Meta Title update to align with keywords We understand the theory behind the writing of the meta title but we are not harnessing the power of keywords in the correct places. H1 is perfect for the bigger message but the meta title needs to be simple / straight to the point

from Returns Management | Simplify Returns with GFS

to Returns Management Services | GFS

obtainable by interpersonal cross referencing linking and sharing

Key Idea Individuals form and maintain their own autonomous shards!

closer peer

link to that private Lcuel Unique Ids that form a Personal Indranet Worspace nidirectionally meaningfully linked strucured deeply meaningfully intentionlly intertwingled spaces enScapes that encapsulate curated associative complexes

reusable repurposable with full provi=enace and history of its own co-evolution and

Conversations that are continuous without being synchronous

People cantered interest based emergent networks

where connecting People Ideas and Reelevant resources

simultaneously scaling mutual learning, synthesis, and Reach. Searches including the ability to exchange searches and results serendioity engine

Pensieve

map CIDs to peers

decopulies identity from where it ives

batching CIDs - that get allocated to the same DHT servers and - sweeping through the keyspace systematically,

we’ve unlocked dramatic efficiency gains.

For a node providing 100k CIDs, - this means a 97% reduction in lookup operations.

Resource-constrained nodes can now handle - hundreds of thousands of CIDs,

while high-capacity nodes can - scale to hundreds of millions and beyond

—all with - smooth, - predictable

resource usage - distributed over time.

This page has some decent rankings for the very specific terms returns management software returns management solution returns platform returns software

We need to look at including in content updates the software, solution and platform words in relation to the returns offering. It does not have to be exact as written above but we need around 10 mentions combined of the above terms to help give it a push it to being more relevant

This is the cruel reality that was the lives of many people of color in the Americas during this time, as a false charge can easily lead to death.

restore or restsart

from - smart-resume

the pronlem of Location Addressing =

why we must distribute the Web

or uh you have some new ideas about what humanity should uh be able to do, you can just

write a protocol uh and then you implement it and if you're right uh and it works then you tell the world and

then it gets deployed and then a lot of people will use it and the world will be a better place

Parresía (Sócrates y Foucault)

IPLD Primer

not turing complete

semantics?

incidental choices that must be made but do not matter pers

data model independent of syntax AST

eLife Assessment

This study provides important evidence for the mechanism underlying KCNC1-related developmental and epileptic encephalopathy. The authors have generated and characterized a new knock-in mouse with a pathogenic mutation found in patients to determine the synaptic and circuit mechanisms contributing to KCNC1-associated epilepsy. They provide convincing evidence for reduced excitability of parvalbumin-positive fast-spiking interneurons, but not in neighboring excitatory neurons, and suggest that this may contribute to seizures and premature death in the mice.

Reviewer #1 (Public review):

Summary:

The authors have created a new model of KCNC1-related DEE in which a pathogenic patient variant (A421V) is knocked into mouse in order to better understand the mechanisms through which KCNC1 variants lead to DEE.

Strengths:

(1) The creation of a new DEE model of KCNC1 dysfunction.

(2) InVivo phenotyping demonstrates key features of the model such as early lethality and several types of electrographic seizures.

(3) The ex vivo cellular electrophysiology is very strong and comprehensive including isolated patches to accurately measure K+ currents, paired recording to measure evoked synaptic transmission, and the measurement of membrane excitability at different timepoint and in two cell types.

(4) 2P imaging relates the cellular dysfunction in PV neurons to epilepsy.

Reviewer #2 (Public review):

Summary:

Wengert et al. generated and comprehensively characterized the Kcnc1 A421V/+ knock-in mouse, which models developmental epileptic encephalopathy. The Kcnc1 gene encodes the Kv3.1 channel subunit, which, similar to the role of BK-channels in some excitatory neurons, facilitates high-frequency firing in inhibitory neurons by accelerating the downward hyperpolarization of individual action potentials. Although various Kcnc1 mutations are linked to developmental epileptic encephalopathies, the functional impact of the A421V mutation remained controversial. To elucidate its effect on the neuronal excitability and neurological functions, the authors generated cre-dependent KI mice and thoroughly characterized them using neonatal neurological assessments, high-quality in vitro electrophysiology, and in vivo imaging/electrophysiology analyses. These studies revealed impaired excitability in the PV+ inhibitory interneurons, correlating with the emergence of epilepsy and premature death. Overall, this study provides strong support for the role of the A421V mutation in disrupting inhibitory function.

Overall, the study is well-designed and conducted at a high quality. The use of a Cre-dependent KI system is effective for maintaining the mutant line despite the premature death phenotype, and may also minimize the phenotype drift that can arise when breeding from mice using milder phenotype manifestation (as ones with severe phenotype often fail to reproduce). The neonatal behavior analysis is thoroughly conducted, and the in vitro electrophysiology studies are of high quality, providing robust insights into the functional impact of the mutation.

One limitation of this study is the demonstration of the trafficking defect of mutant Kv3.1, which relies solely on the fluorescence density, and such analysis often lacks a rigorous quantitative measurement. A biochemical analysis (surface biotinylation or immunoblot using membrane fractionation) will make the conclusion more convincing, although this poses a technical challenge as the Kv3.1 is expressed primarily expressed only in a subset of PV+ cells.

While the study focused on the superficial layer because Kv3.1 is the major channel subunit, some of the neurons co-express Kv3.2, and Kv3.1 and Kv3.2 can form heteromeric channels. It would be interesting to explore whether the mutant Kv3.1 subunits exert a dominant-negative effect on Kv3.2 in these populations.

Reviewer #3 (Public review):

Summary:

Here Wengert et al., establish a rodent model of KCNC1 (Kv3.1) epilepsy by introducing the A421V mutation. The authors perform video-EEG, slice electrophysiology, and in vivo 2P imaging of calcium activity to establish a disease mechanisms involving impairment in the excitability of fast spiking parvalbumin (PV) interneurons in the cortex and thalamic PV cells.

Outside out nucleated patch recordings were used to evaluate the biophysical consequence of the A421V mutation on potassium currents and showed a clear reduction in potassium currents. Similarly action potential generation in cortical PV interneurons was severely reduced. Given that both potassium currents and action potential generation was found to be unaffected in excitatory pyramidal cells in the cortex the authors propose that loss of inhibition leads to hyperexcitability and seizure susceptibility in a mechanism similar to that of Dravet Syndrome.

Strengths:

This manuscript establishes a new rodent model of KCNC1-developmental and epileptic encephalopathy. The manuscript provides strong evidence that parvabumin interneurons are impaired by the Kcnc1-A421V mutation and that cortical excitatory neurons are not impaired. Together, these findings support the conclusion that seizure phenotypes associated with Kcnc1-A421V are caused by impaired cortical inhibition.

Weaknesses:

The manuscript identifies a partial mechanism of disease that leaves several aspects unresolved including the possible role of subcortical regions in the seizure mechanism. Similarly, while the authors identify a reduction in potassium currents and a reduction in PV cell surface expression of Kv3.1 why the A421V missense mutation leads to a more severe phenotype than previously reported loss-of-function mutations in Kv3.1is not clear.

Author response:

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

Reviewer #1 (Public review):           

Summary:

The authors have created a new model of KCNC1-related DEE in which a pathogenic patient variant (A421V) is knocked into a mouse in order to better understand the mechanisms through which KCNC1 variants lead to DEE.  

Strengths:

(1)  The creation of a new DEE model of KCNC1 dysfunction. 

(2)  In Vivo phenotyping demonstrates key features of the model such as early lethality and several types of electrographic seizures. 

(3)  The ex vivo cellular electrophysiology is very strong and comprehensive including isolated patches to accurately measure K+ currents, paired recording to measure evoked synaptic transmission, and the measurement of membrane excitability at different time points and in two cell types.

We thank Reviewer 1 for these positive comments related to strengths of the study.   

Weaknesses:

(1) The assertion that membrane trafficking is impaired by this variant could be bolstered by additional data.

We agree with this comment. However, given the technical challenges of standard biochemical experiments for investigating voltage-gated potassium channels (e.g., antibody quality), the lack of a Kv3.1-A421V specific antibody, and the fact that Kv3.1 is expressed in only a small subset of cells, we did not undertake this approach. However, we did perform additional experiments and analysis to improve the rigor of the experiments supporting our conclusion that membrane trafficking is impaired in the Kcnc1-A421V/+ mouse. 

Such experiments support a highly significant and robust difference in our (albeit imperfect) measurement of the membrane:cytosol ratio of Kv3.1 immunofluorescence between WT and Kcnc1-A421V/+ mice, which is consistent with lack of membrane trafficking (Figure 3). In the revised manuscript, we have added additional data points to this plot and updated the representative example images using improved imaging techniques to better showcase how Kcnc1-A421V/+ PV-INs differ from age-matched WT littermate controls. We think the result is quite clear. Future biochemical experiments perhaps best performed in a culture system in vitro could provide additional support for this conclusion.

(2) In some experiments details such as the age of the mice or cortical layer are emphasized, but in others, these details are omitted.

We apologize for this omission. We have now clarified the age of the mice and cortical layer for each experiment in the Methods and Results sections as well as figure legends.   

(3) The impairments in PV neuron AP firing are quite large. This could be expected to lead to changes in PV neuron activity outside of the hypersynchronous discharges that could be detected in the 2-photon imaging experiments, however, a lack of an effect on PV neuron activity is only loosely alluded to in the text. A more formal analysis is lacking. An important question in trying to understand mechanisms underlying channelopathies like KCNC1 is how changes in membrane excitability recorded at the whole cell level manifest during ongoing activity in vivo. Thus, the significance of this work would be greatly improved if it could address this question.

Yes, the impairments in the neocortical PV-IN excitability are notably severe relative to other PV interneuronopathies that we and others have directly investigated (e.g., Kv3.1 or Kv3.2-/- knockout mice; Scn1a+/- mice). In the revised version of the manuscript, we have now added a more thorough in vivo 2P calcium imaging investigation and analysis of our in vivo 2P calcium imaging data of PV-IN (and presumptive excitatory cell) neural activity (Figure 8 and Supplementary Figure 9, Methods- lines 230-271 Results- lines 630-657, and Discussion lines- 795-814). 

Because of the prominent recruitment of neuropil during presumptive myoclonic seizures, further investigation of individual neuronal excitability in vivo required a slightly different labeling strategy now using a soma-tagged GCaMP8m as well as a separate AAV containing tdTomato driven by the PV-IN-specific S5E2 enhancer. Our new results reveal an increase in the baseline calcium transient frequency in non-PV-INs, and reduced mean transient amplitudes in both non-PV cells and PV-INs. These interesting findings, which are consistent with attenuated PV-IN-mediated perisomatic inhibition leading to disinhibited excitatory cells in the Kcnc1-A421V/+ mice, link our in vivo results to the slice electrophysiology experiments. Of course, there are residual issues with the application of this technique to interneurons and the ability to resolve individual or small numbers of spikes, which likely explains the lack of genotype difference in calcium transient frequency in PV-INs.

(4) Myoclonic jerks and other types of more subtle epileptiform activity have been observed in control mice, but there is no mention of littermate control analyzed by EEG. 

We performed additional experiments as requested and did not observe myoclonic jerks or any other epileptic activity in WT control mice. We have included this data in the revised manuscript (Figure 9C).   

Reviewer #2 (Public review):           

Summary:

Wengert et al. generated and thoroughly characterized the developmental epileptic encephalopathy phenotype of Kcnc1A421V/+ knock-in mice. The Kcnc1 gene encodes the Kv3.1 channel subunit. Analogous to the role of BK channels in excitatory neurons, Kv3 channels are important for the recurrent high-frequency discharge in interneurons by accelerating the downward hyperpolarization of the individual action potential. Various Kcnc1 mutations are associated with developmental epileptic encephalopathy, but the effect of a recurrent A421V mutation was somewhat controversial and its influence on neuronal excitability has not been fully established. In order to determine the neurological deficits and underlying disease mechanisms, the authors generated cre-dependent KI mice and characterized them using neonatal neurological examination, high-quality in vitro electrophysiology, and in vivo imaging/electrophysiology analyses. These analyses revealed excitability defects in the PV+ inhibitory neurons associated with the emergence of epilepsy and premature death. Overall, the experimental data convincingly support the conclusion.

Strengths:

The study is well-designed and conducted at high quality. The use of the Cre-dependent KI mouse is effective for maintaining the mutant mouse line with premature death phenotype, and may also minimize the drift of phenotypes which can occur due to the use of mutant mice with minor phenotype for breeding. The neonatal behavior analysis is thoroughly conducted, and the in vitro electrophysiology studies are of high quality.

We appreciate these positive comments from Reviewer 2. 

Weaknesses:

While not critically influencing the conclusion of the study, there are several concerns.

In some experiments, the age of the animal in each experiment is not clearly stated. For example, the experiments in Figure 2 demonstrate impaired K+ conductance and membrane localization, but it is not clear whether they correlated with the excitability and synaptic defects shown in subsequent figures. Similarly, it is unclear how old mice the authors conducted EEG recordings, and whether non-epileptic mice are younger than those with seizures. 

We have now updated the manuscript to include clear report of age for all experiments including the impaired K⁺ conductance (now Figure 3) and EEG (now Figure 9). There was no intention to omit this information. The recordings of K⁺ conductance impairments in PV-INs from Kcnc1-A421V/+ mice were completed at P1621. Thus, we interpret the loss of potassium current density to be causally linked with the impairments in intrinsic physiological function at that same time-period in neocortical layer II-IV PV-INs and more subtly in PV-positive cells in the RTN and neocortical layer V PVINs.

Mice used in the EEG experiments were P24-48, an age range which roughly corresponded with the midpoint on the survival curve for Kcnc1-A421V/+ mice. Although we saw significant mouse-to-mouse variability in seizure phenotype, no Kcnc1-A421V/+ mice completely lacked epilepsy or marked epileptiform abnormalities, neither of which were seen in WT mice. We did not detect a clear relationship between seizure frequency/type and mouse age. 

