Signup can happen via Mobile Number or Email or Google Oauth. Can we clarify and reword the section accordingly?

Can we hide this section for US?

この辺りは同じファイルを更新している？ので、ファイル名もcaptionに含めると、同いファイルを変更していることが伝わりやすいかなと思いました。 （これ以前のコードも同様に）

作成されるファイルはpythonファイルですが、「sample-script.py」としなくてよいです？ ドキュメントでは、.pyをつけているようでした。 https://docs.astral.sh/uv/guides/scripts/#running-a-script-with-dependencies

が

の方がいいかなー

uvx とバッククォートで囲みたい。以下同様

外部パッケージによって提供されるツール

とか?

.pyはつけない派?

PEP 723で提案されたけどPyPA関連はのちのち変わっていくので、仕様としてはこちらを参照するのが適切です。

(PEPドキュメントの先頭にもそう書いてある)

https://packaging.python.org/en/latest/specifications/inline-script-metadata/#inline-script-metadata

外部

この手順よりは、新規にディレクトリ作成、uv init実行した後に、pyproject.toml, uv.lockだけコピーしてuv sync実行するみたいな手順がいいのではと思った。

実際のユースケースに合わせる

uv pip installした場合はuv addと違ってpyproject.tomlとかuv.lockが更新されないということを書いた方がいいかなぁ

名を

pyproject.tomlの設定を見て、今のuv.lockファイルに存在するパッケージのままか新しいバージョンが存在するかを調べる。

みたいな意味合いだと思います。

あと、これってどっちかというと hoge>4.0.0 とかかいていて、hogeの最新バージョンがでているか(pip list -O)みたいな使い方がメインかなと思ったんですが、そうではない?

pyproject.tomlを書き換えたのはここで例として示しているために必要なだけど、本来はpyproject.toml書き換えたらuv syncの方を使うかなと思ったので(uv 素人なのではずしてたらすいません)

uv.lockファイル

トルでもいいかな(あってもいいけど)

uv lock

uv.lockファイル

uv.lockファイル

ハッシュの例はハッシュ値を省略しつつ1行くらいは載せて欲しいかな

てインストールします。 pyproject.tomlにはdevというセクション(?)が追加され~~

みたいな文章がほしい。

このときもpyproject.tomlの中は書き換えられるよ(ですよね?)とか説明があっていいと思う

依存関係というよりも、「さきほど追加したパッケージが」とかいう説明でよいのではないか

pipコマンドと同様に、とかあってもいいかも

PyPIからダウンロードしてインストールされていることとか触れて欲しい。

依存関係を管理するってちょっとイメージしにくいかなと思いました。

プロジェクトで使用するライブラリを管理する、とか?

コマンド実行後

一文が長いので「利用できます。」で一度切ってもいいかも

なにももう一つの方法なのか

仮想環境下のPythonを実行するもう一つの~

とか?

に

の方がいいかな

さっきとは異なるディレクトリに作ったと言うことですかね。手順で mkdir uv-example がないのでちょっと混乱するかも

では

◯◯な場合に仮想環境を再作成します。

みたいな用途にも触れてくれるとうれしいなと思った。

長いし、トルでも意味は通じるかなと

確認するメッセージが表示されます。

コマンドを

で

でよさそう

仮想環境を有効化した場合は、プロンプトにプロジェクト(ディレクトリ名)が表示されることにも言及して欲しい。

venvは作成したvenv名がプロンプトで表示されていたので

ここで段落を変えてもいいかな

typo: いません

手順にlsを入れるといいかなと。 venvの方にはあった

Similiar to the text, that the second group of aliens are finding the first group, and going to be doing bad things to them. In this article, they are facing millitary forces, same as aliens.

Les grandes maisons d'édition classiques ne sont plus les seules à pouvoir légitimer la publication d'un texte. On voit déjà des livres publiés sans eux. Cela change-t-il quelque chose pour l'auteur et son statut ?

it´s not the flags subpage

it´s not the flags subpage

místě.

it´s not "Stánek", but "Reklamní promo stolek" - corrections needed in all headlines

eLife Assessment

This work significantly advances our understanding of chromatin organization within regions of repetitive sequences in the parasitic protozoan Trypanosoma brucei. Using cutting edge interdisciplinary tools, the authors provide compelling evidence for two discrete types of repetitive DNA element-associated proteins- one set involved in essential centromere function; and, the other involved in glycoprotein antigenic variation via homologous recombination. Thus, these fundamental findings have implications for this parasite's biology, and for therapeutic targeting in kinetoplastid diseases. This work will be exciting to those in the centromere/mitosis and parasite immunity fields.

Reviewer #1 (Public review):

Summary:

Carloni et al. comprehensively analyze which proteins bind repetitive genomic elements in Trypanosoma brucei. For this, they perform mass spectrometry on custom-designed, tagged programmable DNA-binding proteins. After extensively verifying their programmable DNA-binding proteins (using bioinformatic analysis to infer target sites, microscopy to measure localization, ChIP-seq to identify binding sites), they present, among others, two major findings: 1) 14 of the 25 known T. brucei kinetochore proteins are enriched at 177bp repeats. As T. brucei's 177bp repeat-containing intermediate-sized and mini-chromosomes lack centromere repeats but are stable over mitosis, Carloni et al. use their data to hypothesize that a 'rudimentary' kinetochore assembles at the 177bp repeats of these chromosomes to segregate them. 2) 70bp repeats are enriched with the Replication Protein A complex, which, notably, is required for homologous recombination. Homologous recombination is the pathway used for recombination-based antigenic variation of the 70bp-repeat-adjacent variant surface glycoproteins.

Strengths and Weaknesses:

The manuscript was previously reviewed through Review Commons. As noted there, the experiments are well controlled, the claims are well supported, and the methods are clearly described. The conclusions are convincing. All concerns I raised have been addressed except one (minor point #8):

"The way the authors mapped the ChIP-seq data is potentially problematic when analyzing the same repeat type in different genomic regions. Reads with multiple equally good mapping positions were assigned randomly. This is fine when analyzing repeats by type, independent of genomic position, which is what the authors do to reach their main conclusions. However, several figures (Fig. 3B, Fig. 4B, Fig. 5B, Fig. 7) show the same repeat type at specific genomic locations." Due to the random assignment, all of these regions merely show the average signal for the given repeat. I find it misleading that this average is plotted out at "specific" genomic regions.<br /> Initially, I suggested a workaround, but the authors clarified why the workaround was not feasible, and their explanation is reasonable to me. That said, the figures still show a signal at positions where they can't be sure it actually exists. If this cannot be corrected analytically, it should at least be noted in the figure legends, Results, or Discussion.

Importantly, the authors' conclusions do not hinge on this point; they are appropriately cautious, and their interpretations remain valid regardless.

Significance:

This work is of high significance for chromosome/centromere biology, parasitology, and the study of antigenic variation. For chromosome/centromere biology, the conceptual advancement of different types of kinetochores for different chromosomes is a novelty, as far as I know. It would certainly be interesting to apply this study as a technical blueprint for other organisms with mini-chromosomes or chromosomes without known centromeric repeats. I can imagine a broad range of labs studying other organisms with comparable chromosomes to take note of and build on this study. For parasitology and the study of antigenic variation, it is crucial to know how intermediate- and mini-chromosomes are stable through cell division, as these chromosomes harbor a large portion of the antigenic repertoire. Moreover, this study also found a novel link between the homologous repair pathway and variant surface glycoproteins, via the 70bp repeats. How and at which stages during the process, 70bp repeats are involved in antigenic variation is an unresolved, and very actively studied, question in the field. Of course, apart from the basic biological research audience, insights into antigenic variation always have the potential for clinical implications, as T. brucei causes sleeping sickness in humans and nagana in cattle. Due to antigenic variation, T. brucei infections can be chronic.

Comments on revised version:

All my recommendations have been addressed.

Reviewer #2 (Public review):

The Trypanosoma brucei genome, like that of other eukaryotes, contains diverse repetitive elements. Yet, the chromatin-associated proteome of these regions remains largely unexplored. This study represents a very important conceptual and technical advancement by employing synthetic TALE DNA-binding proteins fused to YFP to selectively capture proteins associated with specific repetitive sequences in T. brucei chromatin. The data presented here are convincing, supported by appropriate controls and a well-validated methodology, aligned with current state-of-the-art approaches.

The authors used synthetic TALE DNA binding proteins, tagged with YFP, which were designed to target five specific repeat elements in T. brucei genome, including centromere and telomeres-associated repeats and those of a transposon element. This is in order to identify specific proteins that bind to these repetitive sequences in T. brucei chromatin. Validation of the approach was done using a TALE protein designed to target the telomere repeat (TelR-TALE) that detected many of the proteins that were previously implicated with telomeric functions. A TALE protein designed to target the 70 bp repeats that reside adjacent to the VSG genes (70R-TALE) detected proteins that function in DNA repair and a protein designed to target the 177 bp repeat arrays (177R-TALE) identified kinetochore proteins associated T. brucei mega base chromosomes, as well as in intermediate and mini-chromosomes, which imply that kinetochore assembly and segregation mechanisms are similar in all T. brucei chromosomes.

This study represents a significant conceptual and technical advancement. To the best of our knowledge, it is the first report of employing TALE-YFP for affinity-based detection of protein complexes bound to repetitive genomic sequences in T. brucei. This approach enhances our understanding the organization in these important regions of the trypanosomal chromatin and provides the foundation for investigating the functional roles of associated proteins in parasite biology. These findings will be of particular interest to researchers studying the molecular biology of kinetoplastid parasites and other unicellular organisms, as well as to scientists investigating the roles of repetitive genomic elements in chromatin structure and their functional role in higher eukaryotes.

Importantly, any essential or unique interacting partners identified using the approach employed here, could serve as a potential target for therapeutic intervention in severe tropical diseases cause by kinetoplastids.

use the banner "Nejsi si čímkoliv jistí" on all further web positions

use the banner "Grafický projekt zdarma on all further positions on the web*

what´s the meaning of this text?

Wonder if this needs four cols to align with the other four-col gif pages

This is white, but on the homepage we use coloured text

eLife Assessment

This important study presents an impressive large-scale effort to assess the reproducibility of published findings in the field of Drosophila immunity. The authors analyse 400 papers published between 1959 and 2011, and assess how many of the claims in these papers have been tested in subsequent publications. In a companion article they report the results of experiments to test a subset of the claims that, according to the literature, have not been tested. The present article also explores if various factors related to authors, institutions and journals influence reproducibility in this field. The evidence supporting the claims is solid, but there is considerable scope for strengthening and extending the analysis. The limitations inherent to evaluating reproducibility based on the published literature should also be acknowledged.

Reviewer #1 (Public review):

Summary:

The authors set out on the ambitious task of establishing the reproducibility of claims from the Drosophila immunity literature. Starting out from a corpus of 400 articles from 1959 and 2011, the authors sought to determine whether their claims were confirmed or contradicted by previous or subsequent publications. Additionally, they actively sought to replicate a subset of the claims for which no previous replications were available (although this set was not representative of the whole sample, as the authors focused on suspicious and/or easily testable claims). The focus of the article is on inferential reproducibility; thus, methods don't necessarily map exactly to the original ones.

The authors present a large-scale analysis of the individual replication findings, which are presented in a companion article (Westlake et al., 2025. DOI 10.1101/2025.07.07.663442). In their retrospective analysis of reproducibility, the authors find that 61% of the original claims were verified by the literature, 7.5% were partialy verified, and only 6.8% were challenged, with 23.8% having no replication available. This is in stark contrast with the result of their prospective replications, in which only 16% of claims were successfully reproduced.

The authors proceed to investigate correlates of replicability, with the most consistent finding being that findings stemming from higher-ranked universities (and possibly from very high impact journals) were more likely to be challenged.

