Author response:

The following is the authors’ response to the previous reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

This study utilises fNIRS to investigate the effects of undernutrition on functional connectivity patterns in infants from a rural population in Gambia. fNIRS resting-state data recording spanned ages 5 to 24 months, while growth measures were collected from birth to 24 months. Additionally, executive functioning tasks were administered at 3 or 5 years of age. The results show an increase in left and right frontal-middle and right frontal-posterior connections with age and, contrary to previous findings in high-income countries, a decrease in frontal interhemispheric connectivity. Restricted growth during the first months of life was associated with stronger frontal interhemispheric connectivity and weaker right frontal-posterior connectivity at 24 months of age. Additionally, the study describes some connectivity patterns, including stronger frontal interhemispheric connectivity, which is associated with better cognitive flexibility at preschool age.

Strengths:

The study analyses longitudinal data from a large cohort (n = 204) of infants living in a rural area of Gambia. This already represents a large sample for most infant studies, and it is impressive, considering it was collected outside the lab in a population that is underrepresented in the literature. The research question regarding the effect of early nutritional deficiency on brain development is highly relevant and may highlight the importance of early interventions. The study may also encourage further research on different underrepresented infant populations (i.e., infants not residing in Western high-income countries) or in settings where fMRI is not feasible.

The preprocessing and analysis steps are carefully described, which is very welcome in the fNIRS field, where well-defined standards for preprocessing and analysis are still lacking.

We thank the reviewer for highlighting the strengths of this work.

Weaknesses:

While the study provides a solid description of the functional connectivity changes in the first two years of life at the group level and investigates how restricted growth influences connectivity patterns at 24 months, it does not explore the links between adverse situations and developmental trajectories for functional connectivity. Considering the longitudinal nature of the dataset, it would have been interesting to apply more sophisticated analytical tools to link undernutrition to specific developmental trajectories in functional connectivity. The authors mention that they lack the statistical power to separate infants into groups according to their growing profiles. However, I wonder if this aspect could not have been better explored using other modelling strategies and dimensional reduction techniques. I can think about methods such as partial least squares correlation, with age included as a numerical variable and measures of undernutrition.

We agree with the reviewer that this complex and rich longitudinal dataset would benefit from more sophisticated analytical approaches to characterise developmental trajectories in functional connectivity and to more directly link them to measures of undernutrition. However, conducting such analyses would require substantial additional methodological development, model validation, and careful interpretation, which fall beyond the scope and timeline of the present manuscript. Our aim here was to provide a clear and robust characterisation of functional connectivity changes during the first two years of life and to examine associations with growth outcomes at a specific developmental stage, while ensuring methodological transparency and statistical reliability. Importantly, these more advanced trajectory-based analyses are currently being pursued in the final phase of the BRIGHT project (BRIGHT IMPACT), in collaboration with expert statisticians and data scientists. This ongoing work aims specifically to leverage the longitudinal richness of the dataset to model developmental trajectories and their associations with early-life adversity and nutritional factors. We therefore see the present study as an important foundation for these forthcoming analyses.

Connectivity was assessed in 6 big ROIs. While the authors justify this choice to reduce variability due to head size and optodes placement, this also implies a significant reduction in spatial resolution. Individual digitalisation and co-registration of the optodes to the head model, followed by image reconstruction, could have provided better spatial resolution. This is not a weakness specific to this study but rather a limitation common to most fNIRS studies, which typically analyse data at the channel level since digitalisation and co-registration can be challenging, especially in complex setups like this. However, the BRIGHT project has demonstrated that it is possible and that differences in placement affect activation patterns, which become more localised when data is co-registered at the subject level (Collins-Jones et al., 2021). Could the co-registration of individual data have increased sensitivity, particularly given that longitudinal effects are being investigated?

We agree with the reviewer that the fNIRS community should work toward more precise methods for spatial registration of optodes, not only at the group level but also at the subject level, in order to make more precise inferences about the locations of activations. However, we followed a very thorough offline procedure to model headgear placement based on each participant’s photographs, which we believe complements the coregistration work performed by Collins-Jones in 2021. As reported in the fNIRS data acquisition section “Infants were excluded from further analysis if the band was excessively high over the front above the eyebrows” (line 409, methods section). Moreover channels displacement was measured from the photos, and if it was “equal or greater than 1.6 cm were renumbered, so that each channel was shifted either backward or forward one full channel location in space” (line 413, methods section). While these practices are thoroughly followed in the BRIGHT project, we are aware that they are not part of the standard procedure in many infant fNIRS studies. We hope that this work provides guidance for other researchers on how to coregister infant fNIRS data.

Considering the spatial resolution of fNIRS, which is on the order of centimetres, and the thorough procedure combining fNIRS–MRI coregistration with channel displacement assessment based on photographs, we do not think that individual-level coregistration would have significantly increased the sensitivity of the results.

I believe that a further discussion in the manuscript on the application of global signal regression and its effects could have been beneficial for future research and for readers to better understand the negative correlations described in the results. Since systemic physiological changes affect HbO/HbR concentrations, resulting in an overestimation of functional connectivity, regressing the global signal before connectivity computation is a common strategy in fNIRS and fMRI studies. However, the recommendation for this step remains controversial, likely depending on the case (Murphy & Fox, 2017). I understand that different reasons justify its application in the current study. In addition to systemic physiological changes originating from brain tissue, fNIRS recordings are contaminated by changes occurring in superficial layers (i.e., the scalp and skull). While having short-distance channels could have helped to quantify extracerebral changes, challenges exist in using them in infant populations, especially in a longitudinal study such as the one presented here. The optimal source-detector distance that minimises sensitivity to changes originating from the brain would increase with head size, and very young participants would require significantly shorter source-detector distances (Brigadoi & Cooper, 2015). Thus, having them would have been challenging. Under these circumstances (i.e., lack of short channels and external physiological measures), and considering that the amount the signal is affected by physiological noise (either coming from the brain or superficial tissue) might change through development, the choice of applying global signal regression is justified. Nevertheless, since the method introduces negative correlations in the data by forcing connectivity to average to zero, I believe a further discussion of these points would have enriched the interpretation of the results.

We added a paragraph discussing the choice of using GSR in our pipeline in the discussion of the manuscript as follows: “Importantly, these results remained significant even without GSR, indicating that our findings are not solely driven by preprocessing choices. While the use of GSR in FC studies remains debated (Murphy & Fox, 2017), in the absence of short channels (which are difficult to use reliably with infants (Emberson et al., 2016)) and external physiological measures, applying GSR represented the most appropriate preprocessing option. In fact, failure to correct for systemic physiological fluctuations can, in fact, lead to artificially elevated connectivity estimates in fNIRS data (Abdalmalak et al., 2022)” (line 250, discussion section).

Reviewer #2 (Public review):

Strengths:

The article addresses a topic of significant importance, focusing on early life growth faltering in low-income countries-a key marker of undernutrition-and its impact on brain functional connectivity (FC) and cognitive development. The study's strengths include the laborious data collection process, as well as the rigorous data preprocessing methods employed to ensure high data quality. The use of cutting-edge preprocessing techniques further enhances the reliability and validity of the findings, making this a valuable contribution to the field of developmental neuroscience and global health.

We thank the reviewer for highlighting the strengths of this work.

Weaknesses:

The study fails to fully leverage its longitudinal design to explore neurodevelopmental changes or trajectories, as highlighted by all three reviewers. The revised manuscript still primarily focuses on FC values at a single age stage (i.e., 24 months) rather than utilizing the longitudinal data to investigate how FC evolves over time or predicts cognitive development. Although the authors acknowledge that analyzing changes in FC (ΔFC) would reduce degrees of freedom (to ~30) and risk interpretability, they do not report or discuss these results, even as exploratory findings.

As suggested, we added the table reporting the results of the associations between changes in functional connectivity (DFC) between 5 and 24 months and cognitive flexibility in the supplementary materials (Table SI3). We additionally explored the relationship between changes in growth and cognitive flexibility as suggested by Reviewer #3 and we reported these additional analyses in the text as follows: “We also explored whether changes in growth and changes in functional connectivity between 5 and 24 months were associated with cognitive flexibility at preschool age, but we did not find any significant association (Table SI3 and Table SI4).” (line 213, results section).

Furthermore, the study lacks specificity in identifying which specific brain networks are affected by growth faltering, as the current exploratory analyses mainly provide an overall conclusion that infant brain network development is impacted without pinpointing the precise neural mechanisms or networks involved.

We added this limitation in the discussion as follows: “While the impact of undernutrition on brain development has been documented in LMICs (46), herein, we provided empirical evidence that growth faltering specifically in infants younger than five months of age impacts observable development of functional brain networks in the second year of life. Future studies may be needed to pinpoint which specific brain networks are impacted” (line 279, discussion section).

Reviewer #3 (Public review):

Summary

This study aimed to investigate whether the development of functional connectivity (FC) is modulated by early physical growth, and whether these might impact cognitive development in childhood. This question was investigated by studying a large group of infants (N=204) assessed in Gambia with fNIRS at 5 visits between 5 and 24 months of age. Given the complexity of data acquisition at these ages and following data processing, data could be analyzed for 53 to 97 infants per age group. FC was analyzed considering 6 ensembles of brain regions and thus 21 types of connections. Results suggested that: i) compared to previously studied groups, this group of Gambian infants have different FC trajectory, in particular with a change in frontal inter-hemispheric FC with age from positive to null values; ii) early physical growth, measured through weight-for-length z-scores from birth on, is associated with FC at 24 months. Some relationships were further observed between FC during the first two years and cognitive flexibility, in different ways between 4- and 5-year-old preschoolers, but results did not survive corrections for multiple comparisons.

Strengths

The question investigated in this article is important for understanding the role of early growth and undernutrition on brain and behavioral development in infants and children. The longitudinal approach considered is highly relevant to investigate neurodevelopmental trajectories. Furthermore, this study targets a little studied population from a low-/middle-income country, which was made possible by the use of fNIRS outside the lab environment. The collected dataset is thus impressive and it opens up a wide range of analytical possibilities.

We thank the reviewer for highlighting the strengths of this work.

Weaknesses

Data analyses were constrained by the limited number of children with longitudinal data on NIRS functional connectivity. Nevertheless, considering more advanced statistical modelling approaches would be relevant to further explore neurodevelopmental trajectories as well as relationships with early growth and later cognitive development.

While in this study we selected specific FC and outcome variables based on our hypothesis, the final phase of the BRIGHT project, known as BRIGHT IMPACT, aims to apply advanced statistical models to integrate a range of project variables into a single comprehensive analysis. We have acknowledged this in the discussion as follows: “Applying more advanced statistical modelling methods and structural equation modelling analyses may provide greater insight with further investigations in contexts of adversity and, in turn, establish which outcomes are predicted by FC” (line 309, discussion section).

The abstract and end of the discussion should make it clearer that the associations between FC and cognitive flexibility are results that need to be confirmed, insofar as they did not survive correction for multiple comparisons.

We have acknowledged this in the abstract as follows: “Our results highlight the measurable effects that poor growth in early infancy has on brain development and the possible subsequent impact on pre-school age cognitive development, underscoring the need for early life interventions throughout global settings of adversity”.

We have acknowledged this in the discussion as follows: “While our results are consistent with previous studies, we acknowledge that the significant associations between early FC and later cognitive flexibility do not withstand multiple comparisons. Therefore, we encourage future studies that may replicate these findings with a larger sample” (line 300, discussion section).

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) In Figure 1 B and C the authors should indicate that the results refer to HbO.

We have added the suggested specification in the caption of the figure as suggested.

(2) Figure SI2. Please indicate in the caption that these are the results when pre-processing did not include global signal regression.

We have added the suggested specification in the caption of the figure as suggested.

Reviewer #3 (Recommendations for the authors):

(1) The sentence l529-531 ("To investigate whether FC early in life predicted...") should be more explicit as it is not clear which of the two variables is regressed by the other: is it the measure of cognitive flexibility that is regressed by FC, as the hypothesis suggests? Were other variables considered in the regression model? (For linear regression with only one "prediction" variable, the square root of the coefficient of determination 𝑅2 is equal to the correlation between the two variables.)

Yes, it is the measure of cognitive flexibility that is regressed by FC. We have rephrased it in the text as follows: “we regressed later cognitive flexibility against FC that showed a significant change across the first two years of life”. There were no other variables in the regression model.

(2) A summary table of the statistical results for FC-cognitive flexibility associations should be included as for other analyses, in addition to Figure 3B.

We added a table of the results for the association between FC and cognitive flexibility in the supplementary materials (Table SI2, page 10), matching the same colours of Table 2. We referenced the table in the text in the main manuscript (line 211, result section).

