dataesr/scientific-paragraphs-categorization huggingface dataset:
Little is known about the evolution and biosynthetic function of DNA precursor and the folate metabolism in the Asgard group of archaea. As Asgard occupy a key position in the archaeal and eukaryotic phylogenetic trees, we have exploited very recently emerged genome and metagenome sequence information to investigate these central metabolic pathways. Our genome-wide analyses revealed that the recently cultured Asgard archaeon Candidatus Prometheoarchaeum syntrophicum strain MK-D1 (Psyn) contains a complete folate-dependent network for the biosynthesis of DNA/RNA precursors, amino acids and syntrophic amino acid utilization. Altogether our experimental and computational data suggest that phylogenetic incongruences of functional folate-dependent enzymes from Asgard archaea reflect their persistent horizontal transmission from various bacterial groups, which has rewired the key metabolic reactions in an important and recently identified archaeal phylogenetic group.
Identification of a ThyX orthologue in Candidatus Prometheoarchaeum syntrophicum strain MK-D1 (Psyn) As an initial approach to investigate the Asgard archaeal thymidylate and C1 metabolism, we searched for thyX and thyA homologs in sequence databases for archaeal genomes or metagenome-assembled genomes (MAGs). These studies led to the identification of both thyX and thyA sequences in the genomes of the understudied and diverse group of archaea. Interestingly, our similarity searches identified a ThyX orthologue in Psyn, which is up to 51. 44% identical to bacterial ThyX sequences at the protein sequence level (e. g. Zixibacteria, e-value ≈ 10 -78 or Calditrichaeota bacterium, evalue ≈ 10 -76 ). The observed e-values are very low and are adjusted to the large sequence database size. Therefore, the quality of the observed hits is very high.
As Psyn is the only known example of Asgard archaea that can be cultivated and its genome, lacking thyA, is completely sequenced, we concentrated our efforts on this archaeal ThyX. Transcriptome analyses of RNA extracted from enriched cultures of Psyn indicated that thyX from this strain is expressed [Reads Per Kilobase of transcript, per Million mapped reads (RPKM) value of 225. 37 using the data set from the sequence read archive (SRA) DRR199588]. This gene encodes a protein with a predicted molecular mass of 35, 294 Da. Its genomic environment (Fig. 1b  and Supplementary Figure  1 ) comprises an upstream gene coding for a Nif3-like protein with a length of 259 residues (29,026 Da) and a downstream gene encoding a domain of unknown function DUF2095. Physical association of Psyn thyX with the Nif3 family encoding gene is of interest, as this family of proteins may correspond to GTP cyclohydrolase 1 type 2, which converts GTP to dihydroneopterin triphosphate and may function in folic acid synthesis.
The structurebased sequence alignment of Psyn ThyX with Thermotoga maritima ThyX (PDB structure 5CHP) predicts the conservation of functionally important residues involved in folate-, nucleotide-and flavin-binding (Fig. 1b ). The marked degree of sequence similarity allowed the construction of a high-quality structural model for Psyn ThyX indicating its functional significance as thymidylate synthase and nucleotide and folate binding protein. More specifically, we constructed a model of the ThyX homotetramer based upon PDB structures 1O26, 3N0B and 6J61 as templates and using the protein structure modeling program Modeller  21  (Fig. 1c , left panel). Structural superposition of the model with PDB structure 3GT9 using the Chimera software  22  allowed the addition of FAD, dUMP and folate molecules into the active site of the model (Fig.
Automated prediction of gene transfers between Psyn and bacteria Considering the high level of sequence similarity of Psyn ThyX and bacterial orthologs, we estimated the possible extent of gene transfer between bacteria and Psyn using HGTector. This automated method is well suited for discovering potential recent gene transfers  23  by analyzing the hit distribution patterns from the similarity searches. The method performs a systematic analysis to detect genes that do not appear to have a typical vertical history of the analysed organisms but instead have a putative horizontal origin. This approach efficiently detects atypical genes that are highly likely to be horizontal gene transfers (HGT). As this method uses all sequence data available in the GenBank, it allows exhaustive detection of potential recent gene transfers.
