Critical role for isoprenoids in apicoplast biogenesis by malaria parasites

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1. 

   Version published to 10.7554/elife.73208 on eLife

   Mar 8, 2022
2. 

   eLife

   Feb 24, 2022

   Author Response:

   Reviewer #1:

   The experiments are well designed, generally well controlled, and carefully conducted, and are thoughtfully and appropriately discussed. The authors make conclusions that are well supported by their results.

   When describing the aptamer knockdown of the PPS, the authors explain that the western blot was too noisy for monitoring the knockdown, which is frustrating for the reader and must have been frustrating for the authors. The authors instead counter-intuitively use qRT-PCR to monitor the transcript abundance of the PPS transcript in the aptamer system - this aptamer system is thought to be a modifier of protein, not transcription or transcript abundance. The authors describe that this has been seen once before (using aptamer knockdown of PfFis1), and the authors of that study speculate that the …

   Author Response:

   Reviewer #1:

   The experiments are well designed, generally well controlled, and carefully conducted, and are thoughtfully and appropriately discussed. The authors make conclusions that are well supported by their results.

   When describing the aptamer knockdown of the PPS, the authors explain that the western blot was too noisy for monitoring the knockdown, which is frustrating for the reader and must have been frustrating for the authors. The authors instead counter-intuitively use qRT-PCR to monitor the transcript abundance of the PPS transcript in the aptamer system - this aptamer system is thought to be a modifier of protein, not transcription or transcript abundance. The authors describe that this has been seen once before (using aptamer knockdown of PfFis1), and the authors of that study speculate that the TetR-DOZI aptamer might be degrading the target mRNA. This is a plausible explanation, but it isn't quite clear from the description how this experiment was performed. The authors explain that the knockdown parasites grew normally for three days, but the parasites may be becoming sicker over this period. It's therefore possible that the decrease in PPS mRNA abundance is a product, rather than a cause of the growth defect. Sick or dying parasites could plausibly impact the PPS differently to the two chosen controls, particularly since both control genes chosen have substantially longer half-lives than the PPS mRNA (according to the Shock and DeRisi datasets). I therefore I suggest that this experiment be performed in an IPP rescue scenario (where the parasites aren't dying) with biological replicates. There is no explanation of the replicates here, but the error bars in 6C are implausibly small for real biological replicates.

   To address these concerns, we have added western blot data showing down-regulation of PPS expression in -aTc +IPP conditions, relative to a loading control. We have also repeated the growth assay and RT-qPCR experiment (in biological triplicate) under IPP-rescue conditions. Parasites samples harvested on day 3 of the IPP-rescue assay were analyzed by RT-qPCR and show reduced PPS mRNA abundance that is similar to (and slightly lower than) that observed without IPP supplementation. This similarity is not surprising to us, since the day 3 harvest in the original growth assay (without IPP) was 3 days before observing a parasite growth defect in -aTc conditions. With respect to the mechanism of transcript loss in the aptamer/TetR-DOZI system, the fate of transcripts in this system has not been investigated in depth. However, DOZI is believed to target bound mRNA to P-bodies, which are a known site of mRNA degradation in cells. We have unpublished data with multiple parasite proteins tagged with the aptamer/TetR-DOZI system. In all cases, we see strong reductions in mRNA abundance in -aTc conditions, suggesting that such decreases are a general property of this knockdown system.

   Line 342 "These results directly suggest that apicoplast biogenesis specifically requires synthesis of linear polyprenols containing three or more prenyl groups." - I think that this might be overinterpreting those results - there could be a number of different reasons why polyprenols of different sizes do or don't rescue, including different solubility, diffusion, availability of transporters, predisposition to break down to useable subunits. Perhaps this needs a caveat.

   We have modified the text here to remove “directly” and to acknowledge uncertainty in beta-carotene uptake: “Although it is possible that β-carotene is not taken up efficiently into the apicoplast, rescue by decaprenol, which is similar in size and hydrophobicity to β-carotene, suggests that apicoplast biogenesis specifically requires synthesis of linear polyprenols containing three or more prenyl groups.” We have also added the statement that “this hypothesis is further supported by additional results described in the next two sections”, referring to our identification of an apicoplast-targeted polyprenyl synthase.

