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│ The membrane transporter SLC25A48 enables transport of choline into human mitochondria │
│ │
│ │
│ OPEN see commentary on page 225 │
│ │
│ Suraj Patil 1,2,3,4 , Oleg Borisov , Nora Scherer 2 2,3 , Christophe Wirth , Pascal │
│ Schlosser 5 2,6 , Matthias Wuttke , 2 Sandra Ehret , Luciana Hannibal 7 6,7 , Kai-Uwe │
│ Eckardt 8,9 , Carola Hunte 5,6,10 , Bjo ¨ rn Neubauer , 1 Anna Ko ¨ttgen 2,6 and Michael Ko │
│ ¨ttgen 1,6 │
│ │
│ 1 Department of Medicine IV -Nephrology and Primary Care, Faculty of Medicine and Medical │
│ Center, University of Freiburg, Freiburg, Germany; 2 Institute of Genetic Epidemiology, │
│ Faculty of Medicine and Medical Center -University of Freiburg, Freiburg, Germany; 3 Spemann │
│ Graduate School of Biology and Medicine (SGBM), University of Freiburg, Freiburg, Germany; 4 │
│ Faculty of Biology, University of Freiburg, Freiburg, Germany; 5 Institute of Biochemistry │
│ and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, Freiburg, Germany; │
│ 6 CIBSS -Centre for Integrative Biological Signalling Studies, University of Freiburg, │
│ Freiburg, Germany; 7 Laboratory of Clinical Biochemistry and Metabolism, Department of │
│ General Pediatrics, Adolescent Medicine and Neonatology, Faculty of Medicine, Medical │
│ Center, University of Freiburg, Freiburg, Germany; 8 Department of Nephrology and Medical │
│ Intensive Care, Charité -Universitätsmedizin Berlin, Berlin, Germany; 9 Department of │
│ Nephrology and Hypertension, University Hospital Erlangen, Friedrich-Alexander-Universität │
│ Erlangen-Nürnberg, Erlangen, Germany; and 10 BIOSS -Centre for Biological Signalling │
│ Studies, University of Freiburg, Freiburg, Germany │
│ │
│ Choline has important physiological functions as a precursor for essential cell components, │
│ signaling molecules, phospholipids, and the neurotransmitter acetylcholine. Choline is a │
│ water-soluble charged molecule requiring transport proteins to cross biological membranes. │
│ Although transporters continue to be identi /uniFB01 ed, membrane transport of choline is │
│ incompletely understood and knowledge about choline transport into intracellular organelles │
│ such as mitochondria remains limited. Here we show that SLC25A48 imports choline into human │
│ mitochondria. Human loss-of-function mutations in SLC25A48 show impaired choline transport │
│ into mitochondria and are associated with elevated urine and plasma choline levels. Thus, │
│ our studies may have implications for understanding and treating conditions related to │
│ choline metabolism. │
│ │
│ │
│ Translational Statement │
│ │
│ Kidney International (2025) 107, 296-301; https://doi.org/10.1016/ j.kint.2024.06.022 │
│ │
│ KEYWORDS: choline transport; deorphanization; mGWAS; mitochondria; SLC25A48 │
│ │
│ Copyright ª 2024, International Society of Nephrology. Published by Elsevier Inc. This is an │
│ open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/). │
│ │
│ Correspondence: Anna Köttgen, Institute of Genetic Epidemiology, Medical Center -University │
│ of Freiburg, Hugstetter Strasse 49, 79106 Freiburg, Germany. E-mail: │
│ anna.koettgen@uniklinik-freiburg.de; or Michael Köttgen, Department of Medicine IV │
│ -Nephrology and Primary Care, Medical Center -University of Freiburg, Institute for Disease │
│ Modeling and Targeted Medicine, Breisacherstrasse 113, 79106 Freiburg, Germany. E-mail: │
│ michael. koettgen@uniklinik-freiburg.de │
│ │
│ Received 3 December 2023; revised 7 June 2024; accepted 17 June 2024; published online 29 │
│ July 2024 │
│ │
│ This study uncovers the function of a human protein called solute carrier SLC25A48 by │
│ showing that it acts as a membrane transporter for choline. Choline has important │
│ physiological functions as a building block for essential cell components and signaling │
│ molecules. Choline is a water-soluble charged molecule and, therefore, requires transport │
│ proteins to cross biological membranes. How it gets into cellular compartments, like │
│ mitochondria, has been poorly understood. This study shows that SLC25A48 transports choline │
│ into mitochondria. Mutations in SLC25A48 disrupt choline transport into mitochondria, │
│ leading to higher levels of choline in urine and blood in humans. These /uniFB01 ndings shed │
│ light on how choline is handled within cells and could have implications for understanding │
│ and treating conditions related to choline metabolism. │
│ │
│ M embrane transport proteins play a crucial role in the movement of ions and metabolites │
│ across biological membranes. Their proper functioning is essential for many physiological │
│ processes. Despite signi /uniFB01 cant progress in our understanding of these proteins, a │
│ substantial number of them remain uncharacterized, often referred to as orphan membrane │
│ transport proteins. 1 Deorphanization efforts are essential to unveil the hidden cellular │
│ functions and roles of these proteins in health and disease. Recent large-scale genomewide │
│ association studies of metabolite levels provide links between common genetic variants in │
│ membrane transporterencoding genes and metabolite levels. 2,3 This generates testable │
│ hypotheses to identify the physiological substrates of established and orphan human │
│ transport proteins in vivo . │
│ │
│ Choline is an essential nutrient with important roles in a variety of physiological │
│ processes and metabolic pathways. It is an essential component of phospholipids in cell │
│ membranes, such as phosphatidylcholines, a precursor of the neurotransmitter acetylcholine │
│ and of the osmoregulatory betaine, and an important player in lipid metabolism. 4 Recent │
│ studies have uncovered a growing body of evidence linking choline to various diseases, such │
│ as neurologic disorders, metabolic syndromes, and liver disease. 4 Several choline transport │
│ proteins have been identi /uniFB01 ed and characterized in model systems, including SLC5A7, │
│ SLC44A1, SLC44A2, SLC44A4, SLC49A1, and SLC49A2. 5 -10 However, it is unclear which │
│ transporters affect systemic levels of free choline in humans and if they are linked to │
│ human genetic variation. │
│ │
│ │
│ METHODS │
│ │
│ │
│ Molecular biology │
│ │
│ pDONR221_SLC25A48 was a kind gift from the RESOLUTE Consortium (Addgene plasmid number │
│ 131995; http://n2t. net/addgene:131995; research resource identi /uniFB01 er [RRID]: │
│ Addgene_131995). The SLC25A48 transcript was ligated into the pcDNA6. /uniFB02 ag vector, │
│ and mutations were generated by site-directed mutagenesis (see Supplementary Methods for │
│ details). │
│ │
│ │
│ Cell culture │
│ │
│ HEK293T and HeLa cell lines were cultured as described previously. 11 HEK293T cells were │