Strengths:

(1) The work presents a large-scale, in-depth analysis of a particular field of science that includes authors with deep domain expertise of the field. This is a rare endeavour to establish the reproducibility of a particular subfield of science, and I'd argue that we need many more of these in different areas.

(2) The project was built on a collaborative basis (https://ReproSci.epfl.ch/), using an online database (https://ReproSci.epfl.ch/), which was used to organize the annotations and comments of the community about the claims. The website remains online and can be a valuable resource to the Drosophila immunity community.

(3) Data and code are shared in the authors' GitHub repository, with a Jupyter notebook available to reproduce the results.

Main concerns:

(1) Although the authors claim that "Drosophila immunity claims are mostly replicable", this conclusion is strictly based on the retrospective analysis - in which around 84% of the claims for which a published verification attempt was found. This is in very stark contrast with the findings that the authors replicate prospectively, of which only 16% are verified.

Although this large discrepancy may be explained by the fact that the authors focused on unchallenged and suspicious claims (which seems to be their preferred explanation), an alternative hypothesis is that there is a large amount of confirmation bias in the Drosophila immunity literature, either because attempts to replicate previous findings tend to reach similar results due to researcher bias, or because results that validate previous findings are more likely to be published.

Both explanations are plausible (and, not being an expert in the field, I'd have a hard time estimating their relative probability), and in the absence of prospective replication of a systematic sample of claims - which could determine whether the replication rate for a random sample of claims is as high as that observed in the literature -, both should be considered in the manuscript.

(2) The fact that the analysis of factors correlating with reproducibility includes both prospective and retrospective replications also leads to the possibility of confusion bias in this analysis. If most of the challenged claims come from the authors' prospective replications, while most of the verified ones come from those that were replicated by the literature, it becomes unclear whether the identified factors are correlated with actual reproducibility of the claims or with the likelihood that a given claim will be tested by other authors and that this replication will be published.

(3) The methods are very brief for a project of this size, and many of the aspects in determining whether claims were conceptually replicated and how replications were set up are missing.

Some of these - such as the PubMed search string for the publications and a better description of the annotation process - are described in the companion article, but this could be more explicitly stated. Others, however, remain obscure. Statements such as "Claims were cross-checked with evidence from previous, contemporary and subsequent publications and assigned a verification category" summarize a very complex process for which more detail should be given - in particular because what constitutes inferential reproducibility is not a self-evident concept. And although I appreciate that what constitutes a replication is ultimately a case-by-case decision, a general description of the guidelines used by the authors to determine this should be provided. As these processes were done by one author and reviewed by another, it would also be useful to know the agreement rates between them to have a general sense of how reproducible the annotation process might be.

The same gap in methods descriptions holds for the prospective replications. How were labs selected, how were experimental protocols developed, and how was the validity of the experiments as a conceptual replication assessed? I understand that providing the methods for each individual replication is beyond the scope of the article, but a general description of how they were developed would be important.

(4) As far as I could tell, the large-scale analysis of the replication results was not preregistered, and many decisions seem somewhat ad hoc. In particular, the categorization of journals (e.g. low impact, high impact, "trophy") and universities (e.g. top 50, 51-100, 101+) relies on arbitrary thresholds, and it is unclear how much the results are dependent on these decisions, as no sensitivity analyses are provided.

Particularly, for analyses that correlate reproducibility with continuous variable (such as year of publication, impact factor or university ranking, I'd strongly favor using these variables as continuous variables in the analysis (e.g. using logistic regression) rather than performing pairwise comparisons between categories determined by arbitrary cutoffs. This would not only reduce the impact of arbitrary thresholds in the analysis, but would also increase statistical power in the univariate analyses (as the whole sample can be used in at once) and reduce the number of parameters in the multivariate model (as they will be included as a single variable rather than multiple dummy variables when there are more than two categories).

(5) The multivariate model used to investigate predictors of replicability includes unchallenged claims along with verified ones in the outcome, which seems like an odd decision. If the intention is to analyze which factors are correlated with reproducibility, it would make more sense to remove the unchallenged findings, as these are likely uninformative in this sense. In fact, based on the authors' own replications of unchallenged findings, they may be more likely to belong the "challenged" category than to the "unchallenged" one if they were to be verified.

Reviewer #2 (Public review):

Summary:

Lemaitre et al. conducted an analysis of 400 publications in the Drosophila immunity field (1959-2011), performing both univariable and multivariable analyses to identify factors that correlate with or influence the irreproducibility of scientific claims. Some of the findings are unexpected, for instance, neither the career stage of the PI nor that of the first author appears to matter that much, while others, such as the influence of institutional prestige or publication in "trophy journals," are more predictable. The results provide valuable insight into patterns of irreproducibility in academia and may help inform policies to improve research reproducibility in the field.

Strengths:

This study is based on a large, manually curated dataset, complemented by a companion paper (Westlake et al., 2025. DOI 10.1101/2025.07.07.663442) that provides additional details on experimentally documented cases. The statistical methods are appropriate, and the findings are both important and informative. The results are clearly presented and supported by accessible documentation through the ReproSci project.

Weaknesses:

The analysis is limited to a specific field (immunity) and model system (Drosophila). Since biological context may influence reproducibility -- for example, depending on whether mechanisms are more hardwired or variable -- and the model system itself may contribute to these effects (as the authors note), it remains unclear to what extent these findings generalize to other fields or organisms. The authors could expand the discussion to address the potential scope and limitations of the study's generalizability.

Reviewer #3 (Public review):

Summary:

The authors of this paper were trying to identify how reproducible, or not, their subfield (Drosophilia immunity) was since its inception over 50 years ago. This required identifying not only the papers, but the specific claims made in the paper, assessing if these claims were followed up in the literature, and if so whether the subsequent papers supported or refuted the original claim. In addition to this large manually curated effort, the authors further investigated some claims that were left unchallenged in the literature by conducting replications themselves. This provided a rich corpus of the subfield that could be investigated into what characteristics influence reproducibility.

Strengths:

A major strength of this study is the focus on a subfield, the detailing of identifying the main, major, and minor claims - which is a very challenging manual task - and then cataloging not only their assessment of if these claims were followed up in the literature, but also what characteristics might be contributing to reproducibility, which also included more manual effort to supplement the data that they were able to extract from the published papers. While this provides a rich dataset for analysis, there is a major weakness with this approach, which is not unique to this study.

Weaknesses:

The main weakness is relying heavily on the published literature as the source for if a claim was determined to be verified or not. There are many documented issues with this stemming from every field of research - such as publication bias, selective reporting, all the way to fraud. It's understandable why the authors took this approach - it is the only way to get at a breadth of the literature - however the flaw with this approach is it takes the literature as a solid ground truth, which it is not. At the same time, it is not reasonable to expect the authors to have conducted independent replications for all of the 400 papers they identified. However, there is a big difference trying to assess the reproducibility of the literature by using the literature as the 'ground truth' vs doing this independently like other large-scale replication projects have attempted to do. This means the interpretation of the data is a bit challenging.

Below are suggestions for the authors and readers to consider:

(1) I understand why the authors prefer to mention claims as their primary means of reporting what they found, but it is nested within paper, and that makes it very hard to understand how to interpret these results at times. I also cannot understand at the high-level the relationship between claims and papers. The methods suggest there are 3-4 major claims per paper, but at 400 papers and 1,006 claims, this averages to ~2.5 claims per paper. Can the authors consider describing this relationship better (e.g., distribution of claims and papers) and/or considering presenting the data two ways (primary figures as claims and complimentary supplementary figures with papers as the unit). This will help the reader interpret the data both ways without confusion. I am also curious how the results look when presented both ways (e.g., does shifting to the paper as the unit of analysis shift the figures and interpretation?). This is especially true since the first and last author analysis shows there is varying distribution of papers and claims by authors (and thus the relationship between these is important for the reader).

(2) As mentioned above, I think the biggest weakness is that the authors are taking the literature at face value when assigning if a claim was validated or challenged vs gathering new independent evidence. This means the paper leans more on papers, making it more like a citation analysis vs an independent effort like other large-scale replication projects. I highly recommend the authors state this in their limitations section.

On top of that, I have questions that I could not figure out (though I acknowledge I did not dig super deep into the data to try). The main comment I have is How was verified (and challenged) determined? It seems from the methods it was determined by "Claims were cross-checked with evidence from previous, contemporary and subsequent publications and assigned a verification category". If this is true, and all claims were done this way - are verified claims double counted then? (e.g., an original claim is found by a future claim to be verified - and thus that future claim is also considered to be verified because of the original claim).

Related, did the authors look at the strength of validation or challenged claims? That is, if there is a relationship mapping the authors did for original claims and follow-up claims, I would imagine some claims have deeper (i.e., more) claims that followed up on them vs others. This might be interested to look at as well.

(3) I recommend the authors add sample sizes when not present (e.g., Fig 4C). I also find that the sample sizes are a bit confusing, and I recommend the authors check them and add more explanation when not complete, like they did for Fig 4A. For example, Fig 7B equals to 178 labs (how did more than 156 labs get determined here?), and yet the total number of claims is 996 (opposed to 1,006). Another example, is why does Fig 8B not have all 156 labs accounted for? (related to Fig 8B, I caution on reporting a p value and drawing strong conclusions from this very small sample size - 22 authors). As a last example, Fig 8C has al 156 labs and 1,006 claims - is that expected? I guess it means authors who published before 1995 (as shown in Figure 8A continued to publish after 1995?) in that case, it's all authors? But the text says when they 'set up their lab' after 1995, but how can that be?

(4) Finally, I think it would help if the authors expanded on the limitations generally and potential alternative explanations and/or driving factors. For example, the line "though likely underestimated' is indicated in the discussion about the low rate of challenged claims, it might be useful to call out how publication bias is likely the driver here and thus it needs to be carefully considered in the interpretation of this. Related, I caution the authors on overinterpreting their suggestive evidence. The abstract for example, states claims of what was found in their analysis, when these are suggestive at best, which the authors acknowledge in the paper. But since most people start with the abstract, I worry this is indicating stronger evidence than what the authors actually have.

The authors should be applauded for the monumental effort they put into this project, which does a wonderful job of having experts within a subfield engage their community to understand the connectiveness of the literature and attempt to understand how reliable specific results are and what factors might contribute to them. This project provides a nice blueprint for others to build from as well as leverage the data generated from this subfield, and thus should have an impact in the broader discussion on reproducibility and reliability of research evidence.

does this need copyright or TMs?

use the banner "Grafický návrh zdarma" on all further pages

*the same correction as on previous pages *

use the banner "Grafický návrh zdarma" on all further pages

Naše stany Max v akci: + rename all these headlines on all further pages

estetickou

Stany

maybe a manual line break so this is on one line?

Explore doesn't have a chevron, but on book an assessment the back button does.

needs space

The book an assessment page and book a workshop. seem to have a different spacing/gaps.

eLife Assessment

This study introduces an important approach using selection linked integration (SLI) to generate Plasmodium falciparum lines expressing single, specific surface adhesins PfEMP1 variants, enabling precise study of PfEMP1 trafficking, receptor binding, and cytoadhesion. By moving the system to different parasite strains and introducing an advanced SLI2 system for additional genomic edits, this work provides compelling evidence for an innovative and rigorous platform to explore PfEMP1 biology and identify novel proteins essential for malaria pathogenesis including immune evasion.

Reviewer #1 (Public review):

One of the roadblocks in PfEMP1 research has been the challenges in manipulating var genes to incorporate markers to allow the transport of this protein to be tracked and to investigate the interactions taking place within the infected erythrocyte. In addition, the ability of Plasmodium falciparum to switch to different PfEMP1 variants during in vitro culture has complicated studies due to parasite populations drifting from the original (manipulated) var gene expression. Cronshagen et al have provided a useful system with which they demonstrate the ability to integrate a selectable drug marker into several different var genes that allows the PfEMP1 variant expression to be 'fixed'. This on its own represents a useful addition to the molecular toolbox and the range of var genes that have been modified suggests that the system will have broad application. As well as incorporating a selectable marker, the authors have also used selective linked integration (SLI) to introduce markers to track the transport of PfEMP1, investigate the route of transport and probe interactions with PfEMP1 proteins in the infected host cell.

