Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A pH-correctable, DNA-based fluorescent reporter for organellar calcium

An Author Correction to this article was published on 14 January 2019

This article has been updated

Abstract

It is extremely challenging to quantitate lumenal Ca2+ in acidic Ca2+ stores of the cell because all Ca2+ indicators are pH sensitive, and Ca2+ transport is coupled to pH in acidic organelles. We have developed a fluorescent DNA-based reporter, CalipHluor, that is targetable to specific organelles. By ratiometrically reporting lumenal pH and Ca2+ simultaneously, CalipHluor functions as a pH-correctable Ca2+ reporter. By targeting CalipHluor to the endolysosomal pathway, we mapped lumenal Ca2+ changes during endosomal maturation and found a surge in lumenal Ca2+ specifically in lysosomes. Using lysosomal proteomics and genetic analysis, we found that catp-6, a Caenorhabditis elegans homolog of ATP13A2, was responsible for lysosomal Ca2+ accumulation—an example of a lysosome-specific Ca2+ importer in animals. By enabling the facile quantification of compartmentalized Ca2+, CalipHluor can expand the understanding of subcellular Ca2+ importers.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Design and characterization of CalipHluorLy.
Fig. 2: In vivo sensing characteristics of CalipHluorLy.
Fig. 3: pH and [Ca2+] maps accompanying endosomal maturation.
Fig. 4: Catp-6 facilitates lysosomal Ca2+ accumulation.
Fig. 5: CalipHuormLy maps lysosomal Ca2+ in human cells.

Similar content being viewed by others

Data availability

The data that support the plots within this paper and the findings of this study are available from the corresponding author upon reasonable request.

Change history

  • 14 January 2019

    The originally published paper has been updated to include the following new reference, added as ref. 18: Albrecht, T., Zhao, Y., Nguyen, T. H., Campbell, R. E. & Johnson, J. D. Fluorescent biosensors illuminate calcium levels within defined beta-cell endosome subpopulations. Cell Calcium 57, 263–274 (2015). Subsequent references have been renumbered in the reference list and throughout the text. Minor text changes were made in the sentence in which this new reference is first cited: “Previous attempts used endocytic tracers bearing either pH- or Ca2+-sensitive dyes to serially measure population-averaged pH and apparent Ca2+ in different batches of cells, thus scrambling information from individual endosomes13–17” in the original introduction was changed to “Previous attempts used endocytic tracers bearing either pH- or Ca2+-sensitive dyes13–17 or fluorescent-protein-based sensors18 to serially measure population-averaged pH and apparent Ca2+ in different batches of cells, thus scrambling information from individual endosomes.” These changes have been made in the HTML and PDF versions of the article.

References

  1. Clapham, D. E. Calcium signaling. Cell 131, 1047–1058 (2007).

    Article  CAS  Google Scholar 

  2. Bagur, R. & Hajnóczky, G. Intracellular Ca2+ sensing: its role in calcium homeostasis and signaling. Mol. Cell 66, 780–788 (2017).

    Article  CAS  Google Scholar 

  3. Yang, J., Zhao, Z., Gu, M., Feng, X. & Xu, H. Release and uptake mechanisms of vesicular Ca2+ stores. Protein Cell https://doi.org/10.1007/s13238-018-0523-x (2018).

  4. Parenti, G., Andria, G. & Ballabio, A. Lysosomal storage diseases: from pathophysiology to therapy. Annu. Rev. Med. 66, 471–486 (2015).

    Article  CAS  Google Scholar 

  5. Plotegher, N. & Duchen, M. R. Crosstalk between lysosomes and mitochondria in Parkinson’s disease. Front. Cell Dev. Biol. 5, 110 (2017).

    Article  Google Scholar 

  6. Xu, H., Martinoia, E. & Szabo, I. Organellar channels and transporters. Cell Calcium 58, 1–10 (2015).

    Article  Google Scholar 

  7. Calcraft, P. J. et al. NAADP mobilizes calcium from acidic organelles through two-pore channels. Nature 459, 596–600 (2009).