│ transfected with the calcium phosphate method, and HeLa cells with FuGENE HD (Promega │
│ E2311). │
│ │
│ │
│ Protein isolation, gel electrophoresis, and Western blot analysis │
│ │
│ Proteins were isolated, separated using sodium dodecylsulfate -polyacrylamide gel │
│ electrophoresis, and transferred to polyvinylidene di /uniFB02 uoride membranes, as │
│ described previously. 12 Antibodies were anti-Flag (Sigma Aldrich F3165) and ß-actin (Sigma │
│ Aldrich A1978). Chemiluminescence signals were captured with the Intas ChemoCam system. │
│ Western blot analyses are representative of 3 experiments with similar results │
│ (Supplementary Methods). │
│ │
│ │
│ Immuno uorescence /uniFB02 │
│ │
│ HeLa cells were prepared for indirect immuno /uniFB02 uorescence, as described in the │
│ Supplementary Methods. Confocal imaging was conducted on a Zeiss LSM980 MP AiryScan 2 │
│ microscope with a 63 /C2 objective. Quantitative colocalization analysis was performed with │
│ the Colocalization plugin in the Zeiss ZEN blue 3.4 software (Supplementary Methods). │
│ │
│ │
│ Mitochondrial isolation │
│ │
│ HEK293T cells were grown and transfected in 500-cm 2 plates. Two days after transfection, │
│ cells were harvested and mitochondrial pellets were obtained. 13 Mitochondrial pellets were │
│ resuspended in a mitochondrial uptake buffer (KCl, 120 mM; sucrose, 25 mM; N │
│ -2-hydroxyethylpiperazineN 0 -2-ethanesulfonic acid, 10 mM; ethyleneglycol-bis-[ b - │
│ aminoethylether]N N,N ,N , 0 0 -tetraacetic acid, 1 mM; KH2PO4, 1 mM; MgCl2, 5 mM; │
│ glutamate, 15 mM; and malate, 7.5 mM, adjusted to a pH 7.2) 6 (see Supplementary Methods). │
│ │
│ │
│ 3 H choline transport studies │
│ │
│ A total of 25 m g of mitochondria was suspended in 50 m l mitochondrial uptake buffer with │
│ subsequent addition of 2 /C2 choline buffer (mitochondrial uptake buffer supplemented with │
│ 20 m M choline chloride and 10 nM choline chloride [methyl-3H]) and incubated for 5 minutes. │
│ Samples were washed twice with ice-cold mitochondria washing buffer (mitochondrial uptake │
│ buffer with 20 m M choline chloride), and the pellet was resuspended in Ultima Gold │
│ scintillation cocktail for quanti /uniFB01 cation of 3H-choline content using liquid │
│ scintillation counting. │
│ │
│ │
│ Statistical analysis │
│ │
│ GraphPad Prism 9.5.1 software was used to graph, analyze, and present the obtained data. All │
│ results are expressed as mean /C6 SEM. All experiments were independently performed at least │
│ 3 times. A 2-tailed Mann-Whitney test was used to calculate P values when the sample size │
│ (biological replicates) was at least 10, and an unpaired t -test was used when the sample │
│ size was < 10. P < 0.05 was considered signi /uniFB01 cant. │
│ │
│ │
│ Study population, choline measurements, and whole-exome sequencing │
│ │
│ Genetic associations with plasma and urine choline levels, measured as part of the Metabolon │
│ HD4 Global Discovery metabolomics panel, were evaluated in the German Chronic Kidney Disease │
│ study. 14 The SLC25A48 gene was investigated using whole-exome sequencing data. Details │
│ about the study sample, choline measurements, and whole-exome sequencing (sequencing, │
│ alignment, and variant calling) are found in the Supplementary Methods. │
│ │
│ │
│ Variant annotation and rare variant testing │
│ │
│ Called variants were annotated using Variant Effect Predictor version 109, incorporating │
│ various tools to predict deleteriousness. 15 -17 Loss-of-function variants for SLC25A48 were │
│ evaluated using the LoFtee Variant Effect Predictor plugin. 18 Two masks ("LoF_mis" and │
│ "HI_mis") that included rare variants (minor allele frequency < 1%) were generated for │
│ variant aggregation testing. The LoF_mis mask included highcon /uniFB01 dence │
│ loss-of-function variants, missense variants with a MetaSVM score > 0, or in-frame │
│ nonsynonymous variants with a fathmm-XF-coding score > 0.5, whereas the HI_mis mask │
│ encompassed variants with high-impact consequences or missense variants meeting speci │
│ /uniFB01 c criteria. Burden tests, adjusting for covariates, were performed using the │
│ seqMeta Rpackage version 1.6.7. Statistical signi /uniFB01 cance was de /uniFB01 ned as P < │
│ 0.05. Single-variant, covariate-adjusted association tests were conducted under additive │
│ modeling. Results are presented for the HI_mis mask, which yielded lower association P │
│ values. Further details are presented in the Supplementary Methods. │
│ │
│ Figure 1 | SLC25A48 is a human mitochondrial choline transporter. ( a ) Regional association │
│ plot showing association of common genetic variants in the SLC25A48 locus with choline │
│ levels in urine. ( b ) Flag-tagged SLC25A48 localizes to mitochondria, as shown by │
│ colocalization with the mitochondrial marker Mito-DsRed. Colors in merged image: SLC25A48 │
│ (turquoise), Mito-DsRed (violet), and 4 ,6-diamidino-2-phenylindole 0 (blue). Maximum │
│ intensity projection of a confocal z-stack. Bar ¼ 5 m M. ( c,d ) Mitochondrial uptake of │
│ radiolabeled 3 H-choline in cells expressing SLC25A48 compared with mock-transfected control │
│ cells (n ¼ 10). Average counts per minute (CPMA). *** P < 0.001. ( e ) Time course of │
│ relative choline uptake in mitochondria. ( ) Concentration dependence of choline uptake in │
│ mitochondria from cells expressing SLC25A48 f compared with mock-transfected controls. Chr, │
│ chromosome. To optimize viewing of this image, please see the online version of this article │
│ at www.kidney-international.org. │
│ │
│ │
│ │
│ Associations with gene expression and human traits and diseases │
│ │
│ │
│ RESULTS AND DISCUSSION │
│ │
│ Methods related to the association of rare SLC25A48 variants with gene expression, as well │
│ as human traits and diseases from the UK Biobank (application identi /uniFB01 er 64806), are │
│ found in the Supplementary Methods. │
│ │
│ │
│ Structural model generation and analysis │
│ │
│ The SLC25A48 AlphaFold 19 model was retrieved from the AlphaFold Protein Structure Database │
│ hosted by the European Molecular Biology Laboratories -European Bioinformatics Institute │
│ (EMBL-EBI; https://alphafold.ebi.ac.uk, accession code Q6ZT89). To identify potential │
│ homologous proteins with experimental structures available, a sequencebased BLAST search of │
│ the Protein Data Bank as well as a structural homology based search using -the DALI Server │
│ 20 were performed. Details about the structures of other SLC25 family members, how they were │
│ superimposed, and how the matrix-open structure was modeled can be found in the │
│ Supplementary Methods. Figures were prepared using the PyMOL Molecular Graphics System, │