One of the major strengths of this paper is that the authors have not only put together a robust system for further functional studies, but they have used it to produce a range of interesting findings including:

Co-activation of rif and var genes when in a head-to-head orientation.

The reduced control of expression of var genes in the 3D7-MEED parasite line.

More support for the PTEX transport route for PfEMP1.<br /> Identification of new proteins involved in PfEMP1 interactions in the infected erythrocyte, including some required for cytoadherence.

In most cases the experimental evidence is straightforward, and the data support the conclusions strongly. The authors have been very careful in the depth of their investigation, and where unexpected results have been obtained, they have looked carefully at why these have occurred.

A weakness of the paper is, as mentioned above, that the results are sometimes not as clear as might have been expected, for example, in the requirement for panning modified parasites to produce binding to EPCR. Where this has happened, the authors take a robust and thoughtful approach, and acknowledge that (as in most research) there are more questions to address. Being able to select specific var gene switches using drug markers will provide some useful starting points to understand how switching happens in P. falciparum. However, our trypanosome colleagues might remind us that forcing switches may show us some mechanisms, but perhaps not all.

Despite these sometimes complicated findings, the authors have achieved their aim as stated in the title of the paper, and in doing so have provided an excellent resource to themselves and other researchers in the field to answer some important questions.

Overall, the authors have produced a useful and robust system to support functional studies on PfEMP1, which provides a platform for future studies manipulating the domain content in var genes. They have used this system to produce a range of interesting findings and to support its use by the research community.

Comments on revisions:

I have no further recommendations for changes by the authors. They have addressed my concerns, and the paper reads very well.

Reviewer #2 (Public review):

Summary

Croshagen et al develop a range of tools based on selection-linked integration (SLI) to study PfEMP1 function in P. falciparum. PfEMP1 is encoded by a family of ~60 var genes subject to mutually exclusive expression. Switching expression between different family members can modify the binding properties of the infected erythrocyte while avoiding the adaptive immune response. Although critical to parasite survival and Malaria disease pathology, PfEMP1 proteins are difficult to study owing to their large size and variable expression between parasites within the same population. The SLI approach previously developed by this group for genetic modification of P. falciparum is employed here to selectively and stably activate expression of target var genes at the population level. Using this strategy, the binding properties of specific PfEMP1 variants were measured for several distinct var genes with a novel semi-automated pipeline to increase throughput and reduce bias. Activation of similar var genes in both the common lab strain 3D7 and the cytoadhesion competent FCR3/IT4 strain revealed higher binding for several PfEMP1 IT4 variants with distinct receptors, indicating this strain provides a superior background for studying PfEMP1 binding. SLI also enables modifications to target var gene products to study PfEMP1 trafficking and identify interacting partners by proximity-labeling proteomics, revealing two novel exported proteins required for cytoadherence. Overall, the data demonstrate a range of SLI-based approaches for studying PfEMP1 that will be broadly useful for understanding the basis for cytoadhesion and parasite virulence.

Comments:

While the capability of SLI to active selected var gene expression was initially reported by Omelianczyk et al., the present study greatly expands the utility of this approach. Several distinct var genes are activated in two different P. falciparum strains and shown to modify the binding properties of infected RBCs to distinct endothelial receptors; development of SLI2 enables multiple SLI modifications in the same parasite line; SLI is used to modify target var genes to study PfEMP1 trafficking and determine PfEMP1 interactomes with BioID. Along the way, the authors also demonstrate a new selection marker for P. falciparum transfection (a mutant FNT lactate transporter that provides resistance to the compound BH267.meta). Curiously, Omelianczyk et al activated a single var (Pf3D7_0421300) and observed elevated expression of an adjacent var arranged in a head to tail manner, possibly resulting from local chromatin modifications enabling expression of the neighboring gene. In contrast, the present study observed activation of neighboring genes with head to head but not head to tail arrangement, which may be the result of shared promoter regions. The reason for these differing results is unclear although it should be noted that the two studies examined different var loci.

The IT4var19 panned line that became binding-competent showed increased expression of both paralogs of ptp3 (as well as a phista and gbp), suggesting that overexpression of PTP3 may improve PfEMP1 display and binding. Interestingly, IT4 appears to be the only known P. falciparum strain (only available in PlasmoDB) that encodes more than one ptp3 gene (PfIT_140083100 and PfIT_140084700). PfIT_140084700 is almost identical to the 3D7 PTP3 (except for a ~120 residue insertion in 3D7 beginning at residue 400). In contrast, while the C-terminal region of PfIT_140083100 shows near perfect conservation with 3D7 PTP3 beginning at residue 450, the N-terminal regions between the PEXEL and residue 450 are quite different. This may indicate the generally stronger receptor binding observed in IT4 relative to 3D7 results from increased PTP3 activity due to multiple isoforms or that specialized trafficking machinery exists for some PfEMP1 proteins.

Revisions:

The authors thoughtfully addressed all the reviewer comments.

Reviewer #3 (Public review):

Summary:

The submission from Cronshagen and colleagues describes the application of a previously described method (selection linked integration) to the systematic study of PfEMP1 trafficking in the human malaria parasite Plasmodium falciparum. PfEMP1 is the primary virulence factor and surface antigen of infected red blood cells and is therefore a major focus of research into malaria pathogenesis. Since the discovery of the var gene family that encodes PfEMP1 in the late 1990s, there have been multiple hypotheses for how the protein is trafficked to the infected cell surface, crossing multiple membranes along the way. One difficulty in studying this process is the large size of the var gene family and the propensity of the parasites to switch which var gene is expressed, thus preventing straightforward gene modification-based strategies for tagging the expressed PfEMP1. Here the authors solve this problem by forcing expression of a targeted var gene by fusing the PfEMP1 coding region with a drug selectable marker separated by a skip peptide. This enabled them to generate relatively homogenous populations of parasites all expressing tagged (or otherwise modified) forms of PfEMP1 suitable for study. They then applied this method to study various aspects of PfEMP1 trafficking.

Strengths:

The study is very thorough, and the data are well presented. The authors used SLI to target multiple var genes, thus demonstrating the robustness of their strategy. They then perform experiments to investigate possible trafficking through PTEX, they knockout proteins thought to be involved in PfEMP1 trafficking and observe defects in cytoadherence, and they perform proximity labeling to further identify proteins potentially involved in PfEMP1 export. These are independent and complimentary approaches that together tell a very compelling story.

Weaknesses:

(1) When the authors targeted IT4var19, they were successful in transcriptionally activating the gene, however they did not initially obtain cytoadherent parasites. To observe binding to ICAM-1 and EPCR, they had to perform selection using panning. This is an interesting observation and potentially provides insights into PfEMP1 surface display, folding, etc. However, it also raises questions about other instances in which cytoadherence was not observed. Would panning of these other lines have successfully selected for cytoadherent infected cells? Did the authors attempt panning of their 3D7 lines? Given that these parasites do export PfEMP1 to the infected cell surface (Figure 1D), it is possible that panning would similarly rescue binding. Likewise, the authors knocked out PTP1, TryThrA and EMPIC3 and detected a loss of cytoadhesion, but they did not attempt panning to see if this could rescue binding. The strong selection that panning exerts on parasite populations could result in selection of compensatory changes that enable cytoadherence, which could be very informative, although the analysis could potentially be quite complicated and beyond the scope of the current paper. Nonetheless, these are important concepts to consider when assessing these phenotypes.

(2) The authors perform a series of trafficking experiments to help discern whether PfEMP1 is trafficked through PTEX. While the results were not entirely definitive, they make a strong case for PTEX in PfEMP1 export. The authors then used BioID to obtain a proxiome for PfEMP1 and identified proteins they suggest are involved in PfEMP1 trafficking. However, it seemed that components of PTEX were missing from the list of interacting proteins. Is this surprising and does this observation shed any additional light on the possibility of PfEMP1 trafficking through PTEX? This warrants a comment or discussion.

Comments on revisions:

The authors have responded thoroughly and constructively to suggestions and comments in the initial review. I have no additional comments. This is a great contribution to the literature.

Author response:

The following is the authors’ response to the original reviews.

eLife Assessment:

This study introduces an important approach using selection linked integration (SLI) to generate Plasmodium falciparum lines expressing single, specific surface adhesins PfEMP1 variants, enabling precise study of PfEMP1 trafficking, receptor binding, and cytoadhesion. By moving the system to different parasite strains and introducing an advanced SLI2 system for additional genomic edits, this work provides compelling evidence for an innovative and rigorous platform to explore PfEMP1 biology and identify novel proteins essential for malaria pathogenesis including immune evasion.

Reviewer #1 (Public review):

One of the roadblocks in PfEMP1 research has been the challenges in manipulating var genes to incorporate markers to allow the transport of this protein to be tracked and to investigate the interactions taking place within the infected erythrocyte. In addition, the ability of Plasmodium falciparum to switch to different PfEMP1 variants during in vitro culture has complicated studies due to parasite populations drifting from the original (manipulated) var gene expression. Cronshagen et al have provided a useful system with which they demonstrate the ability to integrate a selectable drug marker into several different var genes that allows the PfEMP1 variant expression to be 'fixed'. This on its own represents a useful addition to the molecular toolbox and the range of var genes that have been modified suggests that the system will have broad application. As well as incorporating a selectable marker, the authors have also used selective linked integration (SLI) to introduce markers to track the transport of PfEMP1, investigate the route of transport, and probe interactions with PfEMP1 proteins in the infected host cell.

What I particularly like about this paper is that the authors have not only put together what appears to be a largely robust system for further functional studies, but they have used it to produce a range of interesting findings including:

Co-activation of rif and var genes when in a head-to-head orientation.

The reduced control of expression of var genes in the 3D7-MEED parasite line.

More support for the PTEX transport route for PfEMP1.

Identification of new proteins involved in PfEMP1 interactions in the infected erythrocyte, including some required for cytoadherence.

In most cases the experimental evidence is straightforward, and the data support the conclusions strongly. The authors have been very careful in the depth of their investigation, and where unexpected results have been obtained, they have looked carefully at why these have occurred.

We thank the reviewer for the kind assessment and the comments to improve the paper.

(1) In terms of incorporating a drug marker to drive mono-variant expression, the authors show that they can manipulate a range of var genes in two parasite lines (3D7 and IT4), producing around 90% expression of the targeted PfEMP1. Removal of drug selection produces the expected 'drift' in variant types being expressed. The exceptions to this are the 3D7-MEED line, which looks to be an interesting starting point to understand why this variant appears to have impaired mutually exclusive var gene expression and the EPCR-binding IT4var19 line. This latter finding was unexpected and the modified construct required several rounds of panning to produce parasites expressing the targeted PfEMP1 and bind to EPCR. The authors identified a PTP3 deficiency as the cause of the lack of PfEMP1 expression, which is an interesting finding in itself but potentially worrying for future studies. What was not clear was whether the selected IT4var19 line retained specific PfEMP1 expression once receptor panning was removed.

We do not have systematic long-term data for the Var19 line but do have medium-term data. After panning the Var19 line, the binding assays were done within 3 months without additional panning. The first binding assay was 2 months after the panning and the last binding assays three weeks later, totaling about 3 months without panning. While there is inherent variation in these assays that precludes detection of smaller changes, the last assay showed the highest level of binding, giving no indication for rapid loss of the binding phenotype. Hence, we can say that the binding phenotype appears to be stable for many weeks without panning the cells again and there was no indication for a rapid loss of binding in these parasites.