The trafficking defect of mutant Kv3.1 proposed in this study is based only on the fluorescence density analysis which showed a minor change in membrane/cytosol ratio. It is not very clear how the membrane component was determined (any control staining?). In addition to fluorescence imaging, an addition of biochemical analysis will make the conclusion more convincing (while it might be challenging if the Kv3.1 is expressed only in PV+ cells).

This relates to comment 3 of Reviewer 1. We agree that, in the initial submission of the manuscript, the evidence from IHC for Kv3.1 trafficking deficits was somewhat subtle. In the revised version of the paper, we have gathered additional replicates of this original experiment with improved imaging quality and clarify how the membrane component was specified, to now show a robust and highly significant (***P<0.001) decrease in membrane:cytosol Kv3.1 ratio. We have also now provided new example images better showcasing the deficits observed in the Kcnc1-A421V/+ mice (Figure 3). The membrane compartment was defined as the outermost 1 micron of the parvalbumin-defined cell soma (drawn blind to the Kv3.1b signal), and, importantly, all analysis was conducted blinded to mouse genotype. These measures help to ensure that the result is robust and unbiased. Nonetheless, we have added a paragraph in the Discussion section highlighting the limitations of our IHC evidence for trafficking impairment (Lines 868-883). 

While the study focused on the superficial layer because Kv3.1 is the major channel subunit, the PV+ cells in the deeper cortical layer also express Kv3.1 (Chow et al., 1999) and they may also contribute to the hyperexcitable phenotype via negative effect on Kv3.2; the mutant Kv3.1 may also block membrane trafficking of Kv3.1/Kv3.2 heteromers in the deeper layer PV cells and reduce their excitability. Such an additional effect on Kv3.2, if present, may explain why the heterozygous A421V KI mouse shows a more severe phenotype than the Kv3.1 KO mouse (and why they are more similar to Kv3.2 KO). Analyzing the membrane excitability differences in the deep-layer PV cells may address this possibility.

We appreciate this thoughtful suggestion. We have now provided data from neocortical layer V PV interneurons in the revised manuscript (Supplementary Figure 5). Abnormalities in intrinsic excitability from neocortical layer V PV-INs in Kcnc1A421V/+ mice were present, but less pronounced than in PV-INs from more superficial cortical layers. These results are consistent with the view that greater relative expression of Kv3.2 “dilutes” the impact of the Kv3.1 A421V/+ variant. More specific determination of whether the A421V/+ variant impairs membrane trafficking and/or gating of Kv3.2 remains unclear. 

We attempted to assess how the mutant Kv3.1 affects Kv3.2 localization, but were unsuccessful due to the lack of reliable antibodies. After immunostaining mouse brain sections with two different anti-Kv3.2 antibodies, only one produced somewhat promising signal (see below). However, even in this case, Kv3.2 staining was successful only once (out of five independent staining experiments) and the signal varied across cortical regions, showing widespread cellular Kv3.2 signal in some areas (b, top panel), and barely detectable signal in others, regardless of Kv3.1 expression. In the remaining four attempts, we detected only ‘fiber-like’ immunostaining signal, further diminishing our confidence in anti-Kv3.2 antibody, although results could be improved with still further testing and refinement which we will attempt. Consequently, this important question remains unsolved in this study. 

Author response image 1.

Immunostaining of Kv3.1 and Kv3.2 in sagittal mouse brain sections. a) An example of intracellular Kv3.2 immunostaining signal, variable across the cortex of a WT mice independent of Kv3.1 expression b) Kv3.2 is detectable intracellularly in most of the cells in the top panel but barely detectable in the lowest panel. c) Representative image of Kv3.2 immunostaining signal in other sagittal mouse brain sections.

We have discussed these important implications and limitations of our results in the Discussion (Lines 868-883). We agree with the Reviewer’s interpretation that an impact on Kv3.1/Kv3.2 heteromultimers across the neocortex may explain why the Kcnc1A421V/+ mouse exhibits a more severe phenotype than Kv3.1-/- or Kv3.2-/- mice (see below), a view which we have attempted to further clarify in the Conclusion.    

In Table 1, the A421V PV+ cells show a depolarized resting membrane potential than WT by ~5 mV which seems a robust change and would influence the circuit excitability. The authors measured firing frequency after adjusting the membrane voltage to -65mV, but are the excitability differences less significant if the resting potential is not adjusted? It is also interesting that such a membrane potential difference is not detected in young adult mice (Table 2). This loss of potential compensation may be important for developmental changes in the circuit excitability. These issues can be more explicitly discussed.

We do not entirely understand this finding and its apparent developmental component. It could be compensatory, as suggested by the Reviewer; however, it is transient and seems to be an isolated finding (i.e., it is not accompanied by compensation in other properties). It is also possible that this change in Kcnc1-A421V/+ PV-INs may reflect impaired/delayed development. We cannot test excitability at a meaningfully later time point as the mice are deceased.

The revised version of the manuscript contains additional data (Supplementary Figure 4) showing that major deficits in intrinsic excitability are still observed even when the resting membrane potential is left unadjusted. These results are further discussed in the Results section (lines 522-523) and the Discussion section (lines 727-731).   

Reviewer #3 (Public review):           

Summary:

Here Wengert et al., establish a rodent model of KCNC1 (Kv3.1) epilepsy by introducing the A421V mutation. The authors perform video-EEG, slice electrophysiology, and in vivo 2P imaging of calcium activity to establish disease mechanisms involving impairment in the excitability of fast-spiking parvalbumin (PV) interneurons in the cortex and thalamic PV cells.

Outside-out nucleated patch recordings were used to evaluate the biophysical consequence of the A421V mutation on potassium currents and showed a clear reduction in potassium currents. Similarly, action potential generation in cortical PV interneurons was severely reduced. Given that both potassium currents and action potential generation were found to be unaffected in excitatory pyramidal cells in the cortex the authors propose that loss of inhibition leads to hyperexcitability and seizure susceptibility in a mechanism similar to that of Dravet Syndrome.  

Strengths: 

This manuscript establishes a new rodent model of KCNC1-developmental and epileptic encephalopathy. The manuscript provides strong evidence that parvabumin-type interneurons are impaired by the A421V Kv3.1 mutation and that cortical excitatory neurons are not impaired. Together these findings support the conclusion that seizure phenotypes are caused by reduced cortical inhibition.

We thank Reviewer 3 for their view of the strengths of the study.

Weaknesses:

The manuscript identifies a partial mechanism of disease that leaves several aspects unresolved including the possible role of the observed impairments in thalamic neurons in the seizure mechanism. Similarly, while the authors identify a reduction in potassium currents and a reduction in PV cell surface expression of Kv3.1 it is not clear why these impairments would lead to a more severe disease phenotype than other loss-of-function mutations which have been characterized previously. Lastly, additional analysis of videoEEG data would be helpful for interpreting the extent of the seizure burden and the nature of the seizure types caused by the mutation.

We agree with this comment(s) from Reviewer 3. We studied neurons in the reticular thalamus and layer V neocortical PV-INs since they are also linked to epilepsy pathogenesis and are known to express Kv3.1. However, for most of the study, we focused on neocortical layer II-IV PV-INs, because these cells exhibited the most robust impairments in intrinsic excitability. Cross of our novel Kcnc1-Flox(A421V)/+ mice to a cerebral cortex interneuron-specific driver that would avoid recombination in the thalamus, such as Ppp1r2-Cre (RRID:IMSR_JAX:012686), could assist in determining the relative contribution of thalamic reticular nucleus dysfunction to overall phenotype as used by (Makinson et al., 2017) to address a similar question; however, we have been unable to obtain this mouse despite extensive effort. There are of course other Kv3.1expressing neurons in the brain, including in the hippocampus, amygdala, and cerebellum, and we have provided additional discussion (Lines 731-736) of this issue.

We further agree with the Reviewer that a major question in the field of KCNC1-related neurological disorders is the mechanistic underpinning of why the KCNC1-A421V variant leads to a more severe disease phenotype than other loss of function KCNC1 variants, and, further, why the mouse phenotype is more severe than the Kcnc1 knockout. Previous results and our own recordings in heterologous systems suggest that the A421V variant is more profoundly loss of function than the R320H variant (Oliver et al., 2017; Cameron et al., 2019; Park et al., 2019), which is consistent with A421V having a more severe disease phenotype. Relative to knockout of Kv3.1, our results are consistent with the view that the A421V exhibits dominant negative activity by reducing surface expression of Kv3.1 and/or Kv3.2 (an effect that would not occur in knockout mice), with a possible additional contribution of impairing gating of those Kv3.1-A421V variant containing Kv3.1/Kv3.2 heteromultimers by inclusion of A421V subunits into the heterotetramer. Our finding that the magnitude of total potassium current was reduced in PV-INs by ~50% is consistent with a combination of these various mechanisms but does not distinguish between them.

In the revised version of the manuscript, we have provided a more complete discussion of these important remaining questions regarding our interpretation of how the severity of KCNC1 disorders relates to the biophysical features of the ion channel variant (lines 868883).

Recommendations for the authors

Reviewer #1 (Recommendations for the authors):          

Major

(1) The authors suggest that the reduced K+ current density in Kcnc1-A421V/+ neurons is due in part to impaired trafficking and cell surface expression of Kv3.1 in these neurons. The data supporting this claim aren't completely convincing. First, it's difficult to visualize a difference in Kv3.1 localization in the images shown in panel H, and importantly, it seems problematic that the method to assess Kv3.1 levels in membrane vs. cytosol relied on using PV co-staining to define the membrane compartment as the outermost 1 um of the PV-defined cell soma. This doesn't seem to be the best method to define the membrane compartment, as the PV signal should be largely cytosolic.

As noted above, we have completed additional data collection to confirm our results, and have performed additional imaging and updated our example images to be more representative of the observed deficits in membrane Kv3.1 expression in the Kcnc1-A421V/+ mice. We attempted to identify a marker to more clearly label the membrane to combine with PV immunocytochemistry but were unable to do so despite some effort. 

Is it possible that in control neurons, the cytosolic PV signal localizes within the membrane-bound Kv3.1 signal, with less colocalization, whereas in Kcnc1-A421V/+ neurons, there would be more colocalization of the cytosolic PV and improperly trafficked Kv3.1.? Could the data be presented in this way showing altered colocalization of Kv3.1 with PV?

We do not entirely understand the nature of this concern. In our experiments, we utilized the PV signal to determine the cell membrane and cytosolic compartments in an unbiased manner using a 1-micron shell traced around/outside the edge of the PV signal to define the membrane compartment, with the remainder of the area (minus the nuclear signal defined by DAPI) defined as the cytosol (see Methods 176-186). Because we did not identify any alterations in PV signal or correlation between PV immunohistochemistry and tdTomato expression in Cre reporter strains between WT and Kcnc1-A421V/+ mice, we believe that our strategy for determining membrane:cytosol ratio of Kv3.1 in an unbiased manner is acceptable (albeit of course imperfect). 

Alternatively, membrane fractionation could be performed on WT vs Kcnc1-A421V/+ neurons, followed by Western blotting with a Kv3.1 antibody to show altered proportions in the cytosolic vs. membrane protein fractions. It's important that these results are convincing, as the findings are mentioned in the Abstract, the Results section, and multiple times in the Discussion, although it is still unclear how much the potential altered trafficking contributes to the decrease in K+ currents versus changes in channel gating.

Multiple technical barriers made it difficult for us to gain direct biochemical evidence for altered trafficking of the A421V/+ Kv3.1 variant (see above). It is not clear how membrane fractionation techniques could be easily applied in this case (at least by us) when PV-INs constitute 3-5% of all neocortical neurons. We further agree (as noted above) that it is difficult to properly disentangle the relative roles of impaired membrane trafficking vs. gating deficits to the observed effect; however, we think that both phenomena are likely occurring. In the revised version of the manuscript, we have more explicitly discussed these limitations in the Discussion section (Lines 868-883).   

(2) More information is needed regarding the age of mice used for experiments for the following results (added to the Results section as well as figure legends):

PV density (Supplementary Figure 1) 

K+ current data (Figure 2A-G)       

Kv3.1 localization (Figure 2H and I)        

RTN electrophysiology (Supplementary Figure 3)

Excitatory neuron electrophysiology (Figure 4)             

In vivo 2P calcium imaging (Figure 7) 

Video-EEG (Figure 8)

We apologize for omitting this critical information. In the revised manuscript, we have provided the age of mice for each of our experiments in the results section, in the figure legend, and in the methods section.   

(3) It's unclear why developmental milestones/behavioral assessments were only done at P5-P10. In the previous publication of another Kcnc1 LOF variant (Feng et al. 2024), no differences were found at P5-P10, and it was suggested in the discussion that this finding was "consistent with the known developmental expression pattern of Kv3.1 in mouse, where Kv3.1 protein does not appear until P10 or later". In that paper, they did find behavioral deficits at 2-4 months. Even though this model is more severe than the previous model, it would be interesting to determine if there are any behavioral deficits at a later time point (especially as they find more neurophysiological impairments at P32P42).

As in our previous study, the lack of clear behavioral deficits in developmental milestones from P5-15 is potentially expected considering the developmental expression of Kv3.1, and we performed these experiments primarily to showcase that the Kcnc1-A421V/+ mice exhibit otherwise normal overall early development (although this could be an artifact of the sensitivity of our testing methods).

For the revised manuscript, we have conducted additional experiments to investigate behavioral deficits in adult Kcnc1-A421V/+ mice. We found cognitive/learning deficits in both Kcnc1-A421V/+ mice relative to WT in both the Barnes maze (Figure 2A-C) and Ymaze (Figure 2D-F). Other aspects of animal behavior including cerebellar-related motor function are likely also impaired at post-weaning timepoints, and will be included in a forthcoming research study focusing on the motor function in these mice.  