    Article  CAS  Google Scholar 

  8. Huang, P. et al. P2X4 forms functional ATP-activated cation channels on lysosomal membranes regulated by luminal pH. J. Biol. Chem. 289, 17658–17667 (2014).

    Article  CAS  Google Scholar 

  9. Kiselyov, K. et al. TRPML: transporters of metals in lysosomes essential for cell survival? Cell Calcium 50, 288–294 (2011).

    Article  CAS  Google Scholar 

  10. Lloyd-Evans, E. On the move, lysosomal CAX drives Ca2+ transport and motility. J. Cell Biol. 212, 755–757 (2016).

    Article  CAS  Google Scholar 

  11. Melchionda, M., Pittman, J. K., Mayor, R. & Patel, S. Ca2+/H+ exchange by acidic organelles regulates cell migration in vivo. J. Cell Biol. 212, 803–813 (2016).

    Article  CAS  Google Scholar 

  12. Morgan, A. J., Davis, L. C. & Galione, A. Imaging approaches to measuring lysosomal calcium. Methods Cell Biol. 126, 159–195 (2015).

    Article  Google Scholar 

  13. Christensen, K. A., Myers, J. T. & Swanson, J. A. pH-dependent regulation of lysosomal calcium in macrophages. J. Cell Sci. 115, 599–607 (2002).

    CAS  PubMed  Google Scholar 

  14. Lloyd-Evans, E. et al. Niemann–Pick disease type C1 is a sphingosine storage disease that causes deregulation of lysosomal calcium. Nat. Med. 14, 1247–1255 (2008).

    Article  CAS  Google Scholar 

  15. Garrity, A. G. et al. The endoplasmic reticulum, not the pH gradient, drives calcium refilling of lysosomes. eLife 5, e15887 (2016).

    Article  Google Scholar 

  16. Sherwood, M. W. et al. Activation of trypsinogen in large endocytic vacuoles of pancreatic acinar cells. Proc. Natl Acad. Sci. USA 104, 5674–5679 (2007).

    Article  CAS  Google Scholar 

  17. Gerasimenko, J. V., Tepikin, A. V., Petersen, O. H. & Gerasimenko, O. V. Calcium uptake via endocytosis with rapid release from acidifying endosomes. Curr. Biol. 8, 1335–1338 (1998).

    Article  CAS  Google Scholar 

  18. Albrecht, T., Zhao, Y., Nguyen, T. H., Campbell, R. E. & Johnson, J. D. Fluorescent biosensors illuminate calcium levels within defined beta-cell endosome subpopulations. Cell Calcium 57, 263–274 (2015).

    Article  CAS  Google Scholar 

  19. Johnson, D. E., Ostrowski, P., Jaumouillé, V. & Grinstein, S. The position of lysosomes within the cell determines their luminal pH. J. Cell Biol. 212, 677–692 (2016).

    Article  CAS  Google Scholar 

  20. Chakraborty, K., Veetil, A. T., Jaffrey, S. R. & Krishnan, Y. Nucleic acid-based nanodevices in biological imaging. Annu. Rev. Biochem. 85, 349–373 (2016).

    Article  CAS  Google Scholar 

  21. Modi, S. et al. A DNA nanomachine that maps spatial and temporal pH changes inside living cells. Nat. Nanotechnol. 4, 325–330 (2009).

    Article  CAS  Google Scholar 

  22. Surana, S., Bhat, J. M., Koushika, S. P. & Krishnan, Y. An autonomous DNA nanomachine maps spatiotemporal pH changes in a multicellular living organism. Nat. Commun. 2, 340 (2011).

    Article  Google Scholar 

  23. Saha, S., Prakash, V., Halder, S., Chakraborty, K. & Krishnan, Y. A pH-independent DNA nanodevice for quantifying chloride transport in organelles of living cells. Nat. Nanotechnol. 10, 645–651 (2015).