Systematic long-term experiments to assess how long the Var19 parasites retain binding would be interesting, but given that the binding-phenotype appears to remain stable over many weeks or even months, this would only make sense if done over a much longer time frame. Such data might arise if the line is used over extended times for a specific project in which case it might be advisable to monitor continued binding. We included a statement in the discussion that the binding phenotype was stable over many weeks but that if long-term work with this line is planned, monitoring the binding phenotype might be advisable: “In the course of this work the binding phenotype of the IT4var19 expressor line remained stable over many weeks without further panning. However, given that initial panning had been needed for this particular line, it might be advisable for future studies to monitor the binding phenotype if the line is used for experiments requiring extended periods of cultivation.”

(2) The transport studies using the mDHFR constructs were quite complicated to understand but were explained very clearly in the text with good logical reasoning.

We are aware of this being a complex issue and are glad this was nevertheless understandable.

(3) By introducing a second SLI system, the authors have been able to alter other genes thought to be involved in PfEMP1 biology, particularly transport. An example of this is the inactivation of PTP1, which causes a loss of binding to CD36 and ICAM-1. It would have been helpful to have more insight into the interpretation of the IFAs as the anti-SBP1 staining in Figure 5D (PTP-TGD) looks similar to that shown in Figure 1C, which has PTP intact. The anti-EXP2 results are clearly different.

We realize the description of the PTP1-TGD IFA data and that of the other TGDs (see also response to Recommendation to authors point 4 and reviewer 2, major points 6 and 7) was rather cursory. The previously reported PTP1 phenotype is a fragmentation of the Maurer’s clefts into what in IFA appear to be many smaller pieces (Rug et al 2014, referenced in the manuscript). The control in Fig. 5D has 13 Maurer’s cleft spots (previous work indicates an average of ~15 MC per parasite, see e.g. the originally co-submitted eLife preprint doi.org/10.7554/eLife.103633.1 and references therein). The control mentioned by the reviewer in Fig. 1C has about 22 Maurer’s clefts foci, at the upper end of the typical range, but not unusual. In contrast, the PTP1-TGD in Fig. 5D, has more than 30 foci with an additional cytoplasmic pool and additional smaller, difficult to count foci. This is consistent with the published phenotype in Rug et al 2014. The EXP1 stained cell has more than 40 Maurer’s cleft foci, again beyond what typically is observed in controls. Therefore, these cells show a difference to the control in Fig. 5 but also to Fig. 1C. Please note that we are looking at two different strains, in Fig. 1 it is 3D7 and in Fig. 5 IT4. While we did not systematically assess this, the Maurer’s clefts number per cell seemed to be largely comparable between these strains (Fig. 10C and D in the other eLife preprint doi.org/10.7554/eLife.103633.1). 

Overall, as the PTP1 loss phenotype has already been reported, we did not go into more experimental detail. However, we now modified the text to more clearly describe how the phenotype in the PTP1-TGD parasites was different to control: “IFAs showed that in the PTP1-TGD parasites, SBP1 and PfEMP1 were found in many small foci in the host cell that exceeded the average number of ~ 15 Maurer’s clefts typically found per infected RBC [66] (Fig. 5D). This phenotype resembled the previously reported Maurer’s clefts phenotype of the PTP1 knock out in CS2 parasites [39].”

(4) It is good to see the validation of PfEMP1 expression includes binding to several relevant receptors. The data presented use CHO-GFP as a negative control, which is relevant, but it would have been good to also see the use of receptor mAbs to indicate specific adhesion patterns. The CHO system if fine for expression validation studies, but due to the high levels of receptor expression on these cells, moving to the use of microvascular endothelial cells would be advisable. This may explain the unexpected ICAM-1 binding seen with the panned IT4var19 line.

We agree with the reviewer that it is desirable to have better binding systems for studying individual binding interactions. As the main purpose of this paper was to introduce the system and provide proof of principle that the cells show binding, we did not move to more complicated binding systems. However, we would like to point out that the CSA binding was done on receptor alone in addition to the CSA-expressing HBEC-5i cells and was competed successfully with soluble CSA. In addition, apart from the additional ICAM1-binding of the Var19 line, all binding phenotypes were conform with expectations. We therefore hope the tools used for binding studies are acceptable at this stage of introducing the system while future work interested in specific PfEMP1 receptor interactions may use better systems, tailored to the specific question (e.g. endothelial organoid models and engineered human capillaries and inhibitory antibodies or relevant recombinant domains for competition).

(5) The proxiome work is very interesting and has identified new leads for proteins interacting with PfEMP1, as well as suggesting that KAHRP is not one of these. The reduced expression seen with BirA* in position 3 is a little concerning but there appears to be sufficient expression to allow interactions to be identified with this construct. The quantitative impact of reduced expression for proxiome experiments will clearly require further work to define it.

This is a valid point. Clearly there seems to be some impact on binding when BirA* is placed in the extracellular domain (either through reduced presentation or direct reduction of binding efficiency of the modified PfEMP1; please see also minor comment 10 reviewer 2). The exact quantitative impact on the proxiome is difficult to assess but we note that the relative enrichment of hits to each other is rather similar to the other two positions (Fig. 6H-J). We therefore believe the BioIDs with the 3 PfEMP1-BirA* constructs are sufficient to provide a general coverage of proteins proximal to PfEMP1 and hope this will aid in the identification of further proteins involved in PfEMP1 transport and surface display as illustrated with two of the hits targeted here.

The impact of placing a domain on the extracellular region of PfEMP1 will have to be further evaluated if needed in other studies. But the finding that a large folded domain can be placed into this part at all, even if binding was reduced, in our opinion is a success (it was not foreseeable whether any such change would be tolerated at all).

(6) The reduced receptor binding results from the TryThrA and EMPIC3 knockouts were very interesting, particularly as both still display PfEMP1 on the surface of the infected erythrocyte. While care needs to be taken in cross-referencing adhesion work in P. berghei and whether the machinery truly is functionally orthologous, it is a fair point to make in the discussion. The suggestion that interacting proteins may influence the "correct presentation of PfEMP1" is intriguing and I look forward to further work on this.

We hope future work will be able to shed light on this.

Overall, the authors have produced a useful and reasonably robust system to support functional studies on PfEMP1, which may provide a platform for future studies manipulating the domain content in the exon 1 portion of var genes. They have used this system to produce a range of interesting findings and to support its use by the research community. Finally, a small concern. Being able to select specific var gene switches using drug markers could provide some useful starting points to understand how switching happens in P. falciparum. However, our trypanosome colleagues might remind us that forcing switches may show us some mechanisms but perhaps not all.

Point noted! From non-systematic data with the Var01 line that has been cultured for extended periods of time (several years), it seems other non-targeted vars remain silent in our SLI “activation” lines but how much SLI-based var-expression “fixing” tampers with the integrity of natural switching mechanisms is indeed very difficult to gage at this stage. We now added a statement to the discussion that even if mutually exclusive expression is maintained, it is not certain the mechanisms controlling var expression all remain intact: “However, it should be noted that it is not known whether all mechanisms controlling mutually exclusive expression and switching remain intact in parasites with SLI-activated var genes.”

Reviewer #2 (Public review):

Summary

Croshagen et al develop a range of tools based on selection-linked integration (SLI) to study PfEMP1 function in P. falciparum. PfEMP1 is encoded by a family of ~60 var genes subject to mutually exclusive expression. Switching expression between different family members can modify the binding properties of the infected erythrocyte while avoiding the adaptive immune response. Although critical to parasite survival and Malaria disease pathology, PfEMP1 proteins are difficult to study owing to their large size and variable expression between parasites within the same population. The SLI approach previously developed by this group for genetic modification of P. falciparum is employed here to selectively and stably activate the expression of target var genes at the population level. Using this strategy, the binding properties of specific PfEMP1 variants were measured for several distinct var genes with a novel semi-automated pipeline to increase throughput and reduce bias. Activation of similar var genes in both the common lab strain 3D7 and the cytoadhesion competent FCR3/IT4 strain revealed higher binding for several PfEMP1 IT4 variants with distinct receptors, indicating this strain provides a superior background for studying PfEMP1 binding. SLI also enables modifications to target var gene products to study PfEMP1 trafficking and identify interacting partners by proximity-labeling proteomics, revealing two novel exported proteins required for cytoadherence. Overall, the data demonstrate a range of SLI-based approaches for studying PfEMP1 that will be broadly useful for understanding the basis for cytoadhesion and parasite virulence.

We thank the reviewer for the kind assessment and the comments to improve the paper.

Comments

(1) While the capability of SLI to actively select var gene expression was initially reported by Omelianczyk et al., the present study greatly expands the utility of this approach. Several distinct var genes are activated in two different P. falciparum strains and shown to modify the binding properties of infected RBCs to distinct endothelial receptors; development of SLI2 enables multiple SLI modifications in the same parasite line; SLI is used to modify target var genes to study PfEMP1 trafficking and determine PfEMP1 interactomes with BioID. Curiously, Omelianczyk et al activated a single var (Pf3D7_0421300) and observed elevated expression of an adjacent var arranged in a head-to-tail manner, possibly resulting from local chromatin modifications enabling expression of the neighboring gene. In contrast, the present study observed activation of neighboring genes with head-to-head but not head-totail arrangement, which may be the result of shared promoter regions. The reason for these differing results is unclear although it should be noted that the two studies examined different var loci.

The point that we are looking at different loci is very valid and we realize this is not mentioned in the discussion. We now added to the discussion that it is unclear if our results and those cited may be generalized and that different var gene loci may respond differently

“However, it is unclear if this can be generalized and it is possible that different var loci respond differently.”

(2) The IT4var19 panned line that became binding-competent showed increased expression of both paralogs of ptp3 (as well as a phista and gbp), suggesting that overexpression of PTP3 may improve PfEMP1 display and binding. Interestingly, IT4 appears to be the only known P. falciparum strain (only available in PlasmoDB) that encodes more than one ptp3 gene (PfIT_140083100 and PfIT_140084700). PfIT_140084700 is almost identical to the 3D7 PTP3 (except for a ~120 residue insertion in 3D7 beginning at residue 400). In contrast, while the C-terminal region of PfIT_140083100 shows near-perfect conservation with 3D7 PTP3 beginning at residue 450, the N-terminal regions between the PEXEL and residue 450 are quite different. This may indicate the generally stronger receptor binding observed in IT4 relative to 3D7 results from increased PTP3 activity due to multiple isoforms or that specialized trafficking machinery exists for some PfEMP1 proteins.

We thank the reviewer for pointing this out, the exact differences between the two PTP3s of IT4 and that of other strains definitely should be closely examined if the function of these proteins in PfEMP1 binding is analysed in more detail. 

It is an interesting idea that the PTP3 duplication could be a reason for the superior binding of IT4. We always assumed that IT4 had better binding because it was less culture adapted but this does not preclude that PTP3(s) is(are) a reason for this. However, at least in our 3D7 PTP3 can’t be the reason for the poor binding, as our 3D7 still has PfEMP1 on the surface while in the unpanned IT4-Var19 line and in the Maier et al., Cell 2008 ptp3 KO (PMID: 18614010)) PfEMP1 is not on the surface anymore. 

Testing the impact of having two PTP3s would be interesting, but given the “mosaic” similarity of the two PTP3s isoforms, a simple add-on experiment might not be informative. Nevertheless, it will be interesting in future work to explore this in more detail.

Reviewer #3 (Public review):

Summary:

The submission from Cronshagen and colleagues describes the application of a previously described method (selection linked integration) to the systematic study of PfEMP1 trafficking in the human malaria parasite Plasmodium falciparum. PfEMP1 is the primary virulence factor and surface antigen of infected red blood cells and is therefore a major focus of research into malaria pathogenesis. Since the discovery of the var gene family that encodes PfEMP1 in the late 1990s, there have been multiple hypotheses for how the protein is trafficked to the infected cell surface, crossing multiple membranes along the way. One difficulty in studying this process is the large size of the var gene family and the propensity of the parasites to switch which var gene is expressed, thus preventing straightforward gene modification-based strategies for tagging the expressed PfEMP1. Here the authors solve this problem by forcing the expression of a targeted var gene by fusing the PfEMP1 coding region with a drug-selectable marker separated by a skip peptide. This enabled them to generate relatively homogenous populations of parasites all expressing tagged (or otherwise modified) forms of PfEMP1 suitable for study. They then applied this method to study various aspects of PfEMP1 trafficking.