(4) In the Results section, it should be more clearly stated which cortical layer/layers are being studied. In some cases, it mentions layers 2-4, and in some, only layer 4, and in others, it doesn't mention layers at all. Toward the beginning of the Results section, the rationale for focusing on layers 2-4 to assess the effects of this variant should be well described and then, for each experiment, it should be stated which cortical layers were assessed. Related to this point, it seems electrophysiology was only done in layer 4; the rationale for this should also be included.

We have now clarified which neocortical layers were under investigation in the study. All PV-INs were targeted in somatosensory layers II-IV, while excitatory neurons were either cortical layer IV spiny stellate cells or pyramidal cells. Paired recordings were also completed in layer IV. We have also more explicitly articulated our rationale for looking at PV-INs in layers II-IV to examine the cellular/circuitlevel impact of Kv3.1 in a model of developmental and epileptic encephalopathy (Lines 487-491). 

(5) Kcnc1-A421V/+ PV neurons showed more robust impairments in AP shape and firing at P32-42 than at P16-21 (Figure 3), and only showed synaptic neurotransmission alterations at P32-42 (Figure 6). Thus, it's unclear why Kcnc1-A421V/+ excitatory neurons were only assessed at P16-21 (Figure 4 and Supplementary Figure 4 related to Figure 5), particularly if only secondary or indirect effects on this population would be expected.

We appreciate this excellent point raised by the Reviewer and we have taken the suggestion to examine excitatory neurons at P32-42 in addition to the earlier juvenile timepoint. Our new results from the later timepoint are similar to our results at P16-21: Excitatory neurons show no statistically significant impairments in intrinsic excitability at either of the two timepoints examined (Supplementary Figure 7). This adds support to our original conclusion that PV-INs represent the major driver of disease pathology across development.   

(6) The 2P calcium imaging experiments are potentially interesting, however, a relationship between these results and the electrophysiology results for PV neurons is lacking. Was there an attempt to assess the frequency and/or amplitude of calcium events specifically in PV neurons, outside of the hypersynchronous discharges, to determine whether there are differences between WT and Kcnc1-A421V/+, as was seen in the electrophysiological analyses? It does seem there are some key differences between the two experiments (age: later timepoint for 2P vs. P16-21 and P32-42, layer: 2/3 vs. 4, and PV marking method: virus vs. mouse line), but the electrophysiological differences reported were quite strong. Thus, it would be surprising if there were no alterations in calcium activity among the Kcnc1-A421V/+ PV neurons.

In our initial experiments, the prominent neuropil GCaMP signal in Kcnc1-A421V/+ mice rendered it difficult to distinguish and accurately describe baseline neuronal excitability in PV-INs and non-PV cells. In our revised manuscript, we utilized a soma-tagged GCaMP8m and separately labeled PV-INs through S5E2-tdTomato. This strategy made it possible to assess the amplitude and frequency of calcium transients in both PV-positive and PV-negative cells in vivo. We have updated the description of our methods (lines 230-271) and our results (lines 630-657) in the revised manuscript.

As noted above, our more detailed analysis of somatic calcium transients in PV-IN and non-PV cells during quiet rest (Figure 8 and Supplementary Figure 9) shows that PV-INs from Kcnc1-A421V/+ mice are abnormally excitable- having reduced transient amplitude relative to WT controls. Interestingly, non-PV cells also exhibited an increased calcium transient frequency and reduced amplitude which is potentially consistent with reduced perisomatic inhibition causing disinhibition in cortical microcircuits. We again highlight that the slow kinetics of GCaMP combined with the calcium buffering and brief spikes of PVINs render quantification of action potential frequency and comparisons between groups difficult.  

(7) As mentioned above, it would be helpful to state the time points or age ranges of these experiments to better understand the results and relate them to each other. For example, the 2P imaging showed apparent myoclonic seizures in 7/7 Kcnc1-A421V/+ mice (recorded for a total of 30-50 minutes/mouse), but the video-EEG showed myoclonic seizures in only 3/11 Kcnc1-A421V/+ mice (recorded for 48-72 hours/mouse). Were these experiments done at very different age ranges, so this difference could be due to some sort of progression of seizure types and events as the mice age? Is it possible these are not the same seizure types (even though they are similarly described)? This discrepancy should be discussed.

Mice in the EEG experiments were between the ages of P24 and 48, slightly younger than the age in which we carried out the in vivo calcium imaging experiments (>P50). Therefore, an age-related exacerbation in myoclonic jerks is possible. 

As is highlighted by the Reviewer, it is interesting that the myoclonic seizures were only detected in a portion of the Kcnc1-A421V/+ mice during EEG monitoring (4/12). We believe that the difference is most likely driven by more sensitive detection of the myoclonic jerk activity and behavior in the 2P imaging of neuropil cellular activity compared to our video-EEG monitoring and 2P imaging of soma-tagged GCaMP. We have occasionally observed repetitive myoclonic jerking in mice that appears highly localized (i.e. one forepaw only) suggesting that the myoclonic seizures exist on a spectra of severity from focal to diffuse. It is therefore possible that myoclonic events and electrographic activity may be slightly underestimated in our video-EEG experiments? 

We have now added a few lines discussing this discrepancy in the Discussion (lines 809814).   

(8) Myoclonic jerks and other types of more subtle epileptiform activity have been observed in control mice. Was video-EEG performed on control mice? These data should be added to Figure 8.

We have added recordings in control WT mice (N=4). We did not detect myoclonic jerks or other epileptiform activity in the control mice (Figure 9).  

Minor

(1) In the first Results section, Line 365, the P value (P<0.001) is different from that in the legend for Figure 1, line 743 (P<0.0001).

We have fixed this discrepancy. 

(2) For Supplementary Figure 1, it would be helpful to show images that span the cortical layers (1-6), as PV and Kv3.1 are both expressed across the cortical layers.

We have updated Supplementary Figure 1 with better example images that span the cortical layers.    

(3) Error bars should be added to the line graphs in Supplementary Figure 2, particularly panels B and C. Some of the differences appear small considering the highly significant p-values (i.e. body weight at P7 and brain weight at P21).

The values shown in Supplementary Figure 2D-E are percentages of mice displaying a particular characteristic, so there is no variance for the data.

Supplementary Figure 2B-C actually do contain error bars plotted as SEM, however, because of the large number of N and small degree of variance in the measurements, the error bars are not apparent in the graphs. This has been noted in the Supplementary Figure 2 legend for clarity. 

(4) In Figure 3, although the Kcnc1-A421V/+ neurons have elevated AP amplitudes relative to WT, the representative traces for P16-21 and P32-42 groups appear strikingly opposite (traces in B in G appear to have much higher amplitudes than those in C and H). As this is one of the three AP phenotypes described, it would be nice to have it reflected in the traces.

We have updated our example traces to better represent our main findings including AP amplitude for both P16-21 and P32-42 timepoints.  

(5) Were any effects on the AHP assessed in the electrophysiology experiments? As other studies have reported the effects of altered Kv3 channel activity on AHP, this parameter could be interesting to report as well.

We have now provided data on the afterhyperpolarization for each condition displayed in the Supplementary data tables. Interestingly, we failed to detect significant differences in AHP between WT and Kcnc1-A421V/+ PV-INs, RTN neurons, or pyramidal cells, although we did identify differences in the dV/dt of the repolarization phase of the AP.   

(6) The figure legend for Figure 7 has errors in the panel labeling (D instead of C, and two Fs).

This error has been corrected in the revised manuscript.

Reviewer #3 (Recommendations for the authors):

Specific comments and questions for the authors:         

(1) Do the authors provide a reason for why the juvenile animals are unaffected by the A421V mutation? Is it that PV cells have not fully integrated at this early time point or that Kv3.1 expression is low? Is the developmental expression profile of Kv3.1 in PV cells known and if so could the authors update the discussion with this information?

We interpret the normal early developmental milestones (P5-P15) to reflect that Kcnc1-A421V/+ mice exhibit the onset of their neurological impairment at the same time that PV-INs upregulate Kv3.1, develop a fast-spiking physiological phenotype, and integrate into functional circuits in the third and fourth postnatal weeks. We have updated the discussion (Line 780-782) with this information and more clearly describe our interpretation of these early-life behavioral experiments.   

(2) I would like to see a more complete analysis of the Video-EEG data that is included in Figure 8. What was the seizure duration and frequency? Were there spike-wave seizure types observed? Were EEG events that involve thalamocortical circuitry affected such as spindles? Was sleep architecture impaired in the model? Were littermate control animals recorded?

Although classical convulsive seizures represent only part of the overall epilepsy phenotype that this mouse exhibits, we agree that reporting seizure duration and frequency is important. We have now included this in our revised manuscript (line 624-626). We have also now added WT control mice to our dataset, and, as expected, we failed to observe any epileptic features in our WT recordings.

In our EEG experiments, we did not record EMG activity in the mouse to allow for unambiguous determination of sleep vs. quiet wakefulness. For that reason, and because we believe it beyond the scope of this particular study, we did not examine sleep-related EEG phenomena such as spindles or sleep architecture. We have, however, added a line in the discussion (line 771-774) suggesting that future studies focus on a more thorough investigation of the EEG activity in these animals. 

(3) The in vivo calcium imaging data shows synchronous bursts in A421V animals which is in agreement with the synchronous bursts observed in the EEG. Overall the analysis of the in vivo calcium imaging data appears to be rudimentary and perhaps this is a missed opportunity. What additional insights were gained from this technically demanding experiment that were not obtained from the EEG recordings?

As noted above, in the revised version of the manuscript, we have conducted additional experiments which allowed us to separately examine PV-IN and non-PV neuron excitability via 2P in vivo calcium imaging. This required an alternative strategy to label individual neuronal somata without contamination by the robust neuropil signal that we observed in the approach undertaken in the original submission. We’ve described the details of this new approach in methods (Lines 230-271) and results section (lines 630-657).

Our new results (Figure 8 and Supplementary Figure 9) reveal that, during quiet rest, neocortical PV-INs from Kcnc1-A421V/+ mice exhibit a reduction in calcium transient amplitude during quiet wakefulness and that non-PV cells exhibit altered transient frequency and amplitude. Overall, we believe that these results are consistent with the view that PV-IN-mediated perisomatic inhibition is compromised in Kcnc1-A421V/+ mice which leads to a downstream hyperexcitability in excitatory neurons within cortical microcircuits.  

(4) The increased severity of seizure phenotypes observed in the A421V model relative to knockout mice is interesting but also confusing given what is known about this mutation. As the authors point out, a possible explanation is that the mutation is acting in a dominant negative manner, where mutant Kv3.1 channels compete with other Kvs that would otherwise be able to partially compensate for the loss of Kv function. Alternatively, the A421V mutation might act by affecting the trafficking of heterotetrameric Kv3 channels to the membrane. Can the authors clarify why a trafficking deficit would produce a different effect than a loss of function mutation? Are the authors proposing that a hypomorphic mutation involving both a partial trafficking deficit and a dominant negative effect of those channels that are properly localized is more severe than a "clean" loss of function? The roughly 50% loss of potassium current absent a change in gating would be expected to behave like a loss-of-function mutation. This might be addressed by comparing the surface expression of the other Kv channels and/or through the use of Kv3.1-selective pharmacology.

These are excellent points raised by the Reviewer. As noted above, we have endeavored to clarify our hypothesis as to the basis of this phenomenon, although the mechanistic basis for the more severe phenotype in the Kcnc1-A421V/+ mouse relative to the Kv3.1 knockout is not entirely clear. Our physiology results and the evidence presented supporting a trafficking impairment, are consistent with dominant negative action of the Kv3.1 A421V variant at the level of channel gating and/or trafficking. To restate, we think the Kcnc1-A421V/+ heterozygous variant is more severe than a Kv3.1 knockout for (at least) three reasons: variant Kv3.1 is incorporated into Kv3.1/Kv3.2 heterotetramers to (1) impair trafficking to the membrane as well as (2) alter the electrophysiological function of those channels that do successfully traffic to the membrane (while Kv3.1 knockout affects Kv3.1 only), and (3) the heterozygous variant may escape compensatory upregulation of Kv3.2 and which is known to occur in Kv3.1 knockout mice.

For example, our data suggests and is consistent with the view that heterotetramers of WT Kv3.1 and Kv3.2 potentially come together with the A421V Kv3.1 subunit in the endoplasmic reticulum and then fail to traffic to the membrane due to the presence of one or more A421V subunit(s), as evidenced by increased Kv3.1 staining in the cytosol in the Kcnc1-A421V/+ mouse relative to WT. This is in contrast to what would occur in the Kv3.1knockout mice as there is no subunit produced from the null allele to impair WT Kv3.2 subunits from forming fully functional Kv3.2 homotetramers to then reach the cell surface and function properly. This is one specific possible mechanism for dominant negative activity.

A non-mutually-exclusive mechanism is that inclusion of one or more Kv3.1 A421V subunits into Kv3 heterotetramers impairs gating and prevents potassium flux such that, even if the tetramer does reach the membrane, that entire tetramer fails to contribute to the total potassium current. This is another possible mechanism for dominant negative function of the A421V subunit.

Experimental elucidation of the precise mechanism of the dominant negative activity of the A421V Kcnc1 variant is beyond the scope of this study; yet, our lab is continuing to work on this. It will likely require dose-response experiments in which various ratios of WT and Kv3.1 A421V subunits are co-expressed in heterologous cells and then recorded for an overall effect on potassium current similar to (Clatot et al., 2017).

In the revised manuscript, we have updated our discussion of these mechanistic considerations for KCNC1-related epilepsy syndromes in lines 868-883 in the Discussion. 

References

Cameron JM et al. (2019) Encephalopathies with KCNC1 variants: genotype-phenotypefunctional correlations. Annals of Clinical and Translational Neurology 6:1263– 1272.

Clatot J, Hoshi M, Wan X, Liu H, Jain A, Shinlapawittayatorn K, Marionneau C, Ficker E, Ha T, Deschênes I (2017) Voltage-gated sodium channels assemble and gate as dimers. Nature Communications 8.