    Article  CAS  Google Scholar 

  24. Modi, S., Nizak, C., Surana, S., Halder, S. & Krishnan, Y. Two DNA nanomachines map pH changes along intersecting endocytic pathways inside the same cell. Nat. Nanotechnol. 8, 459–467 (2013).

    Article  CAS  Google Scholar 

  25. Chakraborty, K., Leung, K. & Krishnan, Y. High lumenal chloride in the lysosome is critical for lysosome function. eLife 6, e28862 (2017).

    Article  Google Scholar 

  26. Toyoshima, C. & Inesi, G. Structural basis of ion pumping by Ca2+-ATPase of the sarcoplasmic reticulum. Annu. Rev. Biochem. 73, 269–292 (2004).

    Article  CAS  Google Scholar 

  27. Schmidt, K., Wolfe, D. M., Stiller, B. & Pearce, D. A. Cd2+, Mn2+, Ni2+ and Se2+ toxicity to Saccharomyces cerevisiae lacking YPK9p the orthologue of human ATP13A2. Biochem. Biophys. Res. Commun. 383, 198–202 (2009).

    Article  CAS  Google Scholar 

  28. Ramonet, D. et al. PARK9-associated ATP13A2 localizes to intracellular acidic vesicles and regulates cation homeostasis and neuronal integrity. Hum. Mol. Genet. 21, 1725–1743 (2012).

    Article  CAS  Google Scholar 

  29. Fares, H. & Greenwald, I. Regulation of endocytosis by CUP-5, the Caenorhabditis elegans mucolipin-1 homolog. Nat. Genet. 28, 64–68 (2001).

    CAS  PubMed  Google Scholar 

  30. Salgado, E. N., Garcia Rodriguez, B., Narayanaswamy, N., Krishnan, Y. & Harrison, S. C. Visualization of Ca2+ loss from rotavirus during cell entry. J. Virol. https://doi.org/10.1128/JVI.01327–18 (2018).

  31. Jewett, J. C., Sletten, E. M. & Bertozzi, C. R. Rapid Cu-free click chemistry with readily synthesized biarylazacyclooctynones. J. Am. Chem. Soc. 132, 3688–3690 (2010).

    Article  CAS  Google Scholar 

  32. Huotari, J. & Helenius, A. Endosome maturation. EMBO J. 30, 3481–3500 (2011).

    Article  CAS  Google Scholar 

  33. Hu, Y.-B., Dammer, E. B., Ren, R.-J. & Wang, G. The endosomal-lysosomal system: from acidification and cargo sorting to neurodegeneration. Transl. Neurodegener. 4, 18 (2015).

    Article  Google Scholar 

  34. Vandecaetsbeek, I., Vangheluwe, P., Raeymaekers, L., Wuytack, F. & Vanoevelen, J. The Ca2+ pumps of the endoplasmic reticulum and Golgi apparatus. Cold Spring Harb. Perspect. Biol. 3, a004184 (2011).

    Article  Google Scholar 

  35. Tharkeshwar, A. K. et al. A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics: the case of NPC1 deficiency. Sci. Rep. 7, 41408 (2017).

    Article  CAS  Google Scholar 

  36. Chapel, A. et al. An extended proteome map of the lysosomal membrane reveals novel potential transporters. Mol. Cell. Proteomics 12, 1572–1588 (2013).

    Article  CAS  Google Scholar 

  37. Lübke, T., Lobel, P. & Sleat, D. E. Proteomics of the lysosome. Biochim. Biophys. Acta 1793, 625–635 (2009).

    Article  Google Scholar 

  38. Brozzi, A., Urbanelli, L., Germain, P. L., Magini, A. & Emiliani, C. hLGDB: a database of human lysosomal genes and their regulation. Database (Oxford) 2013, bat024 (2013).

    Article  Google Scholar 

  39. Schröder, B. A., Wrocklage, C., Hasilik, A. & Saftig, P. The proteome of lysosomes. Proteomics 10, 4053–4076 (2010).

    Article  Google Scholar 

  40. Cao, Q., Yang, Y., Zhong, X. Z. & Dong, X.-P. The lysosomal Ca2+ release channel TRPML1 regulates lysosome size by activating calmodulin. J. Biol. Chem. 292, 8424–8435 (2017).