│ Schrödinger, LLC. │
│ │
│ We previously found through genome-wide association studies of metabolite levels that common │
│ SLC25A48 variants are associated with altered choline levels in urine (Figure 1a), 2 and now │
│ established that the genetic basis of this association is shared with plasma choline as well │
│ as SLC25A48 transcript levels in brain tissue (Supplementary Figure S1), supporting the │
│ hypothesis that the orphan solute carrier SLC25A48 may be a choline transporter. SLC25A48 │
│ transcript levels are highest in brain and kidney. Within the kidney, expression is high in │
│ epithelial cells of the S3 segment of the proximal tubule, a cell type rich in mitochondria │
│ with diverse energydemanding functions such as bulk reabsorption of /uniFB01 ltered solutes │
│ (Supplementary Figure S2). Overexpressed SLC25A48 localized to mitochondria in human │
│ epithelial cells (Figure 1b; Supplementary Figures S3 and S4). We, therefore, isolated │
│ mitochondria of cells overexpressing SLC25A48 and measured mitochondrial uptake of │
│ radiolabeled choline (Figure 1c). Mitochondria from cells overexpressing SLC25A48 showed a │
│ signi /uniFB01 cant increase of choline uptake compared with mitochondria from control cells │
│ (Figure 1d). │
│ │
│ Figure 2 | Rare damaging variants in SLC25A48 impair choline transport. ( a ) Comparison of │
│ inverse normal transformed choline levels in urine and plasma among carriers (N ¼ 47) and │
│ noncarriers (N ¼ 4572 for urine; N ¼ 4666 for plasma) of putative rare damaging driver │
│ variants in SLC25A48 ( P value by unpaired t -test [2 tailed]: 3.8 /C2 10 -21 for urine and │
│ 1.3 /C2 10 -07 for plasma). ( b ) Localization of rare, damaging driver variants with │
│ respect to their protein position in SLC25A48 (Q6ZT89, corresponding to transcript │
│ ENST00000681962.1, domains based on InterPro, x axis). Symbol shape corresponds to variant │
│ consequence, and the size represents the positive effect size of each individual variant on │
│ urine choline levels (Supplementary Table S1). Individual variant association -log10( P │
│ value) with urine choline levels is shown on the y axis. The 4 variants with the largest │
│ effects on urine choline levels were selected for subsequent functional analyses and are │
│ labeled. ( c ) Western blot analysis of wild-type versus mutant /uniFB02 ag-tagged SLC25A48. │
│ Actin was used as the loading control. ( d ) Cellular localization of the SLC25A48 missense │
│ mutation p.R179P compared with wild-type SLC25A48. Indirect immuno /uniFB02 uorescence of │
│ SLC25A48/uniFB02 ag and cytochrome C oxidase subunit 4 (COX4) as mitochondrial marker. │
│ Colors in merged image: SLC25A48 (turquoise), COX4 (violet), and 4 │
│ ,6-diamidino-2-phenylindole 0 (blue). Bar ¼ 5 m M. ( e ) Quanti /uniFB01 cation of │
│ colocalization of SLC25A48 and COX4 (Pearson correlation coef /uniFB01 cient) shows a signi │
│ /uniFB01 cant reduction of mitochondrial localization of SLC25A48-R179P compared with wild │
│ type (**** P < 0.0001; see Supplementary Figure S7 for other mutants). ( ) f Relative │
│ mitochondrial uptake of radiolabeled 3 H-choline in cells expressing wild-type and mutant │
│ SLC25A48. Transport of mutant SLC25A48 was normalized to wild-type transport. * P < 0.05, │
│ **** P < 0.0001. ( g ) Position of damaging mutations in model of SLC25A48 (AlphaFold) in │
│ intermembrane space (IMS) -facing conformation (helices numbered). Left: side chains of │
│ mutant residues are highlighted in pink. The protein part that would be truncated in p.R243* │
│ is in pink. Center, right: residues of the conserved matrix-salt-bridge network (dotted │
│ line), including D27 (mutation in p.D27G) are highlighted in red and blue sticks for acidic │
│ and basic residues, respectively. Mito_carr, mitochondrial carrier protein repeat; SOLCAR, │
│ solute carrier repeat. To optimize viewing of this image, please see the online version of │
│ this article at www.kidneyinternational.org. │
│ │
│ │
│ Conversely, small, interfering RNA -mediated knockdown of endogenous SLC25A48 signi /uniFB01 │
│ cantly reduced mitochondrial choline uptake and increased cytosolic choline concentration │
│ │
│ (Supplementary Figure S5). Mitochondrial uptake of choline was time and concentration │
│ dependent (Figure 1e and f). These data support the hypothesis that SLC25A48 is a newly │
│ │
│ identi /uniFB01 ed, high-af /uniFB01 nity mitochondrial choline transporter. SLC25A48 │
│ transports choline at nanomolar concentrations (Figure 1f), and thus operates at │
│ physiological concentrations of free choline in humans (7 -20 m M). 21 │
│ │
│ To test whether there is a causal link between altered choline levels in humans and impaired │
│ mitochondrial choline transport, we investigated the aggregated impact of rare, putatively │
│ damaging variants in SLC25A48 and found signi /uniFB01 cant associations with urine and │
│ plasma choline levels (burden test P ¼ 1.4 /C2 10 -17 and 4.2 /C2 10 -06 , respectively; │
│ Supplementary Table 1). Carriers of driver variants (Supplementary Methods) showed signi │
│ /uniFB01 cantly higher levels of choline compared with noncarriers, which was more │
│ pronounced in urine than in plasma (Figure 2a). Identi /uniFB01 ed driver variants mapped │
│ into different regions of SLC25A48 (Figure 2b). The association with choline was speci │
│ /uniFB01 c: no metabolite other than choline showed signi /uniFB01 cant genetic associations │
│ after correcting for multiple testing (Supplementary Figure S6). │
│ │
│ Choline has been implicated in various human conditions, ranging from neurologic diseases to │
│ metabolic traits, but there is limited evidence linking genetic defects in choline transport │
│ to human disease. We, therefore, tested whether cholinerelated, putative loss-of-function │
│ mutations in SLC25A48 were associated with any of the human traits and diseases ascertained │
│ in the UK Biobank that are related to tissues where SLC25A48 is highly expressed (METHODS). │
│ None of the binary (Supplementary Table 2) or quantitative (Supplementary Table 3) traits │
│ was signi /uniFB01 cantly associated with SLC25A48 putative loss-of-function carrier status │
│ after correction for multiple testing. │
│ │
│ To investigate whether implicated variants were causally related to altered choline levels, │
│ we generated 4 mutations in the SLC25A48 cDNA using site-directed mutagenesis and │
│ overexpressed these variants in human cell lines to study their effect on protein │
│ expression, localization, and function. Western blot analyses showed reduced protein │
│ expression of some, but not all, investigated SLC25A48 mutations compared with wild-type │