Makinson CD, Tanaka BS, Sorokin JM, Wong JC, Christian CA, Goldin AL, Escayg A, Huguenard JR (2017) Regulation of Thalamic and Cortical Network Synchrony by Scn8a. Neuron 93:1165-1179.e6.

Oliver KL et al. (2017) Myoclonus epilepsy and ataxia due to KCNC1 mutation: Analysis of 20 cases and K+ channel properties. Annals of Neurology 81.

Park J et al. (2019) KCNC1-related disorders: new de novo variants expand the phenotypic spectrum. Annals of Clinical and Translational Neurology 6:1319–1326.

optical sensors are passive - measure reflected soalr radiation in the visible,near-infrared and short-wave infrared regions

Spectrum of reflected radiation depends on whats absorbed by the surface so different surfaces produced individual spectral signatures

In active systems, a specific electromagnetic radiation signal is transmitted from the instrument and the sensor detects the component of the sign that's reflect back Radar * LiDAR

Passive systems detect SW that is reflected or LW which is emitted back

Natural radiation is the measurement source

Fates of waves are its absorbed, scattered or transmitted

classification of electromagnetic spectrum is based on WL

Digital images have numeric values assigned to the pixels representing the intensity of measured quatitys

over 1000 EO sats in orbit, small CubeSats 10cm3 and large multisenores like Nasas terrra sats

EO = earth observation

You must learn, and you must act. Both are needed for habit building and sustainable projects (community commitment to resist black swans).

@RealDidacticus

eLife Assessment

This valuable study provides solid evidence that supports TANGO2 homologs, including HRG-9 and HRG-10, can play a role in cellular bioenergetics and oxidative stress homeostasis. It also challenges the previously reported role of TANGO in heme transport and paves the way for future mechanistic studies addressing the mechanisms of how TANGO2 regulates oxidative stress homeostasis. The strengths include the use of different model systems, genetic tools, behavioral assays and efforts by the authors in using the same reagents to reproduce results of other groups.

Reviewer #1 (Public review):

Sandkuhler et al. re-evaluated the biological functions of TANGO2 homologs in C. elegans, yeast, and zebrafish. Compared to the previously reported role of TANGO2 homologs in transporting heme, Sandkuhler et al. expressed a different opinion on the biological functions of TANGO2 homologs. With the support of some results from their tests, they conclude that 'there is insufficient evidence to support heme transport as the primary function of TANGO2', in addition to the evidence that C. elegans TANGO2 helps counteract oxidative stress.. While the differences are reported in this study, more work is needed to elucidate the intuitive biological function of TANGO2.

Strengths:

(1) This work revisits a set of key experiments, including the toxic heme analog GaPP survival assay, the fluorescent ZnMP accumulation assay, and the multi-organismal investigations documented by Sun et al. in Nature (2022), which are critical for comparing the two works. Meanwhile, the authors also highlight the differences in reagents and methods between the two studies, demonstrating significant academic merit.

(2) This work reported additional phenotypes for the C. elegans mutant of the TANGO2 homologs, including lawn avoidance, reduced pharyngeal pumping, smaller brood size, faster exhaustion under swimming test, and a shorter lifespan. These phenotypes are important for understanding the biological function of TANGO2 homologs, while they were missing from the report by Sun et al.

(3) Investigating the 'reduced GaPP consumption' as a cause of increased resistance against the toxic GaPP for the TANGO2 homologs, hrg-9 hrg-10 double null mutant provides a valuable perspective for studying the biological function of TANGO2 homologs.

(4) The induction of hrg-9 gene expression by paraquat indicates a strong link between TANGO2 and mitochondrial function.

(5) This work thoroughly evaluated the role of TANGO2 homologs in supporting yeast growth using multiple yeast strains and also pointed out the mitochondrial genome instability feature of the yeast strain used by Sun et al.

Weakness:

It is always a challenge to replicate someone else's work, but it is worthwhile to take on the challenge, provide evidence, and raise concerns about it. These authors attempted to replicate the experiment using the same biological material as that used by Sun et al. in Nature (2022), despite some experimental differences between the two studies. This study does not have many technical weaknesses, but it can become a much better project by focusing on the new phenotypes discovered here.

Reviewer #2 (Public review):

This work offers a valuable re-evaluation of earlier claims from other groups about TANGO2 functions and proposes that energy-related and stress-related pathways may be more important to the disorder than previously thought. A key strength of this work is the use of multiple model systems. The authors provide solid data that show how TANGO2 is probably only indirectly involved in heme transport and provide support for alternative mechanisms where TANGO2 is actually directly control. These findings provide valuable information for researchers seeking more accurate therapeutic targets.

Strengths:

The study refutes earlier claims about TANGO2's involvement in heme transport and extends previous findings by implicating TANGO2 in metabolism and oxidative stress, thereby highlighting new aspects of its role in cell physiology. The use of different model systems (Saccharomyces cerevisiae, Caenorhabditis elegans, Danio rerio) to address the main research questions is useful and demonstrates evolutionary conservation of the studied processes. Finally, the results suggest a broader impact than previously described, somewhat supporting the novelty of the study.

Weaknesses:

Although the phenotypic analyses are broad and generally well executed, a key limitation is that the main conclusions mainly rely on these readouts. While informative, sole phenotypic analyses cannot directly demonstrate the underlying molecular mechanisms proposed by the authors. The study includes limited functional or biochemical assays connecting TANGO2 orthologs to the proposed energy and stress pathways. Some observations would benefit from additional orthogonal validation to strengthen the overall interpretation. As a result, the evidence supporting the central mechanistic interpretation remains indirect, although compelling.

Overall, the authors have achieved their stated aims, and their results mainly support their main conclusion (i.e., TANGO2 is unlikely to function in heme transport and is probably linked to energy and stress pathways). However, much of the evidence comes from phenotypic analyses, which limits the strength of the mechanistic claims, leaving the proposed pathways somewhat indirect.

This work is likely to have a valuable impact on the subfield by clarifying that TANGO2 is not involved (at least directly) in heme transport and clarifying its actual role in energy and stress-related processes. By rigorously reassessing and confuting earlier claims from other studies across multiple model systems, the current work will help to guide the future research and therapeutic exploration in the context of TANGO2 deficiencies. This study will provide a solid foundation for more mechanistic insights into TANGO2 function.

Reviewer #3 (Public review):

In this paper, Sandkuhler et al. reassessed the role of TANGO2 as a heme chaperone proposed by Sun et al in a recently published paper (https://doi.org/10.1038/s41586-022-05347-z). Overall, Sandkuhler et al. conclude that the heme-related roles of TANGO2 had been overemphasized by Sun et al. especially because the hrg9 gene does not exclusively respond to different regimens of heme synthesis/uptake but is susceptible to a greater extent to, for example, oxidative stress. Impaired heme trafficking is then interpreted as due to general mitochondrial dysfunction. In recent years, the discussion around the heme-related roles of TANGO2 has been tantalizing but is still far from a definitive consensus. Discrepancies between results and their interpretation are testament to how ambitious the understanding of TANGO2 and the phenotypes associated with TANGO2 defects are.

The work presented by Sandkuhler et al. is methodologically sound, and the authors have appropriately addressed my concerns in the first round of review. Overall, this paper challenges the recent developments in the field in relation to heme trafficking and provides a wider perspective on the biological roles of TANGO2.

Author response:

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

Reviewer #1 (Public review):

(1) A detailed comparison between this work and the work of Sun et al. on experimental protocols and reagents in the main text will be beneficial for readers to assess critically.

We have added a Key Reagents Table outlining the key reagents used in our study. In terms of experimental protocols, we replicated those described by Sun et al. in most instances and described any differences when present. With this resubmission, we included additional ZnMP accumulation experiments in liquid media (see point 3 below).

(2) The GaPP used by Sun et al. (purchased from Frontier Scientific) is more effective in killing the worm than the one used in this study (purchased from Santa Cruz). Is the different outcome due to the differences in reagents? Moreover, Sun et al. examined the lethality after 3-4 days, while this work examined the lethality after 72 hours. Would the extra 24 hours make any difference in the result?

We now cite product vender differences as a possible reason for the observed difference in worm death, as the reviewer suggests, on page 8 (see text below) and include these differences in the Key Reagents Table. We also now stress the fact that our experiments included different doses of GaPP and the use of eat-2 mutants as an additional control, which we believe adds rigor and demonstrates the potency of GaPP in our experiments. We decided on assessment at 72 hours, as we deemed it a less nebulous time point as compared to 3-4 days. Most of the observed worm death occurred earlier in this interval, so we believe it is unlikely that large group differences would emerge after an additional 24 hours.

“Exposing worms to GaPP, a toxic heme analog, we observed that nematodes deficient in HRG-9 and HRG-10 displayed increased survival compared to WT worms, consistent with prior work,[13] though the between-group difference was markedly smaller in our study. We required higher GaPP concentrations to induce lethality, potentially due to product vendor differences, but did observe a clear dose-dependent effect across strains. Although it was previously proposed that the survival benefit seen in worms lacking HRG-9 and HRG-10 resulted from reduced transfer from intestinal cells after GaPP ingestion, our data suggest the reduced lethality is more likely due to decreased environmental GaPP uptake. Supporting this notion, DKO worms exhibited lawn avoidance, reduced pharyngeal pumping, and modestly lower intestinal ZnMP accumulation when exposed to this fluorescent heme analog on agar plates. In liquid media, DKO worms demonstrated higher fluorescence, but only in ZnMP-free conditions, suggesting the presence of gut granule autofluorescence. Furthermore, survival following exposure to GaPP was highest in eat-2 mutants, despite heme trafficking being unaffected in this strain.”

(3) This work reported the opposite result of Sun et al. for the fluorescent ZnMP accumulation assay. However, the experimental protocols used by the two studies are massively different. Sun et al. did the ZnMP staining by incubating the L4-stage worms in an axenic mCeHR2 medium containing 40 μM ZnMP (purchased from Frontier Scientific) and 4 μM heme at 20 ℃ for 16 h, while this work placed the L4-stage worms on the OP50 E. coli seeded NGM plates treated with 40 μM ZnMP (purchased from Santa Cruz) for 16 h. The liquid axenic mCeHR2 medium is bacteria-free, heme-free, and consistent for ZnMP uptake by worms. This work has mentioned that the hrg-9 hrg-10 double null mutant has bacterial lawn avoidance and reduced pharyngeal pumping phenotypes. Therefore, the ZnMP staining protocol used in this work faces challenges in the environmental control for the wild type vs. the mutant. The authors should adopt the ZnMP staining protocol used by Sun et al. for a proper evaluation of fluorescent ZnMP accumulation.

We agree with this comment. As such, we performed the ZnMP assay in liquid media conditions, as now described on page 13:

“For liquid media experiments, three generations of worms were cultured in regular heme (20 uM) axenic media, with the first two generations receiving antibiotic-supplemented media (10 mg/ml tetracycline) and the 3^rd generation cultivated without antibiotic. L4 worms from the 3^rd generation were placed in media containing 40uM ZnMP for 16 hours before being prepared and mounted for imaging as above. Worms were imaged on Zeiss Axio Imager 2 at 40x magnification, with image settings kept uniform across all images. Fluorescent intensity was measured within the proximal region of the intestine using ImageJ.”

In heme-free media, both WT and DKO worms invariably entered L1 arrest, thus we were not able to replicate the results reported by Sun et al. Using media containing heme, we did see an increase in fluorescence, but this was only in the ZnMP-free condition, indicating that the increased signal was attributable to autofluorescence. This is a known phenomenon associated with gut granules in C. elegans in the setting of oxidative stress. The results of these experiments are now summarized on page 6:

“DKO nematodes at the L4 larval stage were previously shown to accumulate the fluorescent heme analog zinc mesoporphyrin IX (ZnMP) in intestinal cells in low-heme (4 µM) liquid media. While attempting to replicate this experiment, we observed that both wildtype and DKO nematodes entered L1 arrest under these conditions. Therefore, to allow for developmental progression, we grew worms on standard OP50 E. coli plates and in media containing physiological levels of heme (20 µM). We then examined whether differences in ZnMP uptake persisted under these basal conditions. DKO worms grown on ZnMP-treated E. coli plates displayed significantly reduced intestinal ZnMP fluorescence compared to N2 (Figure 1B and C). Using basal heme media with ZnMP, there was no significant difference in ZnMP fluorescence between DKO and wildtype nematodes, although DKO worms grown in media without ZnMP exhibited significantly higher autofluorescence (Figure 1D and E). To test whether autofluorescence may have contributed to the higher fluorescent intensities previously reported in heme-deficient DKO worms, we repeated this experiment on agar plates under starved conditions but did not observe a difference between groups (Figure 1B).”

(4) A striking difference between the two studies is that Sun et al. emphasize the biochemical function of TANGO2 homologs in heme transporting with evidence from some biochemical tests. In contrast, this work emphasizes the physiological function of TANGO2 homologs with evidence from multiple phenotypical observations. In the discussion part, the authors should address whether these observed phenotypes in this study can be due to the loss of heme transporting activities upon eliminating TANGO2 homologs. This action can improve the merit of academic debate and collaboration.

Thank you for this suggestion. The following text has been added to the Discussion section (page 9):

“In addition to altered pharyngeal pumping, DKO worms displayed multiple previously unreported phenotypic features, suggesting a broader metabolic impairment and reminiscent of some clinical manifestations observed in patients with TDD. Elucidating the mechanisms underlying this phenotype, and whether they reflect a core bioenergetic defect, is an active area of investigation in our lab. Several C. elegans heme-responsive genes have been characterized, revealing relatively specific defects in heme uptake or utilization rather than broad organismal dysfunction. For example, hrg-1 and hrg-4 mutants exhibit impaired growth only under heme-limited conditions,[23] and hrg-3 loss affects brood size and embryonic viability specifically when maternal heme is scarce.[24] ]By contrast, hrg-9 and hrg-10 mutants exhibit the most severe organismal phenotypes of the hrg family, to date, including reduced pharyngeal pumping, decreased motility, shortened lifespan, and smaller broods, even when fed a heme-replete diet.”

