48 h after transfection, cells were lysed on ice-cold GST buffer (50 mM Tris-HCl, 10% glycerol v/v, 100 mM NaCl, 2 mM MgCl 2 , 1% v/v NP-40, pH 7.
Previous to the assay, cells were washed twice with assay buffer (HBSS with 20 mM HEPES, pH 7. 15) and incubated for 2 h in 30 ml per well of assay buffer with 0. 1% DMSO in the reader at 24°C. Hereafter, the sensor plate was scanned, and a baseline optical signature was recorded before adding 10 ml of the test compound dissolved in assay buffer containing 0. 1% DMSO. Then, DMR responses were monitored along time, and kinetic data were analyzed using EnSpire Workstation Software version 4. 10. Each representative curve shown is the mean of three internal replicates. Phosphoprotein array. Cells transfected with CB1R-GFP and BiP-IR (or control) plasmids were treated with WIN-55212-2 (100 nM final concentration) or vehicle (DMSO, 0. 1% v/v final concentration) as described above for 5 and 15 min.
Samples from two independent experiments were processed separately by using 350 mg of total protein per experimental condition, following the instructions of the Proteome Profiler Human Phospho-Kinase Array Kit (R&D Systems, Bio-techne, #ARY003C). Densitometric analysis of the relative phosphorylation levels versus the corresponding housekeeping controls and between WIN-55212-2/vehicle treatments was performed with ImageJ software and the Protein Array Analyzer toolset. Cellular and subcellular fraction preparations. Membrane preparations for G-protein-coupling assays were obtained from HEK-293T-cell pellets or adult mouse-hippocampus tissue specimens. Frozen samples were thawed at 4°C and homogenized with a glass/Teflon grinder (IKA labortechnik), 10 strokes at maximum speed, in 30 volumes of homogenization buffer (250 mM sucrose, 50 mM Tris-HCl, 1 mM EGTA, 3 mM MgCl 2 , 1 mM DTT, pH 7.
Labeled probes were purified on ProbeQuant G-50 Micro Columns (GE Healthcare). ISH histochemistry procedures were performed as described previously  (Sanabra and Mengod, 2011). For autoradiography, hybridized sections were exposed to Biomax-MR (Kodak) films for 1-10 d at À70°C with intensifying screens. Double in situ-hybridized sections were processed as described previously  (Reyes-Irisarri et al. , 2007). They were exposed in the dark for 4-6 weeks at 4°C. Images from autoradiograms were obtained by using a Wild 420 macroscope (Leica Microsystems) equipped with a digital camera (DXM1200 F, Nikon) and ACT-1 Nikon software. Microphotography was performed with an Olympus BX51 Stereologic Microscope (Olympus) equipped with a digital camera (DP71, Olympus) or with a Carl Zeiss Axioplan microscope equipped with an Olympus XC50 digital camera.
Each mouse was placed in the center of the maze, facing one of the open arms, and the exploratory behavior of the animal was video-recorded for 5 min. The number and duration of entries were measured separately for the open arms and the closed arms. One arm  In all cases, animals were assigned randomly to the different treatment groups, and all experiments were performed in a blinded manner for genotype and pharmacological treatment. All tests were videorecorded for subsequent blinded analysis using Smart3. 0 version 3. 00. 6 Software (Panlab). Experimental design and statistical analyses. Unless otherwise specified, data are presented as mean 6 SEM. Statistical comparisons were conducted by one-way or two-way ANOVA with Tukey's post hoc test, or by Student's t test, as indicated in each case.
All datasets were tested for normality (Kolmogorov-Smirnov's test) and homoscedasticity (Levene's test) before analysis. For clarity, only p values , 0. 05 were considered statistically significant. The sample size for each experiment was estimated on the basis of previous studies conducted by our laboratories using similar protein-interaction, cell-culture, brain-sample, and motorbehavior approaches. Subsequent power analysis was conducted for each parameter by using IBM SPSS software. The number of biological replicates (e. g. , number of mice, number of cell cultures) is provided in the corresponding figure legends. The number of technical replicates (e. g. , number of Y2H assays, number of incubations within each cell culture, number of sections microscopically analyzed per mouse brain, number of behavioral trials per mouse) is provided in the corresponding figure legends or in the corresponding Materials and Methods subsections.
s -. Data extraction and analyses were performed using EthoVision XT 13. 1 software (Noldus, The Netherlands). Swaps between individuals were manually corrected using the track editor module. For behavioural analyses, the arena was virtually separated into two areas: the centre area composed of one half of the surface and the periphery area including the other half; time spent in periphery (s), indicative of thigmotaxis behaviour, was recorded. The velocity of each fish (cm. s -1 ), indicative of individual fish activity, and the interindividual distances (cm), indicative of group cohesion  (Alfonso et al. , 2020) , were also assessed. Variables were averaged over 1 minute every 10 minutes in order to record the kinetics of behavioural responses. The gills were extracted using Trizol according to manufacturer"s recommendations (Invitrogen, Carlsbad, CA, USA).
J o u r n a l P r e -p r o o f Journal Pre-proof normalized by the geometric mean of two housekeeping genes as reference. Seven different reference genes  (eef1-alpha, l13, gapdh, 18S, actb, fau, rpl17)  were tested for all organs and the R function "selectHKs" from the "NormqPCR" package  (Perkins et al. , 2012)  was used to select the best two reference genes, based on their stability across all conditions. For the 4 organs, the function selected the same two reference genes: l13 and fau.