(3) Figure 3B: The legend should precise that these results did not survive corrections for multiple comparisons.

We have specified this in the legend of Figure 3 as suggested.

(4) For the young pre-schooler group, it seems that the age is around 4 years (age mean +/- SD=47.96 +/- 2.77 months) and not 3 years as indicated at several places in the manuscript.

We found only once instance in which we erroneously said that the younger preschoolers were around 3 years. We replaced “Gambian infants from BRIGHT were cross-sectionally assessed at the age of 3 or 5 years for cognitive flexibility” with Gambian infants from BRIGHT were cross-sectionally assessed between the age of 3 and 5 years for cognitive flexibility (line 489, method section).

(5) The authors use the term "intra-hemispheric" connections for the ones within each of the 6 sections. This might be misleading since fronto-posterior connections are also intra-hemispheric ones. Specifying "short-range" or "within-section" connections might be clearer.

As suggested by the reviewer, we replaced “intra-hemispheric” with “intra-hemispheric within section” where appropriate through the whole manuscript.

(6) Abstract: what is the justification for using the term "optimal" for describing developmental trajectories of FC?

The term “optimal” refers to knowledge about typical developmental trajectories, coming especially from fMRI studies, as mentioned in the introduction: “Based on data from fMRI, current models hypothesize that FC patterns mature throughout early development (23–27), where in typically developing brains, adult-like networks emerge over the first years of life as long-range functional connections between pre-frontal, parietal, temporal, and occipital regions become stronger and more selective (28–31). [...]. Importantly, normative developmental patterns may be disrupted and even reversed in clinical conditions that impact development; e.g., increased short-range and reduced long-range FC have been observed in preterm infants (36) and in children with autism spectrum disorder (37, 38)” (line 93-106, introduction).

(7) The confidence interval should be added in Figure SI3.

As suggested, confidence intervals have been added in Figure SI3.

(8) Other scatterplot examples of associations might be added as supplementary information.

As suggested, we added several additional scatterplots to Figure SI3 (with confidence intervals as noted in the comment above) to show other associations between changes in growth and FC at 24 months.

(9) Figure SI6: % in x-axis is still indicated.

We apology for the oversight, all the percentage signs have now been removed from the x-axis tick labels.

(10) The authors might show the (even not significant) results of the associations between changes in growth and cognitive flexibility in supplementary information.

As suggested, we added the table reporting the results of the associations between changes in growth (DWLZ) and cognitive flexibility in the supplementary materials (Table SI3). We additionally explored the relationship between changes in functional connectivity and cognitive flexibility as suggested by Reviewer #2 and we reported these additional analyses in the text as follows: “We also explored whether changes in growth and changes in functional connectivity between 5 and 24 months were associated with cognitive flexibility at preschool age, but we did not find any significant association (Table SI3 and Table SI4).” (line 213, results section).

eLife Assessment

Hoverflies are known for their sexually dimorphic visual systems and exquisite flight behaviors. This valuable study reports how two types of visual descending neurons differ between males and females in their motion- and speed-dependent responses, yet surprisingly, the behavior they control lacks any sexual dimorphism. The results convincingly support these findings, which will be of interest for studies of visuomotor transformations and network-level brain organization.

Reviewer #1 (Public review):

Summary:

Hoverflies are renowned for their striking sexual dimorphism in eye morphology and early visual system physiology, as well as in sexually dimorphic behaviors. Surprisingly, male and female flight behaviors in response to optic flow exhibit only subtle differences. Nicholas et al. investigate the sensorimotor transformation of sexually dimorphic visual information into flight steering commands via descending neurons. Using a combination of intracellular and extracellular recordings, neuroanatomical analysis, and behavioral assays, the authors convincingly demonstrate that descending neurons-particularly at high optic flow velocities-exhibit pronounced sexual dimorphisms, while wing steering responses remain largely monomorphic. The study highlights a very interesting discrepancy between neuronal and behavioral response properties.

More specifically, the authors focused on two types of descending neurons that receive inputs from well-characterized wide-field sensitive tangential cells: OFS DN1 and OFS DN2. Their likely counterparts in Drosophila connect to neck, wing and haltere neuropils. The authors characterized the visual response properties of these two neuronal classes in both male and female hoverflies and identified several interesting differences. They then presented the same set of stimuli, tracked wing beat amplitude and analyzed the sum and the difference of right and left wing beat amplitude as a readout of lift or thrust, and yaw turning, respectively. Behavioral responses showed little to no sexual dimorphism, despite the observed neuronal differences.

Strengths:

I find the question very interesting and the results both convincing and intriguing. A fundamental goal in neuroscience is to link neuronal responses and behavior. The current study highlights that the transformations - even at the level of descending neurons to motoneurons - is complex and less straightforward than one might expect.

Weaknesses:

The authors investigated two types of descending neurons, but it was not clear to me how many other descending neurons are thought to be involved in wing steering responses to wide-field motion. I would suggest providing a more in-depth overview of what is known in hoverflies and Drosophila, since the conclusions drawn from the study would be different if these two types were the only descending neurons involved, as opposed to representing a subset of the neurons conveying visual information to the wing neuropil.

Both neuronal classes have counterparts in Drosophila that also innervate neck motor regions. The authors filled hoverfly DNs in intracellular recordings to characterize their arborization in the ventral nerve cord. In my opinion, these anatomical data could be further exploited and discussed a bit more: is the innervation in hoverflies also consistent with connecting to the neck and haltere motor regions? Are there any obvious differences and similarities to the Drosophila neurons mentioned by the authors? If the arborization also supports a role in neck movements, the authors could discuss whether they would expect any sexual dimorphism in head movements.

Revision comment:

I thank the authors for their detailed replies to my questions and the additional clarifications and analysis included in the paper. All my concerns have been addressed.

Reviewer #2 (Public review):

Summary:

Many fly species exhibit male-specific visual behaviors during courtship while little is known about the circuit underlying the dimorphic visuomotor transformations. Nicholas et al focus on two types of visual descending neurons (DNs) in hoverflies, a species in which only males exhibit high-speed pursuit of conspecifics. They combined electrophysiology and behavior analysis to identify these DNs and characterize their response to a variety of visual stimuli in both male and female flies. The results show that the neurons in both sexes have similar receptive fields but exhibit speed-dependent dimorphic responses to different optic flow stimuli.

Strengths:

Hoverflies, though not a common model system, show very interesting dimorphic behaviors and provide a unique and valuable entry point to explore the brain organization behind sexual dimorphism. The findings here are not only interesting on their own right but will also likely inspire those working in other systems, particularly Drosophila.

The authors employed rigorous morphology, electrophysiology, and behavior methods to deliver comprehensive characterization of the neurons in question. The precision of the measurements allowed for identifying a subtle and nuanced neuronal dimorphism and set a standard for future work in this area.

Weaknesses:

I'd like to thank the authors for the revised manuscript, especially the new analyses and figures. Most of my earlier concerns have been satisfactorily addressed by now. Interested readers are kindly referred to the authors' responses for the discussion of the limitations of this work.

Author response:

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

eLife Assessment

Hoverflies are known for their sexually dimorphic visual systems and exquisite flight behaviors. This valuable study reports how two types of visual descending neurons differ between males and females in their motion- and speed-dependent responses, yet surprisingly, the behavior they control lacks any sexual dimorphism. The results convincingly support these findings, which will be of interest for studies of visuomotor transformations and network-level brain organization.

This statement perfectly recapitulates our findings.

Public Reviews:

Reviewer #1 (Public review):

Summary:

Hoverflies are known for a striking sexual dimorphism in eye morphology and early visual system physiology. Surprisingly, the male and female flight behaviors show only subtle differences. Nicholas et al. investigate the sensori-motor transformation of sexually dimorphic visual information to flight steering commands via descending neurons. The authors combined intra- and extracellular recordings, neuroanatomy, and behavioral analysis. They convincingly demonstrate that descending neurons show sexual dimorphisms - in particular at high optic flow velocities - while wing steering responses seem relatively monomorphic. The study highlights a very interesting discrepancy between neuronal and behavioral response properties.

Thank you for this summary. Most of the statement perfectly recapitulates the main findings of our paper. However, we want to emphasize that some hoverfly flight behaviors are strongly sexually dimorphic, especially those related to courtship and mating. Indeed, only male hoverflies pursue targets at high speed, chase away territorial intruders, and pursue females for mating. However, other flight behaviours, such as those related to optomotor responses and flights between flowers when feeding, are not sexually dimorphic. We have amended the Introduction and Discussion to make the difference between flight behaviors more clear. Please see lines 77 and 305 onwards.

More specifically, the authors focused on two types of descending neurons that receive inputs from well-characterized wide-field sensitive tangential cells: OFS DN1, which receives inputs from so-called HS cells, and OFS DN2, which receives input from a set of VS cells. Their likely counterparts in Drosophila connect to the neck, wing, and haltere neuropils. The authors characterized the visual response properties of these two neuronal classes in both male and female hoverflies and identified several interesting differences. They then presented the same set of stimuli, tracked wing beat amplitude, and analyzed the sum and the difference of right and left wing beat amplitude as a readout of lift or thrust, and yaw turning, respectively. Behavioral responses showed little to no sexual dimorphism, despite the observed neuronal differences.

Thank you for this very nice summary of our work. We want to clarify that LPTC input to DN1 and DN2 has not been shown directly in hoverflies using e.g. dye coupling, or dual recordings. Instead, the presumed HS and VS input is inferred from morphological and physiological DN evidence, and comparisons to similar data in Drosophila and blowflies. We have amended the Introduction to clarify this. Please see line 64 onwards. The rest of the paragraph perfectly recapitulates the main findings of our paper.

Strengths:

I find the question very interesting and the results both convincing and intriguing. A fundamental goal in neuroscience is to link neuronal responses and behavior. The current study highlights that the transformations - even at the level of descending neurons to motoneurons - are complex and less straightforward than one might expect.

Thank you.

Weaknesses:

The authors investigated two types of descending neurons, but it was not clear to me how many other descending neurons are thought to be involved in wing steering responses to wide-field motion. I would suggest providing a more in-depth overview of what is known about hoverflies and Drosophila, since the conclusions drawn from the study would be different if these two types were the only descending neurons involved, as opposed to representing a subset of the neurons conveying visual information to the wing neuropil.

This is a great point. There are around 1000 fly descending neurons identified in Drosophila, of which many could respond to widefield motion, without being specifically tuned to widefield motion. In Drosophila, at least 35 descending neuron types receive input in the part of the brain where the LPTC outputs are located, and at least 29 descending neuron types project to the wing motor neuropil. Thus, it is more than likely that other neurons project visual widefield motion information to the wing neuropil. Furthermore, we only measured wing beat amplitude (WBA) as seen in the horizontal plane, as we were filming from above. As such, other wing angle changes and rotations are not quantified. We have amended our Introduction (see line 53 onwards) and Discussion (see line 320 onwards) to address these important points.

Both neuronal classes have counterparts in Drosophila that also innervate neck motor regions. The authors filled the hoverfly DNs in intracellular recordings to characterize their arborization in the ventral nerve cord. In my opinion, these anatomical data could be further exploited and discussed a bit more: is the innervation in hoverflies also consistent with connecting to the neck and haltere motor regions? Are there any obvious differences and similarities to the Drosophila neurons mentioned by the authors? If the arborization also supports a role in neck movements, the authors could discuss whether they would expect any sexual dimorphism in head movements.

These are all great points. We did not see any clear arborizations to the frontal nerve (FN), where we would expect to find the neck motor neurons (NMNs). In addition, while we did see fine arborizations throughout the length of the thoracic ganglion, we saw no strong outputs projecting directly to the haltere nerve (HN). In the revised version of the MS we have modified figure 4 (morphological characterization) to show a magnification of the thoracic ganglion to clarify this.

There are important differences between the morphology of DN1 and DN2 in hoverflies and DNHS1 and DNOVS2 in Drosophila, in terms of their projections in the thoracic ganglion. For example, In Drosophila DNOVS2, there are several fine branches along the length of the neuron in the thoracic ganglia. Similarly, we found fine branches in Eristalis tenax DN2, however, in addition, we found a wide branch projecting to the area of the thoracic ganglion where the prothoracic and pterothoracic nerves likely get their inputs, which we also found in Eristalis tenax OFS DN1 (Figure 4). This suggests that both neurons could contribute to controlling the wings and/or the forelegs (which is why we quantified the WBA). In Drosophila DNOVS1, there is a similar fat branch to the prothoracic and pterothoracic nerves, Furthermore, while Drosophila DNHS1 and DNOVS2 have different morphology, DN1 and DN2 in Eristalis looked similar. We have modified the Results section to make this clear, see line 193 onwards.