Reviewer #2 (Public review):

(1) The manuscript is written mainly as a criticism of a previously published paper. Although reproducibility in science is an issue that needs to be acknowledged, a manuscript should focus on the new data and the experiments that can better prove and strengthen the new claims.

Thank you for this suggestion. While the primary intent of this study was to replicate key findings from the 2022 publication by Sun et al., the revised manuscript now emphasizes underlying mechanisms more broadly rather than focusing narrowly on that prior publication.

(2) The current presentation of the logic of the study and its results does not help the authors deliver their message, although they possess great potential.

We have attempted to rectify this through substantial revision of the Discussion section and other places throughout the manuscript.

(3) The study is missing experiments to link hrg-9 and hrg-10 more directly to bioenergetic and oxidative stress pathways.

The reviewer is correct in this assertion, but it was not our intent to definitively prove this link or, indeed, the primary mechanism of TANGO2 in the present manuscript. This said, we are actively engaged in this endeavor in our lab and anticipate these data will be published in a separate, forthcoming publication.

We have added additional references pertaining to hrg-9 enrichment as part of the mitochondrial unfolded protein response (page 10) and a comparison of the phenotype observed in hrg-9 and hrg-10 deficient worms versus those lacking other proteins in the hrg family (page 9).

Reviewer #3 (Public review):

(1) The authors stress - with evidence provided in this paper or indicated in the literature - that the primary role of TANGO2 and its homologues is unlikely to be related to heme trafficking, arguing that observed effects on heme transport are instead downstream consequences of aberrant cellular metabolism. But in light of a mounting body of evidence (referenced by the authors) connecting more or less directly TANGO2 to heme trafficking and mobilization, it is recommended that the authors comment on how they think TANGO2 could relate to and be essential for heme trafficking, albeit in a secondary, moonlighting capacity. This would highlight a seemingly common theme in emerging key players in intracellular heme trafficking, as it appears to be the case for GAPDH - with accumulating evidence of this glycolytic enzyme being critical for heme delivery to several downstream proteins.

TANGO2 is essential for mitochondrial health, albeit in a yet unknown capacity. In the absence of TANGO2, defects in heme trafficking may be secondary sequelae of mitochondrial dysfunction. We would point out that prior studies that attempted to show that TANGO2 and its homologs are involved in heme trafficking proposed very different mechanisms (direct binding vs. membrane protein interaction) and relied on artificially low or high heme conditions to produce these effects. We have attempted to address these more clearly in the Discussion section and have added a fifth figure to summarize our current unifying theory for how heme levels and mitochondrial stress may be linked.

(2) The observation - using eat-2 mutants and lawn avoidance behaviour - that survival patterns can be partially explained by reduced consumption, is fascinating. It would be interesting to quantify the two relative contributions.

We have completed additional ZnMP experiments in liquid media at the reviewers’ request. This experimental condition eliminates lawn avoidance as a factor in consumption. Fluorescent intensity was significantly higher in the DKO worms in media lacking ZnMP, indicating increased autofluorescence in DKO worms, while signal was not significantly different in media with ZnMP.

(3) In the legend to Figure 1A it's a bit unclear what the differently coloured dots represent for each condition. Repeated measurements, worms, independent experiments? The authors should clarify this.

The following sentence has been added to the legend for Figure 1:

“Each dot represents the number of offspring laid by one adult worm on one GaPP-treated plate after 24 hours.”

(4) It would help if the entire fluorescence images (raw and processed) for the ZnMP treatments were provided. Fluorescence images would also benefit Figure 1B.

Fluorescent intensity values pertaining to the ZnMP experiments are included in our Extended Data supplement, and we have added representative images to Figure 1, per the reviewer’s request. We thank the reviewer for this helpful suggestion. We would be happy to upload raw images to an open-access repository if deemed necessary by the editorial team.

(5) Increasingly, the understanding of heme-dependent roles relies on transient or indirect binding to unsuspected partners, not necessarily relying on a tight affinity and outdating the notion of heme as a static cofactor. Despite impressive recent advancements in the detection of these interactions (for example https://doi.org/10.1021/jacs.2c06104; cited by the authors), a full characterisation of the hemome is still elusive. Sandkuhler et al. deemed it possible but seem to question that heme binding to TANGO2 occurs. However, Sun et al. convincingly showed and characterised TANGO2 binding to heme. It is recommended that the authors comment on this.

We believe it is plausible that TANGO2 binds heme (as do hundreds of other proteins), especially as it has been shown to bind other hydrophobic molecules. However, we also note that a separate paper examining the role of TANGO2 in heme transport posited that GAPDH is the sole heme binding partner for cytoplasmic transport (https://doi.org/10.1038/s41467-025-62819-2), contradicting the originally posited theory of how TANGO2 functions. This is described in the Discussion section and, as noted above, we have added an additional figure to demonstrate our unifying hypothesis for why TANGO2 may be important in the low-heme state, irrespective of any direct effect on heme trafficking.

Additional comments and revisions:

(1) It was suggested that a triple mutant (eat-2; hrg-9; hrg-10) be tested to determine the primary driver of GaPP toxicity. We appreciate this suggestion, but we offer the following rationale for why these experiments were not pursued. The eat-2 mutant, which lacks a nicotinic acetylcholine receptor subunit in pharyngeal muscles, was included solely as a dietary restriction control to illustrate that reduced GaPP toxicity in the hrg-9/10 double mutant could arise from poor feeding rather than defective heme transport. Both eat-2 and hrg-9/10 mutants exhibit markedly reduced feeding but via different mechanisms. In our assays, GaPP survival was inversely correlated with ingestion rate: eat-2 animals, which feed the least, showed the highest survival, while hrg-9/10 mutants showed intermediate feeding and intermediate survival. Consistent with this, eat-2 worms also displayed the lowest ZnMP accumulation.

(2) GaPP solution was added to NGM plates after seeding with OP50. This is now expressly stated in the Methods section (page 15). We would note that Sun et al. mixed GaPP in with NGM in the liquid phase. We would expect that if there were a difference in GaPP exposure due to these different protocols, worms in our experiment would have received higher GaPP concentrations.

“Standard NGM plates were treated with 1, 2, 5, or 10 µM gallium protoporphyrin IX (GaPP; Santa Cruz) after seeding with OP50. Plates were swirled to ensure an even distribution of GaPP and allowed to dry completely.

(3) The manuscript has been reworked to read as more of an independent study rather than a rebuttal of prior work, though the primary objective of validating prior work remains unchanged.

(4) Several technical details of experiments have been moved from the main text to the materials and methods section.

(5) One reviewer noted that the figure numbering should be adjusted. Numbering does not progress sequentially (i.e., 1A…1B…2A…2B) early in the text, because we have opted to consolidate data pertaining to heme analog experiments in Figure 1 and behavioral data in Figure 2.

(6) “Kingdoms” has been changed to “domains” (page 4).

(7) Example images are now included for Figure 1B, as noted above.

Can we add the business type options that we have? to show which biz types we support

The onboarding journey is a bit different for US. Can we align this to the existing Razorpay US onboarding journey

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

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.

it´s not the flags subpage

it´s not the flags subpage

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.

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

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

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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DOI: 10.1186/s13041-021-00782-x

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @maulamb

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DOI: 10.1182/blood-2025-186

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DOI: 10.1111/acel.70255

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DOI: 10.1038/s41598-025-30030-4

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What is this?

This shows how the end of a major war isn’t always guaranteed to bring the results it has promised, former slaves were still forced into labor.

for - anthropocene - signalling - intrabrain - interbrain - SRG comment - individual / collective gestalt - SRG comment - how information flows from one brain to another - networked language!

for - book - Hierarchy in the Forest - shared struggle against inequality - the most important part of human heritage, intelligence and history - SRG comment - recognizing the sacred in all beings - adjacent to Michel Bauwens and the oscillation of the commons - to - book - publisher's page - Hierarchy in the Forest - The Evolution of Egalitarianism - 2001 - Christopher Boehm - https://hyp.is/_w4TEtZoEfCcjmPIvOEOaQ/www.hup.harvard.edu/books/9780674006911

for - to - book - Goliath's Curse: the History and Future of Societal Collapse - Luke Kemp

for - citizen assemblies & juries - lead to better governance outcomes - can save the world

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  • Deep Humanity interventions

for - definition - dark triad - Narcissism (grandiosity, entitlement, self-adoration), - Machiavellianism (manipulative, strategic self-interest, callousness), and - Psychopathy (impulsivity, lack of empathy/remorse, antisocial behavior). - SRG comment - dark triad - useful for Deep Humanity profile

for - reason for - social shift - from egalitarianism - to power hierarchy - status competition - SRG comment - Goliath's Curse - status competition - Deep Humanity antidote

for - definition - Goliath - the anthropological shift from egalitarianism to power hierarchy over the holocene - to - book publisher's page - Goliath's Curse - Luke Kemp - from - youtube - The Anthropocene Paradigm Shift

for - definition - transformation backlash - resistance experienced when pushing against an existing system - progress trap - polycrisis - climate crisis - transformation backlash

for - metaphor - medical - anthropocene - beyond experts, we need GPs for Anthropocene geophysiology - SRG comment - Is SRG GP for anthropocene?

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eLife Assessment

This study resolves a cryo-EM structure of the GPCR, human GPR30, which responds to bicarbonate and regulates cellular responses to pH and ion homeostasis. Understanding the ligand and the mechanism of activation is important to the field of receptor signaling and potentially facilitates drug development targeting this receptor. Structures and functional assays provide solid evidence for a potential bicarbonate binding site.

Reviewer #1 (Public review):

Summary:

This study resolves a cryo-EM structure of the GPCR, GPR30, in the presence of bicarbonate, which the author's lab recently identified as the physiological ligand. Understanding the ligand and the mechanism of activation is of fundamental importance to the field of receptor signaling. This solid study provides important insight into the overall structure and suggests a possible bicarbonate binding site.

Strengths:

The overall structure, and proposed mechanism of G-protein coupling are solid. Based on the structure, the authors identify a binding pocket that might accommodate bicarbonate. Although assignment of the binding pocket is speculative, extensive mutagenesis of residues in this pocket identifies several that are important to G-protein signaling. The structure shows some conformational differences with a previous structure of this protein determined in the absence of bicarbonate (PMC11217264). To my knowledge, bicarbonate is the only physiological ligand that has been identified for GPR30, making this study an important contribution to the field. However, the current study provides novel and important circumstantial evidence for the bicarbonate binding site based on mutagenesis and functional assays.

Weaknesses:

Bicarbonate is a challenging ligand for structural and biochemical studies, and because of experimental limitations, this study does not elucidate the exact binding site. Higher resolution structures would be required for structural identification of bicarbonate. The functional assay monitors activation of GPR30, and thus reports on not only bicarbonate binding, but also the integrity of the allosteric network that transduces the binding signal across the membrane. However, biochemical binding assays are challenging because the binding constant is weak, in the mM range.

The authors appropriately acknowledge the limitations of these experimental approaches, and they build a solid circumstantial case for the bicarbonate binding pocket based on extensive mutagenesis and functional analysis. However, the study does fall short of establishing the bicarbonate binding site.

Reviewer #2 (Public review):

Summary:

In this manuscript, "Cryo-EM structure of the bicarbonate receptor GPR30," the authors aimed to enrich our understanding of the role of GPR30 in pH homeostasis by combining structural analysis with a receptor function assay. This work is a natural development and extension of their previous work on Nature Communications (PMID: 38413581). In the current body of work, they solved the cryo-EM structure of the human GPR30-G-protein (mini-Gsqi) complex in the presence of bicarbonate ions at 3.15 Å resolution. From the atomic model built based on this map, they observed the overall canonical architecture of class A GPCR and also identified 3 extracellular pockets created by ECLs (Pockets A-C). Based on the polarity, location, size, and charge of each pocket, the authors hypothesized that pocket A is a good candidate for the bicarbonate binding site. To identify the bicarbonate binding site, the authors performed an exhaustive mutant analysis of the hydrophilic residues in Pocket A and analyzed receptor reactivity via calcium assay. In addition, the human GPR30-G-protein complex model also enabled the authors to elucidate the G-protein coupling mechanism of this special class A GPCR, which plays a crucial role in pH homeostasis.

Strengths:

As a continuation of their recent Nature Communications publication, the authors used cryo-EM coupled with mutagenesis and functional studies to elucidate bicarbonate-GPR30 interaction. This work provided atomic-resolution structural observations for the receptor in complex with G-protein, allowing us to explore its mechanism of action, and will further facilitate drug development targeting GPR30. There were 3 extracellular pockets created by ECLs (Pockets A-C). The authors were able to filter out 2 of them and hypothesized that pocket A was a good candidate for the bicarbonate binding site based on the polarity, location, and charge of each pocket. From there, the authors identified the key residues on GPR30 for its interaction with the substrate, bicarbonate. Together with their previous work, they mapped out amino acids that are critical for receptor reactivity.

Weaknesses:

When we see a reduction of a GPCR-mediated downstream signaling, several factors could potentially contribute to this observation: 1) a reduced total expression of this receptor due to the mutation (transcription and translation issue); 2) a reduced surface expression of this receptor due to the mutation (trafficking issue); and 3) a dysfunctional receptor that doesn't signal due to the mutation. In the current revision, based on the gating strategy, the surface expression of the HA-positive WT GPR30-expressing cells is only 10.6% of the total population, while the surface expression levels of the mutants range from 1.89% (P71A) to 64.4% (D111A). Combining this information with the functional readout in Figure 3F and G, as well as their previous work, the authors concluded that mutations at P71, E115, D125, Q138, C207, D210, and H307 would decrease bicarbonate responses. Among those sites,

E115, Q138, and H307 were from their previous Nature Comm paper.

Authors claim P71 and C207 make a structural-stability contribution, as their mutations result in a significant reduction in surface expression: P71A (1.89%) and C207A (2.71%). However, compared to 10.6% of the total population in the WT, (P71A is 17.8% of the WT, and C207A is 25.6% of the WT), this doesn't rule out the possibility that the mutated receptor is also dysfunctional: at 10 mM NaHCO3, RFU of WT is ~500, RFU of P71 and C207 are ~0.