   Line 361 " the cytosolic enzyme, PF3D7_1128400" - I don't think we know the localisation of this protein based on the published data. The Gabriel et al study makes it clear the protein isn't apicoplast or mitochondrial, but it is punctate at stages in a pattern that doesn't look to me to be a straightforward cytosolic localisation (and the original authors don't describe it as cytosolic).

   We agree that the localization of PF3D7_1128400 requires further investigation. The Gabriel study, which (surprisingly) is the only study we found that has examined localization of this protein by microscopy, observed diffuse signal in trophozoites consistent with cytoplasmic localization, in additional to focal, punctate signals in schizonts that were distinct from the apicoplast or mitochondrion. The authors described their results as, “Analysis by fluorescence microscopy of live parasites confirms expression along the intra-erythrocytic cycle and shows FPPS/GGPPS localization throughout the cytoplasm and also forming spots, which increase in number as parasites mature from trophozoite to schizont stages.” For simplicity we referred to FPPS/GGPPS localization as cytoplasmic but agree that available data suggest more a complex localization that requires further studies to understand. We have modified the text to indicate that available data suggests a complex cellular distribution that includes both the cytoplasm and additional sub-cellular foci outside the apicoplast and mitochondrion.

   Line 423 "with strong prediction of an apicoplast-targeting transit peptide but uncertainty in the presence of a signal peptide". I don't think this describes well the bioinformatic analysis of the N-terminus. Although the experimental data are convincing that this is an apicoplast-targeted protein, bioinformatically this would not be predicted as an apicoplast protein. There is no obvious signal peptide, and "uncertainty" is too vague a descriptor. None of the versions of signalP, nor psort, predict this as possessing a signal peptide (which by definition means that PlasmoAP absolutely rejects it), and there is no obvious hydrophobic segment at the N-terminus that we would normally expect of a signal peptide. The toxoplasma hyperlopit doesn't suggest that the Toxoplasma orthologue is apicoplast, and the protein isn't found in the Boucher et al apicoplast proteome. This is somewhat of a mystery. It doesn't diminish the solid localisation data, with the excellent complementary data from IFA as well as the doxycycline+IPP experiment, but it should be pointed out clearly that this localisation isn't to be expected from the sequence analysis.

   We thank the reviewer for this perspective and agree that SignalP is unable to identify a signal peptide at the N-terminus of PPS. We have modified the text to remove our description of “uncertainty” and explicitly state that SignalP is unable to identify a canonical signal peptide at the N-terminus of PPS.

   We note that multiple proteins detected in the Boucher et al. apicoplast proteome also lack an identifiable signal peptide by SignalP yet are clearly imported into the apicoplast. These proteins include the key MEP pathway enzymes DXR (PF3D7_1467300) and IspD (PF3D7_0106900), holo ACP synthase (PF3D7_0420200), FabB/F (PF3D7_0626300), and the E1 subunit of pyruvate dehydrogenase (PF3D7_1446400). Thus, apicoplast import despite lack of identifiable signal peptide by SignalP is not unique to PPS but general to multiple (if not numerous) apicoplast-targeted proteins. These observations suggest to us that protein N-termini in Plasmodium can have sequence properties compatible with ER targeting that are broader and more heterogenous than other eukaryotic organisms that comprise the training sets upon which SignalP is currently based. It remains a future challenge to fully understand these properties.

   With respect to the lack of PPS detection in the Boucher et al. apicoplast proteome, PPS appears to have a very low expression level and unusual solubility properties that require overnight extraction of parasite pellets in 2% SDS (or LDS) for detection. In our experience, the RIPA extraction conditions (which contained 0.1% SDS) used in the Boucher et al. study are insufficient to solubilize PPS, which may explain lack of PPS detection in their study.

   To explicitly address these questions regarding PPS targeting to the apicoplast, we have added a new section to the Discussion to explore PPS targeting in the absence of a recognizable signal peptide, its unusual solubility properties and lack of detection in the Boucher et al. proteome, and planned future studies to further test, refine, and understand targeting determinants.