In addition, to investigate this further, our revised version of the MS includes analysis of the movement of different body parts (the head angle, fore- and hindleg extension) to investigate this further, and to look for sexual dimorphism. Unfortunately, however, this did not include the halteres, as they cannot be seen well in the videos. The new data can be seen in Figure 7.

Reviewer #2 (Public review):

Summary:

Many fly species exhibit male-specific visual behaviors during courtship, while little is known about the circuit underlying the dimorphic visuomotor transformations. Nicholas et al focus on two types of visual descending neurons (DNs) in hoverflies, a species in which only males exhibit high-speed pursuit of conspecifics. They combined electrophysiology and behavior analysis to identify these DNs and characterize their response to a variety of visual stimuli in both male and female flies. The results show that the neurons in both sexes have similar receptive fields but exhibit speed-dependent dimorphic responses to different optic flow stimuli.

This statement perfectly recapitulates the main findings of our paper. As mentioned above, while hoverfly flight behaviors related to courtship and mating are strongly sexually dimorphic, other flight behaviours, such as those related to optomotor responses and flights between flowers when feeding, are not. We have amended the Introduction and Discussion to make the difference between flight behaviors more clear. Please see lines 77 and 305 onwards.

Strengths:

Hoverflies, though not a common model system, show very interesting dimorphic behaviors and provide a unique and valuable entry point to explore the brain organization behind sexual dimorphism. The findings here are not only interesting on their own right but will also likely inspire those working in other systems, particularly Drosophila.

Thank you.

The authors employed rigorous morphology, electrophysiology, and behavior methods to deliver a comprehensive characterization of the neurons in question. The precision of the measurements allowed for identifying a subtle and nuanced neuronal dimorphism and set a standard for future work in this area.

Thank you.

Weaknesses:

Cell-typing using receptive field preferred directions (RFPDs): if I understood correctly, this classification method mostly relies on the LPDs near the center of the receptive field (median within the contour in Fig.1). I have two concerns here. First, this method is great if we are certain there are only two types of visual DNs as described in the manuscript. But how certain is this? Given the importance of vision in flight control, I would expect many DNs that transmit optic flow information to the motor center. I'd also like to point out that there are other lobula plate tangential cells (LPTCs) than HS and VS cells, which are much less studied and could potentially contribute to dimorphic behaviors.

This is very true, and important. As mentioned above, in Drosophila there are 35 descending neuron types with inputs on the dorsal surface of the brain (labelled DNp1-35), suggesting that they could receive input from LPTCs. However, only 3 of these have been shown physiologically and morphologically to receive LPTC input, in blowflies and Drosophila (DNHS1, DNOVS1, DNOVS2). Note that in both blowflies and fruitflies DNOVS1 gives graded responses, and no action potentials, meaning that we would not be able to record from it using extracellular electrophysiology.

We previously used clustering techniques to show that in Eristalis, we can reliably distinguish two types of optic flow sensitive DNs from extracellular electrophysiological data, based on a range of receptive field parameters, and we think that these correspond to DNHS1 and DNOVS2 in Drosophila (Nicholas et al, J Comp Physiol A, 2020, cited in paper). As mentioned above in response to Reviewer 1, this does not mean that there are no other neurons that could respond to widefield optic flow, and which might be involved in the WBA we recorded in the paper. However, the point of this paper was not to conclusively show that there are only two optic flow sensitive descending neurons. The point was to say that there are two quite distinct optic flow sensitive neurons that have similar receptive fields in males and females, while their velocity response functions differ between males and females.

We have modified the Introduction (see lines 53 and 64 onwards) and Discussion to make these important points clear to the Reader, including a mention of the 45-60 LPTCs that exist in the lobula plate, and what their role might be.

Second, this method feels somewhat impoverished given the richness of the data. The authors have nicely mapped out the directional tuning for almost the entire visual field. Instead of reducing this measurement to 2 values (center and direction), I was wondering if there is a better method to fully utilize the data at hand to get a better characterization of these DNs. As the authors are aware, local features alone can be ambiguous in characterizing optic flows. What's more, taking into account more global features can be useful for discovering potentially new cell types.

This is a great point, and we did analyse other receptive field properties in this study (shown in previous supp fig 1). In addition, and as mentioned above, we have published a clustering analysis across receptive field properties of these neurons (Nicholas et al, J Comp Physiol A, 2020, cited in paper). The point that we attempted to make in this paper was that by using two strikingly simple metrics, we can reliably distinguish which of the two neuron types we are recording from simply based on azimuthal location and overall directional preference. This makes automated analysis very straightforward. Indeed, we now use this routinely to ID what neuron we are recording from computationally, rather than making a human-based assumption.

However, we agree that this needs to be shown, and that further in depth analysis was warranted. Therefore, we have provided additional receptive field analysis and clustering (see new supplementary figure 1) and associated text. We also want to highlight that all data is uploaded to Data Dryad for anyone interested in doing additional in-depth analyses.

Line 131, it wasn't clear to me why full-screen stimuli were used for comparison here, instead of the full receptive field maps. Male flies exhibit sexual dimorphic behaviors only during courtship, which would suggest that small-sized visual stimuli (mimicking an intruder or female conspecific) would be better suited to elicit dimorphic neuronal responses. A similar comment applies to the later results as well. Based on the receptive field mapping in Figure 1, I'm under the impression that these 2 DN types are more suited to detect wide-field optic flows, those induced by self-motion as mentioned in the manuscript. The results are still very interesting, but it's good to make this point clear early on to help set appropriate expectations. Conversely, this would also suggest that there are other visual DN types that are responsible for the courtship-related sexually dimorphic behaviors.

Thank you for mentioning these important points. Our reasoning for using full-screen stimuli for the analysis on line 131 was that since we used the small sinusoidal gratings for mapping the receptive fields, and to subsequently classify the neurons, it would be unfair to use the same data to investigate potential sexual dimorphism. I.e., we selected neurons that fulfilled certain criteria, and then we cannot rightfully use the same criteria to determine differences. This was not explicitly mentioned in the paper, so we have modified the text to make this clear to the Reader, see lines 142 onwards.

However, in Supp Figure 2d/e we show that there are no striking receptive field differences between males and females in terms of receptive field center nor directional preference. In Supp Figure 2f we also show that there is no difference between male and female receptive field height and width. We have modified the text to draw the Reader’s attention to this figure, and also mention the additional analysis done in response to the comment above.

As a side note, I personally expected at least DN1 to have a smaller receptive field in males, as the hoverfly HSN is strikingly sexually dimorphic (Nordström et al, Curr Biol 2008). However, while optic flow sensitive DNs do respond to small objects (see e.g. the J Comp Physiol paper mentioned above) we did not detect any obvious sexual dimorphism in receptive field properties. Indeed, we think that a different subset of DNs control parts of target pursuit behavior (target selective DNs (TSDNs)). This is now addressed in the modified version of the paper, see line 89-92.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) I think that the additional measurement of head turns in response to some of the stimuli that showed the strongest sexual dimorphism would be very interesting, but I fully acknowledge that this might be beyond the scope of the current paper or technically too challenging, requiring additional cameras and a whole new tracking software, etc.

We have added an additional figure to the paper, with associated text, showing the response of the head, fore- and hindlegs to the same stimuli, as far as we could extract them with only one camera filming from above. The new data can be found in the new figure 7, and associated text.

(2) Are the onset measurements for WBD comparable across flight manoeuvres, given that they are limited to a single projection plane?

This is a great point, and we have now added this caveat in the text, see line 261-262.

(3) Line 62 - typo: DNp15 not NDp15.

Thank you, fixed.

Reviewer #2 (Recommendations for the authors):

(1) Related to a comment earlier, in the Introduction, it is mentioned that there are 3 optic flow-sensitive DNs in Drosophila and blowfly. However, I don't see convincing evidence for this in the cited references, none of which have exclusively surveyed all the DNs.

We have revised this to say that 3 neuron have been identified morphologically and physiologically, but that does not mean that there are no others. Please see line 60 onwards.

(2) Line 142 and Supplementary Figure 3, this is stated in the next section, but I think it's better to make it clear that DN2 in females has a higher spontaneous rate before mentioning the starfield. Please also specify if the stationary starfield affects the DN2 rate at all in the female flies.

Great points. We now describe the spontaneous rate before mentioning the responses to moving starfield stimuli, and highlight that there is no difference between no stimulus (pre-stimulation) and a stationary stimulus. Please see lines 155 onwards.

(3) Line 34, 'redress' should be 'to address'.

Thank you, fixed.

(4) Line 59, a bit unclear to me what this sentence is trying to say. Also, I wouldn't say LPTCs are 'indirect' in the sensorimotor transformation -- it's a necessary link in this pathway, no?

That was indeed a strange sentence. We have simplified it to the following: “LPTCs project to the inferior posterior slope[6], where they synapse with descending neurons[7,8]. In Drosophila at least 35 descending neuron types have their inputs in the posterior surface of the brain (named DNp1-35) [9].”

(5) Figures:

This is a formatting problem. The figure legends are separated from the figures, and there are no titles on the figures to indicate which one is which.

We are sorry about this. We have added labels to the figures.

Figure 1: What kind of geographic projections are these? The azimuth axis is not labeled.

These stimuli were not perspective corrected, and therefore the RF maps simply reflect the visual monitor. We have clarified this in the figure legend, including mentioning that the axis label is the same for elevation and azimuth.

Figure 2a: The error bars are not aligned to the angular axis.

These have now been aligned.

Supplement Figure 2b: I'm not sure why there are two measurements at each stimulus orientation. The bottom panel is confusing -- what do you mean by 'receptive field location'? And what does this red arrow/line mean in the bottom panel?

Thank you for pointing this out. The figure was supposed to help the reader understand our transformations, so it’s great to know that it needed further explanation. To address this, we have added extra text and panel labels, please see lines 520 onwards.

(6) Methods:

Line 356: Maybe a picture or schematic drawing would be helpful to explain the setup. For instance, it's unclear what 32 degrees here refers to.

This is a great suggestion, and a pictogram explaining the set-up can now be seen in Supplementary Fig. 6b.

Line 404: What does it mean that 'spatially interpolate 10 times'?

This sentence has been changed to “After subtracting the spontaneous rate, calculated for 0.8 s preceding stimulus onset (dotted line, inset, Fig. 1b, e), we interpolated the resulting local maximum responses to a ten-fold higher spatial resolution (colour coding, Fig. 1a, d).”

Line 405: How to determine the center from the 50% contour?

We have modified the Methods to explain how this was done, please see lines 478 onwards.

Line 408: Please explain more explicitly how LPD and LMS are computed.

We have modified the Methods to explain how this was done, please see lines 488 onwards.

Line 418: Is reference 42 correct? I could be wrong, but this reference seems to be talking about target-selective DNs rather than optic flow-sensitive DNs?

Yes, this reference is correct. In a supp figure to ref 42, we show data from optic flow sensitive neurons, but not their receptive fields. Thanks for checking.

Line 426: Are the full-screen stimuli presented in 8 directions too? Do I understand correctly that the preferred direction vector for the full-screen stimuli is extracted from a cosine fit, which is slightly different from the 'receptive field preferred direction' in the receptive field mapping measurement, which is the median of all the 'local preferred direction' (which are from the cosine fit)?

We have modified the text to make this clear, please see lines 519 onwards, as well as the receptive field analysis, please see lines 474 onwards.

The mule represent being controlled like Janie is being treated in her marriage

Joe thinks that Janie is poised and that she has no right to be at all upset when he has done so much for her.

This paragraph is talking about how Janie’s day was feeling boring but when she started to listen to the people on the porch it made things better and more interesting for her.

Yes

Chapter 10 we see the breathe of fresh air as tea cake meets her emotional need and they have this almost instant bond as they play checkers together and drink and he even walks her home we just see the man of Janie’s childhood dreams almost a real love blossoming.

She realizes this isn’t the relationship she wants anymore after being a victim to Jody’s abusive tendency’s.

Years of rough and falling marriage has worn Angie out. She takes the verbal abuse and does nothing about it.

In this small paragraph it’s talking about how Janie’s doesn’t really understand her worth and that people want to reach something easier instead of living their hard lives.

This shows Janie’s inner freedom and new beginning while also being at the funeral and not showing happiness.

She showed up to the funeral looking sad but deep down she was happy that she was finally free.

After Joe’s death she had gained freedom and felt free to what ever.

Janie is searching for the meaning in life

Janie burning her headrags is one of the most symbolic acts in the novel. For years, Joe forced her to tie up her hair so no other man could admire it. By burning the rags and letting her hair down, she destroys his control over her body and image. Even more powerful. she throws away all the hairpins. She's not just changing a style, she's burying Joe's version of her.

Janie knows that these men have no actual interest in her, but her money and property. She prefers being independent and isn’t focused on marriage or romantic relationships right now.