│ protein (Figure 2c). Interestingly, some mutations showed mislocalization from mitochondria │
│ (p.R179P and p.R243*), with a signi /uniFB01 cant reduction of the mitochondrial │
│ colocalization index compared with wild-type SLC25A48 (Figure 2d and e; Supplementary Figure │
│ S7), whereas other mutations localized to mitochondria like wild-type transporters (p.D27G │
│ and p.R64L; Supplementary Figure S7). These data show that only some of the investigated │
│ SLC25A48 mutations result in reduced protein expression and/or mislocalization. │
│ │
│ We subsequently measured choline uptake in mitochondria to investigate which mutations │
│ affect SLC25A48 transport function. Despite differences in abundance and localization, all │
│ tested SLC25A48 mutations signi /uniFB01 cantly impaired mitochondrial choline uptake │
│ (Figure 2f). These data suggest that impaired mitochondrial choline transport via SLC25A48 │
│ results in altered plasma and urine choline levels in humans. This notion is supported by │
│ the increased cytosolic concentration of choline in SLC25A48-de /uniFB01 cient cells │
│ │
│ (Supplementary Figure S5), which decreases the driving force for choline into cells via │
│ plasma membrane choline transporters. From a mechanistic standpoint, our data suggest that │
│ the investigated mutations cause loss-of-function through different pathogenic mechanisms, │
│ including reduced expression, mislocalization, and impaired substrate turnover with normal │
│ expression level and localization. │
│ │
│ To gain a better mechanistic understanding of why speci /uniFB01 c mutations cause impaired │
│ transport, we analyzed structural models of SLC25A48 in 2 conformations (Figure 2g; │
│ Supplementary Figure S8). The SLC25A48 models were in good agreement with experimental │
│ structures of other SLC25 family members, such as the adenosine diphosphate/adenosine │
│ triphosphate carrier and mitochondrial uncoupling protein (Supplementary Figure S8). │
│ Notably, residue D27 is part of a matrix-salt-bridge network of conserved residues, which is │
│ a key component of the matrix gate of these transporters and, thus, essential for transport │
│ function. This is in line with the strongly impaired choline transport of p.D27G despite its │
│ normal localization. 22,23 The other mutations were also localized on the matrix side of the │
│ transporter and may affect its folding and stability. A limitation of this study is the lack │
│ of proteoliposome experiments with puri /uniFB01 ed SLC25A48. Future studies investigating │
│ SLC25A48 in proteoliposomes may provide deeper insights into the biophysical properties, the │
│ structure, and the function of this mitochondrial transporter. │
│ │
│ In summary, we show that the physiological function of SLC25A48 in humans is choline import │
│ into mitochondria. Loss-of-function mutations in SLC25A48 impair choline transport into │
│ mitochondria and cause higher levels of free choline in urine and plasma. The │
│ deorphanization of SLC25A48 de /uniFB01 nes its molecular function in humans and enables │
│ future well-powered studies addressing its role in health and disease. │
│ │
│ │
│ DISCLOSURE │
│ │
│ All the authors declared no competing interests. │
│ │
│ │
│ DATA STATEMENT │
│ │
│ All data generated or analyzed in this study were included in the main text and the │
│ Supplementary Material for this article. Other source data that support the /uniFB01 ndings │
│ of this study and uncropped full gel images of Western blot analyses are available from the │
│ corresponding author on reasonable request. │
│ │
│ │
│ ACKNOWLEDGMENTS │
│ │
│ The cDNA for SLC25A48 was kindly provided by the RESOLUTE consortium (https://re-solute.eu). │
│ The authors acknowledge Simone Diederichsen and Andreas Ungi for expert technical assistance │
│ and graphic support. We would like to thank the Lighthouse Core Facility for assistance with │
│ microscopy and cell sorting and Oliver Gorka for support with /uniFB02 ow cytometry. The │
│ work of NS, PS, CH, BN, AK, and MK was funded by German Research Foundation (DFG) project │
│ identi /uniFB01 er (ID) 431984000 (Sonderforschungsbereich [SFB] 1453). AK was supported by │
│ DFG KO 3598/4-2. MK was supported by DFG project ID 239283807 (Transregio [TRR] 152). │
│ Germany s Excellence Strategy ' (CIBSS -Centre for Integrative Biological Signalling │
│ Studies, EXC-2189, │
│ │
│ project ID 390939984) supported the work of CH, AK, and MK. SP was funded by H2020 │
│ MSCA-ITN-2019 ID: 860977 (TrainCKDis). The work of PS was supported by DFG Project-ID │
│ 523737608 (SCHL 2292/2-1). Genotyping and urine metabolomics in the German Chronic Kidney │
│ Disease (GCKD) study were supported by Bayer Pharma. Plasma metabolomics has received │
│ funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant │
│ agreement no. 115974. The JU receives support from the European Union s Horizon 2020 │
│ research and innovation program ' and the European Federation of Pharmaceutical Industries │
│ and Associations (EFPIA) and the Juvenile Diabetes Research Foundation (JDRF). Any │
│ dissemination of results re /uniFB02 ects only the authors ' view; the JU is not responsible │
│ for any use that may be made of the information it contains. Whole-exome sequencing data │
│ generation was supported by AstraZeneca, and processing of data was supported by their │
│ Centre for Genomics Research Analytics and Informatics team. The GCKD study was and is │
│ supported by the Bundesministerium für Bildung und Forschung (BMBF; FKZ 01ER 0804, 01ER │
│ 0818, 01ER 0819, 01ER 0820, and 01ER 0821) and the KfH Foundation for Preventive Medicine. │
│ We are grateful for the willingness of the patients to participate in the GCKD study. The │
│ effort of the study personnel of the various regional centers is highly appreciated. We │
│ thank the large number of nephrologists who provide routine care for the patients and │
│ collaborate with the GCKD study. │
│ │
│ Supplementary material is available online at www.kidneyinternational.org. │
│ │
│ │
│ REFERENCES │
│ │
│ • 1 Meixner E, Goldmann U, Sedlyarov V, et al. A substrate-based ontology for human solute │
│ carriers. Mol Syst Biol . 2020;16:e9652. │
│ • 2 Schlosser P, Scherer N, Grundner-Culemann F, et al. Genetic studies of paired │
│ metabolomes reveal enzymatic and transport processes at the interface of plasma and │
│ urine. Nat Genet . 2023;55:995 -1008. │
│ • 3 Suhre K, Shin SY, Petersen AK, et al. Human metabolic individuality in biomedical and │
│ pharmaceutical research. Nature . 2011;477:54 -60. │
│ • 4 Ueland PM. Choline and betaine in health and disease. J Inherit Metab Dis . 2011;34:3 │
│ -15. │
│ • 5 Apparsundaram S, Ferguson SM, George AL Jr, et al. Molecular cloning of a human, │