    Article  CAS  Google Scholar 

  41. Sahoo, N. et al. Gastric acid secretion from parietal cells is mediated by a Ca2+ efflux channel in the tubulovesicle. Dev. Cell 41, 262–273 (2017).

    Article  CAS  Google Scholar 

  42. Bargal, R. et al. Identification of the gene causing mucolipidosis type IV. Nat. Genet. 26, 118–123 (2000).

    Article  CAS  Google Scholar 

  43. Schaheen, L., Dang, H. & Fares, H. Basis of lethality in C. elegans lacking CUP-5, the mucolipidosis type IV orthologue. Dev. Biol. 293, 382–391 (2006).

    Article  Google Scholar 

  44. C. elegans Deletion Mutant Consortium. Large-scale screening for targeted knockouts in the Caenorhabditis elegans genome. G3 (Bethesda) 2, 1415–1425 (2012).

    Article  Google Scholar 

  45. Schaheen, L., Patton, G. & Fares, H. Suppression of the cup-5 mucolipidosis type IV-related lysosomal dysfunction by the inactivation of an ABC transporter in C. elegans. Development 133, 3939–3948 (2006).

    Article  CAS  Google Scholar 

  46. Fares, H. & Greenwald, I. Genetic analysis of endocytosis in Caenorhabditis elegans: coelomocyte uptake defective mutants. Genetics 159, 133–145 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. van Veen, S. et al. Cellular function and pathological role of ATP13A2 and related P-type transport ATPases in Parkinson’s disease and other neurological disorders. Front. Mol. Neurosci. 7, 48 (2014).

    PubMed  PubMed Central  Google Scholar 

  48. Schöndorf, D. C. et al. iPSC-derived neurons from GBA1-associated Parkinson’s disease patients show autophagic defects and impaired calcium homeostasis. Nat. Commun. 5, 4028 (2014).

    Article  Google Scholar 

  49. Usenovic, M., Tresse, E., Mazzulli, J. R., Taylor, J. P. & Krainc, D. Deficiency of ATP13A2 leads to lysosomal dysfunction, α-synuclein accumulation, and neurotoxicity. J. Neurosci. 32, 4240–4246 (2012).

    Article  CAS  Google Scholar 

  50. Bras, J., Verloes, A., Schneider, S. A., Mole, S. E. & Guerreiro, R. J. Mutation of the parkinsonism gene ATP13A2 causes neuronal ceroid-lipofuscinosis. Hum. Mol. Genet. 21, 2646–2650 (2012).

    Article  CAS  Google Scholar 

  51. Estrada-Cuzcano, A. et al. Loss-of-function mutations in the ATP13A2/PARK9 gene cause complicated hereditary spastic paraplegia (SPG78). Brain 140, 287–305 (2017).

    Article  Google Scholar 

  52. Grynkiewicz, G., Poenie, M. & Tsien, R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450 (1985).

    CAS  PubMed  Google Scholar 

  53. Collot, M. et al. CaRuby-Nano: a novel high affinity calcium probe for dual color imaging. eLife 4, e05808 (2015).

    Article  Google Scholar 

  54. Moore, D. & Dowhan, D. Purification and concentration of DNA from aqueous solutions. Curr. Protoc. Mol. Biol. 59, 2.1.1–2.1.10 (2002).

    Google Scholar 

  55. Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Kamath, R. S. & Ahringer, J. Genome-wide RNAi screening in Caenorhabditis elegans. Methods 30, 313–321 (2003).

    Article  CAS  Google Scholar 

  57. Evangelidis, G. D. & Psarakis, E. Z. Parametric image alignment using enhanced correlation coefficient maximization. IEEE Trans. Pattern Anal. Mach. Intell. 30, 1858–1865 (2008).