She now has freedom to do things that she wants without Joe stark verbally abusing her

Janie is finally free since Joe has died, she can finally do what she has been wanting to do.

Janie don’t really feel sad like everyone expects, and you can tell she actually likes the freedom she has now. It shows she’s finally living for herself instead of doing what other people want

This had to be a freeing experience for Janie and finally being able to be her true self instead of having to hide who she is.

She pretended to be sad during the funeral

She showed up to the funeral looking sad but deep down she was happy that she was finally free.

This is showing how loyal the town’s people were to stark and how they were a little suspicious of Janie.

Ch 9 We see the community rebuke her by believing the rumor that she is posing Jody. We also see the freedom she has when he dies we see her let her hair down as a symbol of independence and freedom

She is free from joes control. She feels relieved, she is able to be herself and express herself how she wants.

With Joe’s death, Janie is now free from the abuse and can be more like herself.

She realized that she was free from the controlling man that joe was and she was going to begin to find herself.

She felt free and that she could be herself

Janie finally lets everything out and tells Jody how she’s really been feeling, and it’s sad because he still doesn’t really hear her even at the end. You can tell she’s tired of being quiet and is starting to stand up for herself after all those years.

I can see why the concept of "parent involvement" was changed to "family engagement" and I think the term is all the better for the word change. Because its true that there are some ML students who may be living with extended family rather than with their parents, they could be living with an aunt and uncle or their house could involve both their parents and their extended family all under one roof. And having the ML students family "engaged" instead of "involved" sends a more positive message of wanting to give the family a chance to not only be included in their own child's education but also the class as a whole. An ML student's family sharing their experiences can benefit the non-ML students as well and help them understand the culture their ML students come from.

Ensure the AI agent is answering accurately

drawn to cardiff itself due to the increasing number of catholics, Irish immigrants encouraged to build the docks in (), further flocking due to the famine etc. As such, it is plausible to argue that Bute saw Castell Coch as a bastion of 'the faith', the architectural style was from a time of Catholicism within Wales, Bute further emphasising his desire to draw back to () with the placement of a statue of the 'madonna and child'.

Many still disagree, but the form it took was scholarly --> the restoration of castell coch in the gothic style was highly due to it's place as a manifestation of decades of scholarship, buting an understanding of the past materialised in the present

Version 3 of this preprint has been peer-reviewed and recommended by Peer Community in Microbiology.<br /> See the peer reviews and the recommendation.

make process parentheticals like this tooltips

eLife Assessment

This study provides valuable insight into the role of actin protrusions in mediating early pre-endoyctic steps of human papillomavirus entry at the cell surface. Using state-of-the-art microscopy in an immortalized keratinocyte model, the authors present convincing evidence that filopodia actively promote the transfer of heparin sulfate-coated virions from the extracullar matrix to the viral entry factor CD151. These findings provide a strong framework for future studies aimed at further resolving the dynamics of virion transfer and receptor engagement.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The editors have determined that the authors adequately addressed the prior reviewer comments.]

Summary:

The author's goal was to arrest PsV capsids on the extracellular matrix using cytochalasin D. The cohort was then released and interaction with the cell surface, specifically with CD151 was assessed.

Note on previous revisions:

The authors did an excellent job in their revision to include data from the effect of proteolytic priming on their observed virion transfer to the cell body. All other minor issues were addressed adequately.

The work could be especially critical to understanding the process of in vivo infection.

Reviewer #2 (Public review):

Review of the previous version:

The study design involves infecting HaCaT cells (immortalised keratinocytes mimicking basal cells of a target tissue) and observing virus localization with and without actin polymerization inhibition by cytochalasin D (cytoD) to analyze virion transfer from the ECM to the cell via filopodial structures, using cellular proteins as markers.

In the context of the model system, the authors stress in the revised version the importance of using HaCaT cells as a relevant 'polarized' cell model for infection. The term 'polarized' is used in the cell biological literature for epithelial cells to describe a strict apical vs. basolateral demarcation of the plasma membrane with an established diffusion barrier of the tight junction. However, HaCat cells do not form tight junctions. In squamous epithelia, such barriers are only found in granular layers of the epithelium. The published work cited in support of their claims either does not refer to polarity or only in the context of other cells such as CaCo-2 cells.

Overall, the matter of polarity would be important, if indeed the virus could only access cell-associated HSPGs as primary binding receptor, or the elusive secondary receptor via the ECM in the used model system (HaCaT cells), if they would locate exclusively basolaterally. This is at least not the case for binding, as observed in several previous publications (just two examples: Becker et al, 2018, Smith et al., 2008). With only a rather weak attempt at experimental verification of their model system with regards to polarity of binding, the authors then go on to base their conclusions on this unverified assumption.

This is one example of several in the manuscript, where claims for foundational premises, observations, and/or conclusions remain undocumented or not supported by experimental data.

Another such example is the assumption of transfer of the virus from ECM to the tetraspanin CD151. Here, the conclusions are based on the poorly documented inability of the virus to bind to the cell body, which is in stark contrast to several previous publications, and raises questions. Thus, association with CD151 likely occurs both from ECM derived virus AND virus that binds to cells, so that any conclusions on the mode of association is possible only in live cell data (which is not provided). Overall, their proposed model thus remains largely unsubstantiated with regards to receptor switching.

There are a number of important additional issues with the manuscript:

First, none of the inhibitors have been tested in their system for efficacy and specificity, but rely on published work in other cell types. This considerably weakens the confidence on the conclusion drawn by the authors.

Second, the authors aim to study transfer from ECM to the cell body and effects thereof. However, there are still substantial amounts of viruses that bind to the cell body compared to ECM-bound viruses in close vicinity to the cells. This is in part obscured by the small subcellular regions of interest that are imaged by STED microscopy, or by the use of plasma membrane sheets. This remains an issue despite the added Supple. Fig. 1, where also only sub cellular regions are being displayed. As a consequence the obtained data from time point experiments is skewed, and remains for the most part unconvincing, largely because the origin of virions in time and space cannot be taken into account. This is particularly important when interpreting the association with HS, the tetraspanin CD151, and integral alpha 6, as the low degree of association could be originating from cell bound and ECM-transferred virions alike.

Third, the use of fixed images in a time course series also does not allow to understand the issue of a potential contribution of cell membrane retraction upon cytoD treatment due to destabilisation of cortical actin. Or, of cell spreading upon cytoD washout. The microscopic analysis uses an extension of a plasma membrane stain as marker for ECM bound virions, this may introduce a bias and skew the analysis.

Fourth, while the use of randomisation during image analysis is highly recommended to establish significance (flipping), it should be done using only ROIs that have a similar density of objects for which correlations are being established. For instance, if one flips an image with half of the image showing the cell body, and half of the image ECM, it is clear that association with cell membrane structures will only be significant in the original. But given the high density of objects on the plasma membrane, I am not convinced that doing the same by flipping only the plasma membrane will not also obtain similar numbers than the original.

Author response:

The following is the authors’ response to the previous reviews

eLife Assessment

This study provides valuable insight into the role of actin protrusions in mediating early pre-endoyctic steps of human papillomavirus entry at the cell surface. Using state-of-the-art microscopy in an immortalized keratinocyte model, the authors present mostly solid evidence that filopodia actively promote the transfer of heparin sulfate-coated virions from the extracullar matrix to the viral entry factor CD151. Remaining gaps in the mechanistic model could be further supported by including a more expansive analysis of the fixed microscopy samples and live cell imaging to distinguish virion transfer from direct binding.

We thank the editorial team for the improved eLife assessment. Regarding the remaining gap, we agree that it is not clear why the large majority of the virions indeed are transferred and not directly binding virions.

Public Reviews:

Reviewer #1 (Public review):

Summary:

The author's goal was to arrest PsV capsids on the extracellular matrix using cytochalasin D. The cohort was then released and interaction with the cell surface, specifically with CD151 was assessed.

The model that fragmented HS associated with released virions mediates the dominant mechanism of infectious entry has only been suggested by research from a single laboratory and has not been verified in the 10+ years since publication. The authors are basing this study on the assumption that this model is correct, and these data are referred to repeatedly as the accepted model despite much evidence to the contrary. The discussion in lines 65-71 concerning virion and HSPG affinity changes is greatly simplified. The structural changes in the capsid induced by HS interaction and the role of this priming for KLK8 and furin cleavage has been well researched. Multiple laboratories have independently documented this. If this study aims to verify the shedding model, additional data needs to be provided.

Comment of the authors: the above paragraph is copied from the very first review and describes the situation before revision.

Note on revisions:

The authors did an excellent job in their revision to include data from the effect of proteolytic priming on their observed virion transfer to the cell body. All other minor issues were addressed adequately.

We are grateful that the referee acknowledges that we addressed all issues adequately.

The work could be especially critical to understanding the process of in vivo infection. 

We agree, and would like to point out that a similar comment was raised by the reviewing editor assigned to our original submission, John Schiller. For unknown reasons, he was no longer involved in the evaluation of the revision.

Reviewer #2 (Public review):

The study design involves infecting HaCaT cells (immortalised keratinocytes mimicking basal cells of a target tissue) and observing virus localization with and without actin polymerization inhibition by cytochalasin D (cytoD) to analyze virion transfer from the ECM to the cell via filopodial structures, using cellular proteins as markers.

In the context of the model system, the authors stress in the revised version the importance of using HaCaT cells as a relevant 'polarized' cell model for infection. The term 'polarized' is used in the cell biological literature for epithelial cells to describe a strict apical vs. basolateral demarcation of the plasma membrane with an established diffusion barrier of the tight junction. However, HaCat cells do not form tight junctions. In squamous epithelia, such barriers are only found in granular layers of the epithelium. The published work cited in support of their claims either does not refer to polarity or only in the context of other cells such as CaCo-2 cells.

We thank the reviewer for this important clarification and fully agree. HaCaT cells do not form tight junctions and therefore do not fulfill the classical definition of polarized epithelial cells with a strict apical basolateral diffusion barrier. In response to this comment, we have removed the term “polarized” in reference to HaCaT cells throughout the revised manuscript. Our intention was not to imply classical epithelial polarity, but rather to emphasize that HaCaT cells represent a functionally relevant keratinocyte model that recapitulates key early steps of HPV infection observed in vivo, particularly abundant ECM deposition enabling for strong virion binding to the ECM.

We now state on line 120: “PsVs that bind to the ECM at sites distal from the cell body are unable to establish direct contact with entry receptors, until the cell migrates onto them or they are transported along cell protrusions towards the cell body (Schelhaas et al., 2008; Smith et al., 2008). Both cell migration and protrusion transport depend on actin dynamics (Schaks et al., 2019). We aimed for blocking these active recruitment mechanisms in HaCaT cells, a cell line that is widely used as a cell culture model for HPV infection. They resemble primary keratinocytes in several key aspects: they are not virally transformed and produce large amounts of ECM, promoting interactions between viruses and ECM components and thereby facilitating infection (Bienkowska-Haba et al., 2018; Gilson et al., 2020). In addition, subconfluent HaCaT cells form filopodia and filopodial transport is used for the recruitment of ECM-bound virus particles to the cell body (Schelhaas et al., 2008, Smith et al., 2008). Together, these features make HaCaT cells a suitable model for studying active PsV recruitment from the ECM to the cell surface.”

Overall, the matter of polarity would be important, if indeed the virus could only access cell-associated HSPGs as primary binding receptor, or the elusive secondary receptor via the ECM in the used model system (HaCaT cells), if they would locate exclusively basolaterally.

We apologize for not having stressed enough that virions bind as well directly to the not imaged, upper cell membrane. To make clear that HaCaT cells are still a suitable model for studying active recruitment, throughout the manuscript, we worked on the following issues (this is an outline, for details see below):

(1) We now discuss adequately that virions reach cell surface receptors either by passive diffusion or by active transport mechanisms, the latter involving actin dynamics (filopodial transport and cell migration), to which we refer in the revised manuscript as active recruitment.

(2) We explain why the large majority of virions in the microscopic assay are actively recruited virions.

(3) We explain the difference between biochemical infection assays that do not differentiate between passive and active recruitment, and microscopic assays studying the basal cell membrane and by this primarily actively recruited virions

This is at least not the case for binding, as observed in several previous publications (just two examples: Becker et al, 2018, Smith et al., 2008). With only a rather weak attempt at experimental verification of their model system with regards to polarity of binding, the authors then go on to base their conclusions on this unverified assumption.

We agree with the reviewer that strict epithelial polarity would only be relevant if HPV binding or receptor accessibility were confined to the basolateral membrane, which is not the case in HaCaT cells, as shown previously (e.g., Becker et al., 2018; Smith et al., 2008). However, our conclusions do not rely on strictly polarity-dependent binding.