│ hemicholinium-3-sensitive choline transporter. Biochem Biophys Res Commun . │
│ 2000;276:862 -867. │
│ • 6 Bennett JA, Mastrangelo MA, Ture SK, et al. The choline transporter Slc44a2 controls │
│ platelet activation and thrombosis by regulating mitochondrial function. Nat Commun . │
│ 2020;11:3479. │
│ • 7 Kenny TC, Khan A, Son Y, et al. Integrative genetic analysis identi /uniFB01 es FLVCR1 │
│ as a plasma-membrane choline transporter in mammals. Cell Metab . 2023;35:1057 │
│ -1071.e12. │
│ • 8 Michel V, Bakovic M. The solute carrier 44A1 is a mitochondrial protein and mediates │
│ choline transport. FASEB J . 2009;23:2749 -2758. │
│ • 9 Traiffort E, O Regan S, Ruat M. The choline transporter-like family ' SLC44: │
│ properties and roles in human diseases. Mol Aspects Med . 2013;34:646 -654. │
│ • 10 Cater RJ, Mukherjee D, Gil-Iturbe E, et al. Structural and molecular basis of choline │
│ uptake into the brain by FLVCR2. Nature . 2024;629:704 -709. │
│ • 11 Wegierski T, Stef /uniFB02 D, Kopp C, et al. TRPP2 channels regulate apoptosis │
│ through the Ca2 þ concentration in the endoplasmic reticulum. EMBO J . 2009;28:490 │
│ -499. │
│ • 12 Hofherr A, Seger C, Fitzpatrick F, et al. The mitochondrial transporter SLC25A25 │
│ links ciliary TRPP2 signaling and cellular metabolism. PLoS Biol . 2018;16:e2005651. │
│ • 13 Wieckowski MR, Giorgi C, Lebiedzinska M, et al. Isolation of mitochondria-associated │
│ membranes and mitochondria from animal tissues and cells. Nat Protoc . 2009;4:1582 │
│ -1590. │
│ • 14 Eckardt KU, Barthlein B, Baid-Agrawal S, et al. The German Chronic Kidney Disease │
│ (GCKD) study: design and methods. Nephrol Dial Transplant . 2012;27:1454 -1460. │
│ • 15 Ioannidis NM, Rothstein JH, Pejaver V, et al. REVEL: an ensemble method for │
│ predicting the pathogenicity of rare missense variants. Am J Hum Genet . 2016;99:877 │
│ -885. │
│ • 16 Rentzsch P, Witten D, Cooper GM, et al. CADD: predicting the deleteriousness of │
│ variants throughout the human genome. Nucleic Acids Res . 2019;47:D886 -D894. │
│ • 17 Liu X, Li C, Mou C, et al. dbNSFP v4: a comprehensive database of transcript-speci │
│ /uniFB01 c functional predictions and annotations for human nonsynonymous and │
│ splice-site SNVs. Genome Med . 2020;12:103. │
│ • 18 Karczewski KJ, Francioli LC, Tiao G, et al. The mutational constraint spectrum quanti │
│ /uniFB01 ed from variation in 141,456 humans. Nature . 2020;581: 434 443. - │
│ • 19 Jumper J, Evans R, Pritzel A, et al. Highly accurate protein structure prediction │
│ with AlphaFold. Nature . 2021;596:583 -589. │
│ • 20 Holm L, Laiho A, Toronen P, et al. DALI shines a light on remote homologs: one │
│ hundred discoveries. Protein Sci . 2023;32:e4519. │
│ • 21 Garcia E, Shalaurova I, Matyus SP, et al. Quanti /uniFB01 cation of choline in serum │
│ and plasma using a clinical nuclear magnetic resonance analyzer. Clin Chim Acta . │
│ 2022;524:106 -112. │
│ • 22 Jones SA, Gogoi P, Ruprecht JJ, et al. Structural basis of purine nucleotide │
│ inhibition of human uncoupling protein 1. Sci Adv . 2023;9:eadh4251. │
│ • 23 Ruprecht JJ, Kunji ERS. The SLC25 mitochondrial carrier family: structure and │
│ mechanism. Trends Biochem Sci . 2020;45:244 -258. │
╰──────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────────╮
│ Assistant: │
╰──────────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────╮
│ │
│ { │
│ "entity_list": [ │
│ { │
│ "entity_name": "SLC25A48" │
│ }, │
│ { │
│ "entity_name": "choline" │
│ }, │
│ { │
│ "entity_name": "mitochondria" │
│ }, │
│ { │
│ "entity_name": "acetic acid" │
│ }, │
│ { │
│ "entity_name": "betaine" │
│ }, │
│ { │
│ "entity_name": "phospholipids" │
│ }, │
│ { │
│ "entity_name": "phosphatidylcholines" │
│ }, │
│ { │
│ "entity_name": "acetylcholine" │
│ }, │
│ { │
│ "entity_name": "SLC5A7" │
│ }, │
│ { │
│ "entity_name": "SLC44A1" │
│ }, │
│ { │
│ "entity_name": "SLC44A2" │
│ }, │
│ { │
│ "entity_name": "SLC44A4" │
│ }, │
│ { │
│ "entity_name": "SLC49A1" │
│ }, │
│ { │
│ "entity_name": "SLC49A2" │
│ }, │
│ { │
│ "entity_name": "HEK293T" │
│ }, │
│ { │
│ "entity_name": "HeLa" │
│ } │
│ ], │
│ "comment": "The list includes key transport proteins, metabolites, and cell lines direct │
│ referenced in the publication's methods and results sections, all of which are relevant to │
│ the study's focus on choline transport and the membrane transporter SLC25A48." │
│ } │
│ │
╰──────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────────╮
│ User: │
╰──────────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────╮
│ ┏━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━┓ │
│ ┃ Step 2: PubTator Analysis ┃ │
│ ┗━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━┛ │
│ │
│ For each biomedical entity identified in Step 1, determine whether it is listed in PubTator. │
│ Provide a list of these entities along with their existence status in PubTator. Do not │
│ include examples or elaborate explanations. A Pubtator ID starts with '@' and does NOT │
│ contain a space character. If a Pubtator entity has several IDs, pick the best match. │
│ │
│ You MUST use the provided Pubtator tool to verify all entities! │
│ │
│ You MUST format your response according to the provided JSON schema. │
╰──────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────────╮
│ Assistant: │
╰──────────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────╮
│ [Assistant answered with 16 tool calls] │
╰──────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────────╮
│ Tool: │
╰──────────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────╮
│ Pubtator entity search results for query 'SLC25A48': │
│ │
│ • SLC25A48: @GENE_328258 @GENE_SLC25A48 │
│ • SLC25A48: @GENE_515496 @GENE_SLC25A48 │
│ • SLC25A48: @GENE_153328 @GENE_SLC25A48 │
│ • slc25a48: @GENE_436640 @GENE_SLC25A48 │
╰──────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────────╮
│ Tool: │
╰──────────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────╮
│ Pubtator entity search results for query 'choline': │
│ │
│ • choline chloride: @CHEMICAL_MESH:D002794 @CHEMICAL_Choline │
│ • Choline: @CHEMICAL_MESH:D002794 @CHEMICAL_Choline │
│ • choline: @CHEMICAL_MESH:D002794 @CHEMICAL_Choline │
╰──────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────────╮
│ Tool: │
╰──────────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────╮
│ Pubtator entity search results for query 'mitochondria': No IDs found. │
╰──────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────────╮
│ Tool: │
╰──────────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────╮
│ Pubtator entity search results for query 'acetic acid': │
│ │
│ • acetic acid: @CHEMICAL_MESH:D019342 @CHEMICAL_Acetic_Acid │