Both scenarios suggest that the present-day thymidylate metabolism in Eukarya and Asgard archaea originated in bacteria. It was recently proposed that the contribution of Asgard to the present eukaryotic gene repertoire is very limited  41. However, widespread recent LGTs events from bacteria to Asgard displacing the original archaeal genes would underestimate the contribution of the current day Asgard to the eukaryotic gene repertoire. In turn, our study on the thymidylate metabolism might represent only the tip of an iceberg of functional bacterial genes in Asgard genomes. Taken together, our data support the idea that Asgard archaea harbor highly mosaic genomes that have received many bacterial genes, even for central metabolic processes such as the folate metabolism. We have also experimentally demonstrated that some of the transferred Psyn genes, like Psyn thyX, are fully functional in vivo and in vitro.
Materials and Methods Bioinformatics methods and genome-wide analyses. The complete genome sequence available for the Candidatus Prometheoarchaeum syntrophicum strain MK-D1 (Psyn) chromosome (NZ_CP042905. 1) was used for bioinformatics analyses. This genome is 4,278 Mb with a GC content of 31. 2%. RPKM values for gene expression were determined using the Geneious Prime® 2021. 1. 1 (Build 2021-03-12). Metabolic reconstructions were performed using the RAST server and KEGG databases. These analyses were complemented with highly sensitive manual similarity searches using HHpred  42.
Structural alignments and structure modelling. PROfile Multiple Alignment with predicted Local Structures and 3D constraints (PROMALS3D) was used to align the Psyn ThyX sequence with the Thermotoga (T. ) maritima ThyX structure (PDB 5CHP). PROMALS3D (http://prodata. swmed. edu/promals3d/promals3d. php) aligns multiple protein sequences and/or structures, with enhanced information from database searches, secondary structure prediction, and 3D structures. Input sequences and structures used FASTA and PDB formats, respectively. (red: alpha-helix, blue: beta-strand). A model of the Psyn ThyX homotetramer was constructed with the protein structure modeling program Modeller using PDB structures 1O26, 3N0B, and 6J61 as templates  21. Ten initial models were constructed and the best one was chosen using molpdf and DOPE score, and by evaluating the stereochemical quality with PROCHECK.
Automated prediction of LGT. Automated analyses of potential LGT events were performed using a HGTector  23. This method first performs Diamond all-against-all similarity searches of each protein-coding sequence of Psyn. The NCBI genome database was downloaded and compiled (on August 2021) on a local machine. The output of the similarity searches was passed to the analysis program HGTector where the taxonomy of the self-group was defined as Candidatus Prometheoarchaeum syntrophicum (NCBI TaxID: 2594042). The close-group was automatically inferred by HGTector as the "superkingdom Archaea" (TaxID: 2157) that contains 1062 taxa in the reference database. From the statistical distribution of close and distal hits, HGTector predicted a list of potentially transferred genes. The output of the program is a scatter plot, where the horizontal axis represents the close score, the vertical axis represents the distal score, and potential LGT genes are colored in yellow.
We slightly modified the source code which was made publicly available at: https://github. com/cgneo/neoHGT. Phylogenetic analyses. For phylogenetic analyses, we constructed a database with a backbone of 183 thyX sequences that cover all the major bacterial and archaeal phyla derived from a previous study  10. These were submitted to BLASTP, with an e-value cutoff of 1e-05, against the Psyn genome, as well as against the 140 metagenomic bins larger than 2Mb assigned to Asgard archaea publicly available in the Genbank database. We used similar approaches with the other folate enzymes with 355 ThyA, 53 FTHFS, 40 MetH, 58 PurH, 70 FolA, 85 FolD, 33 MTHFR and 49 MTHFS sequences to find homologous sequences in Asgard genomes. Homologs found in Asgard genomes were then used as seeds for reciprocal BLAST searches against an NR database (15/01/2021 version) to find the best homolog outside Asgards.