Strengths:

The study is very thorough, and the data are well presented. The authors used SLI to target multiple var genes, thus demonstrating the robustness of their strategy. They then perform experiments to investigate possible trafficking through PTEX, they knock out proteins thought to be involved in PfEMP1 trafficking and observe defects in cytoadherence, and they perform proximity labeling to further identify proteins potentially involved in PfEMP1 export. These are independent and complimentary approaches that together tell a very compelling story.

We thank the reviewer for the kind assessment and the comments to improve the paper.

Weaknesses:

(1)  When the authors targeted IT4var19, they were successful in transcriptionally activating the gene, however, they did not initially obtain cytoadherent parasites. To observe binding to ICAM-1 and EPCR, they had to perform selection using panning. This is an interesting observation and potentially provides insights into PfEMP1 surface display, folding, etc. However, it also raises questions about other instances in which cytoadherence was not observed. Would panning of these other lines have been successfully selected for cytoadherent infected cells? Did the authors attempt panning of their 3D7 lines? Given that these parasites do export PfEMP1 to the infected cell surface (Figure 1D), it is possible that panning would similarly rescue binding. Likewise, the authors knocked out PTP1, TryThrA, and EMPIC3 and detected a loss of cytoadhesion, but they did not attempt panning to see if this could rescue binding. To ensure that the lack of cytoadhesion in these cases is not serendipitous (as it was when they activated IT4var19), they should demonstrate that panning cannot rescue binding.

These are very important considerations. Indeed, we had repeatedly attempted to pan 3D7 when we failed to get the SLI-generated 3D7 PfEMP1 expressor lines to bind, but this had not been successful. The lack of binding had been a major obstacle that had held up the project and was only solved when we moved to IT4 which readily bound (apart from Var19 which was created later in the project). After that we made no further efforts to understand why 3D7 does not bind but the fact that PfEMP1 is on the surface indicates this is not a PTP3 issue because loss of PTP3 also leads to loss of PfEMP1 surface display. Also, as the parent 3D7 could not be panned, we assumed this issue is not easily fixed in the SLI var lines we made in 3D7.

Panning the TGD lines: we see the reasoning for conducting panning experiments with the TGD lines. However, on second thought, we are unsure this should be attempted. The outcome might not be easily interpretable as at least two forces will contribute to the selection in panning experiments with TGD lines that do not bind anymore:

Firstly, panning would work against the SLI of the TGD, resulting in a tug of war between the TGD-SLI and binding. This is because a small number of parasites will loop out the TGD plasmid (revert) and would normally be eliminated during standard culturing due to the SLI drug used for the TGD. These revertant cells would bind and the panning would enrich them. Hence, panning and SLI are opposed forces in the case of a TGD abolishing binding. It is unclear how strong this effect would be, but this would for sure lead to mixed populations that complicate interpretations. 

The second selecting force are possible compensatory changes to restore binding. These can be due to different causes: (i) reversal of potential independent changes that may have occurred in the TGD parasites and that are in reality causing the binding loss (i.e. such as ptp3 loss or similar, the concern of the reviewer) or (ii) new changes to compensate the loss of the TGD target (in this case the TGD is the cause of the binding loss but for instance a different change ameliorates it by for instance increasing PfEMP1 expression or surface display). As both TGDs show some residual binding and have VAR01 on the surface to at least some extent, it is possible that new compensatory changes might indeed occur that indirectly increase binding again. 

In summary, even if more binding occurs after panning of the lines, it is not clear whether this is due to a compensatory change ameliorating the TGD or reversal of an unrelated change or are counter-selections against the SLI. To determine the cause, the panned TGD lines would need to be subjected to a complex and time-consuming analysis (WGS, RNASeq, possibly Maurer’s clefts phenotype) to find out whether they were SLI-revertants, or had an unrelated chance that was reverted or a new compensatory change that helps binding. This might be further muddled if a mix of cells come out of the selection that have different changes of the options indicated above. In that case, it might even require scRNASeq to make sense of the panning experiment. Due to the envisaged difficulty in interpreting the outcome, we did not attempt this panning.

To exclude loss of ptp3 expression as the reason for binding loss (something we would not have seen in the WGS if it is only due to a transcriptional change), we now carried out RNASeq with the TGD lines that have a binding phenotype. While we did not generate replicas to obtain quantitative data, the results show that both ptp3 copies were expressed in these TGDs comparable to other parasite lines that do bind with the same SLI-activated var gene, indicating that the effect is not due to ptp3 (see response to point 4 on PTP3 expression in the Recommendations for the authors). While we can’t fully exclude other changes in the TGDs that might affect binding, the WGS did not show any obvious alterations that could be responsible for this. 

(2) The authors perform a series of trafficking experiments to help discern whether PfEMP1 is trafficked through PTEX. While the results were not entirely definitive, they make a strong case for PTEX in PfEMP1 export. The authors then used BioID to obtain a proxiome for PfEMP1 and identified proteins they suggest are involved in PfEMP1 trafficking. However, it seemed that components of PTEX were missing from the list of interacting proteins. Is this surprising and does this observation shed any additional light on the possibility of PfEMP1 trafficking through PTEX? This warrants a comment or discussion.

This is an interesting point and we agree that this warrants to be discussed. A likely reason why PTEX components are not picked up as interactors is that BirA* is expected to be unfolded when it passes through the channel and in that state can’t biotinylate. Labelling likely would only be possible if PfEMP1 lingered at the PTEX translocation step before BirA* became unfolded to go through the channel which we would not expect under physiological conditions. We added the following sentences to the discussion: “While our data indicates PfEMP1 uses PTEX to reach the host cell, this could be expected to have resulted in the identification of PTEX components in the PfEMP1 proxiomes, which was not the case. However, as BirA* must be unfolded to pass through PTEX, it likely is unable to biotinylate translocon components unless PfEMP1 is stalled during translocation. For this reason, a lack of PTEX components in the PfEMP1 proxiomes does not necessarily exclude passage through PTEX.”

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Most of my comments are in the public section. I would just highlight a few things:

(1) In the binding studies section you talk about "human brain endothelial cells (HBEC-5i)". These cells do indeed express CSA but this is a property of their immortalisation rather than being brain endotheliium, which does not express CSA. I think this could be confusing to readers so I think you might want to reword this sentence to focus on CSA expressing the cell line rather than other features.

We thank the reviewer for pointing this out, we now modified the sentence to focus on the fact these are CSA expressing cells and provided a reference for it.

(2) As I said in the public section, CHO cells are great for proof of concept studies, but they are not endothelium. Not a problem for this paper.

Noted! Please also see our response to the public review.

(3) I wonder whether your comment about how well tolerated the Bir3* insertion is may be a bit too strong. I might say "Nonetheless, overall the BirA* modified PfEMP1 were functional."

Changed as requested.

(4) I'm not sure how you explain the IFA staining patterns to the uninitiated, but perhaps you could explain some of the key features you are looking for.

We apologise for not giving an explanation of the IFA staining patterns in the first place. Please see detailed response to public review of this reviewer (point 3 on PTP1-TGD phenotype) and to reviewer 2 (Recommendations to the authors, points 6 and 7 on better explaining and quantifying the Maurer’s clefts phenotypes). For this we now also generated parasites that episomally express mCherry tagged SBP1 in the TGD parasites with the reduced binding phenotype. This resulted in amendments to Fig. S7, addition of a Fig. S8 and updated results to better explain the phenotypes. 

This is a great paper - I just wish I'd had this system before.

Thank you!

Reviewer #2 (Recommendations for the authors):

Major Comments

(1) Does the RNAseq analysis of 3D7var0425800 and 3D7MEEDvar0425800 (Figure 1G, H) reveal any differential gene expression that might suggest a basis for loss of mutually exclusive var expression in the MEED line?

We now carried out a thorough analysis of these RNASeq experiments to look for an underlying cause for the phenotype. This was added as new Figure 1J and new Table S3. This analysis again illustrated the increased transcript levels of var genes. In addition, it showed that transcripts of a number of other exported proteins, including members of other gene families, were up in the MEED line. 

One hit that might be causal of the phenotype was sip2, which was down by close to 8-fold (pAdj 0.025). While recent work in P. berghei found this ApiAP2 to be involved in the expression of merozoite genes (Nishi et al., Sci Advances 2025(PMID: 40117352)), previous work in P. falciparum showed that it binds heterochromatic telomere regions and certain var upstream regions (Flück et al., PlosPath 2010 (PMID: 20195509), now cited in the manuscript). The other notable change was an upregulation of the non-coding RNA ruf6 which had been linked with impaired mono-allelic var expression (Guizetti et al., NAR 2016 (PMID: 27466391), now also cited in the manuscript). While it would go beyond this manuscript to follow this up, it is conceivable that alterations in chromosome end biology due to sip2 downregulation or upregulation of ruf6 are causes of the observed phenotype

We now added a paragraph on the more comprehensive analysis of the RNA Seq data of the MEED vs non-MEED lines at the end of the second results section.

(2) Could the inability of the PfEMP1-mDHFR fusion to block translocation (Fig 2A) reflect unique features of PfEMP1 trafficking, such as the existence of a soluble, chaperoned trafficking state that is not fully folded? Was a PfEMP1-BPTI fusion ever tested as an alternative to mDHFR?

This is an interesting suggestion. The PfEMP1-BPTI was never tested. However, a chaperoned trafficking state would likely also affect BPTI. Given that both domains (mDHFR and BPTI) in principle do the same when folded and would block when the construct is in the PV, it is not so likely that using a different blocking domain would make a difference. Therefore, the scenario where BPTI would block when mDHFR does not, is not that probable. The opposite would be possible (mDHFR blocking while BPTI does not, because only the latter depends on the redox state). However, this would only happen if the block  occurred before the construct reaches the PV.

At present, we believe the lacking block to be due to the organization of the domains in the construct. In the PfEMP1-mDHFR construct in this manuscript the position of the blocking domain is further away from the TMD compared to all other previously tested mDHFR fusions. Increased distance to the TMD has previously been found to be a factor impairing the blocking function of mDHFR (Mesen-Ramirez et al., PlosPath 2016 (PMID: 27168322)). Hence, our suspicion that this is the reason for the lacking block with the PfEMP1-mDHFR rather than the type of blocking domain. However, the latter option can’t be fully excluded and we might test BPTI in future work.

(3) The late promoter SBP1-mDHFR is 2A fused with the KAHRP reporter. Since 2A skipping efficiency varies between fusion contexts and significant amounts of unskipped protein can be present, it would be helpful to include a WB to determine the efficiency of skipping and provide confidence that the co-blocked KAHRP in the +WR condition (Fig 2D) is not actually fused to the C-terminus of SBP1-mDHFR-GFP.

Fortunately, this T2A fusion (crt_SBP1-mDHFR-GFP-2A-KAHRP-mScarlet^epi) was used before in work that included a Western blot showing its efficient skipping (S3 A Fig in MesenRamirez et al., PlosPath 2016). In agreement with these Western blot result, fluorescence microscopy showed very limited overlap of SBP1-mDHFR-GFP and KAHRP-mCherry in absence of WR (Fig. 3B in Mesen-Ramirez et al., PlosPath 2016 and Fig. 2 in this manuscript) which would not be the case if these two constructs were fused together. Please note that KAHRP is known to transiently localize to the Maurer’s clefts before reaching the knobs (Wickham et al., EMBOJ 2001, PMID: 11598007), and therefore occasional overlap with SBP1 at the Maurer’s clefts is expected. However, we would expect much more overlap if a substantial proportion of the construct population would not be skipped and therefore the co-blocked KAHRP-mCherry in the +WR sample is unlikely to be due to inefficient skipping and attachment to SBP1-mDHFR-GFP.