    Article  Google Scholar 

  58. Sauvola, J. & Pietikäinen, M. Adaptive document image binarization. Pattern Recognit. 33, 225–236 (2000).

    Article  Google Scholar 

  59. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  Google Scholar 

  60. Engelstein, M. et al. An efficient, automatable template preparation for high throughput sequencing. Microb. Comp. Genomics 3, 237–241 (1998).

    Article  CAS  Google Scholar 

  61. Vandeventer, P. E. et al. Multiphasic DNA adsorption to silica surfaces under varying buffer, pH, and ionic strength conditions. J. Phys. Chem. B 116, 5661–5670 (2012).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Kuriyan and M. Zajac for valuable comments. We thank the Integrated Light Microscopy facility at the University of Chicago, the Caenorhabditis Genetic Center for strains, and Ausubel Lab for Arhinger Library RNAi clones. We thank the Krainc lab at Northwestern University (Chicago, IL, USA) for the fibroblast cells harboring mutations in ATP13A2 (L6025). HDF cells were a kind gift from the lab of J. Rowley (University of Chicago, Chicago, IL, USA). This work was supported by the University of Chicago Women’s Board; a Pilot and Feasibility award from an NIDDK center grant no. P30DK42086 to the University of Chicago Digestive Diseases Research Core Center; MRSEC grant no. DMR-1420709; the National Center for Advancing Translational Sciences of the National Institutes of Health through grant no. 1UL1TR002389-01 that funds the Institute for Translational Medicine, Chicago Biomedical Consortium, with support from the Searle Funds at The Chicago Community Trust, C-084; and University of Chicago start-up funds to Y.K. Y.K. is a Brain Research Foundation Fellow.

Author information

Authors and Affiliations

Authors

Contributions

K.C. and Y.K. designed the project. N.N. synthesized and designed the calcium dye. N.N., K.C., A.S., E.Z., and K.L. performed experiments. J.D. provided key resources. N.N., K.C., A.S., and Y.K. analyzed the data. K.C. and Y.K wrote the paper. All authors discussed the results and gave inputs on the manuscript.

Corresponding authors

Correspondence to Kasturi Chakraborty or Yamuna Krishnan.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 In vitro characterization of Rhod-5F.

(a) Chemical structure of Rhod-5F. (b) Fluorescence emission spectra of Rhod-5F(green) and Alexa Fluor 647 (red) with increasing [Ca2+] upon exciting Rhod-5F and Alexa Fluor 647 at 560 nm and 650 nm, respectively. (c) Normalized O/R ratio of Rhod-5F/Alexa Fluor 647 with increasing [Ca2+] at pH 7.2 and 5.5. Error bar represents mean + s.e.m. of three independent experiments.

Supplementary Figure 2 Characterization of CalipHluorLy and CalipHluor.

(a) Gel showing the conjugation of Rhod-5F to D2-DBCO strand. Gels were visualized in EtBr and TMR channels. (b) Native PAGE showing formation of CalipHluorLy. Gels were visualized in Alexa Fluor 488, TMR and Alexa Fluor 647 channels. (c) Schematic of working principle of CalipHluor. pH-induced FRET changes between Alexa Fluor 488 (donor, green sphere) and Alexa Fluor 647 (acceptor, red star) is used to report pH ratiometrically. A Ca2+ sensitive fluorophore (Rhod-5F, yellow diamond) and Alexa Fluor 647 report Ca2+ (at a given pH) ratiometrically by direct excitation of each dye. (d) Gel showing the conjugation of Rhod-5F to O3-DBCO strand. Gels were visualized in EtBr and TMR channels. (e) Native PAGE showing formation of CalipHluor. Gels were visualized in Alexa Fluor 488, TMR and Alexa Fluor 647 channels. (f) Emission spectra of CalipHluor at pH values ranging from 7.5 to 5.0 upon excitation at 488 nm. (g) Normalized ratio of fluorescence intensity of donor to that of acceptor (D/A) of CalipHluor as a function of pH. (D λex = 495 nm, λem = 520 nm; A λex = 495 nm, λem = 665 nm). Gels were performed twice independently. Error bar represents mean + S.E.M of three independent experiments.