Identical sequences (same identifying code or 100% sequence similarity) were finally removed to generate the final dataset. For analysis of the genomic environment of the Psyn thyX gene, we collected all the genes located 15 kb downstream and 15 kb upstream of the Psyn thyX genes and searched for homologs with BLASTP against an NR database (15/01/2021 version). The first BLASTP hit was retrieved, as well as the percentages of the archaeal sequences in the taxonomy profile of the first top 100 hits. Phylogenetic analyses were performed using the obtained protein sequence data sets (see above) that were aligned using MAFFT v7. 388 with default settings  45. Identical sequences belonging to the same phylum or genus were removed to retain a single representative sequence. Ambiguously aligned sites were removed using trimAl v1.
4. rev15  46  with the "-automated1" and "-phylip" options. The phylogenetic position of the Asgard sequences was then inferred with PhyML v. 1. 8. 1  47  using the best substitution models as determined by Smart Model Selection  48  , and 1000 bootstrapped data sets were used to estimate the statistical confidences of the nodes. Trees were then visualized using the FigTree software (https://github. com/rambaut/figtree/releases). Raw data including BLAST outputs, table with the accession numbers of each Asgard sequences, alignment files, alignments used for the phylogenies, Newick tree files and additional information can be downloaded at http://gofile. me/2ppPR/HvmT893hn. Design and synthesis of Psyn ThyX expression plasmid. pTwist-Psyn-ThyX plasmid (3370bp), carrying the synthetic Psyn thyX gene of 897bp was synthesized by Twist Biosciences (https://www.
For immunoblotting detection, after protein transfer, the nitrocellulose membranes (Bio-Rad) were treated with 20% blocking buffer (Li-Cor Biosciences, Lincoln, US) followed by incubation with anti-His-tag mouse primary antibody (dilution1/5000, Bio-Rad) and revelation with a IRDye 800CW Goat anti-mouse IgG secondary antibody (dilution 1/10000, Li-Cor Biosciences) using a ChemiDoc TM Touch Imaging System (Bio-Rad). Additional information. Supplementary information for this manuscript includes the supplementary figures 1-9 and the supplementary table  1. orange designates a partial copy. Psyn is indicated with an asterisk and its gene distribution is framed in black. The (meta)genomic assembly and gene names are in supplementary table 1 (sheet "distribution of folate genes"). The scatter plot was created by the HGTector.
A range of novel 2-ME analogues have been designed in silico in our laboratory using the Lamarckian genetic algorithm in AutoDockTools4 with the prepare_ligand4py script (Figure  1 )  [19]. The rationale behind the design was three-fold. Firstly, the aim was to obtain an increased cytotoxicity by C2 and C3 modifications, the efficacy of which has been demonstrated in a number of commercially available cancer cell lines, including the MES-SA/DX5 multidrug resistant sarcoma cells  [20]. Secondly, an improved pharmacokinetic profile was envisioned via two mechanisms, namely evasion of the 17β-hydroxysteroid dehydrogenase-mediated metabolism through D-ring modifications, and increasing the binding affinity to erythrocytic carbonic anhydrase II (CAII) to bypass the first-pass hepatic metabolism by addition of a sulpha moiety.
The Estrone Analogues Increase LC3B Expression and Induce Apoptosis, a Phenomenon Which Is Partially Attenuated If Autophagy Is Blocked Induction of autophagic processes and apoptotic cell death were further investigated. Annexin V quantification demonstrated a significantly increased number of cells undergoing apoptosis with a concomitant decrease in cell viability after a 24 h-exposure to both drugs, a trend not yet visible after a 2 h-exposure (Supplementary data section S2. 2). Induction of autophagic process was then quantified by measurement of the fluorescence intensity resulting from the intracellular binding of the anti-LC3B/MAP1LC3B antibody conjugated to Alexa Fluor 488. A statistically significant elevation of LC3B in both HeLa and MDA-MB-231 cells after 24 h indicated the possible involvement of autophagy in response to both compounds (Supplementary data section S2.