(4) Does comparison of RNAseq from the various 3D7 and IT4 lines in the study provide any insight into PTP3 expression levels between strains with different binding capacities? Was the expression level of ptp3a/b in the IT4var19 panned line similar to the expression in the parent or other activated IT4 lines? Could the expanded ptp3 gene number in IT4 indicate that specialized trafficking machinery exists for some PfEMP1 proteins (ie, IT4var19 requires the divergent PTP3 paralog for efficient trafficking)?

PTP3 in the different IT4 lines that bind:

In those parasite lines that did bind, the intrinsic variation in the binding assays, the different binding properties of different PfEMP1 variants and the variation in RNA Seq experiments to compare different parasite lines precludes a correlation of binding level vs ptp3 expression. For instance, if a PfEMP1 variant has lower binding capacity, ptp3 may still be higher but binding would be lower than if comparing to a parasite line with a better binding PfEMP1 variant. Studying the effect of PTP3 levels on binding could probably be done by overexpressing PTP3 in the same PfEMP1 SLI expressor line and assessing how this affects binding, but this would go beyond this manuscript.

PTP3 in panned vs unpanned Var19:

We did some comparisons between IT4 parent, and the IT4-Var19 panned and unpanned

(see Author response table 1). This did not reveal any clear associations. While the parent had somewhat lower ptp3 transcript levels, they were still clearly higher than in the unpanned Var19 line and other lines had also ptp3 levels comparable to the panned IT4-Var19 (see Author response table 2) 

PTP3 in the TGDs and possible reason for binding phenotype:

A key point is whether PTP3 could have influenced the lack of binding in the TGD lines (see also weakness section and point 1 of public review of reviewer 3: ptp3 may be an indirect cause resulting in lacking binding in TGD parasites). We now did RNA Seq to check for ptp3 expression in the relevant TGD lines although we did not do a systematic quantitative comparison (which would require 3 replicates of RNASeq), but we reasoned that loss of expression would also be evident in one replicate. There was no indication that the TGD lines had lost PTP3 expression (see Author response table 2) and this is unlikely to explain the binding loss in a similar fashion to the Var19 parasites. Generally, the IT4 lines showed expression of both ptp3 genes and only in the Var19 parasites before panning were the transcript levels considerably lower:

Author response table 1.

Parent vs IT4-Var19 panned and unpanned

Author response table 2.

TGD lines with binding phenotype vs parent

The absence of an influence of PTP3 on the binding phenotype in the cell lines in this manuscript (besides Var19) is further supported by its role in PfEMP1 surface display. Previous work has shown that KO of ptp3 leads to a loss of VAR2CSA surface display (Maier et al., Cell 2008). The unpanned Var19 parasite also lacked PfEMP1 surface display and panning and the resulting appearance of the binding phenotype was accompanied by surface display of PfEMP1. As both, the EMPIC3 and TryThra-TGD lines had still at least some PfEMP1 on the surface, this also (in addition to the RNA Seq above) speaks against PTP3 being the cause of the binding phenotype. The same applies to 3D7 which despite the poor binding displays PfEMP1 on the host cell surface (Figure 1D). This indicating that also the binding phenotype in 3D7 is not due to PTP3 expression loss, as this would have abolished PfEMP1 surface display. 

The idea about PTP3 paralogs for specific PfEMP1s is intriguing. In the future it might be interesting to test the frequency of parasites with two PTP3 paralogs in endemic settings and correlate it with the PfEMP1 repertoire, variant expression and potentially disease severity. 

(5) The IT4var01 line shows substantially lower binding in Figure 5F compared with the data shown in Figure 4E and 6F. Does this reflect changes in the binding capacity of the line over time or is this variability inherent to the assay?

There is some inherent variability in these assays. While we did not systematically assess this, we had no indication that this was due to the parasite line changing. The Var01 line was cultured for months and was frozen down and thawed more than once without a clear gradual trend for more or less binding. While we can’t exclude some variation from the parasite side, we suspect it is more a factor of the expression of the receptor on the CHO cells the iRBCs bind to. 

Specifically, the assays in Fig. 6F and 4E mentioned by the reviewer both had an average binding to CD36 of around 1000 iE/mm2, only the experiments in Fig. 5F are different (~ 500 iE/mm2) but these were done with a different batch of CHO cells at a different time to the experiments in Fig. 6F and 4E. 

(6) In Figure S7A, TryThrA and EMPIC3 show distinct localization as circles around the PfEMP1 signal while PeMP2 appears to co-localize with PfEMP1 or as immediately adjacent spots (strong colocalization is less apparent than SBP1, and the various PfEMP1 IFAs throughout the study). Does this indicate that TryThrA and EMPIC3 are peripheral MC proteins? Does this have any implications for their function in PfEMP1 binding? Some discussion would help as these differences are not mentioned in the text. For the EMPIC3 TGD IFAs, localization of SBP1 and PfEMP1 is noted to be normal but REX1 is not mentioned (although this also appears normal).

We apologise for the lacking description of the candidate localisations and cursory description of the Maurer’s clefts phenotypes (next point). Our original intent was to not distract too much from the main flow of the manuscript as almost every part of the manuscript could be followed up with more details. However, we fully agree that this is unsatisfactory and now provided more description (this point) and more data (next point).

Localisation of TryThrA and EMPIC3 compared to PfEMP1 at the Maurer’s clefts: the circular pattern is reminiscent of the results with Maurer’s clefts proteins reported by McMillan et al using 3D-SIM in 3D7 parasites (McMillan et al., Cell Microbiology 2014 (PMID: 23421990)). In that work SBP1 and MAHRP1 (both integral TMD proteins) were found in foci but REX1 (no TMD) in circular structures around these foci similar to what we observed here for TryThrA and EMPIC3 which both also lack a TMD. The SIM data in McMillan et al indicated that also PfEMP1 is “more peripheral”, although it did only partially overlap with REX1. The conclusion from that work was that there are sub-compartments at the Maurer’s clefts. In our IFAs (Fig. S7A) PfEMP1 is also only partially overlapping with the TryThrA and EMPIC3 circles, potentially indicating similar subcompartments to those observed by 3D-SIM. We agree with the reviewer that this might be indicative of peripheral MC proteins, fitting with a lack of TMD in these candidates, but we did not further speculate on this in the manuscript.

We now added enlargements of the ring-like structures to better illustrate this observation in Fig. S7A. In addition, we now specifically mention the localization data and the ring like signal with TryThrA and EMPIC3 in the results and state that this may be similar to the observations by McMillan et al., Cell Microbiology 2014.

We also thank the reviewer for pointing out that we had forgotten to mention REX1 in the EMPIC3-TGD, this was amended.  

(7) The atypical localization in TryThrA TGD line claimed for PfEMP1 and SBP1 in Fig S7B is not obvious. While most REX1 is clustered into a few spots in the IFA staining for SBP1 and REX1, SBP1 is only partially located in these spots and appears normal in the above IFA staining for SBP1 and HA. The atypical localization of PfEMP1-HA is also not obvious to me. The authors should clarify what is meant by "atypical" localization and provide support with quantification given the difference between the two SBP1 images shown.

We apologise for the inadequate description of these IFA phenotypes. The abnormal signal for SBP1, REX1 and PfEMP1 in the TryThrA-TGD included two phenotypes found with all 3 proteins: 

(1) a dispersed signal for these proteins in the host cell in addition to foci (the control and the other TGD parasites have only dots in the host cell with no or very little detectable dispersed signal). 

(2) foci of disproportionally high intensity and size, that we assumed might be aggregation or enlargement of the Maurer’s clefts or of the detected proteins.

The reason for the difference between the REX1 (aggregation) phenotype and the PfEMP1 and SBP1 (dispersed signal, more smaller foci) phenotypes in the images in Fig. S7B is that both phenotypes were seen with all 3 proteins but we chose a REX1 stained cell to illustrate the aggregation phenotype (the SBP1 signal in the same cell is similar to the REX1 signal, illustrating that this phenotype is not REX1 specific; please note that this cell also has a dispersed pool of REX1 and SBP1). 

Based on the IFAs 66% (n = 106 cells) of the cells in the TryThrA-TGD parasites had one or both of the observed phenotypes. We did not include this into the previous version of the manuscript because a description would have required detouring from the main focus of this results section. In addition, IFAs have some limitations for accurate quantifications, particularly for soluble pools (depending on fixing efficiency and agent, more or less of a soluble pool in the host cell can leak out). 

To answer the request to better explain and quantify the phenotype and given the limitations of IFA, we now transfected the TryThrA-TGD parasites with a plasmid mediating episomal expression of SBP1-mCherry, permitting live cell imaging and a better classification of the Maurer’s clefts phenotype. Due to the two SLI modifications in these parasites (using up 4 resistance markers) we had to use a new selection marker (mutated lactate transporter PfFNT, providing resistance to BH267.meta (Walloch et al., J. Med. Chem. 2020 (PMID: 32816478))) to transfect these parasites with an additional plasmid. 

These results are now provided as Fig. S8 and detailed in the last results section. The new data shows that the majority of the TryThrA-TGD parasites contain a dispersed pool of SBP1 in the host cell. About a third of the parasites also showed disproportionally strong SBP1 foci that may be aggregates of the Maurer’s clefts. We also transfected the EMPIC3-TGD parasites with the FNT plasmid mediating episomal SBP1-mCherry expression and observed only few cells with a cytoplasmic pool or aggregates (Fig. S8). Overall these findings agree with the previous IFA results. As the IFA suggests similar results also for REX1 and PfEMP1, this defect is likely not SBP1 specific but more general (Maurer’s clefts morphology; association or transport of multiple proteins to the Maurer’s clefts). This gives a likely explanation for the cytoadherence phenotype in the TryThrA-TGD parasites. The reason for the EMPIC3-TGD phenotype remains to be determined as we did not detect obvious changes of the Maurer’s clefts morphology or in the transport of proteins to these structures in these experiments. 

Minor comments

(1) Italicized numbers in parenthesis are present in several places in the manuscript but it is not clear what these refer to (perhaps differently formatted citations from a previous version of the manuscript). Figure 1

legend: (121); Figure S3 legend: (110), (111); Figure S6 legend: (66); etc.

We thank the reviewer for pointing out this issue with the references, this was amended.

(2) Figure 5A and legend: "BSD-R: BSD-resistance gene". Blasticidin-S (BS) is the drug while Blasticidin-S deaminase (BSD) is the resistance gene.

We thank the reviewer for pointing this out, the legend and figure were changed.

(3) Figure 5E legend: µ-SBP1-N should be α-SBP1-N.

This was amended.

(4) Figure S5 legend: "(Full data in Table S1)" should be Table S3.

This was amended.

(5) Figure S1G: The pie chart shows PF3D7_0425700 accounts for 43% of rif expression in 3D7var0425800 but the text indicates 62%.

We apologize for this mistake, the text was corrected. We also improved the citations to Fig. S1G and H in this section.

(6) "most PfEMP1-trafficking proteins show a similar early expression..." The authors might consider including a table of proteins known to be required for EMP1 trafficking and a graph showing their expression timing. Are any with later expressions known?