   With respect to Toxoplasma, T. gondii appears to also express two polyprenyl synthase homologs, TGME49_224490 and TGME49_269430, that are ~30% identical (in homologous regions) to PF3D7_1128400 (FPPS/GGPPS) and PF3D7_0202700 (PPS), respectively. TGME49_224490 appears to be targeted to the mitochondrion in T. gondii (based on MitoProt and HyperLOPIT analysis), in contrast to its P. falciparum homolog, PF3D7_1128400, which localizes to the cytoplasm and other cellular foci outside the mitochondrion. TGME49_269430 does not appear to target the apicoplast in T. gondii (based on HyperLOPIT data), which contrasts with our determination of apicoplast targeting for the P. falciparum homolog, PF3D7_0202700. These differing localizations may suggest distinct cellular roles for these homologs in T. gondii compared to P. falciparum. We are also aware of a recent study (Pubmed 34896149) showing that loss of MEP pathway activity in T. gondii (due to loss of apicoplast ferredoxin) does not impact apicoplast biogenesis, in contrast to our observations in P. falciparum based on FOS treatment, DXS deletion, and PPS knockdown. These distinct phenotypes further suggest differences in isoprenoid utilization and metabolism between T. gondii and P. falciparum that remain to be understood. We have added a new section to the Discussion to address these considerations.

   The section after line 344 "Iterative condensation of DMAPP with IP…", up until line 377 doesn't sit well within the section that has the heading "Apicoplast biogenesis requires polyprenyl isoprenoid synthesis". I suggest either creating a separate subheading for this material, or moving it into the start of the subsequent section "Localization of an annotated polyprenyl synthase to the apicoplast.".

   We thank the reviewer for this suggestion, which we have followed. We have moved the referenced text to the beginning of the subsequent section to better align the text with that section heading.

   Reviewer #2:

   Minor comments:

   The authors emphasize that this study reveals a previously unnoted interconnection between apicoplast maintenance and pathways that produce an output from the apicoplast to serve the cell. But is the prevailing view really that these two are separate? Isn't the interconnection already clear from many other studies and observations? E.g., the fatty acids produced inside the apicoplast provide membrane- and lipid- precursors for the rest of the cell as well as for the apicoplast itself (Botte et al., PNAS, 2013) (although not essential in Plasmodium blood stages). Other pathways that function inside the apicoplast such as the Fe-S cluster synthesis are critical to support enzymes that provide exported metabolites (e.g., IPP synthesis, IspG/H) and function in maintenance (e.g., MiaB) (Gisselberg et al., PLoSPath, 2013). Perhaps the authors could tone this conclusion down and acknowledge that maintenance and output are interconnected in other cases, which have been acknowledged in the literature.

   We thank the reviewer for this perspective and agree that in Toxoplasma as well as in mosquito- and liver-stage Plasmodium there are multiple apicoplast outputs (i.e., metabolic products exported from the apicoplast) that contribute to parasite fitness, including IPP, fatty acids, and coproporphyrinogen III. To clarify, we are specifically referring to blood-stage Plasmodium in our manuscript, when heme and fatty acid synthesis are dispensable and where the prior literature has intensely focused on IPP as the key essential output of the blood-stage apicoplast and consistently stated that IPP is not required for organelle maintenance.

   We agree that prior work has firmly established that apicoplast housekeeping functions (e.g., synthesis of proteins and Fe-S clusters) are required for organelle maintenance and to support IPP synthesis. However, our work is the first to demonstrate in blood-stage Plasmodium that the reverse is also true- that IPP as an essential apicoplast output is also required for organelle maintenance and that apicoplast maintenance and IPP synthesis are thus reciprocally dependent. We have modified the Discussion section to clarify these points and to explicitly acknowledge that apicoplast maintenance and other metabolic outputs may also be interdependent in Toxoplasma and other Plasmodium stages.

   Could the authors elaborate more on the leader sequence predicting apicoplast localization for the PPS characterized here and discuss why it might have been missed in previous detailed study of apicoplast localised proteins (Boucher et al., PlosBiol, 2018)?

   Please see our response above to Reviewer #1.

   Could the authors discuss conservation of the PPS gene(s) in other Apicomplexa with (e.g., T. gondii) and without (e.g., Cryptosporidium spp.) an apicoplast? This could be relevant for other people in the field and could give further insights into the enzyme's role in apicoplast maintenance.