The Estrone Analogues Increase LC3B Expression and Induce Apoptosis, a Phenomenon Which Is Partially Attenuated if Autophagy Is Blocked Induction of autophagic processes and apoptotic cell death were further investigated. Annexin V quantification demonstrated a significantly increased number of cells undergoing apoptosis with a concomitant decrease in cell viability after a 24 h-exposure to both drugs, a trend not yet visible after a 2 h-exposure (Supplementary data Section S2. 2). Induction of autophagic process was then quantified by measurement of the fluorescence intensity resulting from the intracellular binding of the anti-LC3B/MAP1LC3B antibody conjugated to Alexa Fluor 488. A statistically significant elevation of LC3B in both HeLa and MDA-MB-231 cells after 24 h indicated the possible involvement of autophagy in response to both compounds (Supplementary data Section S2.
In accordance with this concept, an increased p27 Kip1 and p15 INK4B expression was observed in this study, both which are involved in G 1 /S cell cycle progression  [40]. Additionally, MTAs may affect many homeostatic mechanisms within interphase cells, such as their ability to correctly traffic components to various subcellular compartments, as well as their ability to move and migrate. Autophagy is one such process which requires intact microtubule dynamics for the trafficking of autophagosomes to the lysosomes and degradation of their content after fusion  [41, 42]. Previously published data indicated that autophagy may play a role in the intracellular response within neoplastic cells exposed to ESE-16 in vitro  [24, 27, 43]. In this study, LC3B quantification via flow cytometry was used as a screening assay based on previously published data correlating an increased level of the protein with an increased number of autophagic vesicles.
1% containing 0. 3% bovine serum albumin (BSA). Rinsed slides were mounted and viewed with a Zeiss Axio Imager Z1 microscope controlled by Axiovision software (Carl Zeiss, Oberkochen, Germany) using a X63 oil objective. Images were captured using an Ocra R2 N/B camera (Hamamatsu, Hamamatsu, Japan).
Time-Lapse Imaging HeLa Kyoto cells expressing EGFP-alpha-tubulin and H2B-mcherry were seeded on 2-well glass-slides (Ibidi, Gräfelfing, Germany, #80297) at a density of 7000 cells per well and allowed to grow for 24 h prior to imaging. After treatment with 0. 4 µM ESE-16, the slide was placed on a heated stage (37 • C) at 5% CO 2. Images were acquired every 150 s by a spinning disk confocal laser microscope (Andromeda iMIC) with a focus stabilizer (Field Electron and Ion Company (FEI), Hillsboro, OR, USA). The microscope was equipped with a Plan-Apochromat 10×/0. 45 M27 and iXon electron multiplying charge coupled device camera (Andor). Image sequences were acquired using the FEI Live acquisition software v. 2. 7. 0. 16 (FEI) and the cellular responses were analysed manually.
To amplify the signal, slides were incubated with Bio-Rad amplification reagent (Bio-Rad, Watford, UK) for 15 min at RT. The arrays were washed with TBS-T, probed with Alexa647-Streptavidin ((Thermo Fisher Scientific, Waltham, MA, USA) diluted in TBS-T-BSA for 1 h at RT and washed again in TBS-T. For staining of total protein, arrays were incubated for 15 min in 7% acetic acid and 10% methanol, rinsed twice in water, incubated for 10 min in Sypro Ruby (Thermo Fisher Scientific, Waltham, MA, USA) and rinsed again. The processed slides were dried by centrifugation and scanned using a GenePix 4000B microarray scanner (Molecular Devices, San Jose, CA, USA). Spot intensity was determined with MicroVigene software (VigeneTech Inc. , Carlisle, MA, USA). All primary antibodies used in RPPA had previously been tested by Western Blotting to assess their specificity for the protein of interest.