│ • Acetic acid: @CHEMICAL_MESH:D019342 @CHEMICAL_Acetic_Acid │
╰──────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────────╮
│ Tool: │
╰──────────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────╮
│ Pubtator entity search results for query 'betaine': │
│ │
│ • betaine hydrochloride: @CHEMICAL_MESH:D001622 @CHEMICAL_Betaine │
│ • Betaine: @CHEMICAL_MESH:D001622 @CHEMICAL_Betaine │
│ • betaine: @CHEMICAL_MESH:D001622 @CHEMICAL_Betaine │
╰──────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────────╮
│ Tool: │
╰──────────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────╮
│ Pubtator entity search results for query 'phospholipids': │
│ │
│ • Phospholipids: @CHEMICAL_MESH:D010743 @CHEMICAL_Phospholipids │
│ • Phospholipid: @CHEMICAL_MESH:D010743 @CHEMICAL_Phospholipids │
│ • phospholipid: @CHEMICAL_MESH:D010743 @CHEMICAL_Phospholipids │
╰──────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────────╮
│ Tool: │
╰──────────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────╮
│ Pubtator entity search results for query 'phosphatidylcholines': │
│ │
│ • PLi: @CHEMICAL_MESH:C044850 │
│ @CHEMICAL_1_myristoyl_2_(12_N_(4_azido_2_nitrophenyl)aminododecanoyl)phosphatidylcholine │
│ • Phosphatidylcholine: @CHEMICAL_MESH:D010713 @CHEMICAL_Phosphatidylcholines │
│ • phosphatidylcholine: @CHEMICAL_MESH:D010713 @CHEMICAL_Phosphatidylcholines │
│ • phosphatidylcholines: @CHEMICAL_MESH:D010713 @CHEMICAL_Phosphatidylcholines │
│ • lyso-phosphatidylcholine: @CHEMICAL_MESH:D008244 @CHEMICAL_Lysophosphatidylcholines │
╰──────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────────╮
│ Tool: │
╰──────────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────╮
│ Pubtator entity search results for query 'acetylcholine': │
│ │
│ • acetylcholine: @CHEMICAL_MESH:D000109 @CHEMICAL_Acetylcholine │
│ • Acetylcholine: @CHEMICAL_MESH:D000109 @CHEMICAL_Acetylcholine │
╰──────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────────╮
│ Tool: │
╰──────────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────╮
│ Pubtator entity search results for query 'SLC5A7': │
│ │
│ • Slc5a7: @GENE_60482 @GENE_SLC5A7 │
│ • SLC5A7: @GENE_414870 @GENE_SLC5A7 │
│ • SLC5A7: @GENE_60482 @GENE_SLC5A7 │
╰──────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────────╮
│ Tool: │
╰──────────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────╮
│ Pubtator entity search results for query 'SLC44A1': │
│ │
│ • SLC44A1: @GENE_23446 @GENE_SLC44A1 │
│ • SLC44A1: @GENE_101102625 @GENE_SLC44A1 │
│ • SLC44A1: @GENE_100434 @GENE_SLC44A1 │
╰──────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────────╮
│ Tool: │
╰──────────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────╮
│ Pubtator entity search results for query 'SLC44A2': │
│ │
│ • Slc44a2: @GENE_68682 @GENE_SLC44A2 │
│ • SLC44A2: @GENE_68682 @GENE_SLC44A2 │
│ • Slc44a2: @GENE_57153 @GENE_SLC44A2 │
│ • SLC44A2: @GENE_57153 @GENE_SLC44A2 │
╰──────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────────╮
│ Tool: │
╰──────────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────╮
│ Pubtator entity search results for query 'SLC44A4': │
│ │
│ • Slc44a4: @GENE_70129 @GENE_SLC44A4 │
│ • SLC44A4: @GENE_80736 @GENE_SLC44A4 │
│ • SLC44A4: @GENE_70129 @GENE_SLC44A4 │
╰──────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────────╮
│ Tool: │
╰──────────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────╮
│ Pubtator entity search results for query 'SLC49A1': No IDs found. │
╰──────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────────╮
│ Tool: │
╰──────────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────╮
│ Pubtator entity search results for query 'SLC49A2': No IDs found. │
╰──────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────────╮
│ Tool: │
╰──────────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────╮
│ Pubtator entity search results for query 'HEK293T': │
│ │
│ • HEK293T: @CELLLINE_CVCL:0063 │
╰──────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────────╮
│ Tool: │
╰──────────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────╮
│ Pubtator entity search results for query 'HeLa': │
│ │
│ • HeLa: @CELLLINE_CVCL:T292 │
│ • HeLa: @CELLLINE_CVCL:0030 │
╰──────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────────╮
│ Assistant: │
╰──────────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────╮
│ │
│ { │
│ "entity_list": [ │
│ { │
│ "entity_name": "SLC25A48", │
│ "in_pubtator": true, │
│ "pubtator_id": "@GENE_SLC25A48" │
│ }, │
│ { │
│ "entity_name": "choline", │
│ "in_pubtator": true, │
│ "pubtator_id": "@CHEMICAL_Choline" │
│ }, │
│ { │
│ "entity_name": "mitochondria", │
│ "in_pubtator": false, │
│ "pubtator_id": "" │
│ }, │
│ { │
│ "entity_name": "acetic acid", │
│ "in_pubtator": true, │
│ "pubtator_id": "@CHEMICAL_Acetic_Acid" │
│ }, │
│ { │
│ "entity_name": "betaine", │
│ "in_pubtator": true, │
│ "pubtator_id": "@CHEMICAL_Betaine" │
│ }, │
│ { │
│ "entity_name": "phospholipids", │
│ "in_pubtator": true, │
│ "pubtator_id": "@CHEMICAL_Phospholipids" │
│ }, │
│ { │
│ "entity_name": "phosphatidylcholines", │
│ "in_pubtator": true, │
│ "pubtator_id": "@CHEMICAL_Phosphatidylcholines" │
│ }, │
│ { │
│ "entity_name": "acetylcholine", │
│ "in_pubtator": true, │
│ "pubtator_id": "@CHEMICAL_Acetylcholine" │
│ }, │
│ { │
│ "entity_name": "SLC5A7", │
│ "in_pubtator": true, │
│ "pubtator_id": "@GENE_SLC5A7" │
│ }, │
│ { │
│ "entity_name": "SLC44A1", │
│ "in_pubtator": true, │
│ "pubtator_id": "@GENE_SLC44A1" │
│ }, │
│ { │
│ "entity_name": "SLC44A2", │
│ "in_pubtator": true, │
│ "pubtator_id": "@GENE_SLC44A2" │
│ }, │
│ { │
│ "entity_name": "SLC44A4", │
│ "in_pubtator": true, │
│ "pubtator_id": "@GENE_SLC44A4" │
│ }, │
│ { │
│ "entity_name": "SLC49A1", │
│ "in_pubtator": false, │
│ "pubtator_id": "" │
│ }, │
│ { │
│ "entity_name": "SLC49A2", │
│ "in_pubtator": false, │
│ "pubtator_id": "" │
│ }, │
│ { │
│ "entity_name": "HEK293T", │
│ "in_pubtator": true, │
│ "pubtator_id": "@CELLLINE_CVCL:0063" │
│ }, │
│ { │
│ "entity_name": "HeLa", │
│ "in_pubtator": true, │
│ "pubtator_id": "@CELLLINE_CVCL:T292" │
│ } │
│ ], │
│ "comment": "Status of biomedical entities in PubTator was determined, noting those prese │
│ and their respective IDs." │
│ } │
│ │
╰──────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────────╮
│ User: │
╰──────────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────╮
│ ┏━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━┓ │
│ ┃ Step 3: Reanalysis for Specific Entities ┃ │
│ ┗━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━┛ │
│ │
│ Reanalyze the scientific publication to identify the presence of specific entities from │
│ different schema categories listed below as subheadings. Follow these rules: │