   Please see our response above to Reviewer #1. Polyprenyl synthases are diverse enzymes that perform a variety of cellular functions, whose specific roles can differ between organisms. Although the two Plasmodium prenyl synthases show preferential homology with each of two different prenyl synthase homologs in Toxoplasma and Cryptosporidium (CPATCC_003578 and CPATCC_001801), the differing localizations of these homologs in each parasite suggest differing cellular roles. The differing dependence of apicoplast biogenesis on MEP pathway activity in T. gondii and P. falciparum and the absence of an apicoplast in Cryptosporidium further support differences in isoprenoid utilization and metabolism in these organisms. We have added a new section to the Discussion to address these considerations.

   Reviewer #3:

   The paper is very nicely written and was a true pleasure to read. The introduction is concise yet dense with all relevant background of our current understanding of functioning of the apicoplast in relation to IPP production and utilization. The rational of the experiments and the interpretation of the results are presented clearly and everything is discussed well in the context of the current understanding of the field. The main conclusion of the paper that isoprenoid is not solely essential for critical functions elsewhere in the cell, such as prenylation-dependent vesicular trafficking but also for apicoplast biogenesis via its processing by an essential polyprenyl synthase conserved with plants and bacteria is well substantiated and very exciting. The authors demonstrate an equally beautiful and clever use of available and newly generated genetic mutants in combination with complementary pharmacological interventions and metabolic supplementation. There are no true major weaknesses that could jeopardize the conclusions or change the interpretation of the results. However, the authors do consistently perform statistical analyses on data obtained from individual cells obtained in no more than two independent experiments, which in my humble opinion does not qualify for statistical analysis. That said, the results are so clear-cut that no statistics are required to convince me, or to quote Ernest Rutherford: '"If your experiment needs statistics, you ought to have done a better experiment."

   We thank the reviewer for these positive comments and suggestions. For growth assays, we have performed a third biological replicate and updated those figures and the indicated statistical analyses. For microscopy experiments, we have removed p values.

   Read the original source
3. 

   eLife

   Feb 24, 2022

   Evaluation Summary:

   This is an excellent, innovative and high quality study that reveals an essential role for isoprenoids within the Plasmodium apicoplast, and demonstrates a likely polyprenol synthase that is required for apicoplast biogenesis. This is an important finding for understanding apicoplast and isoprenoid biology in general, and is significant because synthesis of isoprenoids appears to be the only essential role for the apicoplast in asexual intraerythrocytic stages.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

   Read the original source
4. 

   eLife

   Feb 24, 2022

   Reviewer #1 (Public Review):

   The experiments are well designed, generally well controlled, and carefully conducted, and are thoughtfully and appropriately discussed. The authors make conclusions that are well supported by their results.

   When describing the aptamer knockdown of the PPS, the authors explain that the western blot was too noisy for monitoring the knockdown, which is frustrating for the reader and must have been frustrating for the authors. The authors instead counter-intuitively use qRT-PCR to monitor the transcript abundance of the PPS transcript in the aptamer system - this aptamer system is thought to be a modifier of protein, not transcription or transcript abundance. The authors describe that this has been seen once before (using aptamer knockdown of PfFis1), and the authors of that study speculate that the TetR-DOZI …

   Reviewer #1 (Public Review):

   The experiments are well designed, generally well controlled, and carefully conducted, and are thoughtfully and appropriately discussed. The authors make conclusions that are well supported by their results.

   When describing the aptamer knockdown of the PPS, the authors explain that the western blot was too noisy for monitoring the knockdown, which is frustrating for the reader and must have been frustrating for the authors. The authors instead counter-intuitively use qRT-PCR to monitor the transcript abundance of the PPS transcript in the aptamer system - this aptamer system is thought to be a modifier of protein, not transcription or transcript abundance. The authors describe that this has been seen once before (using aptamer knockdown of PfFis1), and the authors of that study speculate that the TetR-DOZI aptamer might be degrading the target mRNA. This is a plausible explanation, but it isn't quite clear from the description how this experiment was performed. The authors explain that the knockdown parasites grew normally for three days, but the parasites may be becoming sicker over this period. It's therefore possible that the decrease in PPS mRNA abundance is a product, rather than a cause of the growth defect. Sick or dying parasites could plausibly impact the PPS differently to the two chosen controls, particularly since both control genes chosen have substantially longer half-lives than the PPS mRNA (according to the Shock and DeRisi datasets). I therefore I suggest that this experiment be performed in an IPP rescue scenario (where the parasites aren't dying) with biological replicates. There is no explanation of the replicates here, but the error bars in 6C are implausibly small for real biological replicates.