Species specific secondary antibodies labelled with horseradish peroxidase were used to obtain a signal, and densitometry analysis was done for semi-quantification using Image-J software (NIH, Bethesda, MD, USA). Blots were adjusted against loading controls and expressed as fold decrease or increase as compared to DMSO vehicle-control cells. As the expression of cofilin and HSP 90 remained relatively stable within cells exposed to the compounds under investigation, either or both of them were used as a loading control standard.
Statistical Analysis Quantitative studies: RPPA and flow cytometry. Data obtained from three independent experiments are shown as the mean standard deviation (SD) and were analysed for significance using the analysis of variance (ANOVA)-single factor model followed by a two-tailed Student's t-test. Means are presented in bar charts, with T-bars referring to standard deviations. p Values < 0. 05 were regarded as statistically significant and have been indicated by an asterisk (*). For flow cytometry, data from at least 10,000 cells were analysed employing Kaluza ® flow analysis software version 1. 3 (Beckman Coulter, Brea, CA, USA). Qualitative studies: microscopy and Western blots. Morphological studies by means of microscopy were conducted and a minimum of 5 representative images were captured from each sample.
00132, ***p < 2e -16 for WT, and ***p = 4e -06 for S81L; n = 11, 9, 9, and 5 for w 1118 , GFP, WT, and S81L, respectively. Samples were collected from two to three independent experiments). Scale bar = 200 μm. b Expression of C2C10H WT with two copies of C2C10H S81L in muscle tissues did not exacerbate abnormal muscle and mitochondrial phenotypes. Scale bar = 10 μm. c ATP levels in each of the indicated genotypes were measured. Data are shown as mean ± SD (one-way ANOVA and post hoc Dunnett test, two-sided, ***p = 2. 2e -07 for W and *p = 0. 0234 for S81L; n = 4 independent experiments). d HeLa cells were co-transfected with Myc-tagged CHCHD10 S59L and FLAG-tagged CHCHD10 WT. Empty vector (EV) was used as a control. mTagRFP-T-Mito-7 was co-transfected to visualize mitochondria. Representative images of HeLa cells stained with anti-Myc (green) and anti-FLAG (gray) antibodies 24 h after transfection.
Scale bar = 20 μm. i Quantification of TOM20 signal length (mitochondria) from CHCHD10 WT and CHCHD10 S59L transfected HeLa, CHCHD10 KO , and CHCHD2 / 10 DKO HeLa. Data are shown as mean ± SD (one-way ANOVA and post hoc Dunnett test, two-sided, ***p = 3. 3e -16, <2e -16, and <2e -16 for HeLa, C10 KO and C2/C10 KO, respectively; n = 20 cells from three independent experiments). j CHCHD10 KO and CHCHD2/10 DKO HeLa cells were transfected with EV, FLAG-tagged CHCHD10 WT, and CHCHD10 S59L. After 24 h, mitochondrial respiration for each transfectant was measured via Seahorse XF Cell Mito Stress tests. A representative graph of a single experiment is shown (mean ± SD). Actual statistical analyses were performed with three (C10 KO) and five (C2/ C10 KO) independent experiments (one-way ANOVA and post hoc Dunnett test, two-sided, comparison with EV, **p = 0.
b HeLa cells transfected with FLAG-tagged CHCHD10 WT or CHCHD10 S59L with TARDBP were subjected to sequential protein extraction with RIPA and urea buffers. Immunoblotting was performed with anti-TDP-43 or anti-actin (loading control) antibodies. Data are shown as mean ± SD (one-way ANOVA and post hoc Tukey's test, two-sided, ***p = 0. 000738 for WT, ***p = 0. 000533 for S59L + WT; n = 3 independent experiments). c, d HeLa cells were co-transfected with FLAG-tagged TARDBP WT or disease-causing mutants combined with empty vector (EV), CHCHD10 WT , or CHCHD10 S59L. After 24 h, mitochondria were fractionated. Immunoblotting was conducted with anti-TDP-43 (arrowhead indicates transfected TDP-43), anti-HSP60, and anti-TOM20 (loading controls) antibodies. Data are shown are mean ± SD (one-way ANOVA and post hoc Dunnett test, two-sided, *p = 0.