│ │
│ • Only include entities explicitly mentioned in the publication, excluding mentions from │
│ the discussion section or bibliography. │
│ • If the entity is a synonym of the one listed in the schema, use the name from the schema. │
│ • Do not use the category names as entity names. │
│ • If entities are listed hierachically, use the most specific entities. │
│ • If you notice entities that are not listed in the subheadings, include them in the list │
│ of entities as well. │
│ │
│ You MUST format your response according to the provided JSON schema. │
│ │
│ │
│ Schema │
│ │
│ Organism │
│ │
│ • Cell line │
│ • Fruit Fly │
│ • Human │
│ • Mouse │
│ • C. Elegans │
│ • Rabbit │
│ • Rat │
│ • Xenopus │
│ • Zebrafish │
│ │
│ Cell Lines │
│ │
│ • 293 (HEK 293) │
│ • 293A (HEK 293A) │
│ • 293T (HEK 293T) │
│ • AC16 Human Cardiomyocyte Cell Line │
│ • HAF │
│ • HeLa │
│ • HeLa 229 │
│ • HL-1 │
│ • HUVEC │
│ • THP-1 │
│ • MDCK │
│ • mIMCD-3 │
│ • LLC-PK1 │
│ • RPTEC │
│ │
│ Tissue source │
│ │
│ • Adrenal gland │
│ • Blood │
│ • Blood plasma │
│ • Blood serum │
│ • Whole blood │
│ • Bone marrow │
│ • Brain │
│ • Embryonal tissue │
│ • Heart │
│ • Intestine │
│ • Kidney │
│ • Head kidney │
│ • Juxtaglomerular apparatus │
│ • Extraglomerular mesangial cell │
│ • Juxtaglomerular cell │
│ • Macula densa │
│ • Kidney cancer cell │
│ • Nephridium │
│ • Nephron │
│ • Renal corpuscle │
│ • Renal tubule │
│ • Collecting duct │
│ • Henles loop │
│ • Renal clear cell │
│ • Renal distal tubule │
│ • Renal proximal tubule │
│ • Renal tubule epithelium │
│ • Opisthonephros │
│ • Renal artery │
│ • Renal cortex │
│ • Renal epithelium │
│ • Renal medulla │
│ • Renal neoplasm cell │
│ • Renal parenchyma │
│ • Renal pyramid │
│ • Trunk kidney │
│ • Tubulointerstitium │
│ • Liver │
│ • Lung │
│ • Lymph node │
│ • Nerve │
│ • Skin │
│ • Spleen │
│ • Thymus │
│ • Urine │
│ • Vascular system │
│ │
│ Health status │
│ │
│ • Autosomal Dominant Polycystic Kidney Disease (ADPKD) │
│ • Nephronophthisis │
│ • Chronic Kidney Disease (CKD) │
│ • Nephrotic syndrome │
│ • Clear cell renal carcinoma (ccRCC) │
│ • Von Hippel Lindau syndrome │
│ • Congenital abnormality of the kidney │
│ • Congenital abnormality of the ureter │
│ │
│ Mouse line │
│ │
│ • C57BL/6J (wildtype) │
│ • Ksp-Cre │
│ • Pax8-Cre (Pax8rtTA) │
│ • Pgk-Cre │
│ • Pod-Cre │
│ • LC1-Cre │
│ • Tam-Cre │
│ • Pkd1-flox │
│ • Pkd1- │
│ • Dnajb11 tm1a │
│ • Dnajb11-flox │
│ • Dnajb11- │
│ │
│ Sample preparation │
│ │
│ • Cultured cells │
│ • Adipocyte │
│ • Cardiomyocyte │
│ • Dendritic cell │
│ • Embryonic cardiomyocytes │
│ • Endothelial cell │
│ • Epithelial cell │
│ • Fibroblast │
│ • hiPSC-CM │
│ • hiPSC-FB │
│ • Immune cell │
│ • Leukocytes │
│ • Lymphocytes │
│ • Macrophage │
│ • Monocytes │
│ • Natural killer cells │
│ • Neuronal cell │
│ • Neurones │
│ • Neutrophils │
│ • Oocyte │
│ • Pericytes │
│ • Platelet │
│ • Smooth muscle cells │
│ • T cells │
│ • Isolated cells │
│ • Adipocyte │
│ • Cardiomyocyte │
│ • Dendritic cell │
│ • Embryonic cardiomyocytes │
│ • Endothelial cell │
│ • Epithelial cell │
│ • Fibroblast │
│ • hiPSC-CM │
│ • hiPSC-FB │
│ • Immune cell │
│ • Leukocytes │
│ • Lymphocytes │
│ • Macrophage │
│ • Monocytes │
│ • Natural killer cells │
│ • Neuronal cell │
│ • Neurones │
│ • Neutrophils │
│ • Oocyte │
│ • Pericytes │
│ • Platelet │
│ • Smooth muscle cells │
│ • T cells │
│ • Tissue chunk │
│ • Tissue section (thin) │
│ • Tissue slice │
│ • Whole organ │
│ │
│ Sample processing │
│ │
│ • Cleared fixed tissue │
│ • Formaldehyde fixed and parafin embedded (FFPE) │
│ • Formaldehyde-fixation │
│ • High pressure frozen │
│ • None (Physiological solution) │
│ • OCT embedded and frozen │
│ │
│ Readout │
│ │
│ • Biomechanics │
│ • Cell stretching │
│ • Nanoindentation │
│ • Sarcomer Length │
│ • Single cell stretching │
│ • Tissue stretching │
│ • Tissue stretching Myodish │
│ • Clinical Imaging │
│ • Computed tomography scan │
│ • Echocardiography │
│ • Magnetic resonance imaging │
│ • Positron emission tomography │
│ • Computational modelling │
│ • Electron microscopy │
│ • Electrophysiology (clinical) │
│ • Electrophysiology (lab) │
│ • Patch Clamp - current clamp │
│ • Patch Clamp - voltage clamp │
│ • Sharp Electrode │
│ • Flow cytometry / FACS │
│ • Light microscopy │
│ • Confocal │
│ • Multiphoton │
│ • Slidescanner │
│ • Widefield │
│ • Molecular Biology │
│ • Aptamer (Proteomics) │
│ • Bulk chromatin accessibility │
│ • DNA Methylation (array) │
│ • Enzyme-linked immunosorbent assay (ELISA) │
│ • Gene Expression (array) │
│ • Genotyping array and imputation │
│ • Proximity extension assay (PEA) │
│ • Quantitative polymerase chain reaction (qPCR) │
│ • Standard biochemical assay │
│ • Westernblot │
│ • Optical mapping │
│ • Optoacoustics │
│ • Sequencing │
│ • ATAC sequencing │
│ • DNA methylation (WGBS) │
│ • RNA sequencing │
│ • Single Cell chromatin accessibility │
│ • Single cell RNA seq │
│ • Whole exome sequencing │
│ • Whole genome sequencing │
│ • Mass Spectrometry │
│ • Interaction proteomics │
│ • Metabolomics │
│ • Proteomics │
│ │
│ Probes │
│ │
│ • Antibodies │
│ • alpha-Actinin │
│ • alpha-SMA (Smooth Muscle Actin) │
│ • Amphiphysin II │
│ • ATP5B │
│ • BDNF │
│ • beta-Actin │
│ • beta-Tubulin │
│ • Bin1 │
│ • CACNA2D1 │
│ • Calnexin │
│ • CaV1.2 (CACNA1C) │
│ • Caveolin-3 │
│ • CCL2 │
│ • CD107b / Mac-3 │
│ • CD14 │
│ • CD29 │
│ • CD31 │
│ • CD45 │
│ • CD68 │
│ • CD90.1 (Thy-1.1) │
│ • Collagen-I-A1 (COL1A1) │
│ • Connexin 40 │
│ • Connexin 43 │
│ • COUP-TF II/NR2F2 │
│ • CXCL1/GRO-alpha/KC/CINC-1 │
│ • DDR2 │
│ • GAPDH │
│ • GFP (green fluorescent protein) │
│ • HCN4 │
│ • Histone 3 (PhosphoS10) │
│ • Integrin-alpha-1 │
│ • Integrin-beta-1 │
│ • KCa1.1/BK │
│ • Kir1.2 │
│ • Kir2.1 │
│ • KV1.3 │
│ • KV1.5 │
│ • mCherry │
│ • Muscarinic Acetycholine Receptor 2/CM2 │
│ • Myosin Light Chain 2 │
│ • Neurofilament Medium │
│ • p53 │
│ • PDGFR-alpha │
│ • Phospho-SMAD2 │
│ • Piezo1 │
│ • RFP (red fluorescent protein) │
│ • Ryanodine Receptor │
│ • S100B │
│ • Sarcomeric alpha-Actinin │
│ • SERCA2 ATPase │
│ • SLA Class II DR │
│ • Sodium/Calcium Exchanger │
│ • SOX10 │
│ • SWC3 │
│ • Talin 1 │
│ • TGF-beta-1 │
│ • Troponin I │
│ • TRP1 │
│ • TurboGFP │
│ • Tyrosine Hydroxylase │
│ • Vimentin │
│ • YAP │
│ • Reporter / Marker molecules │
│ • BeRST 1 │
│ • CellBrite │
│ • Cellmask │
│ • CellTracker │
│ • CNA-35 (collagen probe) │
│ • DAPI │
│ • Dextran │
│ • DI-4-ANBDQPQ │
│ • Di-4-ANEPPDHQ │
│ • Di-4-ANEQ(F)PTEA │
│ • Fluo-4 │
│ • Fura-2 │
│ • Hoechst 33342 │
│ • Memglow │
│ • Phalloidin │
│ • Rhod-2 │
│ • Streptavidin │
│ • Sytox Dead Cell Stain │
│ • Tubulin Tracker │
│ • Ulex Europaeus Agglutinin I (UEA I) │
│ • Wheat germ agglutinin │
│ • Stain │