   Line 342 "These results directly suggest that apicoplast biogenesis specifically requires synthesis of linear polyprenols containing three or more prenyl groups." - I think that this might be overinterpreting those results - there could be a number of different reasons why polyprenols of different sizes do or don't rescue, including different solubility, diffusion, availability of transporters, predisposition to break down to useable subunits. Perhaps this needs a caveat.

   Line 361 " the cytosolic enzyme, PF3D7_1128400" - I don't think we know the localisation of this protein based on the published data. The Gabriel et al study makes it clear the protein isn't apicoplast or mitochondrial, but it is punctate at stages in a pattern that doesn't look to me to be a straightforward cytosolic localisation (and the original authors don't describe it as cytosolic).

   Line 423 "with strong prediction of an apicoplast-targeting transit peptide but uncertainty in the presence of a signal peptide". I don't think this describes well the bioinformatic analysis of the N-terminus. Although the experimental data are convincing that this is an apicoplast-targeted protein, bioinformatically this would not be predicted as an apicoplast protein. There is no obvious signal peptide, and "uncertainty" is too vague a descriptor. None of the versions of signalP, nor psort, predict this as possessing a signal peptide (which by definition means that PlasmoAP absolutely rejects it), and there is no obvious hydrophobic segment at the N-terminus that we would normally expect of a signal peptide. The toxoplasma hyperlopit doesn't suggest that the Toxoplasma orthologue is apicoplast, and the protein isn't found in the Boucher et al apicoplast proteome. This is somewhat of a mystery. It doesn't diminish the solid localisation data, with the excellent complementary data from IFA as well as the doxycycline+IPP experiment, but it should be pointed out clearly that this localisation isn't to be expected from the sequence analysis.

   The section after line 344 "Iterative condensation of DMAPP with IP...", up until line 377 doesn't sit well within the section that has the heading "Apicoplast biogenesis requires polyprenyl isoprenoid synthesis". I suggest either creating a separate subheading for this material, or moving it into the start of the subsequent section "Localization of an annotated polyprenyl synthase to the apicoplast.".

   Read the original source
5. 

   eLife

   Feb 24, 2022

   Reviewer #2 (Public Review):

   Okada et al., investigated the role of isoprenoids for the Plasmodium falciparum apicoplast (morphology, elongation, inheritance of the organelle). Most known functions of isoprenoids (ubiquinone-, heme A- and dolichol-synthesis as well as protein prenylation) are found outside of the apicoplast. To investigate a putative role of isoprenoids for the apicoplast itself, the authors elegantly use drug treatments at specific time points throughout the intraerythrocytic life cycle combined with rescue through metabolite supplementation. A role of isoprenoids for the apicoplast has previously been suggested but had not been investigated in this detail. By generating a MiaA-KO line, which presents no defect in intraerythrocyctic Plasmodium, the authors demonstrate convincingly that isoprenoids must have other …

   Reviewer #2 (Public Review):

   Okada et al., investigated the role of isoprenoids for the Plasmodium falciparum apicoplast (morphology, elongation, inheritance of the organelle). Most known functions of isoprenoids (ubiquinone-, heme A- and dolichol-synthesis as well as protein prenylation) are found outside of the apicoplast. To investigate a putative role of isoprenoids for the apicoplast itself, the authors elegantly use drug treatments at specific time points throughout the intraerythrocytic life cycle combined with rescue through metabolite supplementation. A role of isoprenoids for the apicoplast has previously been suggested but had not been investigated in this detail. By generating a MiaA-KO line, which presents no defect in intraerythrocyctic Plasmodium, the authors demonstrate convincingly that isoprenoids must have other critical functions within the apicoplast, besides tRNA prenylation, until this study the only known function for the apicoplast. In their search to identify the essential role of isoprenoids for the apicoplast, the authors investigate the role of a putative second polyprenyl synthase (PPS), besides a known enzyme inside the cytosol, which is expected to synthesize the longer chain isoprenoids, that contribute to the aforementioned roles of isoprenoids outside the apicoplast. The authors demonstrate that this enzyme is found inside the apicoplast (based on co-localization and 'fragmented localization' during apicoplast disruption). An inducible knock-down using the aptamer/TetR-DOZI system, uncovers that the PPS is required for apicoplast elongation and inheritance and apicoplast structure can only be rescued through supplementation with the very long chain isoprenoid, decaprenol (C50). Finally, the authors perform mass spectrometry combined with stable isotope labelling, to demonstrate that the phytoene, β-carotene is not synthesised by the PPS, but may originate from the serum. Thus, the newly identified apicoplast PPS likely acts inside the apicoplast where it synthesises very long chain isoprenoids which are critical for the apicoplast itself. What function they play remains unclear, but the authors speculate that the isoprenoids contribute to the membrane structure of the organelle.