Supplementary Figure 3 In vivo performance of CalipHluorLy.

Representative pseudo color images of coelomocytes labeled with CalipHluorLy and clamped at the indicated (a) pH and (b) free [Ca2+] at pH 5.5. Scale bar 5 μm. Experiments were repeated three times independently with similar results.

Supplementary Figure 4 Comparison of in vitro and in vivo pH and Ca2+ calibration profile of CalipHluorLy.

(a-d) D/A ratios of CalipHluorLy as a function of pH clamped at different amounts of added [Ca2+]. (e-h) Normalized O/R ratios of CalipHluorLy as a function of free [Ca2+] clamped at different pH points. For in vivo n = 10 worms; 15 cells and 50 endosomes were quantified; in vitro n = 2. Error bar represents mean + s.e.m.

Supplementary Figure 5 Endocytic trafficking of CalipHluorA647 in coelomocytes.

(a-c) Representative confocal images taken 5 min, 17 min, and 60 min following injection of CalipHluorA647 in worms expressing GFP::RAB-5, GFP::RAB-7 and LMP-1::GFP. Scale bar 5 μm. Experiment was performed once. n = 10 worms.

Supplementary Figure 6 Lethality rescue and RNAi controls.

a) catp-6 rescues lethality of cup-5 +/−. Representative images showing the number of progeny of cup-5 +/− worms in plates containing RNAi bacteria of mrp-4 (positive control), clh-6, catp-6, catp-5 and e.v. (control). Experiments were repeated twice independently with similar results. b) RT-PCR analysis of total RNA isolated from C. elegans pre- and post-RNAi. Lanes correspond to PCR-amplified cDNA of the indicated gene product isolated from wild type without RNAi treatment (denoted by gene name) and the corresponding dsRNA-fed worms (denoted as. ‘ gene name) c) Representative images of worms expressing LMP-1::GFP (green) in the background of various indicated RNAi, which were injected with CalipHluorLyA647 (red) and imaged 60 mins post-injection. Scale bar: 5 μm. d) Quantification of colocalization between the CalipHluorLyA647 and GFP in LMP-1::GFP worms. n = 10 cells; error bars represent mean + s.e.m.

Supplementary Figure 7 Characterization of CalipHluormLy.

a) Schematic of the working principle of CalipHluormLy. An Oregon Green based pH sensor (green sphere), an ion insensitive Alexa Fluor 647 (red star) and a Ca2+ sensitive fluorophore (Rhod-5F, yellow diamond). Calibration curves comparing in vitro (red) and on beads (orange) calibration at; b) pH 4.6 and c) pH 5.1. Comparison of d) Kd and e) Fold change (FC) in O/R of CalipHluorLy (pink) and CalipHluormLy (black). f) Representative images Comparison of g) Fold change (FC) in O/R and h) Kd of CalipHluormLy in vitro (pink), on beads (orange) and in cellulo (gray). (n = 5 cells; 30 endosomes; n = 60 beads). Experiments were performed three times independently. *Error is obtained from Hill equation fit. Error bars represent mean + s.e.m. Scale bar: 10 µm.

Supplementary Figure 8 Uptake of CalipHluormLy in fibroblast cells.

a)-b) CalipHluormLy internalization by primary human skin fibroblasts is competed out by excess maleylated BSA (mBSA, 10 μM), revealing that uptake is mediated by scavenger receptors. Cells are imaged in Alexa Fluor 647 channel. AF: autofluorescence. Scale bar: 10 µm. Experiments were performed in triplicate. Error bars indicate the mean of three independent experiments +/− s.e.m. (n = 25 cells).

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1–8, Supplementary Note 1 and Supplementary Tables 1–3

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Narayanaswamy, N., Chakraborty, K., Saminathan, A. et al. A pH-correctable, DNA-based fluorescent reporter for organellar calcium. Nat Methods 16, 95–102 (2019). https://doi.org/10.1038/s41592-018-0232-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41592-018-0232-7

This article is cited by

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research