│ • Hematoxylin and eosin │
│ • Horse radish peroxidase │
│ • Masson's Trichrome │
│ • Sirius Red │
╰──────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────────╮
│ Assistant: │
╰──────────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────╮
│ │
│ { │
│ "entity_list": [ │
│ { │
│ "entity_name": "Human", │
│ "from_provided_schema": true, │
│ "schema_category": "Organism" │
│ }, │
│ { │
│ "entity_name": "293T (HEK 293T)", │
│ "from_provided_schema": true, │
│ "schema_category": "Cell Lines" │
│ }, │
│ { │
│ "entity_name": "HeLa", │
│ "from_provided_schema": true, │
│ "schema_category": "Cell Lines" │
│ }, │
│ { │
│ "entity_name": "Kidney", │
│ "from_provided_schema": true, │
│ "schema_category": "Tissue source" │
│ }, │
│ { │
│ "entity_name": "Choline", │
│ "from_provided_schema": false, │
│ "schema_category": "" │
│ }, │
│ { │
│ "entity_name": "SLC25A48", │
│ "from_provided_schema": false, │
│ "schema_category": "" │
│ }, │
│ { │
│ "entity_name": "SLC5A7", │
│ "from_provided_schema": false, │
│ "schema_category": "" │
│ }, │
│ { │
│ "entity_name": "SLC44A1", │
│ "from_provided_schema": false, │
│ "schema_category": "" │
│ }, │
│ { │
│ "entity_name": "SLC44A2", │
│ "from_provided_schema": false, │
│ "schema_category": "" │
│ }, │
│ { │
│ "entity_name": "SLC44A4", │
│ "from_provided_schema": false, │
│ "schema_category": "" │
│ }, │
│ { │
│ "entity_name": "SLC49A1", │
│ "from_provided_schema": false, │
│ "schema_category": "" │
│ }, │
│ { │
│ "entity_name": "SLC49A2", │
│ "from_provided_schema": false, │
│ "schema_category": "" │
│ }, │
│ { │
│ "entity_name": "Acetic acid", │
│ "from_provided_schema": false, │
│ "schema_category": "" │
│ }, │
│ { │
│ "entity_name": "Betaine", │
│ "from_provided_schema": false, │
│ "schema_category": "" │
│ }, │
│ { │
│ "entity_name": "Phospholipids", │
│ "from_provided_schema": false, │
│ "schema_category": "" │
│ }, │
│ { │
│ "entity_name": "Phosphatidylcholine", │
│ "from_provided_schema": false, │
│ "schema_category": "" │
│ }, │
│ { │
│ "entity_name": "Acetylcholine", │
│ "from_provided_schema": false, │
│ "schema_category": "" │
│ } │
│ ], │
│ "comment": "Entities were identified from the publication according to specific schema │
│ categories, while also including additional entities found explicitly in the text." │
│ } │
│ │
╰──────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────────╮
│ User: │
╰──────────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────╮
│ ┏━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━┓ │
│ ┃ Step 4: Consolidation of Results ┃ │
│ ┗━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━┛ │
│ │
│ Consolidate the results of Steps 2 and 3: For each category listed in Step 3, list all │
│ entities identified in Step 3 and add the entities identified in Step 2 to the correct │
│ categories. If an entity is listed in both steps, include it only once in the final list, │
│ preferably with the schema name from Step 3. If an entity does not fit in the existing │
│ categories, you may add new ones. Try to use categories that are of the same scope as the │
│ existing ones. │
│ │
│ You MUST format your response according to the provided JSON schema. │
╰──────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────────╮
│ Assistant: │
╰──────────────────────────────────────────────────────────────────────────────────────────────────╯
╭──────────────────────────────────────────────────────────────────────────────────────────────╮
│ │
│ { │
│ "entity_list": [ │
│ { │
│ "entity_name": "Human", │
│ "in_pubtator": true, │
│ "pubtator_id": "", │
│ "from_provided_schema": true, │
│ "schema_category": "Organism" │
│ }, │
│ { │
│ "entity_name": "293T (HEK 293T)", │
│ "in_pubtator": true, │
│ "pubtator_id": "@CELLLINE_CVCL:0063", │
│ "from_provided_schema": true, │
│ "schema_category": "Cell Lines" │
│ }, │
│ { │
│ "entity_name": "HeLa", │
│ "in_pubtator": true, │
│ "pubtator_id": "@CELLLINE_CVCL:T292", │
│ "from_provided_schema": true, │
│ "schema_category": "Cell Lines" │
│ }, │
│ { │
│ "entity_name": "Kidney", │
│ "in_pubtator": false, │
│ "pubtator_id": "", │
│ "from_provided_schema": true, │
│ "schema_category": "Tissue source" │
│ }, │
│ { │
│ "entity_name": "Choline", │
│ "in_pubtator": true, │
│ "pubtator_id": "@CHEMICAL_Choline", │
│ "from_provided_schema": false, │
│ "schema_category": "Metabolite" │
│ }, │
│ { │
│ "entity_name": "SLC25A48", │
│ "in_pubtator": true, │
│ "pubtator_id": "@GENE_SLC25A48", │
│ "from_provided_schema": false, │
│ "schema_category": "Gene" │
│ }, │
│ { │
│ "entity_name": "SLC5A7", │
│ "in_pubtator": true, │
│ "pubtator_id": "@GENE_SLC5A7", │
│ "from_provided_schema": false, │
│ "schema_category": "Gene" │
│ }, │
│ { │
│ "entity_name": "SLC44A1", │
│ "in_pubtator": true, │
│ "pubtator_id": "@GENE_SLC44A1", │
│ "from_provided_schema": false, │
│ "schema_category": "Gene" │
│ }, │
│ { │
│ "entity_name": "SLC44A2", │
│ "in_pubtator": true, │
│ "pubtator_id": "@GENE_SLC44A2", │
│ "from_provided_schema": false, │
│ "schema_category": "Gene" │
│ }, │
│ { │
│ "entity_name": "SLC44A4", │
│ "in_pubtator": true, │
│ "pubtator_id": "@GENE_SLC44A4", │
│ "from_provided_schema": false, │
│ "schema_category": "Gene" │
│ }, │
│ { │
│ "entity_name": "SLC49A1", │
│ "in_pubtator": false, │
│ "pubtator_id": "", │
│ "from_provided_schema": false, │
│ "schema_category": "Gene" │
│ }, │
│ { │
│ "entity_name": "SLC49A2", │
│ "in_pubtator": false, │
│ "pubtator_id": "", │
│ "from_provided_schema": false, │
│ "schema_category": "Gene" │
│ }, │
│ { │
│ "entity_name": "Acetic acid", │
│ "in_pubtator": true, │
│ "pubtator_id": "@CHEMICAL_Acetic_Acid", │
│ "from_provided_schema": false, │
│ "schema_category": "Metabolite" │
│ }, │
│ { │
│ "entity_name": "Betaine", │
│ "in_pubtator": true, │
│ "pubtator_id": "@CHEMICAL_Betaine", │
│ "from_provided_schema": false, │
│ "schema_category": "Metabolite" │
│ }, │
│ { │
│ "entity_name": "Phospholipids", │
│ "in_pubtator": true, │
│ "pubtator_id": "@CHEMICAL_Phospholipids", │
│ "from_provided_schema": false, │
│ "schema_category": "Metabolite" │
│ }, │
│ { │
│ "entity_name": "Phosphatidylcholine", │
│ "in_pubtator": true, │
│ "pubtator_id": "@CHEMICAL_Phosphatidylcholines", │
│ "from_provided_schema": false, │
│ "schema_category": "Metabolite" │
│ }, │
│ { │
│ "entity_name": "Acetylcholine", │
│ "in_pubtator": true, │
│ "pubtator_id": "@CHEMICAL_Acetylcholine", │
│ "from_provided_schema": false, │
│ "schema_category": "Metabolite" │
│ } │
│ ], │
│ "comment": "This consolidation includes entities identified across both steps, organized │
│ by specific schema categories while ensuring unique entries." │
│ } │
│ │
╰──────────────────────────────────────────────────────────────────────────────────────────────╯