Most exported proteins are expressed early, which is nicely shown in Marti et al 2004 (cited for the statement) in a graph of the expression timing of all PEXEL proteins (Fig. 4B in that paper). PNEPs also have a similar profile (Grüring et al 2011, also cited for that statement), further illustrated by using early expression as a criterion to find more PNEPs (Heiber et al., 2013 (PMID: 23950716)). Together this includes most if not all of the known PfEMP1 trafficking proteins. The originally co-submitted paper (Blancke-Soares & Stäcker et al., eLife preprint doi.org/10.7554/eLife.103633.1) analysed several later expressed exported proteins

(Pf332, MSRP6) but their disruption, while influencing Maurer’s clefs morphology and anchoring, did not influence PfEMP1 transport. However, there are some conflicting results for Pf332 (referenced in Blancke-Soares & Stäcker et al). This illustrates that it may not be so easy to decide which proteins are bona fide PfEMP1 trafficking proteins. We therefore did not add a table and hope it is acceptable for the reader to rely on the provided 3 references to back this statement.

(7)  Figure S1J: The predominate var in the IT4 WT parent is var66 (which appears to be syntenic with Pf3D7_0809100, the predominate var in the 3D7 WT parent). Is there something about this locus or parasite culture conditions that selects for these vars in culture? Is this observed in other labs as well?

This is a very interesting point (although we are not certain these vars are indeed syntenic, they are on different chromosomes). As far as we know at least Pf3D7_0809100 is commonly a dominant var transcribed in other labs and was found expressed also in sporozoites (Zanghì et al. Cell Rep. 2018). However, it is unclear how uniform this really is. For IT4 we do not know in full but have also here commonly observed centromeric var genes to be dominating transcripts in unselected parasite cultures. It is possible that transcription drifts to centromeric var genes in cultured parasites. However, given the anecdotal evidence, it is unknown to which extent this is related to an inherent switching and regulation regiment or a consequence of faulty regulation following prolonged culturing.

(8) Figure 4B, C: Presumably the asterisks on the DNA gels indicate non-specific bands but this is not described in the legend. Why are non-specific bands not consistent between parent and integrated lanes?

We apologize for not mentioning this in the legend, this was amended.

It is not clear why the non-specific bands differ between the lines but in part this might be due to different concentrations and quality of DNA preps. A PCR can also behave differently depending on whether the correct primer target is present or not. If present, the PCR will run efficiently and other spurious products will be outcompeted, but in absence of the correct target, they might become detectable.  

Overall, we do not think the non-specific bands are indications of anything untoward with the lines, as for instance in Fig. 4B the high band in the 5’ integration in the IT4 line (that does not occur anywhere else) can’t be due to a genomic change as this is the parental line and does not contain the plasmid for integration. In the same gel, the ori locus band of incorrect size (likely due to crossreaction of the primers to another var gene which due to the high similarity of the ATS region is not always fully avoidable), is present in both, the parent IT4 and the integrant line which therefore also is not of concern. In C there are a couple of bands of incorrect size in the Integration line. One of these is very faint and both are too large and again therefore are likely other vars that are inefficiently picked up by these primers. The reason they are not seen in the parent line is that there the correct primer binding site is present, which then efficiently produces a product that outcompetes the product derived from non-optimal matching primer products and hence appear in the Int line where the correct match is not there anymore. For these reasons we believe these bands are not of any concern.  

(9) Figure 4C: Is there a reason KAHRP was used as a co-marker for the IFA detecting IT4var19 expression instead of SBP1 which was used throughout the rest of the study?

This is a coincidence as this line was tested when other lines were tested for KAHRP. As there were foci in the host cell we were satisfied that the HA-tagged PfEMP1 is produced and the localization deemed plausible. 

(10) Figure 6: Streptavidin labeling for the IT4var01-BirA position 3 line is substantially less than the other two lines in both IFA and WB. Does the position 3 fusion reduce PfEMP1 protein levels or is this a result of the context or surface display of the fusion? Interestingly, the position 3 trypsin cleavage product appears consistently more robust compared with the other two configurations. Does this indicate that positioning BirA upstream of the TM increases RBC membrane insertion and/or makes the surface localized protein more accessible to trypsin?

It is possible that RBC membrane insertion or trypsin accessibility is increased for the position 3 construct. But there could also be other explanations:

The reason for the more robustly detected protected fragment for the position 3 construct in the WB might also be its smaller size (in contrast to the other two versions, it does not contain BirA*) which might permit more efficient transfer to the WB membrane. In that case the more robust band might not (only) be due to better membrane insertion or better trypsin accessibility.

The lower biotinylation signal with the position 3 construct might also be explained by the farther distance of BirA* to the ATS (compared to position 1 and 2), the region where interactors are expected to bind. The position 1 and 2 constructs may therefore generally be more efficient (as closer) to biotinylate ATS proximal proteins. Further, in the final destination (PfEMP1 inserted into the RBC membrane) BirA* would be on the other side of the membrane in the position 3 construct while in the position 1 and 2 constructs BirA* would be on the side of the membrane where the ATS anchors PfEMP1 in the knob structure. In that case, labelling with position 3 would come from interactions/proximities during transport or at the Maurer’s clefts (if there indeed PfEMP1 is not membrane embedded) and might therefore be less.

Hence, while alterations in trypsin accessibility and RBC membrane insertion are possible explanations, other explanations exist. At present, we do not know which of these explanations apply and therefore did not mention any of them in the manuscript. 

Reviewer #3 (Recommendations for the authors):

(1) In the abstract and on page 8, the authors mention that they generate cell lines binding to "all major endothelial receptors" and "all known major receptors". This is a pretty allencompassing statement that might not be fully accepted by others who have reported binding to other receptors not considered in this paper (e.g. VCAM, TSP, hyaluronic acid, etc). It would be better to change this statement to something like "the most common endothelial receptors" or "the dominant endothelial receptors", or something similar.

We agree with the reviewer that these statements are too all-encompassing and changed them to “the most common endothelial receptors” (introduction) and “the most common receptors” (results).

(2) The authors targeted two rif genes for activation and in each case the gene became the most highly expressed member of the family. However, unlike var genes, there were other rif genes also expressed in these lines and the activated copy did not always make up the majority of rif mRNAs. The authors might wish to highlight that this is inconsistent with mutually exclusive expression of this gene family, something that has been discussed in the past but not definitively shown.

We thank the reviewer for highlighting this, we now added the following statement to this section: “While SLI-activation of rif genes also led to the dominant expression of the targeted rif gene, other rif genes still took up a substantial proportion of all detected rif transcripts, speaking against a mutually exclusive expression in the manner seen with var genes.”

(3) In Figure 6, H-J, the authors display volcano plots showing proteins that are thought to interact with PfEMP1. These are labeled with names from the literature, however, several are named simply "1, 2, 3, 4, 5, or 6". What do these numbers stand for?

We apologize for not clarifying this and thank the reviewer for pointing this out. There is a legend for the numbered proteins in what is now Table S4 (previously Table S3). We now amended the legend of Figure 6 to explain the numbers and pointing the reader to Table S4 for the accessions.

In the others this row has a BG fill, and the white text clashes with the neon here.

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Check all assessment button spellings as I presume this is cloned

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Nav - Should "GTC - Home" just be "Home"

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Last button is a hard fail on accessibility contrast checker

Alle kosten die nodig zijn om een asset in gebruiksklare staat te brengen, moet je capitalizen.

Le Chagrin et la Pitié : Analyse d'un Film Révolutionnaire

Résumé Exécutif

Ce document de synthèse analyse le film documentaire Le Chagrin et la Pitié de Marcel Ophuls, en s'appuyant sur les perspectives et témoignages présentés dans le documentaire d'ARTE. Sorti en 1971, Le Chagrin et la Pitié a provoqué une rupture fondamentale dans la mémoire collective française concernant la période de l'Occupation.

Les points essentiels sont les suivants :

• Destruction du Mythe Résistancialiste : Le film a été le premier à confronter frontalement et à déconstruire le mythe gaulliste d'une France majoritairement unie dans la Résistance.

Il a révélé une réalité bien plus complexe, faite de collaboration, d'attentisme, d'ignorance volontaire et d'actes héroïques isolés.

• Une Méthodologie d'Interview Novatrice : Marcel Ophuls a développé un art de l'interview unique, mêlant douceur apparente, humour et questions incisives.

En transformant les témoins en "personnages" au sens fort, il a créé une "dramaturgie du témoignage" qui expose les ambiguïtés et les contradictions de la période.

• Censure et Succès Paradoxal : Initialement conçu pour la télévision, le film a été refusé par l'ORTF, la télévision d'État, au motif qu'il "détruit des mythes dont la France a encore besoin".

Cette censure a paradoxalement amplifié son impact, le transformant en un événement culturel majeur lors de sa sortie en salles, où il a connu un immense succès public.

• Un Catalyseur de Mémoire : Le film a déclenché un débat public sans précédent sur la responsabilité de l'État français et de citoyens français dans la collaboration et la déportation des Juifs.

Il a ouvert la voie à de nouvelles œuvres cinématographiques et aux travaux d'historiens comme Robert Paxton.

• Héritage Politique et Sociétal Durable : L'onde de choc du film a eu des répercussions à long terme, influençant la société française dans son rapport à son passé.

Son héritage est perceptible jusque dans le discours de Jacques Chirac en 1995, reconnaissant officiellement la responsabilité de l'État français dans la Shoah, un discours considéré comme un prolongement direct du travail de mémoire initié par le film.

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Un Séisme Cinématographique et Culturel

Le 14 avril 1971, une petite salle de cinéma du Quartier Latin à Paris projette pour la première fois Le Chagrin et la Pitié.

Ce documentaire, réalisé par Marcel Ophuls, alors âgé de 42 ans, offre une "vision décapante" des années d'Occupation, loin de la mythologie héroïque officielle.

Produit par les télévisions allemande et suisse, il est rapidement acclamé à l'international, acheté par 27 pays et sélectionné aux Oscars.

En France, cependant, l'accueil est radicalement différent. L'ORTF (Office de Radiodiffusion-Télévision Française) refuse d'acheter et de diffuser ce qu'elle considère comme un "film hérétique".

Cette décision déclenche de violentes controverses et érige le film en symbole d'un "duel entre la génération post-68 et le pouvoir".

Le film devient célèbre, paradoxalement, parce qu'on ne l'a pas montré à la télévision.

Le titre lui-même, inspiré par le témoignage d'un résistant qui confie que ses sentiments les plus fréquents furent "le chagrin et la pitié", est décrit comme "extraordinairement romanesque" et "impitoyable", reflétant la complexité d'une période où les lignes morales étaient brouillées.

La Genèse du Projet : De l'ORTF à l'Exil

Le Parcours de Marcel Ophuls

Rien ne prédestinait Marcel Ophuls à briser le mythe gaulliste, si ce n'est son parcours personnel.

Né en Allemagne en 1927, fils du cinéaste Max Ophuls, il fuit le nazisme avec sa famille pour la France, puis pour Hollywood.

Devenu citoyen américain, il rentre en France après-guerre.

Après des débuts comme assistant réalisateur et une amitié avec François Truffaut, il connaît un échec commercial qui le pousse, "très à contre-cœur" et pour des "raisons alimentaires", à rejoindre la télévision française en 1966.

L'Invention d'un Style

À l'ORTF, au sein de l'équipe de l'émission "Zoom", Ophuls développe son style.

Utilisant les nouvelles technologies légères (caméra 16mm, enregistreur Nagra), il pratique un "journalisme subjectif", allant à la rencontre des Français (femmes, ouvriers, jeunes) et perfectionnant ce qui est décrit comme un "art de l'interview".

Du Projet Interrompu à la Production Indépendante

Le projet initial est une suite à deux émissions sur Munich 1938, visant à explorer les conséquences de l'Occupation. Le mouvement de Mai 68 et la grève qui s'ensuit à l'ORTF interrompent le projet.

Ophuls, ainsi que les producteurs André Harris et Alain de Sédouy, sont licenciés.

Le groupe trouve refuge auprès d'une nouvelle société de production suisse et Ophuls convainc la télévision allemande (NDR) de financer 70% du film.

Le tournage est lancé au printemps 1969, né de la censure et de la nécessité de trouver du travail ailleurs.