The authors also interpret "The D125ECL1A mutant has lost its activity but is located on the surface" and only mention "D125 is unlikely to be a bicarbonate binding site, and the mutational effect could be explained due to the decreased surface expression". Again, compared to 10.6% of the total population in the WT, D125A (3.94%) is 37.2% of the WT. At 10 mM NaHCO3, the RFU of the WT is ~500, the RFU of D125 is ~0. This doesn't rule out the possibility that the mutated receptor is also dysfunctional. It is not clear why D125A didn't make it to the surface.

Other mutants that the authors didn't mention much in their text: D111A (64.4%, 607.5% of WT surface expression), E121A (50.4%, 475.5% of WT surface expression), R122 (41.0%, 386.8% of WT surface expression), N276A (38.9%, 367.0% of WT surface expression) and E218A (24.6%, 232.1% of WT surface expression) all have similar RFU as WT, although the surface expression is about 2-6 times more. On the other hand, Q215A (3.18%, 30% of WT surface expression) has similar RFU as WT, with only a third of the receptor on the surface.

Altogether, the wide range of surface expression across the different cell lines, combined with the different receptor function readouts, makes the cell functional data only partially support their structural observations.

Reviewer #3 (Public review):

Summary

GPR30 responds to bicarbonate and plays a role in regulating cellular pH and ion homeostasis. However, the molecular basis of bicarbonate recognition by GPR30 remains unresolved. This study reports the cryo-EM structure of GPR30 bound to a chimeric mini-Gq in the presence of bicarbonate, revealing mechanistic insights into its G-protein coupling. Nonetheless, the study does not identify the bicarbonate-binding site within GPR30.

Strengths

The work provides strong structural evidence clarifying how GPR30 engages and couples with Gq.

Weaknesses

Several GPR30 mutants exhibited diminished responses to bicarbonate, but their expression levels were also reduced. As a result, the mechanism by which GPR30 recognizes bicarbonate remains uncertain, leaving this aspect of the study incomplete.

Author response:

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

The parts of the text that have been changed.The major changes are as follows:

We re-analyzed the dataset and improved the local resolution of the extracellular region (Author response image 1).

We re-modeled based on the improved density and canceled the bicarbonate model based on comments from all reviewers.

We performed calcium assay using cell lines stably expressing the mutants, whose surface expression levels were analyzed by fluorescence-activated cell sorting (FACS)<br /> (Figure 3F, G and Figure 3–figure supplement 1-3).

Thus, we significantly revised our discussion of the extracellular binding pocket and the result of the mutational study. In the revised manuscript, we speculate that H307 is a candidate for the bicarbonate binding site.

Author response image 1.

Figure Comparison of local resolution between re-analyzed and previous maps.A Side and top view of the re-analyzed receptor-focused map of GPR30 colored by local resolution. B Side and top view of the previous receptor-focused map of GPR30 colored by local resolution

Reviewer #1 (Public Review):

Summary:

This study resolves a cryo-EM structure of the GPCR, GPR30, which was recently identified as a bicarbonate receptor by the authors' lab. Understanding the ligand and the mechanism of activation is of fundamental importance to the field of receptor signaling. However, the main claim of the paper, the identification of the bicarbonate binding site, is only partly supported by the structural and functional data, leaving the study incomplete.

Strengths:

The overall structure, and proposed mechanism of G-protein coupling seem solid. The authors perform fairly extensive unbiased mutagenesis to identify a host of positions that are important to G-protein signaling. To my knowledge, bicarbonate is the only physiological ligand that has been identified for GPR30, making this study a particularly important contribution to the field.

Weaknesses:

Without higher resolution structures and/or additional experimental assessment of the binding pocket, the assignment of the bicarbonate remains highly speculative. The local resolution is especially poor in the ECL loop region where the ligand is proposed to bind (4.3 - 4 .8 Å range). Of course, sometimes it is difficult to achieve high structural resolution, but in these cases, the assignment of ligands should be backed up by even more rigorous experimental validation.The functional assay monitors activation of GPR30, and thus reports on not only bicarbonate binding, but also the integrity of the allosteric network that transduces the binding signal across the membrane. Thus, disruption of bicarbonate signaling by mutagenesis of the putative coordinating residues does not necessarily mean that bicarbonate binding has been disrupted. Moreover, the mutagenesis was apparently done prior to structure determination, meaning that residues proposed to directly surround bicarbonate binding, such as E218, were not experimentally validated. Targeted mutagenesis based on the structure would strengthen the story.

Moreover, the proposed bicarbonate binding site is surprising in a chemical sense, as it is located within an acidic pocket. The authors cite several other structural studies to support the surprising observation of anionic bicarbonate surrounded by glutamate residues in an acidic pocket (references 31-34). However, it should be noted that in general, these other structures also possess a metal ion (sodium or calcium) and/or a basic sidechain (arginine or lysine) in the coordination sphere, forming a tight ion pair. Thus, the assigned bicarbonate binding site in GPR30 remains an anomaly in terms of the chemical properties of the proposed binding site.

Thank you for your insightful comments. Based on the weaknesses you pointed out, we reconstructed the receptor based on the improved density and removed the bicarbonate model. We performed calcium assays using cell lines stably expressing the variant based on the structure.

Reviewer #2(Public Review):

Summary:

In this manuscript, "Cryo-EM structure of the bicarbonate receptor GPR30," the authors aimed to enrich our understanding of the role of GPR30 in pH homeostasis by combining structural analysis with a receptor function assay. This work is a natural development and extension of their previous work (PMID: 38413581). In the current body of work, they solved the first cryo-EM structure of the human GPR30-G-protein (mini-Gsqi) complex in the presence of bicarbonate ions at 3.21 Å resolution. From the atomic model built based on this map, they observed the overall canonical architecture of class A GPCR and also identified 4 extracellular pockets created by extracellular loops (ECLs) (Pockets A-D). Based on the polarity, location, and charge of each pocket, the authors hypothesized that pocket D is a good candidate for the bicarbonate binding site. To verify their structural observation, on top of the 10 mutations they generated in the previous work, the authors introduced another 11 mutations to map out the essential residues for the bicarbonate response on hGPR30. In addition, the human GPR30-G-protein complex model also allowed the authors to untangle the G-protein coupling mechanism of this special class A GPCR that plays an important role in pH homeostasis.

Strengths:

As a continuation of their recent Nature Communication publication (PMID: 38413581), this study was carefully designed, and the authors used mutagenesis and functional studies to confirm their structural observations. This work provided high-resolution structural observations for the receptor in complex with G-protein, allowing us to explore its mechanism of action, and will further facilitate drug development targeting GPR30. There were 4 extracellular pockets created by ECLs (Pockets A-D). The authors were able to filter out 3 of them and identified that pocket D was a good candidate for the bicarbonate binding site based on the polarity, location, and charge of each pocket. From there, the authors identified the key residues on GPR30 for its interaction with the substrate, bicarbonate. Together with their previous work, they carefully mapped out nine amino acids that are critical for receptor reactivity.

Weaknesses:

It is unclear how novel the aspects presented in the new paper are compared to the most recent Nature Communications publication (PMID: 38413581). Some areas of the manuscript appear to be mixed with the previous publication. The work is still impactful to the field. The new and novel aspects of this manuscript could be better highlighted.

I also have some concerns about the TGFα shedding assay the authors used to verify their structural observation. I understand that this assay was also used in the authors' previous work published in Nature Communications. However, there are still several things in the current data that raised concerns:

Thank you for your insightful comments. Based on the weaknesses you pointed out, we highlighted the new and novel aspects of this manuscript could be better highlighted.l. We performed calcium assays using cell lines stably expressing the variant based on the structure.

(1) The authors confirmed the "similar expression levels of HA-tagged hGPR30" mutants by WB in Supplemental Figure 1A and B. However, compared to the hGPR30-HA (~6.5 when normalized to the housekeeping gene, Na-K-ATPase), several mutants of the key amino acids had much lower surface expression: S134A, D210A, C207A had ~50% reduction, D125A had ~30% reduction, and Q215A and P71A had ~20% reduction. This weakens the receptor reactivity measured by the TGFα shedding assay.

Since the calcium assay data is included in the main figure, the TGFα shedding assay and WB expression quantification data are Figure 3. –– supplement figure 1-4, but we included an explanation of the expression levels in the figure caption.

(2) In the previous work, the authors demonstrated that hGPR30 signals through the Gq signaling pathway and can trigger calcium mobilization. Given that calcium mobilization is a more direct measurement for the downstream signaling of hGPR30 than the TGFα shedding assay, pairing the mutagenesis study with the calcium assay will be a better functional validation to confirm the disruption of bicarbonate signaling.

According to the suggestion, we performed calcium assay using cell lines stably expressing the mutants (Figure 3F, G and Figure 3–figure supplement 1-3).

(3) It was quite confusing for Figure 4B that all statistical analyses were done by comparing to the mock group. It would be clearer to compare the activity of the mutants to the wild-type cell line.

Thank you for your comment. As you mentioned, the comparisons are made between wild-type GPR30 and mutants in the revised manuscript (Figure 3G, Figure 3.—figure supplement 4B)

Additional concerns about the structural data include

(1) E218 was in close contact with bicarbonate in Figure 4D. However, there is no functional validation for this observation. Including the mutagenesis study of this site in the cell-based functional assay will strengthen this structural observation.

We cancelled the bicarbonate model, and we performed mutation analysis targeting all residues facing the binding pocket using cell lines that stably express variants including E218A.

(2) For the flow chart of the cryo-EM data processing in Supplemental data 2, the authors started with 10,148,422 particles after template picking, then had 441,348 Particles left after 2D classification/heterogenous refinement, and finally ended with 148,600 particles for the local refinement for the final map. There seems to be a lot of heterogeneity in this purified sample. GPCRs usually have flexible and dynamic loop regions, which explains the poor resolution of the ECLs in this case. Thus, a solid cell-based functional validation is a must to assign the bicarbonate binding pocket to support their hypothesis.

We re-analyzed the dataset and improved the local resolution of the extracellular region (Author response image 1) and cancelled the bicarbonate model. Yet, as suggested by the reviewer, solid cell-based functional validation is efficient to analyze the receptor function response to bicarbonate. Thus, we performed mutation analysis targeting all residues facing the binding pocket using cell lines stably expressing the mutants, whose surface expression levels were analyzed by FACS (Figure 3F, G and Figure 3.––figure supplement 1-3).

Reviewer #3 (Public Review):

Summary:

GPR30 responds to bicarbonate and regulates cellular responses to pH and ion homeostasis. However, it remains unclear how GPR30 recognizes bicarbonate ions. This paper presents the cryo-EM structure of GPR30 bound to a chimeric mini-Gq in the presence of bicarbonate. The structure together with functional studies aims to provide mechanistic insights into bicarbonate recognition and G protein coupling.

Strengths:

The authors performed comprehensive mutagenesis studies to map the possible binding site of bicarbonate.

Weaknesses:

Owing to the poor resolution of the structure, some structural findings may be overclaimed.

Based on EM maps shown in Figure 1a and Figure Supplement 2, densities for side chains in the receptor particularly in ECLs (around 4 Å) are poorly defined. At this resolution, it is unlikely to observe a disulfide bond (C130ECL1-C207ECl2) and bicarbonate ions. Moreover, the disulfide between ECL1 and ECL2 has not been observed in other GPCRs and the published structure of GPR30 (PMID: 38744981). The density of this disulfide bond could be noise.

The authors observed a weak density in pocket D, which is accounted for by the bicarbonate ions. This ion is mainly coordinated by Q215 and Q138. However, the Q215A mutation only reduced but not completely abolished bicarbonate response, and the author did not present the data of Q138A mutation. Therefore, Q215 and Q138 could not be bicarbonate binding sites. While H307A completely abolished bicarbonate response, the authors proposed that this residue plays a structural role. Nevertheless, based on the structure, H307 is exposed and may be involved in binding bicarbonate. The assignment of bicarbonate in the structure is not supported by the data.

Thank you for your insightful comments. Based on the weaknesses you pointed out, we reconstructed the receptor based on the improved density and removed the bicarbonate model. We performed calcium assays using cell lines stably expressing the variant based on the structure.

Reviewer #1 (Recommendations For The Authors):

(1) The experimental validation of the bicarbonate binding could be strengthened by developing an assay that directly monitors bicarbonate binding (rather than GPCR signaling)

We agree that a direct binding assay for bicarbonate would be highly attractive (i.e. Filter binding assay using 14C-HCO₃⁻). However, the weak affinity of bicarbonate ions (in the mM range) would make reliable radioisotope-based detection impossible due to minimal specific receptor occupancy and high non-specific background and thus it is highly challenging and there are limitations to what can be done in this structural paper.

and determining a structure at comparable resolution in the absence of bicarbonate. In addition, all residues that are proposed to be located adjacent to the bicarbonate should be mutated and functionally validated.

We re-modeled the receptor based on the improved density and canceled the bicarbonate model. We performed calcium assay using cell lines stably expressing the mutants (Figure 3F, G and Figure 3.–figure supplement 1-3).

(2) What are the maps contoured in Figure 4D? The legend should describe this. Is 218 within the map region shown, or is there no density for its sidechain?

We removed the corresponding figure and cancelled the bicarbonate model.

(3) The contour level of the maps in Figure 1 - Figure Supplement 2 should also be indicated. Are these all contoured at the same level?

Thank you for your comment. We re-analyzed the same data set and obtained new density maps and models. We reworked Figure 1 and Figure 1. figure supplement 2; the contour level of the map for Figure 1 and composite map for the Figure 1. figure supplement 2 is the same, 7.65. 

(4) Regarding the cited structures of bicarbonate-binding proteins, for three of the four cited structures, the bicarbonate is actually coordinated by positive ligands, with the Asp/Glu playing a more peripheral role:

Capper et al: Overall basic cavity with tight bidentate coordination by Arg. The Glu is 5-6 Å away.