We added the following paragraphs clarifying that (i) in HaCaT cells PsVs also bind by passive diffusion to the upper cell membrane and that (ii) at the basal membrane the large majority of imaged PsVs is actively recruited.

Line 332: “…, the lower PCC at 0 min/CytD suggests that without active recruitment less PsVs reach CD151. At 30 min after CytD, the PCC has reached the level of 0.1 as in the control, which is in line with the idea of fast recruitment as observed in Figure 4. To follow how the basal cell membrane is populated with PsVs over time, as additional analysis we determined the PsVs per µm² in ROIs placed in the cell body region. At 0 min, CytD reduces the PsV density to 19 - 33%, albeit the effect is not significant, and at 180 min/CytD the same PsV density as in the control is reached (Supplementary Figure 6A and B). Overall, under CytD there was a trend towards less PsVs present (Supplementary Figure 6A and B). Hence, both Figure 5C and Supplementary Figure 6A and B suggest that active virion transport is required to reach efficiently the basal membrane.”

Line 447: “Throughout all experiments, we observe at 0 min/CytD only few PsVs at the basal membrane (Figure 1A, Supplementary Figure 6A and B; see also PCC at 0 min between PsVs an CD151 in Figure 5C), suggesting that in the absence of active recruitment the access to the basal membrane via passive diffusion is limited. We wondered, how many PsVs may bind to the cell membrane without a diffusion barrier? For this reason, we incubated EDTA detached HaCaT cells in suspension with PsVs for 1 h at 4 °C, followed by re-attachment for 1 h. Under these conditions, we find, despite of a shorter incubation time (1 h versus 5 h), a roughly 3-fold larger PsV density (1.7 PsVs/µm² (Supplementary Figure 6D)) than the highest density observed in the other experiments. However, it should be noted that values of the different experiments cannot be directly compared. Aside from the different treatments, another difference lies in the size of the imaged membrane. The re-attachment of cells is not complete after 1 h (compare size of adhered membranes in Supplementary Figure 6A and 1A), wherefore the membranes are likely strongly ruffled, which results in the underestimation of the membrane area. As a result, we overestimate the PsVs per µm² adhered membrane (please note that we cannot re-attach cells for longer times as we then lose PsVs due to endocytosis). In any case, the experiment suggests that PsVs bind more efficiently to membrane surface receptors without a diffusion barrier. We conclude that in our assay PsVs cannot readily bypass the active PsV recruitment by diffusing directly to the basal cell membrane, which is plausible, because to make this happen a 55 nm large PsV must diffuse through the narrow gap between glass-coverslip and adhered cell.”

Line 538: “The analyzed PsVs hardly bind to the basal cell surface directly by diffusion (Supplementary Figure 6, compare PsV maxima density at 0 min/CytD in A and B to C). Therefore, the actin-driven virion transport would play a decisive role in HPV infection if cells would form a monolayer with a disruption at which ECM is present and that is approached by PsVs, a scenario similar to in vivo infection. In addition, cell migration could establish contact between PsVs and the cell surface.”

Line 548: “…that can readily bind to the upper cell membrane. We are not aware of a PsV translocation mechanism from the upper to the basal membrane. Therefore, in our assay, PsVs bound to the upper membrane are not expected to show up at the basal membrane. Comparing 0 min of control and CytD (Supplementary Figure 6A and B), we find that compared to the control 19 - 33% of the PsVs reach the basal membrane in the absence of active transport, or in other words, most likely by passive diffusion. Actually, the range from 19 – 33% must be a strong overestimate as PsVs in the control are in transit and many actively recruited PsVs are already internalized during the 5 h incubation period. For this reason, we propose that most likely much less than 10% of the PsVs reach the basal membrane by diffusion. Moreover, in the absence of the diffusion barrier, the density of bound PsVs is strongly increased (Supplementary Figure 6D), showing indirectly that at the basal membrane the binding sites are difficult to access without active recruitment. Taken together, we propose the large majority of PsVs analyzed in our assay are ECM bound and actively recruited to the basal cell membrane.”

This is one example of several in the manuscript, where claims for foundational premises, observations, and/or conclusions remain undocumented or not supported by experimental data.

Another such example is the assumption of transfer of the virus from ECM to the tetraspanin CD151. Here, the conclusions are based on the poorly documented inability of the virus to bind to the cell body, which is in stark contrast to several previous publications, and raises questions.

We hope with the above changes we made clear that virions can also directly bind to the cell body. We also added a paragraph discussing differences between biochemical and microscopic assays.

Line 568: “In this scenario, sub-confluent HaCaT cells, or even better single HaCaT cells, would be an ideal model system for the microscopic study of these very early infection steps that involve ECM attachment and subsequent active recruitment, as supposed to occur during in vivo infection of basal keratinocytes after binding of virions to the basement membrane (Bienkowska-Haba et al., 2018; Day and Schelhaas, 2014; Kines et al., 2009; Schiller et al., 2010). In contrast, in biochemical infection assays, virions diffusing to HSPGs on the cell surface, and by this bypassing active recruitment, are assayed together with the actively recruited virions. Should cells secrete little ECM and are grown to confluency, the passively binding virions are supposed to strongly dominate the infection rate in a biochemical infection assay.”

There are a number of important additional issues with the manuscript:

First, none of the inhibitors have been tested in their system for efficacy and specificity, but rely on published work in other cell types. This considerably weakens the confidence on the conclusion drawn by the authors.

We use inhibitors CytD, blebbistatin, leupeptin and furin inhibitor I. The below references are examples reporting the usage of the inhibitors on HaCaT cells studied in the context of HPV infection.

Furin inhibitor I:

Cruz et al., Cleavage of the HPV16 Minor Capsid Protein L2 during Virion Morphogenesis Ablates the Requirement for Cellular Furin during De Novo Infection. Viruses, 2015; doi.org/10.3390/v7112910

Cytochalasin D/Blebbistatin:

Schelhaas et al., Human papillomavirus type 16 entry: retrograde cell surface transport along actinrich protrusions. PLoS Pathog., 2008. doi: 10.1371/journal.ppat.1000148.

Smith et al., Virus activated filopodia promote human papillomavirus type 31 uptake from the extracellular matrix. Virology, 2009; doi.org/10.1016/j.virol.2008.08.040 and

Leupeptin/Furin inhibitor I:

Cerqueira et al., Kallikrein-8 Proteolytically Processes Human Papillomaviruses in the Extracellular Space To Facilitate Entry into Host Cells. J. Virology, 2015; doi.org/10.1128/jvi.00234-15

Moreover, the reversible inhibitory effect of CytD the key inhibitor, used in this study on transport and infection is validated in this study. However, we discuss this data now in the context of directly binding virions more critically.

Line 485: “Hence, the infection assay suggests that the treatment is largely reversible and only slightly harmful, if at all. However, the luciferase infection assay does not distinguish between actively recruited PsVs and PsVs that bind passively by diffusion to the upper membrane. The latter fraction likely dominates the total infection rate and should be less affected by CytD than the fraction of actively recruited PsVs. Therefore, if the infection pathway of a small fraction of actively recruited PsVs is irreversibly inhibited, we may not be able to detect this effect on the background of unaffected passively binding PsV.”

Second, the authors aim to study transfer from ECM to the cell body and effects thereof. However, there are still substantial amounts of viruses that bind to the cell body compared to ECM-bound viruses in close vicinity to the cells.

Regarding direct binding to the cell body, please see our detailed reply above.

This is in part obscured by the small subcellular regions of interest that are imaged by STED microscopy, or by the use of plasma membrane sheets. This remains an issue despite the added Supple. Fig. 1, where also only sub cellular regions are being displayed. As a consequence the obtained data from time point experiments is skewed, and remains for the most part unconvincing, largely because the origin of virions in time and space cannot be taken into account. This is particularly important when interpreting the association with HS, the tetraspanin CD151, and integral alpha 6, as the low degree of association could be originating from cell bound and ECM-transferred virions alike.

We hope with the above explanations it is plausible that the imaged virions primarily reach the basal membrane by active recruitment.

Third, the use of fixed images in a time course series also does not allow to understand the issue of a potential contribution of cell membrane retraction upon cytoD treatment due to destabilisation of cortical actin. Or, of cell spreading upon cytoD washout. The microscopic analysis uses an extension of a plasma membrane stain as marker for ECM bound virions, this may introduce a bias and skew the analysis.

The referee is correct in pointing out that cell spreading after CytD wash off would affect our analysis, e.g. by increasing the overlap between PsVs and the cell body although no active recruitment via filopodial transport and cell migration occurs. An argument speaking against this possibility is the lack of increase in the PCC between PsVs and F-actin after CytD removal, if the protease inhibitor leupeptin was present (Figure 2B and D). Leupeptin prevents PsV/phalloidin overlap despite restored actin polymerization after washout of both inhibitors, suggesting that priming is required for increased PsV–actin association and is too slow to change PCC within 60 min. These results support that the observed overlap reflects active, priming-dependent recruitment rather than cell morphology changes.

We state on line 252: “Moreover, the experiment suggests that without PsV priming the PCC between PsV-L1 and F-actin does not increase, for instance, due to cell spreading after CytD removal.”

On line 494, we state “However, we assume that this is rather unlikely, as cell spreading would increase the PCC between PsVs and F-actin under a condition where PsVs are not-primed (and therefore not actively recruited) but cell spreading occurs, which is not the case in Figure 2B and D (CytD/leupeptin).”

Fourth, while the use of randomisation during image analysis is highly recommended to establish significance (flipping), it should be done using only ROIs that have a similar density of objects for which correlations are being established. For instance, if one flips an image with half of the image showing the cell body, and half of the image ECM, it is clear that association with cell membrane structures will only be significant in the original. But given the high density of objects on the plasma membrane, I am not convinced that doing the same by flipping only the plasma membrane will not also obtain similar numbers than the original.

Regarding the association of PsVs with CD151 and HS, we corrected for random background with reference to a calibration line that describes the random background association in dependence of the density of objects. We now refer to this issue on line 343: “…, the fraction of PsVs closely associated with CD151 is around 10% (Figure 5D, control), after correction for random background association, for which we used a calibration line based on the same density of PsVs in flipped images (see Supplementary Figure 7).”

In the legend of Supplementary Figure 7 we state: “…The fraction of closely associated PsVs (PsV-L1 maxima with a distance ≤ 80 nm to the next nearest CD151 maximum) in the Control of Figure 5 was analyzed on original and flipped images (for an example of a flipped image see Supplementary Figure 5A)…on flipped images, we often find values more than half of the values of the original images, demonstrating that many PsVs have a distance ≤ 80 nm to CD151 merely by chance, in the following referred to as background association…We take the altogether 24 fraction values obtained on flipped images (12 values from Control and CytD each), and plot the fraction of closely associated PsVs against the average CD151 maxima density in the respective images. As can be seen in (C), the fraction increases with the maxima density, as the chance of a distance ≤ 80 nm increases with the maxima density. The fitted linear regression line describes how the background association depends from the maxima density. As a result, the background association (y) can be calculated for any maxima density (x) with the equation y = 2.04 • x. The CytD/0 min condition may be overcorrected, if it includes many images with CD151 flipped onto peripheral PsVs that actually are distal to CD151 (for an example ROI see Supplementary Figure 5A). On the other hand, PsVs right at the cell border, where CD151 staining tends to be strong (Supplementary Figure 5A), after flipping have less CD151 than before, contributing to undercorrection.”

When omitting the CytD/0 min values, we obtain essentially the same calibration line.

Recommendations for the authors:

Reviewer #2 (Recommendations for the authors):

There are further issues that are not pertaining to the study design that I find important.

Fig.1

There are few, if any, filopodia in untreated cells. It would be good to quantify their abundance to substantiate that resting HaCat cells are indeed a good model for filopodial transport bs. membrane retraction / spreading.

We see filopodia in untreated HaCaT cells (although quite variable in abundance, please see control cells in e.g. Figure 3 and 8 and Supplementary Figure 2).

In HaCat ECM the virus binds also to laminin-332 for a good part. Would this not also confound the analysis?

We agree with the reviewer that in HaCaT-derived ECM, virus binding is not restricted to heparan sulfate (HS), and that laminin-332 represents an additional relevant binding partner. Indeed, viruses bound to laminin-332 may likewise be transported toward the cell body via laminin-binding integrins. We therefore consider laminin-332 to act as a parallel attachment factor alongside HS rather than as a mutually exclusive alternative.

However, the primary aim of this study was not to comprehensively map all ECM binding partners, but to analyze the actin-dependent transport of ECM-bound virus particles. HS was chosen as a representative and well-characterized ECM marker for initial virus attachment. Importantly, inhibition of actin dynamics by cytochalasin D blocks this transport process downstream of initial binding. Thus, irrespective of whether the virus is initially bound to HS, laminin-332, or both, the readout reflects interference with the same actin-dependent transport mechanism.