Une Méthodologie Révolutionnaire : La Dramaturgie du Témoignage

L'Art de l'Interview

Marcel Ophuls rejette l'étiquette de "cinéma vérité", qu'il juge "horriblement prétentieux".

Sa méthode consiste à créer une "dramaturgie du témoignage" où les personnes interrogées deviennent de véritables personnages.

Il aborde ses sujets "en douceur, en rigolant", utilisant parfois l'"humour juif" pour désarmer, mais son approche est fondamentalement sans concession.

Il laisse ses témoins "dérouler leurs pensées", manifestant une forme de respect pour leur parole tout en maintenant une distance critique, voire un "manque d'empathie".

Cette approche permet de révéler les fissures, les non-dits et les justifications a posteriori.

Témoignages Emblématiques

Le film est construit autour d'une mosaïque de témoignages qui, mis en regard, créent une vision polyphonique et troublante de la France occupée.

• Le choix de Clermont-Ferrand comme microcosme s'est avéré judicieux en raison de sa proximité avec Vichy et de la présence de toutes les facettes de l'époque :

*pétainisme, * Milice, et * Résistance.

Témoin(s)

Rôle / Statut

Thème Principal du Témoignage

Les frères Klein

Commerçants

La banalité de l'antisémitisme et le manque de solidarité. Leur annonce dans Le Moniteur pour se déclarer "catholique" et non "juif" est une séquence phare.

René de Chambrun

Gendre de Pierre Laval

La défense sophistique de Vichy, argumentant que le régime aurait sauvé une partie des Juifs français.

Ophuls le confronte directement à la caméra sur le droit moral d'un État à "choisir entre deux groupes humains".

Christian de la Mazière

Ancien de la Waffen-SS française

L'engagement fasciste assumé ("jeune fasciste").

Son témoignage, qualifié de "glaçant" et "authentique", crève l'écran et met mal à l'aise toutes les consciences.

Il conclut le film par un appel à la prudence adressé à la jeunesse de 68.

Pierre Mendès France

Homme politique, résistant

La dignité face à la persécution.

Son récit de l'arrestation de son père et de la naissance de sa fille, qu'il n'avait jamais vue, est un moment d'émotion intense.

Les frères Grave

Paysans résistants

L'héroïsme ordinaire et modeste. Leur témoignage sur les débuts de la résistance en Auvergne, où ils chantaient L'Internationale car Pétain avait annexé La Marseillaise, illustre l'engagement populaire.

Claude Lévi-Strauss

Anthropologue

Le regard extérieur et moral. Il juge sévèrement l'État français pour avoir "renié le droit d'asile traditionnel de la France" en livrant des ressortissants qu'il devait protéger.

Témoins des "tondues"

Spectateurs de la Libération

La violence misogyne de l'épuration.

La séquence, associée à une chanson de Brassens, est qualifiée de "transgressive" et a profondément marqué les féministes émergentes de l'époque.

La Censure et la Controverse : Un Mythe Intouchable

Le Refus de l'ORTF

La direction de la télévision d'État justifie sa décision de ne pas diffuser le film par une phrase devenue célèbre :

"Ce film détruit des mythes dont la France a encore besoin."

Cette déclaration révèle une volonté explicite du pouvoir politique de maintenir une version officielle de l'Histoire, occultant les aspects les plus sombres de la période.

L'Opposition de Simone Veil et des Résistants

Une opposition significative est venue de figures respectées, notamment Simone Veil.

Ayant elle-même survécu à la déportation, elle estimait que le film "entachait de collaboration l'ensemble de la société française" et ne rendait pas justice aux nombreux Français courageux qui, sans être des résistants armés, avaient aidé des Juifs.

Les commentateurs du documentaire suggèrent que sa position, bien que sincère, a servi de paravent aux "pétinistes à Légion d'honneur" de l'ORTF.

De nombreux anciens résistants ont également fait pression, craignant que le film ne donne une "mauvaise image de la France".

L'Affaire Bousquet

Le film expose la présence au sommet de la société de figures de la collaboration.

Une séquence montre René Bousquet, secrétaire général de la police de Vichy et organisateur de la rafle du Veld'Hiv, devenu après-guerre un puissant directeur de la Banque d'Indochine.

La banque a contacté les producteurs suisses pour leur demander de supprimer le passage en échange de contreparties financières, ce que ces derniers ont refusé.

Cette affaire illustre à quel point les responsables de l'époque étaient encore en poste et influents.

L'Héritage d'un Film-Événement

Une Rupture Mémorielle

Le Chagrin et la Pitié a provoqué un "basculement mémoriel".

Il a forcé la société française à regarder en face la collaboration de l'État et le comportement d'une partie de sa population.

Pour la première fois, la parole se libère, comme en témoigne le nombre sans précédent de lettres envoyées au journal Le Monde en 1971, où les citoyens débattent avec passion de la période.

Le film a rendu impossible de "remettre la poussière sous le tapis".

Impact International

Aux États-Unis, le film sort en 1972 dans un contexte marqué par la guerre du Vietnam et le scandale du Watergate.

La critique américaine y voit un miroir, posant la question : "Dans des circonstances comparables, avons-nous bien agi ?".

Le film change également la perception américaine de la Libération, révélant que les GIs ont débarqué non seulement dans un pays occupé, mais aussi dans un pays qui avait "sereinement organisé sa collaboration avec l'occupant".

Un Catalyseur pour l'Histoire et le Cinéma

Le film est considéré comme un "facilitateur" qui a permis l'émergence d'autres œuvres traitant de l'Occupation sous un angle critique, comme Lacombe Lucien de Louis Malle ou Monsieur Klein de Joseph Losey.

Il a également préparé le terrain pour l'accueil du livre de l'historien américain Robert Paxton, La France de Vichy, qui, par une approche archivistique, confirmait les conclusions du film.

Ophuls et Paxton sont vus comme partageant le même "esprit" en osant juger Vichy.

Vers la Reconnaissance Politique

L'impact du film s'étend sur plusieurs décennies. Le débat qu'il a ouvert est considéré comme une étape essentielle menant à la reconnaissance officielle de la responsabilité de la France.

Un intervenant établit une continuité directe : "Il n'y a pas de discours de Chirac en 1995 s'il n'y a pas le chagrin à la pitié."

Ce discours, où Jacques Chirac déclare que "la folie criminelle de l'occupant a été secondée par des Français, secondée par l'État français", marque l'aboutissement du processus de mémoire que le film avait brutalement initié 24 ans plus tôt.

C'est la preuve qu'un film, "somme toute assez rare", peut "changer les choses" et "changer des vies".

DOI: 10.64898/2025.12.02.689353

Resource: (BD Biosciences Cat# 340449, RRID:AB_400425)

Curator: @dhovakimyan1

SciCrunch record: RRID:AB_400425

What is this?

DOI: 10.64898/2025.12.02.689353

Resource: (BD Biosciences Cat# 340548, RRID:AB_400054)

Curator: @dhovakimyan1

SciCrunch record: RRID:AB_400054

What is this?

DOI: 10.3390/cells10051026

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @maulamb

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.3390/cells10051026

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @maulamb

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.3390/cells10051026

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @maulamb

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.3390/cells10051026

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @maulamb

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1371/journal.pgen.1009112

Resource: RRID:BDSC_997

Curator: @maulamb

SciCrunch record: RRID:BDSC_997

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DOI: 10.1186/s13058-025-02176-6

Resource: (Abcam Cat# ab137560, RRID:AB_2877175)

Curator: @dhovakimyan1

SciCrunch record: RRID:AB_2877175

What is this?

DOI: 10.1186/s13048-025-01907-9

Resource: RRID:IMSR_ORNL:BALB-CRL

Curator: @dhovakimyan1

SciCrunch record: RRID:IMSR_ORNL:BALB-CRL

What is this?

DOI: 10.1186/s13041-021-00782-x

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @maulamb

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1186/s13041-021-00782-x

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @maulamb

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1186/s13041-021-00782-x

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @maulamb

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1186/s13041-021-00782-x

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @maulamb

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1186/s13041-021-00782-x

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @maulamb

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1186/s13041-021-00782-x

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @maulamb

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1186/s13041-021-00782-x

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @maulamb

SciCrunch record: RRID:SCR_006457

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DOI: 10.1182/blood-2025-186

Resource: Mutant Mouse Regional Resource Center (RRID:SCR_002953)

Curator: @AleksanderDrozdz

SciCrunch record: RRID:SCR_002953

What is this?

DOI: 10.1111/eci.13174

Resource: (Bio-Rad Cat# MCA341R, RRID:AB_2291300)

Curator: @dhovakimyan1

SciCrunch record: RRID:AB_2291300

What is this?

DOI: 10.1111/eci.13174

Resource: (Cell Signaling Technology Cat# 9621, RRID:AB_330304)

Curator: @dhovakimyan1

SciCrunch record: RRID:AB_330304

What is this?

DOI: 10.1111/acel.70255

Resource: Mutant Mouse Regional Resource Center (RRID:SCR_002953)

Curator: @AleksanderDrozdz

SciCrunch record: RRID:SCR_002953

What is this?

DOI: 10.1101/2025.09.30.676706

Resource: (MMRRC Cat# 034829-JAX,RRID:MMRRC_034829-JAX)

Curator: @AleksanderDrozdz

SciCrunch record: RRID:MMRRC_034829-JAX

What is this?

DOI: 10.1093/g3journal/jkaa066

Resource: RRID:BDSC_34621

Curator: @maulamb

SciCrunch record: RRID:BDSC_34621

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Curator: @maulamb

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DOI: 10.1093/g3journal/jkaa066

Resource: RRID:BDSC_25793

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DOI: 10.1093/g3journal/jkaa066

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What is this?

DOI: 10.1093/g3journal/jkaa066

Resource: RRID:BDSC_36658

Curator: @maulamb

SciCrunch record: RRID:BDSC_36658

What is this?

DOI: 10.1093/g3journal/jkaa066

Resource: RRID:BDSC_55883

Curator: @maulamb

SciCrunch record: RRID:BDSC_55883

What is this?

DOI: 10.1093/g3journal/jkaa066

Resource: RRID:BDSC_57013

Curator: @maulamb

SciCrunch record: RRID:BDSC_57013

What is this?

DOI: 10.1093/g3journal/jkaa066

Resource: RRID:BDSC_29379

Curator: @maulamb

SciCrunch record: RRID:BDSC_29379

What is this?

DOI: 10.1093/g3journal/jkaa066

Resource: RRID:BDSC_57014

Curator: @maulamb

SciCrunch record: RRID:BDSC_57014

What is this?

DOI: 10.1093/g3journal/jkaa066

Resource: RRID:BDSC_33046

Curator: @maulamb

SciCrunch record: RRID:BDSC_33046

What is this?

DOI: 10.1093/g3journal/jkaa066

Resource: RRID:BDSC_34092

Curator: @maulamb

SciCrunch record: RRID:BDSC_34092

What is this?

DOI: 10.1093/g3journal/jkaa066

Resource: RRID:BDSC_27517

Curator: @maulamb

SciCrunch record: RRID:BDSC_27517

What is this?

DOI: 10.1093/g3journal/jkaa066

Resource: RRID:BDSC_57011

Curator: @maulamb

SciCrunch record: RRID:BDSC_57011

What is this?

DOI: 10.1093/g3journal/jkaa066

Resource: RRID:BDSC_29435

Curator: @maulamb

SciCrunch record: RRID:BDSC_29435

What is this?

DOI: 10.1093/g3journal/jkaa066

Resource: RRID:BDSC_28368

Curator: @maulamb

SciCrunch record: RRID:BDSC_28368

What is this?

DOI: 10.1093/g3journal/jkaa066

Resource: RRID:BDSC_34982

Curator: @maulamb

SciCrunch record: RRID:BDSC_34982

What is this?