Koropatkin et al: Two structures. The first, solved at pH 5, is proposed to have carbonic acid bound. The second, solved at pH 8, shows carbonate in a complex with calcium, with the calcium coordinated by carboxylates.

Wang et al: The bicarbonate is coordinated by a lysine and a sodium ion. The sodium is coordinated by carboxylates.

The authors should more thoughtfully discuss the unusual properties of this binding site with regard to the previous literature. Is it possible that bicarbonate binds in complex with a metal ion? Could this possibility be experimentally tested?

We cancelled the bicarbonate model.

(5) As a structure of GPR30 has been recently published by another group (PMID: 38744981), it would be valuable to discuss structural similarities and differences and discuss how bicarbonate activation and activation by the chloroquine ligand identified by the other group might both be accommodated by this structure.

Thank you for your valuable comment. We compared the structure presented by another group and added our discussion, as “During the revision of this manuscript, the structures of apo-GPR30-G_q (PDB 8XOG) and the exogenous ligand Lys05-bound GPR30-G_q (PDB 8XOF) were reported [42]. We compared our structure of GPR30 in the presence of bicarbonate with these structures. In the extracellular region, the position of TM5 in GPR30 in the presence of bicarbonate is similar to that in apo-GPR30. In contrast, the position of TM6 is shifted outward relative to that of apo-GPR30, resembling the conformation observed in Lys05-bound GPR30 (Figure 6A, B). Additionally, the position of ECL1 is also shifted outward compared to that of apo-GPR30 (Figure 6B). In the GPR30 structure in the presence of bicarbonate, ECL2 was modeled, suggesting differences in structural flexibility. These findings indicate that the structure of GPR30 in the presence of bicarbonate is different from both the apo structure and the Lys05-bound structure, demonstrating that the structure and the flexibility of the extracellular domain of GPR30 change depending on the type of ligand. Furthermore, focusing on the interaction with G_q, the αN helix of G_q is not rotated in the structure bound to Lys05, in contrast to the characteristic bending of the αN helix in our structure (Figure 6C, D). Although it is necessary to consider variations in experimental conditions, such as salt concentration, the differences in the G_q binding modes suggest that the downstream signals may change in a ligand-dependent manner.” (lines 249-266).

Reviewer #2 (Recommendations For The Authors):

(1) It is highly recommended that the authors carefully go through the "insights into bicarbonate binding" section. The results of the new findings in this paper were blended in with the results from the previous work: the importance of E115, Q138, and H307 in the receptor-bicarbonate interaction was shown in the Nature Communication paper but the authors didn't make it clear, which added a little confusion.

We emphasized this fact in the main text (lines 130-132).

(2) It would be nice for the authors to add some content about the physiological concentration of HCO3 or refer more to their previous work about the rationale for selecting the bicarbonate dose in their functional assay.

Thank you for your comment. The physiological concentration of bicarbonate is 22-26 mM in the extracellular fluid, including interstitial fluid and blood, and 10-12 mM in the intracellular fluid. The bicarbonate concentration alters in various physiological and pathological conditions – metabolic acidosis in chronic kidney disease causes a drop to 2-3 mM, and metabolic alkalosis induced by severe vomiting increases HCO₃^- concentrations more than 30 mM. Thus, our present and previous works clearly show that GPR30 is activated by physiological concentrations of bicarbonate, whether it is localized intracellularly or on the membrane, and that GPR30 can be deactivated or reactivated in various pathophysiological conditions. We added this in the discussion section (lines 267-278).

(3) In Figure 3A, in the legend, the authors mentioned: "black dashed lines indicate hydrogen bonds". No hydrogen bond was noted in the figure.

We totally corrected Figure 3.

(4) Figure 3B, it would be helpful for the authors to denote the meaning of the blue-white-red color coding in the legend.

We removed the figure.

(5) Supplemental Figure 3: since AF3 was released on May 3rd, it would be awesome in the revision version if the authors would update this to the AF3 model.

The AF2 model has been replaced with the AF3. (Figure 2–figure supplement 2A-C). The AF2 and AF3 models are almost identical, and they form incorrect disulfide bonds. This confirms the usefulness of the experimental structural determination in this study.

(6) Supplemental Figure 4: it wasn't clear to me if the expression experiments were repeated multiple times or if there was any statistical analysis for the expression level was done in this study.

We performed the expression experiment by western blotting once and did not perform statistical analyses. We performed repeated FACS analyses of HEK cells stably expressing N-terminally HA-tagged wild-type or mutant GPR30s to analyze their membrane and whole-cell expressions during revision (Figure 3.–figure supplement 1-3). Using these stable cells, we performed calcium assays using cell lines stably expressing the mutants (Figure 3F, G and Figure 3–figure supplement 1-3).

(7) Supplemental Figure 4: Also, is there a reason for the authors to compare the expression level of hGPR30 to the housekeeping gene NA-K-ATPase rather than the total loaded protein? Traditionally housekeeping genes have been used as loading controls to semiquantitatively compare the expression of target proteins in western blots. However, numerous recent studies show that housekeeping proteins can be altered due to experimental conditions, biological variability across tissues, or pathologies. A consensus has developed for using total protein as the internal control for loading. An editorial from the Journal of Biological Chemistry reporting on "Principles and Guidelines for Reporting Preclinical Research" from the workshop held in June 2014 by the NIH Director's Office, Nature Publishing Group, and Science stated, "It is typically better to normalize Western blots using total protein loading as the denominator".

Thank you for your instructive comment. We evaluated western blotting with the same amount of total protein loaded 20 µg for whole-cell lysate and 1.5 µg for cell surface protein (Figure 3.–figure supplement 3C-F).

Reviewer #3 (Recommendations For The Authors):

The claim about this disulfide should be removed unless the authors can provide mass spec evidence.

Thank you for your crucial comments. Firstly, C130 is a residue of TM3, not ECL1, so our misprint has been corrected to C130^3.25. C207^ECL2, located at position 45.50, is the most conserved residue in ECL2, and it forms a disulfide bond with cysteine at position 3.25 (PMID: 35113559). The paper was additionally cited regarding the preservation of the bond of C130^3.25-C207^ECL2 (line 103). Indeed, disruption of this disulfide bond by the C207^ECL2 A mutation resulted in a marked reduction in receptor activity. In addition, the data set was re-analyzed to improve the local resolution of the extracellular region, and it was shown that the density of ECL2 is not noise (Figure 2. ––figure supplement 2). We are confident about the presence of the disulfide bond, based on the structural analysis data and the conservation.

The highly flexible extracellular region is greatly affected by experimental conditions and ligands, so we speculate that the ECL2 and the disulfide bond was not observed in other reported structures of GPR30. Then, we have added the following content to the discussion, as “In the GPR30 in the presence of bicarbonate, ECL2 was modelled, suggesting differences in structural flexibility.” (lines 256-257).

The authors should remove the assignment of bicarbonate in the structure, and tone down the binding site of bicarbonate.

We cancelled the bicarbonate model.

Minor:

(1) The potency of bicarbonate for GPR30 is in the mM range. Although the concentration of bicarbonate in the serum can reach mM range, how about its concentration in the tissues? Given its low potency, it may be not appropriate to claim GPR30 is a bicarbonate receptor at this point, but the authors can claim that GPR30 can be activated by or responds to bicarbonate.

The physiological concentration of bicarbonate is 22-26 mM in the extracellular fluid, including interstitial fluid and blood, and 10-12 mM in the intracellular fluid. Therefore, GPR30 is activated by physiological concentrations of bicarbonate in the tissues. Also, the bicarbonate concentration alters in various physiological and pathological conditions – metabolic acidosis in chronic kidney disease causes a drop to 2-3 mM, and metabolic alkalosis induced by severe vomiting increases HCO3- concentrations more than 30 mM. Thus, our work clearly shows that GPR30 is activated by physiological concentrations of bicarbonate, whether it is localized intracellularly or on the membrane, and that GPR30 can be deactivated or reactivated in various pathophysiological conditions. According to the reasons above, we claim GPR30 is a bicarbonate receptor (lines 267-278).

(2) The description that there is no consensus on a drug that targets GPR30 is not accurate, since lys05 has been reported as an agonist of GPR30 and their structure is published (PMID: 38744981). The published structures of GPR30 should be introduced in the paper.

We added the discussion about the structural comparison with the Lys05-bound structure (Figure 6, lines 249-266)

(3) BW numbers in Figure 4A should be shown.

We added BW numbers in the figures of the mutational studies.

for - book - Goliath's curse - author - Luke Kemp - SRG comment - a naughty social superorganism! - from - youtube - The Anthropocene Paradigm Shift - https://hyp.is/a1E9ZtQ4EfCEQs_6Hskbmw/www.youtube.com/watch?v=9ggpzwSI1qY

This line gives me confidence because I often feel discouraged when my first drafts are messy. The chapter explains that rough drafts are normal and even useful because they help writers discover their ideas. This encourages me to focus on getting thoughts onto the page first and worry about clarity and concision later during revision. It makes the writing process feel less stressful.

This sentence stands out to me because it challenges the idea that academic writing must sound fancy to be good. I sometimes worry that my writing isn’t “academic” enough, but this chapter reminds me that precision is more important than sounding sophisticated. Clear writing shows clear thinking, and readers care more about understanding the ideas than being impressed by complicated sentences.

Need to add quizzes

The answer can not be saved. Any correct answer provided?

R0:

Reviewer #1:

This sub study was nested in a factorial randomized controlled trial (RCT) in women aged 18–30 years. Participants included in this study were randomized to receive either a preconception intervention package or routine care until early childhood. The design strategy involved a reasonable sample size justification to show superiority. The sample needed for the study objectives was well justified with power considerations. However, the investigators do note that the sample size, while adequate for detecting moderate effect sizes, may have been insufficient to identify smaller but clinically meaningful differences. The descriptives are informative as seen in Tables 1 and 2.

1. Please define IQR in the footnote of Table 2 or put a descriptive section in the ‘Analysis Plan’ paragraph.

Generalized linear models (GLMs) with a Gaussian family and identity link function were used to estimate mean differences in CRP, AGP, IGF-1, and IGFBP3 concentrations. To estimate risk ratios for inflammatory status between infants in the intervention and routine care groups, GLMs with a binomial family and log link function were employed. Final models were adjusted for place of birth. There are several considerations needing clarification.

There are four endpoints. Therefore,

1. Some consideration of multiple comparison p-value adjustment should have been discussed.

Also, with respect to model content,

1. Exactly how was adjustment by birthplace incorporated into the models?

The overall conclusions follow from the analyses performed and results seen in Table 3. The strengths and limitations are reasonably described in the ‘Discussion’ section. As an added point, however,

4.There is a gap between the manuscript text and the supplement supporting information proposal Version 2.0. Was there any attempt to explore the mediation analysis discussed in that proposal?

Reviewer #2:

1. Overall Assessment This study reports a well-designed randomized controlled trial. It investigates the impact of an integrated intervention on infant biomarkers related to inflammation and growth like CRP, AGP, IGF-1, IGFBP3. The research addresses a significant question in maternal and child health. However, the discussion sections can be improved with detailed explanation on biological plausibility. Also, the implications of this study can be broadly elaborated.
2. Originality and Relevance The research topic appears to be original and highly relevant. The novelty in this study is integrated interventions across different stages right from preconception to 2 years of early child development. The intervention is policy-relevant and aligns well as per Goal-2 and Goal-4 of SDG-2030. The concept is innovative and similar integrated frameworks are reported in the literature. The specific distinct approach of this study needs to be articulated.
3. Scientific Rigor and Methodology This randomized controlled design follows standard protocols and manuscript is well-aligned as per CONSORT guidelines. Please elaborate on randomization process, blinding, and control of confounders. The sample size calculations appear to be powered for anthropometric assessments. For biomarker outcomes, sample size calculations need to be refined/justified.
4. Results and Interpretation The results of this study report no significant differences in biomarkers between intervention and control groups. The null findings can be discussed with possible biological explanations like timing of assessment, nutritional variability, breastfeeding. Subgroup analysis by maternal or infant characteristics can be helpful.
5. Discussion and Implications There is a scope to elaborate the discussion section by linking the pathways of maternal interventions with infant biomarker responses. Implications of this study for public health, including integration into maternal and child health programs, can be discussed highlighting the need for long-term follow-up.
6. Presentation and Clarity The manuscript is well-written and well-organized as per required guidelines. However, most of the references are quite older and references from 2022 onwards are missing. More recent Citations can be included from year 2023-2025.
7. Ethical and Data Considerations All the ethical procedures are described clearly including IEC and CTRI. Data availability through Open Access links is provided.
8. Conclusion and Recommendation This well-executed trial can be good evidence for understanding the biological outcomes of integrated maternal-child interventions.

Recommendation: Minor Revision.