Data are shown as mean ± SD (two-sided t test, ***p = 1. 26e -06 and 8. 98e -15 for S59L and Tom20, respectively; n = 20 cells from three independent experiments). Scale bar = 20 μm. h HeLa cells expressing CHCHD10 S59L were treated with a control peptide (scPM, 2 μM) or TDP-43 inhibitor (PM1, 2 μM). Mitochondrial respiration was measured by Seahorse XF Cell Mito Stress tests. A representative graph of a single experiment is shown (mean ± SD). Actual statistical analyses were performed with four independent experiments (one-way ANOVA and post hoc Dunnett test, twosided, comparison with EV, *p = 0. 024685; Detailed information on statistical analyses is available in Supplementary Fig. 9f ). i HEK293 cells were transfected with FLAG-tagged CHCHD10 WT or CHCHD10 S59L with/without HA-tomato-tagged TARDBP.
Scale bar = 200 μm. b RNAi-mediated knockdown of PINK1 in muscles rescued C2C10H S81L -induced muscle degeneration and mitochondrial defects. Representative images from three independent experiments. Scale bar = 20 μm. c ATP levels in thoraxes from 10-day-old flies were measured. Data are shown as mean ± SD (two-sided t test, ***p = 1. 22e -05 for male and 0. 000157 for female; n = 4 independent experiments). d, e HeLa cells were transfected with siRNAs targeting PINK1 or MFN2. After 18 h, the cells were transfected with CHCHD10 S59L and Mito-RFP. Representative images of transfected HeLa cells immunostained with antibodies against FLAG. Scale bar = 20 μm. Quantification of Mito-RFP and CHCHD10 S59L -FLAG signal length. Data are shown as mean ± SD (one-way ANOVA and post hoc Dunnett test, two-sided, comparison with EV; ***p < 2e -16; n = 20 cells from three independent experiments).
f Mitochondrial respiration was measured by Seahorse XF Cell Mito Stress tests 24 h after CHCHD10 S59L transfection into siRNA-treated cells. Graphs of a single representative experiment are shown (mean ± SD). Actual statistical analyses were performed with 8 independent experiments (one-way ANOVA and post hoc Dunnett test, two-sided, comparison with EV, ***p = 0. 00034, 0. 00031 and **p = 0. 0055 for basal, ATP and maximal level, respectively; detailed information on statistical analyses is available in Supplementary Fig. 9g ). g RT-qPCR analysis of PINK1 in siRNAtransfected fibroblasts. Results are shown as fold change of PINK1 mRNA expression in siPINK1 transfected fibroblasts relative to siControl transfected fibroblasts. Data were normalized to β-2 macroglobulin (B2M) or 28S. Results shown are mean ± SEM from two independent experiments.
1% Triton X-100, and blocked with 5% bovine serum albumin (BSA) in PBS. Primary antibodies were diluted in 5% BSA in PBS and incubated overnight at 4 °C. Samples were then rinsed three times with PBS-Tween-20 and incubated with secondary antibodies for 1. 5 h at room temperature. Coverslips were mounted onto microscope slides with Prolong Diamond Antifade Reagent with DAPI (4′,6-diamidino-2-phenylindole) (Invitrogen). Samples were observed with an LSM 710 confocal microscope (Carl Zeiss) or Nikon Crest X-light 2 spinning disc confocal microscope (Nikon). Co-localization was analyzed with co-localization tool of ZEN software (Carl Zeiss). For patient primary fibroblast imaging, cells were incubated in a 100 nM solution of MitoTracker red (Invitrogen) for 15 min at 37 °C. The medium was replaced by DMEM supplemented with 10% FBS and penicillin (100 U/ml)/streptomycin (0.