Consequently, the presence of laminin-332 binding does not confound our analysis, as the experimental outcome is determined by inhibition of transport rather than by the specific ECM attachment factor. Nonetheless, we acknowledge laminin-332 as an important parallel interaction partner and had already mentioned it the first version of the manuscript, but removed the sentence during the last revision, that has now been added again. On line 593 we state: “Finally, not all PsVs bound to the ECM are expected to bind to HS but could also bind to laminin 332 (Culp et al., 2006).”

Fig.2

Would benefit from live cell analysis. There are considerable amounts of virions on the cell body, which partially contradicts statements from Fig. 1. The fast transfer to the cell body after cyto D washout is based on the assumption that filopodia formation and transport along them (and not membrane extension) occurs quickly. Is this reasonable? Does membrane extension and migration occur between 0 min and later time points?

Regarding membrane extension after CytD removal, that in the analysis may be indistinguishable from active recruitment transfer, please see our reply above (no PCC increase between PsV-L1 and F-actin after CytD removal if leupeptin is employed). Regarding migration, we now included this possibility as an active recruitment mechanism that may occur in parallel to filopodial transport (please see our reply above).

Fig.4

How are the subcellular ROIs chosen? Is there not a bias by not studying a full cell?

In Figure 4 we are specifically interested in the time course of PsV diminishment from the cell periphery. The ROIs are generated with reference to the membrane staining, using the cell body delineation as a starting point. For details about how ROIs are generated, please see legend of Figure 4 and materials and methods.

Fig. 5/6

The data needs a better analysis on correlation by using randomisation as explained above.

Please see our reply above. The association between PsVs and CD151 or HS has been corrected using a calibration line based on the same density of objects.

Fig. 8. Why does blebbistatin block the transport only partially? Previous work on actin retrograde flow suggests that in the absence of myosin II function the transport stops completely. Would this not be a concern, when interpreting the city D data?

Is the referee referring to Schelhaas et al., 2008 that we cite in the paper? In this paper, in HeLa cells blebbistatin reduced the directed particle motion by 82%, but not completely.

Suppl. Fig. 1A, B: Intented to adress the issue of viruses binding to the cell body, it unfortunately falls short. It would have been better to analyse complete cells rather than ROIs, or better even, a comprehensive analysis of cell islets (boundary cells vs. central cells, with cell body to cell periphery).

This experiment addresses the increase in PsV density resulting from active recruitment. Outlining entire cells would include also PsVs close to the cell edge that have not been actively recruited.

Regarding cell islets (we call them patches of confluent cells as islets may be confused with e.g. more structured Langerhans islets), there are hardly any PsVs at the basal membrane. We state on line 135: “Frequently, we observe patches of confluent cells which are common to HaCaT cells. Cells at the center of these patches are dismissed during imaging, because hardly any PsVs are bound to their basal membrane, indicating that PsVs do rather not reach this area by passive diffusion. Instead, we focus on isolated HaCaT cells or cells at the periphery of cell patches. At these cells, we find more PsVs per cell than one would expect from the employed ≈ 50 viral genome equivalents (vge) per cell, indicating that PsVs are unequally distributed between the cells.”

Is the difference between untreated and cytoD treated significant?

We stated in the Figure legend that the difference is not significant (the exact p value is p = 0.089). We now have revised the Figure (previously Supplementary Figure 1A and B, now Supplementary Figure 6A and B), showing the PsV density at the basal membrane over time, also for the experiment shown in Figure 6. The now revised Figure (Supplementary Figure 6A and B) is discussed together with the re-attachment experiment (Supplementary Figure 6C and D), in order to compare the PsV accessibility to the cell membrane with and without diffusion barrier. Please see our reply above (paragraph starting at line 447).

many videos that used to have captions enabled are no longer do

💻/thinkpad/🧊/me/📓/2026/04/29

pdweb.archive

This article introduces us ti the CIDER technique, which is a design evaluation method used to help technology creators create an inclusive product. There are 5 stages of CIDER, which all focus on correcting implicit biasas. Firstly, there is the Critique step where learners analyze a specific technology to indentify implicit assumptions made about users' disabilities and resources. Then, learners choose 1 assumption to write a scenario where user is excluded, in order to sympathize and thoroughly understand real experience of users. The third phase requires learners to brainstorm ways to redesign the technology to remove that issue. Subsequently, the instructer compiles a list of all assumptions identified and shows learners what they've overlooked. Finally, learners select a new assumption from that shared list and repeat the second and third steps to broaden their understanding of diverse biases. This CIDER technique helps avoid overgeneralized personas and build confidence for students who work on professional inclusive design.

This article supports the chapter's definition of disability as a social construction. It states the difference between the medical model (the problem belongs to the individual with a disability), and the social model (the disability was caused by the way society was developed). I believe the social model is more convincing because it makes the designers of society responsible for changing barriers rather than making individuals who have disabilities accountable for those barriers. Examples such as the staircase at Walmart or the reach on grocery shelves support this belief. If we would design products differently many disabilities would cease to be present.

One source listed is Handbook of Augmentative and Alternative Communication by Denise C. DeCoste. This source talks about ways people can communicate if they have trouble speaking. It explains that assistive communication tools can be simple, like picture boards, or more advanced, like devices that speak for the user. A key detail is that these tools can be changed to fit each person’s needs, which shows that assistive technology is made to help people in different ways depending on what they need.

Most people can relate to feeling like they have short attention span or the feeling of being hyperactive. But can most people relate to experiencing a state of hyperfixation? It seems like one of the more unique and less talked about symptoms of ADHD. It is something that I feel I can absolutely relate to, despite not being diagnosed. I could list numerous short-term hobbies Ive had over the years. For example in high school I became interested in building lamps. Another time, baking bread. Most recently, astronomy- I bought a Celestron Star-Hopper 8in Dobsonian telescope on facebook marketplace and have been using it to look at the moon every chance I get. It weighs nearly 100 pounds and is 4ft long. It seems like theres constantly something I am intently researching or obsessing over. But if someone asked me what I enjoy doing? Im not sure if I would have a good answer.

This article explains that disabilities may not only be applied to people with Physical or Mental disabilities but by our society. Disabilities are looked down upon by society through inaccessible buildings or negative attitudes contradicting the medical model.

I looked at the source about assistive technology, and I think it connects to the chapter because it shows how tools can help people work around barriers. Assistive technology can include things like screen readers, hearing aids, wheelchairs, or other devices that help people do daily tasks. This made me think that disability is not only about what a person can or cannot do. It is also about whether society gives them the right support. A tool that seems small to one person can make a big difference for someone else. This source helped me understand that access is not just about being “nice.” It is about making sure people can take part in school, work, and daily life.

Social Model of Disability, referenced in this chapter, emphasizes the difference between individuals' impairment and barriers to accessibility imposed on people by society. Instead of treating disability as a flaw or a deficiency, Social Model of Disability states that individuals become disabled through social and physical barriers rather than impairment. Thus, people are disabled by society, not their disability; that is to say, disability becomes a barrier imposed by society. To me, it seemed to be an interesting approach, especially taking into account social media which either promotes or eliminates barriers.

I thought that this brief article on color blindness was interesting, especially the statistic about the probability of male versus female color blindness. To me, that kind of makes sense because all of the people I know who are color blind are males, but I just didn't think the difference was that drastic. I also thought it was interesting that color blindness tends to be with a couple of specific colors, and not all colors in general.

Color blindness is a topic that is rarely discussed because some people do not realize that they have it until they encounter a certain situation. My uncle often confuses colors and once he said that he wanted to buy the lipstick color that his wife liked but it was quite difficult for him because lipstick colors often have different shades like red, which has many shades of red. I have seen a few devices that help them distinguish between different colors like glasses for example. They will have difficulty if they work in fields related to art. This disability most commonly defines men.

This article in the New York Times by Meg Miller and Ilaria Parogni details "alt text"- pieces of digital text (typically with read-aloud functionality) attached to images which they describe. These alt-texts are described to be useful tools for those with varying degrees of impaired vision- but are often quite limited by the fact that most images posted online simply lack them- and by the fact that AI generated alt-texts (while allowing for them to be more widely generated) are often lacking in quality. However, the article still notes that immense progress on this front is being made.

I have a friend that is color blind but our friend group a lot of times forgets that he is color blind. The first time it affected us however was when we were playing a minigame that required standing on the correct colored tiles and my friend struggled a bit with it but we all still had a good time. It's not often seen as a big disability but I can see how such an impairment can make certain tasks harder, especially with more severe color blindness. One thing I saw in the wiki page was the stuff on the color blind glasses. While it may help some people differentiate colors better, I heard that it was a big scam and don't actually give people normal vision. I remember when Logan Paul wore them and he pretended to have an eye-opening experience but he later said in an interview that it did nothing for him at all.

When reading this, like the title suggested, I thought this woman was someone who had something like a superpower. As I read through the article, I realized that because of this superpower of hers, some settings might be overstimulating due to excessive color (from her perspective). That's when I came to the further realization that some people would consider this a disability as she might need some accommodation to be comfortable in some spaces. This situation made me realize that "disabilities" aren't necessarily always visible and aren't negative. It also made me realize there are so many types of "disabilities" that I might not have ever even heard of.

I found this source super cool. It talks about a small percentage of women who can see four base colors instead of three. It's super interesting and reminds me of the Psych 101 class I took in the fall where one of the topics we looked at were how do we know if everyone sees the same colors.

This source explains that disability is not just about a person’s condition, but also about how society is designed. It compares the medical model, which focuses on fixing the person, with the social model, which focuses on fixing barriers in the environment. One key point is that things like inaccessible buildings or websites can create disability even if the person’s condition doesn’t change. This idea is important because it shifts responsibility to designers and society, not just individuals.

The reason I chose this source specifically (to look into) is because I almost was colour blind. It's a long story but pretty much I stabbed myself in the eye and barely missed a spot that is a big part in seeing colours (atleast what the doctor said), so I was curious on finding out more today. I found it interesting that most colour blindness was onset from genetics/ inherited. Also, colour blindness tends to not be all colours, but rather certain ones. For example, red green is the most common form of colour blindness. Now, this isn't full on blindness to red and green, but rather the inability (or difficulty) to distinguish those hues.

This is so true, yet it remains an unsolved problem for designers and programmers. Since there are thousands types of users with varied needs, disabilities, and preferences, it is really hard to design a platform that takes all those aspects into account. Because developers usually have limited resources to satisfy each individual, they often end up prioritizing the majority just to get the product launched. This creates a cycle where the same groups of people are left behind over and over again.

I don't understand this concept of "we won't include people who are actually in need of the product". I feel like you need opinions of that group to first of all cater to their needs and what they struggle with, they can also exactly point out what works and what doesn't. there is this company that designs for people with limited mobility and since they interegate actual feedback and have the group they cater for in the team, they can come up with solution for mostly every problem the consumer can

I think this goes back to what we were discussing earlier in the course- particularly on programmed bias in social media. In that earlier topic, we discussed how social media designers, mostly unconsciously, program in their own biases into social media- particularly the algorithms they make- leading to prejudiced outcomes. Here, it's a similar situation where non-disabled designers create non-accessible social media sites because they are simply not likely to consider such things.

Diversity should be important in both who the product is designed for and who is designing those products. I feel recently big companies prioritize diversity and accessibility in a way that seems performative and just to please their consumers criticisms about diversity and accessibility. Able-bodied people are still designing things for disabled people, and it doesn't work because they don't have the perspective and experience of disability to create solutions for them. Designers should be diverse so they can create based on experience rather than assumption.

Kubb Lawn Game Introduction

Master the art of the Viking-inspired kubb lawn game with this comprehensive guide. Covering essential rules, equipment, and winning strategies for all skill levels. Explore tactics to dominate the pitch and become a "King of the Pitch" this summer by reading on.

Master the Art of Strategy: Your Ultimate Guide to the Kubb Lawn Game!

Master "Viking Chess" with the addictive kubb lawn game. A strategic, fun activity for all ages. Discover the rules and learn to topple the king now.

the country now gained +7,747 native-born citizens in the year 2024.

The Netherlands and Luxembourg are common destinations – suggesting that cross-border commuting also plays a role.

This follows a common pattern of migration flows: The ones that are on the move belong to the younger working-age cohort, they are not

the second largest absolute outflow in our dataset. Second only to Germany, and up

people born there. It's only a part of the whole migration story, eg. Germany's

its largest in decades

While native-born immigration to Sweden remained rather stable lately, the emigration numbers rose significantly.

comparison: Cyprus, Denmark, France, Greece, Hungary, Ireland, Malta, Poland, and Portugal.