Reviewer #3:

This study is a secondary analysis of the WINGS factorial randomized controlled trial evaluating the effects of a multidomain, integrated intervention delivered from preconception through early childhood on infant biomarkers of inflammation and growth (CRP, AGP, IGF-1, IGFBP3) at 3 and 6 months of age. This study links the integrated intervention to specific changes in inflammatory and growth-related biomarkers like CRP, AGP, IGF-1 and IGFBP3. The study addressed the biologically relevant and policy-important question related to early-life interventions in low-resource settings The findings indicate no significant differences in these biomarkers between the intervention and control groups, except for a transient decrease in IGFBP3 at 3 months, which was not sustained at 6 months. The authors conclude that while the intervention improved growth outcomes in the parent trial, it did not significantly influence early-life inflammation or IGF axis biomarkers. The manuscript is well-written, clearly articulated and follows the required CONSORT Guidelines. Major Comments 1. Rationale and Framing • Biological rationale connecting integrated maternal–child interventions (nutrition, WASH, psychosocial care) with the specific biomarkers studied (CRP, AGP, IGF-1, IGFBP3), needs clarity • Clarify why these markers and 3- and 6-month time points were selected, especially since primary growth outcomes were reported at 24 months in the main WINGS paper. • A concise conceptual model or figure showing hypothesized pathways could help readers follow the mechanistic logic. 2. Study Power and Sample • The power calculation is based on CRP only. Please justify the adequacy of the sample size for detecting meaningful differences in IGF-1 and IGFBP3, given their biological variability in infancy. • Power calculations are based on LAZ outcomes from the primary WINGS study rather than biomarker data. This needs justification. 3. Statistical Analysis and results • Tables 2 and 3 could be simplified to highlight group comparisons more effectively. • Adjustment only for the place of delivery seems limited. • The author may consider other covariates, such as mothers’ BMI, socioeconomic indicators, or exposure to infections, in the analysis. In case they are intentionally excluded from the analysis, explain their exclusion. • It would be useful to include effect size interpretation (e.g., percentage change or standardized mean difference) to better convey the biological relevance of null findings. 4. Interpretation of Findings • However, cautious interpretation of the null findings is needed. Aspects such as biological plausibility, contextual limitations, and future implications for longitudinal research require further elaboration. • The discussion acknowledges the absence of significant effects, but can be deepened if the authors discuss the following issues o Address low baseline inflammation as a potential ceiling effect. o Note that intervention effects might appear later in life (after 6 months). o Acknowledge that non-inflammatory mechanisms (caregiving, infection prevention, psychosocial stimulation) might explain the positive growth outcomes in the primary trial. • Expand the comparison with similar trials—such as SHINE (Zimbabwe), ELICIT (Tanzania), and MAL-ED studies—that examined inflammation and growth factor pathways. • The trial was conducted in a single urban Indian setting, which limits extrapolation to rural or diverse socioeconomic contexts. The discussion should acknowledge this limitation more explicitly and suggest strategies for replication in varied environments. 5. Policy and Program Implications • The conclusion is based on the non-significant findings of biomarkers. Whereas the short duration of biomarker assessment may oversimplify complex biological processes. More elaborate discussion is needed on possible confounders like infections, duration, and type of breastfeeding.

Minor Comments 1. Abstract: Conclude with a stronger statement about contribution: e.g., “These findings add to the understanding of biological mechanisms underlying integrated early-life interventions in LMICs.” 2. Tables: Present only adjusted results in the main text; unadjusted data may be submitted as supplementary files. Ensure all tables include units (mg/L, ng/mL) and consistent decimal formatting. 3. CONSORT Diagram: Please include the number of exclusions, losses to follow-up, and reasons for non-participation in Figure 1 for transparency. 4. Discussion: Add a short note acknowledging that biomarker variability in early infancy is high and may obscure subtle intervention effects. 5. References: Consider citing more recent literature (published within the last 3 years) that links microbiome–inflammation–growth relationships in infants. 6. Language and Formatting: Ensure consistency in abbreviations (e.g., IGFBP3 vs IGF-BP3). Use consistent phrasing for “preconception, pregnancy, and early childhood interventions, growth-related biomarkers, and growth factor profiles” throughout.

Overall Recommendations: Minor–to–Moderate Revision This is a robust, well-implemented study addressing an important mechanistic question within global child health. Although the results are null, they offer valuable insights into early-life biology and integrated program evaluation. Strengthening the biological framing, contextual discussion, and presentation of adjusted analyses will substantially enhance the manuscript’s impact and readability.

Advocating for the approach to teaching language and analyzes code-switching in different cultures instead of teaching standard English and making it seem like there's a correct or wrong way.

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Shows how broad code-meshing is and covers different languages, dialects, and ways of communication. Shows that it's used across different contexts.

Defines code-switching, also introduces code meshing as his preference when discussing the different dialects that come together in different ways academically, such as writing.

taking time to reflect helps me understand what I learned during the writing process. When I look back at the steps I took-reading, researching, and writing-I can see what I understood well and what was harder for me. Reflection also helps me notice how my ideas changed and improved. It makes the whole assignment feel more meaningful, because I can see my progress and what I learned along the way.

what barriers prevent many from returning to their homes?

50% of an entire nation cannot survive without humanitarian aid, showing the collapse of local systems

what challenges do the countries face in providing long-term care?

What specific conflicts or crises are driving this projected increase in West and Central Africa

nearly half of refugee children lack access to education, but the data spuriously shows that refugee students often have high exam pass rates when given the chance

This number is very high, even larger than the population of many countries. It shows how widespread forced displacement has become globally

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Explains why people code-switch, it's apart of their identity and English is helpful but Dine is the center of her.

While writing poetry she primarily writes in English but uses Dine to express and go more into depth about the meaning of what she's writing. She uses code-switching to add more meaning to her poems.

Writing in Dine allowed her to create a different form of poetry and highlighted her language switching between English and Dine.

Code-switching occurred on the radio and created community as there was a Native audience and representation. It was a way to accept herself and culture as she wasn't in her oppressive school environment.

Switching from her native language to silence in order to avoid physically being punished. The power dynamic is apparent when it comes to the different linguistics.

Represents her inability to pronounce certain words in English and how she was required to code-switch in order to avoid feeling discriminated against.

Being Native American, I completely understand relate to where she's coming from.

She speaks English & Navajo making her bilingual and hinting at the fact that she felt different having to be more American around peers.

Does uncertainty about how to react affect everyone equally, or are some people more likely to overcome this situation than others?

Even though more helpers should increase the chance that help will be given, the psychological effect actually decreases it

the text shows how human judgment becomes less sharp when we assume someone else will take charge

it goes against common intuition which most people assume that having more bystanders should increase the chance of someone helping

An example of attachment.

The pressure Elena receives from her relatives shows how cultural norms strongly shape the expectations around marriage and family. It made me think about what different cultures defined as the right age to marry or have children.

Do you see the huge increase between children that live with their mother rather than children that live with their father how does it go from 21% to 4%! Men need to start being Held accountable and not congratulated just for the little stuff that they do when there’s way more to the job.

I actually come from a single parent household, but When I turned about 11, my family turned into a blended family. When I was 11, it was about 2017 So I am the statistic!

I feel like thisall has to do with The decline in marriage. When you’re married, you can’t just Up and leave. You have to reconsider and try to work things out because to get divorced is way harder than to just break up.

This gives a more recent explanation of what a digital divide is in our modern context. In my own classroom (High School Art) there are some students who have Pinterest boards full of artistic inspiration and tons of saved art posts on Instagram that they can use as reference or inspiration when we are working on projects, but some students do not know how to use those same platforms in they way that the first type of student does. Some students are very familiar with using digital art programs as well, even on a rudimentary level, but some students have never even used a program such as MS Paint, so the gap of understanding digital art terms for exaple is widened for those students.

The people that are just OK with cohabitation rather than marriage surprise me but then it makes sense when we get to the next Section and they talk about one partner or many. The reason why more people are OK with cohabitation is because it’s less commitment rather than actually proposing a marriage, people want to still feel like they have the option to leave.(so they don’t feel trapped) It’s like they’re afraid of not having freedom or just afraid of the commitment in general.

I like how the bureau is basically telling us that the definition of family has shifted overtime to something much brighter than just husband, wife and child. Being family is about an emotional connection, shared, living and economic cooperation. Rather than just the institutional structure

This shows fake empathy, just like the beautiful aliens who don't actually support the first group of aliens.

So does blaming a kind of tragedy make it easier for people to accept harming the innocent people?

There is a connection between nice guys and violence. If cruelty doesn't come from obvious people, then is "niceness" just a fake thing, and does it make the actions worse?

It is an ironic observation, where they focus on comfort for agents who cause harm but not the people to arrest and harm.

This shows a strange kind of way of problem solving and contrasts with the place inside the job fair.

This is an interesting way of proving someone is normal, where he talked about a small and common thing.

Why can the agents show kindness to a dog, but not to the people they arrest?

His role is interesting where he did not directly participate but did help in the slaughter of the Jews. In this process, however, he should know that he’s supporting mass killing.

This quote is interesting because it uses Eichmann's own words to directly challenge Arendt's theory.

It is interesting because it explains the causes of these evil actions, where people act evil because they lack the ability for empathy and thinking from another perspective.

It connects to the text because it shows that people are failing to understand the radical evil, just like the killing done by the aliens.

So when determining if someone is evil or not, should we actually focus on their characters or what they do and their consequences?

It indicates that people without consciousness are just monsters and considered evil, but why should consciousness be valued so much that it simply determines if someone is a monster or not?

It is interesting because it contrasts terrifying and normal and suggests that normal people can also be the ones who causes danger and be evil.

I’m learning to shift my mindset from doubt to possibility by asking myself “why not?” when new opportunities show up. Instead of focusing on everything that could go wrong, I’m trying to look for reasons to take the chance. At the heart of it, I’m working on not rejecting myself before I even begin.

some suffering is viewed as less urgent or less valid based on where it occurs or who is affected, even though the situation is equally tragic

Why are visa applications so complex and difficult to navigate during times of war?

Is the treatment of refugees consistent across the West, or is it depending more on things like race or religion.

when people assume war is “normal” for certain regions, they become less sensitive to the suffering of those living there

It highlights the disconnect between public expressions of compassion and actual government policies that make it harder for refugees to get help.

How can journalists film senes in battle fields? Do they use drones, or just simply film the victims after war

Is there any Western powerful countries that gives political support to either side publicly?

Next Step is to see if all this will work in Agregore

That would enable anyone with IPFS Desktop and Agregore browser to be a create and share documents on the IndyWeb

We need 1s with Peergos Account to be able to share

secret links and establish trusted collaboation between poeple they know

Use IPFS and IPNS for

me/indy)Pad and documents under me

save manually on corresponding Peergos space

download it and upload it to IPFS Desktop

to get this document describing the work towards this goal

Just a sentence level thing but I think you are missing an "I" here!

• Researchers analyzed 66 samples of bread, cereals, pasta, and flour from 16 countries, including Poland.
• "Forever chemicals" — trifluoroacetic acid (TFA), a pesticide degradation product — were found in 54 samples (82%).
• Average concentration: 80 mg/kg.
• Highest levels: 360 mg/kg in breakfast cereal (Ireland) and 340 mg/kg in whole-grain bread (Belgium).
• The Polish sample (toast bread) contained 60 mg/kg of TFA.
• Wheat-based products had higher TFA concentrations than oat-based ones.
• Over 30 pesticide active substances used in European agriculture break down into TFA.
• TFA has previously been detected in surface water, groundwater, tap water, bottled water (in two-thirds of samples), and even wine — a phenomenon observed since the 1990s.
• No safety standards currently exist for TFA.
• In Germany, TFA is classified as potentially harmful to fertility and fetal development, though confirmed toxicity occurs only in animals at much higher concentrations than found in the environment.

I love that you added both the paper and the presentation!

In the story, people are encountering this bystander effect. Although they saw the pain of aliens, due to perceptions of others, they choose to not help even it's not moral.

responsibilities beyond individual hesitation?

gloss

from-HAMT

lazy grow

I love the use of a video! Makes it fun to learn about through a different way

Maybe above this paragraph label it "CNA Reflection" so that the reader knows that it is not just a continuation of the context paragraph

I assume this is where the welcome video will be? Make sure in the welcome video you invite them to look at your "about me" page for the welcome paragraph/introduction

Hypothesis mission

enable conversation over the world's knowledge to

More explanation on webbs account of protests against miss america

Backs up Webbs accounts of women in her organizations being left to do the "shit work" such as cooking, keeping records, etc.

Many things are incorrect. Earlier, it's stated that stereoisomers are geometric isomers. Additionally, the visuals are not helpful.

Good call out!

Minor point- I'm finding this difficult to read, especially the tables.

How did you account for spatial mixing that may occur with the given analyzed spot size? It's possible that a neighboring cell signal could be contributing to the target cell.

I appreciate that this is an important and sometimes tricky problem, but well worth doing! Did you consider using an accuracy metric for the registration?

Similar comment here regarding overinterpretation of the Raman peaks. Unless you use another complementary technique that is truly capable of molecular structure/identity characterization, this is an overstatement. However, I think your overall point still stands regarding lipid-associated C-C modes as relevant signatures and markers.

Could you explain how you are justifying the molecular assignments here? I understand these peaks can be tied to specific vibrations (e.g. 1120-1140 cm-1 can be C-C stretching) which molecules such as lipids may have, but using this as the basis for identifying a specific molecule seems to be an overinterpretation. Other fatty acids, for example, may have very similar peaks, within the error of the instrument especially.

It would be helpful to explain the possible biological significance or interpretation of this difference, as it feels a little inscrutable at first glance.

Does this include cells from all tissue and cell types? This seems like a major result, but the differences in performance are modest, so it would be informative to cross-validate on one of these dimensions (e.g. train on lung and predict for skin, and vice versa)

What is the spectral resolution and sampling rate of this system? The datasheet for this spectrograph lists a resolution of 3-6 cm^-1, and if the spectra have 873 dimensions and cover 600-1800 cm^-1, then the spectral sampling rate is around 1.4 cm^-1. Assuming these numbers are roughly correct, this makes it hard to interpret figures that highlight differences between Raman intensities at wavenumbers closer than either of these values (e.g. 1643, 1644, and 1645 cm^-1 in Extended data Fig 11).

Calling this "enhanced predictive power for skin" feels a bit misleading, as the AUC of 0.53 in lung corresponds to no predictive power.

I might be missing something, but it seems like this is a bit circular, as the features (DEGs and DPs) are selected using the same marker (p21+/-) that the model is predicting, so the feature space is "pre-aligned" to the prediction task.

Small comment: this is the first time the abbreviation "DP" is used, but what it stands for is not explained. Is it equivalent to the abbreviation "DPP" used in the methods section?

It seems like it would be important to normalize the mean Raman intensities by cell area or volume, otherwise changes in Raman intensity could be due to changes in cell area rather than a true change in the overall abundance of raman-active molecules or bonds.

Syria

i need the paper

The chemical energy conversion is key here. This is central to organic molecule survival.