   The conclusions of this paper are well supported by the data presented. The data is outlined and structured in a very clear manner. The paper reports numerous findings which greatly enhance our understanding of apicoplast biology. The study could have been further improved through a biochemical characterization of the apicoplast PPS or through metabolomic analyses of the ko-strain, but these are beyond the scope of this study.

   Minor comments:

   The authors emphasize that this study reveals a previously unnoted interconnection between apicoplast maintenance and pathways that produce an output from the apicoplast to serve the cell. But is the prevailing view really that these two are separate? Isn't the interconnection already clear from many other studies and observations? E.g., the fatty acids produced inside the apicoplast provide membrane- and lipid- precursors for the rest of the cell as well as for the apicoplast itself (Botte et al., PNAS, 2013) (although not essential in Plasmodium blood stages). Other pathways that function inside the apicoplast such as the Fe-S cluster synthesis are critical to support enzymes that provide exported metabolites (e.g., IPP synthesis, IspG/H) and function in maintenance (e.g., MiaB) (Gisselberg et al., PLoSPath, 2013). Perhaps the authors could tone this conclusion down and acknowledge that maintenance and output are interconnected in other cases, which have been acknowledged in the literature.

   Could the authors elaborate more on the leader sequence predicting apicoplast localization for the PPS characterized here and discuss why it might have been missed in previous detailed study of apicoplast localised proteins (Boucher et al., PlosBiol, 2018)?

   Could the authors discuss conservation of the PPS gene(s) in other Apicomplexa with (e.g., T. gondii) and without (e.g., Cryptosporidium spp.) an apicoplast? This could be relevant for other people in the field and could give further insights into the enzyme's role in apicoplast maintenance.

   Read the original source
6. 

   eLife

   Feb 24, 2022

   Reviewer #3 (Public Review):

   The paper is very nicely written and was a true pleasure to read. The introduction is concise yet dense with all relevant background of our current understanding of functioning of the apicoplast in relation to IPP production and utilization. The rational of the experiments and the interpretation of the results are presented clearly and everything is discussed well in the context of the current understanding of the field. The main conclusion of the paper that isoprenoid is not solely essential for critical functions elsewhere in the cell, such as prenylation-dependent vesicular trafficking but also for apicoplast biogenesis via its processing by an essential polyprenyl synthase conserved with plants and bacteria is well substantiated and very exciting. The authors demonstrate an equally beautiful and clever …

   Reviewer #3 (Public Review):

   The paper is very nicely written and was a true pleasure to read. The introduction is concise yet dense with all relevant background of our current understanding of functioning of the apicoplast in relation to IPP production and utilization. The rational of the experiments and the interpretation of the results are presented clearly and everything is discussed well in the context of the current understanding of the field. The main conclusion of the paper that isoprenoid is not solely essential for critical functions elsewhere in the cell, such as prenylation-dependent vesicular trafficking but also for apicoplast biogenesis via its processing by an essential polyprenyl synthase conserved with plants and bacteria is well substantiated and very exciting. The authors demonstrate an equally beautiful and clever use of available and newly generated genetic mutants in combination with complementary pharmacological interventions and metabolic supplementation. There are no true major weaknesses that could jeopardize the conclusions or change the interpretation of the results. However, the authors do consistently perform statistical analyses on data obtained from individual cells obtained in no more than two independent experiments, which in my humble opinion does not qualify for statistical analysis. That said, the results are so clear-cut that no statistics are required to convince me, or to quote Ernest Rutherford: '"If your experiment needs statistics, you ought to have done a better experiment."

   Read the original source
7. 

   Version published to 10.1101/2021.08.19.456988v1 on bioRxiv

   Aug 19, 2021