The willingness to migrate is high among German talents

data about the share of university graduates

As the migrants are predominantly working-age, and they mostly leave for high-salary economies, all data

This Eurostat dataset contains information about the age profile and the destinations of these migrants - but not about education levels.

The top three destinations for German migrants

with an extremely high share of families with children among the leaving persons

After only slightly net negative migration numbers for domestically born citizens in the 2010s, the losses

has only reached passport holders yet

the 2024 spike may be exaggerated, as it is partly due to

nevertheless, the underlying brain-drain trend remains clear

2024). But it's an important and often overlooked part: The

drain: people a country invested in from day 1 opt to continue (or start) their career somewhere else. It's a widespread trend in Europe - 17 of 19 measured countries suffer net losses.

Float Bridges Introduction

Float bridges, or "roadways on water," use advanced engineering and buoyancy principles to support heavy traffic over deep water without requiring traditional seabed pillars. Explore the evolution of these infrastructure marvels and the science that prevents them from sinking, from their origins as temporary military tools to record-breaking permanent structures.

Imagine driving on water! Discover how float bridges defy gravity using engineering marvels, transforming aquatic travel. Explore the full story here.

bur

religion

background for the castle

Industrialisation had aided it, but also threaghtened the continuation of building!!!

same in castell coch

relation to st lucius

as seen in castell coch!

evident in castell coch??

SLAYYYY works well with castell coch, the building was in the style he prefered?

as seen at castell coch??

SLAYYYY this shows how, while there were clear catholic taints to it, which was seen by Bute! not everyone saw it as catholic, with ruskin managing to get rid of the papal label associated with it, with a far greater array of anglican, and even dissenter, churches build

Did this innfluence castell coch? Enabled them to build it at a more affordable price?

links to religion! He himself wasn't very religious, so this was bute's innfluence and shows how religion wasn't a requisite for engaging with the style, although it was typically advertised as such

clear that his collection of illuminated manuscripts innfluenced the interior, it very much gvies that vibes!

link to industrialisation - immense wealth was needed!

This is the fella that bute met - a highly educated and well travelled man like himself!

This new way of thinking about disability has totally changed my perspective. Prior to now, I never thought about the fact that what we define as a "disability" in large part will depend upon what a society believes are things that people should be able to do. The example with tetrachromats shows this so clearly. Tetrachromats (as far as I know) literally see colors better than everyone else, but since our society does not build itself based off of a tetrachromatic's abilities they provide absolutely no advantages. So, the question now is this; If our society were to build itself around tetrachromats, would the rest of us be considered "color impaired/disabled"? This relates to something I've observed lately; A lot of the structure and design that make up the physical world (and many other aspects of society), seem to default back to a fairly narrow idea of what constitutes a "normal" human body. As shown in both the examples of staircases and grocery shelves, much of the social definition of disabilities (the limitations caused by them) is socially constructed through design rather than the actual individual. I believe that there is an ethically relevant issue here too; Since the definitions of disability are socially constructed, it seems reasonable to conclude that society also has some level of obligation/responsibility to either create barriers to disablement, or eliminate existing ones through its design decisions.

I had no idea that tetrachromacy was a condition some people live with. Throughout my life Ive often questioned whether everyone experiences color the same way, or if we all have a unique perception of how the different colors appear. For example, could a blue book appear blue to one person, and yellow to another? I would reason about it, and come to the conclusion that even if a color appears different to different people, it doesn't matter, as the quantifiable characteristics of that color are the same between people. Light colors appear light and dark colors appear dark. Colors mix with each other in predictable ways. This excerpt seems to have in some ways answered that original question for me, and proven me wrong. In the past I failed to consider changes in human anatomy. Some people simply cant see all the colors, or colors appear washed out. Evidently, some people can even see more colors than me.

I think this first sentence is important because it shows that disability depends a lot on the situation. A person may be able to do many things, but one part of society can make them seem “unable.” For example, if a classroom only gives information through small printed text, then students who cannot see it clearly will have a harder time. The problem is not only with the student. The problem is also with how the classroom is set up. This made me think that society should not assume everyone has the same body, mind, or needs. A better system should give people different ways to access the same thing.

Unfortunately I had seen this in real life but never thought of it this way. I have bad vision but I seen menu, instructions, guidelines,.. written so tiny that I blame myself when I can't read. Yet after learn about this definition i realize how everyone normalize this behavior. I would really appreciate if more people learn about this definition so there's no more "sorry, we're not handicap friendly".

I think I disagree with this definition of "disability" because there are disabilities that are definitely objective and not a subjectively defined thing based on society's expectation. There's just a medical model versus the social model of what being disabled means. Disabilities could be a result of the body or mind but it can also be because society failed to accommodate different bodies and minds. A bird born without wings is not disabled because society expects a bird to fly. Even without society, the bird will die on its own. The bird is objectively disabled. In the end, society can partially define disabilities while others are objective disabilities.

the golem is allowed to do as it wishes, it pushes the prince’s sealed sarcophagus through the portal into Elysium. Three solars meet the golem on the other side, open the sarcophagus, welcome the prince into the afterlife, and take the Nether Scroll of Azumar into safekeeping. This leaves the party to fight it out with the dracolich and there is no reward.

Usethis bit. the three flameskulls are waiting at the gap between the buildings

DOI: 10.1016/j.cmet.2026.04.001

Resource: RRID:IMSR_JAX:012849

Curator: @nmaralla

SciCrunch record: RRID:IMSR_JAX:012849

What is this?

DOI: 10.1016/j.cmet.2026.04.001

Resource: RRID:IMSR_JAX:007914

Curator: @nmaralla

SciCrunch record: RRID:IMSR_JAX:007914

What is this?

Author response:

We are particularly encouraged by the consensus that our study provides a substantial resource and that the bioinformatic framework is biologically grounded and convincing, while appropriately noting that further experimental validation will be required. We fully agree with this point. As clarified in the revised manuscript, the lineage relationships we describe are inferred from integrative transcriptomic analyses and are intended to provide a mechanistic and conceptual framework rather than definitive proof of cellular origin. We have further strengthened the Discussion to explicitly acknowledge these limitations and outline future directions, including lineage tracing and functional validation studies.

At the same time, we respectfully note that such experimental validation would require a substantial extension of this work and likely 2–3 years of additional studies, including development of appropriate model systems. We believe these efforts represent an important next phase of investigation rather than a revision-level addition to the current manuscript. Our primary goal here is to present a high-resolution human transcriptomic resource and a coherent framework that identifies biologically plausible epithelial intermediates linking normal fallopian tube hierarchy to malignant states.

Given the reviewers’ positive evaluation and recognition of the value and rigor of the dataset and analyses, we respectfully request consideration to proceed with publication as an eLife Version of Record without further experimental revision. We believe that the timely dissemination of these findings will provide a useful resource for the field and help guide the experimental studies needed to test the hypotheses generated here.

Reviewer #2 (Public review):

Summary:

The authors used single-nuclei sequencing of benign fallopian tubes and ovarian cancer to delineate the plausible cell of origin of high-grade serous ovarian cancer.

Strengths:

These substantial data provide the field with significant research resources to examine additional features in normal fallopian tubes and ovarian cancers. The highly detailed bioinformatic analysis, rooted in a strong biological framework, is convincing. The methodology was appropriate and used validated methodology based on biological relevance (region selection and transcriptomics analysis).

The authors propose a convincing model of epithelial progenitor cells and their localisation in high-grade serous ovarian cancers. These findings are important and useful.

Weaknesses:

Overall, the weaknesses are clearly stated in the discussion. The study provides a novel framework for future study, and proposes a model which will require validation.

Within the ovarian cancer field, the endometrioid and clear cell histotypes are thought to arise from ciliated or secretory cells. Typically these are thought to be from the cervix or uterus. This concept was not mentioned in the work.

Further, in the ovarian cancer field, stemness is judged by some classic assays - aldehyde assays looking at ALDH1A1 and spheroid-producing ability. These were not mentioned - could these be useful in a population of fallopian tube epithelial cells, or would other assays/markers be more appropriate?

The choice of ES2 and OVCAR was not sufficiently justified, as ES2 is widely regarded as a clear cell ovarian cancer cell line in many research circles. Additionally, I did not see confirmation of gene knockdown by Western blot or qPCR.

PGR loss through copy number variant was surprising, as this was a marker. So would the marker be lost through one of these mechanisms randomly or specifically?

Reviewer #1 (Public review):

Summary:

Using comprehensive profiling of normal and cancerous tissue via bulk and single-cell RNA sequencing, the authors identified that high-grade serous ovarian cancer is likely to originate from the epithelial progenitor cells from the distal fimbrial region of the fallopian tube, where it has been previously shown to be most prone to ovulatory stress and other microenvironmental influences. The authors also included a CNV analysis to identify hotspots in HGSOCs.

The findings are preliminary, but the resource on its own has great potential and can be used for developing methods for early detection, stratification and treatment.

The main limitation of this study is that the lineage is purely inferred from bioinformatics analysis. More validation work is required, perhaps using cell models / other model organisms.

Strengths and weaknesses:

The authors investigated the origin of high-grade serous ovarian cancer, which is one of the deadliest. They performed comparative analysis using both bulk and single-nucleus RNA sequencing between cancerous and normal tissues (fallopian tube and ovaries) and identified a population of epithelial progenitor cells from the distal fimbrial region that are exposed to ovulatory stress, as the most plausible cells of origin. The extensive profiling of the molecular signatures can also be used for early detection and stratification for treating the disease.

Previous studies have shown that HGSOCs likely originated from the epithelial lining of the fallopian tubes (PMID 32349388). The bulk RNAseq data is confusing in that neither the overall correlation of the transcriptome nor the sample clustering (Figure 1) supports the idea that the HGSOCs are close to the fallopian tube. The authors could perform a more comprehensive marker gene-based analysis to demonstrate their relationship.

The authors also performed a comprehensive analysis of single-cell datasets on both normal and cancerous tissue in humans. From there, they performed a combination of RNA velocity, PAGA and pseudotime, etc, to try and delineate the relationship amongst related cell populations. It would be helpful if the authors could clarify why they applied this particular suite of tools (explaining the differences between tools and bioinformatic approaches) to assist the broader readership who may not be familiar with this type of single-cell bioinformatic analysis.

It also seems to me that the authors did not account for patient effect when they performed the data integration (this point is discussed in the text). This may explain at least partially why the clusters are segregated by patient samples. Another explanation is that it could be due to uneven sampling, as only very few cells (1000s) were captured from each of the tumour samples, and this is clear when a dramatic difference can be seen in their cellular composition.

The trajectory analysis of normal and cancer single-cell data should also include other cells to prevent confirmation bias, as these analyses would only consider relationships amongst the cells available in the model.

As the authors indicated in the limitations, the cell lineage in the studies is largely inferred from the bioinformatics analysis. Experimental lineage tracing via other experimental models (organoids/animal models) would be required.

Despite these limitations, this study will serve as an important resource for the scientific community. I would also suggest that the authors should share this resource via additional portals in addition to the GEO data deposit (e.g. the HCA, or single-cell portals such as at the Broad Institute or CellXGene Discover).

eLife Assessment

This valuable study reports a substantial single-cell RNAseq and bulk RNAseq dataset from multiple high-grade serous ovarian cancers, including a single-cell atlas of human fallopian tube epithelium. The bioinformatic analysis investigating the lineage and location of epithelial progenitor cells is convincing, although this will require experimental validation. The work also provides a resource to examine additional features of normal fallopian tubes and ovarian cancers, and for developing methods for early detection and tumour stratification.

22:15 "wenn die krise länger dauert"<br /> die krise wird gar nicht mehr aufhören, die krise ist der neue normalzustand

Abstract

This is an initial summary report of - a project

taking a new and systematic approach to - improving the intellectual effectiveness of - the individual human being.

A detailed conceptual framework explores - the nature of the system

composed of - the individual and - the tools, - concepts, and - methods

that match his - basic capabilities to - his problems.

One of the tools that - shows the greatest immediate promise is - the computer,

when it can be harnessed for - direct - on-line assistance,

integrated with - new concepts and - methods.

rhyme rheme to reason with

x

x

The article explains that some women carry a genetic variation that gives them a fourth type of cone cell in their eyes called tetrachromacy. This can potentially allow them to perceive millions more colors than the average person.

This is the test comment

Test

Consider tweaking this so it's not so AI ;-)

Again, very AI-typical and this structure features quite a bit on the page already. It's not this, but this. You don't need this, but this.

I'd remove this and get straight into the introducing the Club section

I'd consider rewording this because it sounds very AI ;-)

Masking is another term which clearly emphasizes on the harsh reality that we live in an environment which is very much unwilling to accommodate its inhabitants. If someone feels like he or she needs to disguise their own self just to make their way in the environment, then such an act reflects the society rather than the individual who suffers from a disability. In my opinion, the phrase "mental or physical toll" stands out because society admires individuals who "push through" but does not question why they have to push through.

I think that this is important to note because often times something that is labelled as "accessible" is only referring to physically accessible. But like this chapter says, there are many other disabilities that are invisible from the outside look of someone. Even though designers may think that there structure is accessible to all, it really is hard to make everything work for everyone, given many different conditions.

I think this should make us pause and think about all the consequences of not making the world more flexible and adaptive to disabled people. There are mental health consequences and there are physical consequences when disabled people have to constantly put extra effort into fitting in with normal people to be comfortable. As an alternative solution, society can change the design of settings, tools etc. to be usable by more kinds of people rather than only by people considered "normal".

I find this interesting. With the rise of the internet, etc, I bet theres been alot of decisions that have had to be made in regard to disability access. The text to speech / annotate is a good example of this. However, when it comes to things that have made things not accessible, not much comes to mind. I think this is clearly because I have not been in the position where I am thinking about accessibility, so I'm oblivious to what is actually going on. It shows the importance of perspective, and all the considerations / time thinking about perspective that developers (hopefully!) go through. With more time and resources for accesibility designers, hopefully we can make the relationship less complicated in regards to accessibility design.

With the rise of ADHD diagnosis in the US, many people speculate that the increase in social media use and frequent internet access has led to many more people being diagnosed with adhd. I find it interesting that the potential problem causing these issues also offers solutions, such as providing accessible jobs.

I found this cool because when I think of health, I don't think of politics. This shows how closely health is connected to politics & how CHWs help support communities in using their voice to create change

I think this is important to note because it is good to know the difference between activists & organizers. I realized that organizing is more about creating lasting impacts & changes in a community

Social media is a huge way to spread information. It is important that false or incorrect information is not spread around, causing people to get the wrong thoughts & feelings about certain things.

This made me realize that even the smallest bit of financial help can reduce stress & actually improve health & well being

This is important because many people have mixed feelings about these terms, so having read the definitions, I think it could help people have a better understanding

This really helped me realize that public health isn't just treating an illness, but it is also about fairness & equal opportunities

This helped me see the prevention process at different levels & how it can happen at different stages of health, kind of like a before, during & after type of thing

On-the-job training is actually really helpful because it gives CHWs real experience & allows people from the community to enter the field. I think it's good to come into it with certain experience, but being on the job allows you to gain ongoing education & experience

I realized that understanding this is really important because without the right competencies, it would be hard to perform the core roles effectively, even if you know what your job is

This is the first time I heard about Hypothesis, but it does appear to have some interesting functionalities.

định nghĩa tẩy chay

Recently I have been asking this question a lot to myself because I recently started to take an interest in reading I use to not be an avid reader but now I have slowly starting to read and enjoy each book I read have couple on hand and there are more that I want to read but each time I get book I have to decide one or the other and maybe get the other one a different time or if its something I am not interested in.

It clarifies that exigency isn’t just about importance—it’s about urgency and audience. It pushes you to think about why the issue matters right now, in this specific place and to these specific people, which makes your argument more focused and meaningful instead of too general.

Sclerosis literally means hardening. It describes a process where healthy, soft tissue is replaced by hard, scar - like connective tissue, usually due to chronic inflammation or injury.

Sclerosis in the Brain The Attack: Your immune system mistakenly attacks the myelin sheath (the insulation created by glial cells)

The Damage: This is stripping away of myelin is called demyelination

The Sclerosis: as the body tries to heal, it leaves behind tough, fibrous scar tissue called plaques or lesions

The Result: Because these hard scares replace the smooth myelin, the electrical signals (thoughts/movements) get slowed down, distorted, or blocked

The Numbers Game: We aren't smarter because our brain is built with "special" ingredients; we're smarter because we have a higher total number of neurons (around 86 billion) than other animals.

The 1:1 Ratio: New research shows humans have about one glial cell for every one neuron, just like other primates. This proves our brain follows the same "standard" blueprint as monkeys and apes - we just have a larger, denser version of it .

Humans aren't built different from other primates; we just have more neurons (86 billion). 1:1 glia to neuron ratio proves we follow the same scaling rules as other primates but our higher neuron count in the cortex is what drives our intelligence.

These questions are aspects to her community problem that need to be solved in order to deal with the problem.

Here, she uses her knowledge from being in her position in the community to try to bring people together against the problem her community faces.

This section shows how important her position in the community is, as there are people who are deceptive, so being in her position means not only having to go against people trying to hide Native things, as well as those who actively deceive people.

This displays how she views her position in the community, she and others like her are the only ones that are able to have their voices heard, which shows the responsibility necessary to be in her position.

This sentence shows where she views herself in the community, and her reaction to this situation supports where she positions herself.

Non- Neuronal cells that form the primary support system of the brain and spinal cord. While neurons are the cells that send electrical signals, glia ensure those neurons have a stable, healthy environment to function properly

The Endocrine System is a network of glands and organs that produce and release hormones directly into the bloodstream to regulate essential bodily functions, including metabolism, growth, reproduction, mood, and sleep

Physiological Definition: The normal, healthy functioning of living organisms and their parts, encompassing physical and chemical processes

type of method to use for wp3..there are not efficient support systems for those with disabilities so we as a society need to be more accommodating and realize how important and beneficial they are

Specific to general: After you finish a series, don't you feel inspired or motivated to follow the path of your favorite character? something like this...

general to specific example

i feel like this type of organization starts with a stoey/example. Use example below as a guide

more common, usually what I do. Start off with claim and then provide evidence that supports the claim

with simlink. - The subsequent process using these outputs as their inputs will have them simlinked to the storeDir location

Me gusta mucho esta idea porque hay que recordar que aprender no siempre ocurre leyendo o resolviendo incanzablemente ejercicios en un papel sino que esto se entiende más cuando se ejecuta en la práctica, práctica en la que eventualmente saldrán errores, claro está. Y es que es precisamente de esto que se trata, ese circulo o ciclo en el que pensamos, lo intentamos, fallamos y buscamos mejorar**. Claro que la parte teórica siempre va a ser fundamental y necesaria porque la practica se convertiría en repetición de comprensión vacío.

Aprender un lenguaje de programación (o cualquier otra cosas) unicamente por moda definitivamente es algo contraproducente, si una persona se limita simplemente a memorizar comandos si la tendencia cambia va a tener que empezar desde cero ya sea en ese o en otro y sumado a eso es cómo lo que mencioné en mi hedgedoc sobre la metacognición, repetir y repetir sin entender que es lo que pasa no genera verdadero conocimiento (si, llegaríamos al punto de aprender por repetición más no por entendimiento y esto no genera un conocimiento o saber tácito y propio)

Suele suceder con frecuencia, casi nunca se puede resolver un problema grande de una sentada (por ponerlo de una forma coloquial) ya que esto, sumado a que siempre queremos que todo salga perfecto pues es en ultimas imposible ... Siempre es mejor poco a poco siendo conscientes del proceso y su creación sin caer en el error ¿Cual? en el de postergar las cosas por quedarse estancado "mejorarse"

Esto de que cualquiera puede aprender programación suena lindo y bien intencionado pero no basta solo con querer, hay que ser realistas al tener en cuenta que hay factores que las personas no tienen, cómo por ejemplo: tiempo o acceso a herramientas. Claro que la programación (y muchas otras cosas en la educación y en la vida) no debería ser algo exclusivo de ciertos nichos o personas ya que esto debería promoverse para varios sectores de la sociedad pero teniendo en cuenta que habilidades se tienen para posteriormente ir mejorandolas.

Y es que es cierto, tanto la programación cómo muchísimos campos del conocimiento y de la vida, se tratan de eso ... ensayo y error hasta que se ejecute la linea y funcione pero no siempre quiere decir que porque sirva esté bien hecho, porque si las personas creen que el aprendizaje (al menos para estos casos en específico) es el "mejor", van a estar obligados a buscar la respuesta más rapida para que su sistema funcione si o si, sin detenerse a inspeccionar, corregir o al menos preguntarsen ¿cómo funciona?, ¿por qué no está funcionando?

Claro que es bien sabido que programar está en muchas profesiones y esto demuestra una vez más que la tecnología (cómo lo hemos visto en clase y fuera de ella) es que hace parte de nuestras vidas. Si quiero ser crítico con lo que muchas veces las personas vean que la programación es solo para conseguir trabajo o conseguir mejor salario (que si bien también es debatible) me parece muy hedónico, ya que debería verse cómo lo que es (o cómo al menos creo verlo) … creación, aprendizaje continuo, pensamiento lógico y el saber del funcionamiento de las cosas.

The plan!

Would be fun to try

such as ext.args within modules.config

What other level is there?

The Typewriter Revolution blog: Legendary!<br /> by [[Richard Polt]]<br /> accessed on 2026-04-28T13:31:05

https://www.facebook.com/groups/TypewriterCollectors/posts/10163553004084678/

Ames Supply Company apparently sold replacement green colored key legends for replacing the originals if necessary.

This Royal 10 has some as an example.

For those curious about some of the history behind these, check out: <br /> - https://typewriterdatabase.com/1960-Ames_Gen_Cat_10-March.misc-supplies.manual <br /> - https://writingball.blogspot.com/2016/10/legendary.html

Case#: Patient 2 is a 24-year-old Japanese man. Birth weight was 1900 g (~4.2 lbs) and mental and motor development were both normal. He had graduated from high school. Physical examination demonstrated a height of 157.0 cm, body weight of 45.3 kg.

DiseaseAssertion: MPS1-S

FamilyInfo: He was born from nonconsanguineous, young and healthy parents. He had a healthy elder brother and an affected twin brother (Patient 1).

CasePresentingHPOs: Inguinal hernia, bronchial asthma, systolic ejection heart murmur, umbilical hernia, joint contractures, spastic gait, hypoesthesia, positive Romberg sign, Babinski signs, mild aortic valve stenosis (HP:0000023, HP:0002099, HP:0031664, HP:0001537, HP:0002828, HP:0002064, HP:0033748, HP:0002403, HP:0003487, HP:0001650)

CaseHPOFreeText: Admitted to the hospital due to a 3-month history of progressive gait disturbance, onset was 6 months after the development of gait disturbance in Patient 1. Exaggeration of deep tendon reflexes was slight in the upper extremities, and marked in the lower extremities. Radiographies of chest and cervical spine showed similar findings to those in Patient 1. CSF examinations demonstrated an elevated protein level (342 mg/dL) without pleocytosis. WAIS-III demonstrated an overall IQ of 75, verbal IQ of 67 and performance IQ of 90.

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CaseEnzymeAssay: The patient showed IDUA activity from peripheral leukocytes < 0.9 nmol/mg protein/h (normal range; 29.8–89.8 nmol/mg protein/h), and that of their mother showed 19.9 nmol/mg protein/h.

CaseUrineGAGs: Urine chemistry examination demonstrated increased excretion of uronic acid (51.7 mg/g creatinine).

CaseERT: Yes, with laronidase

CaseBMT: N/A

Variant1: c.164dup (p.Leu56AlafsTer7) (c.252insC - in paper but nomenclature is not current)

Variant1ClinVarID: 855487

Variant1CAID: CA355945969

Variant2: c.1121C>A (p.Thr374Asn) (c.1209C>A - in paper but nomenclature is not current)

Variant2ClinVarID: 4078984

Variant2CAID: CA355963378

AdditionalVariants: N/A

ParentalGenotype: Mother: c.164dup; Father: c.1121C>A

PreviouslyPublished N/A

Case#: Patient 1 is a 24-year-old Japanese man. His birth weight was 2300 g (~5 lbs) and he had graduated from a vocational school. Physical examination demonstrated a height of 156.6 cm (mean height of Japanese male at age 24 is 170.9 ± 6.0 (SD) cm, body weight of 45.8 kg (mean body weight of Japanese male at age 24 is 62.6 ± 9.8 (SD) kg according to the National Health and Nutrition Survey in Japan, 2006). He demonstrated overall intelligence quotient (IQ) of 101, verbal IQ of 93 and performance IQ of 112.

DiseaseAssertion: MPS1-S

FamilyInfo: He was born from nonconsanguineous, young and healthy parents. He had a healthy elder brother and an affected twin brother (Patient 2).

CasePresentingHPOs: Inguinal hernia (treated by surgical repair in childhood and again at age 20), systolic ejection heart murmur, umbilical hernia, scissor gait, Babinski sign, mild aortic valve stenosis, severe cervical cord compression (HP:0000023, HP:0031664, HP:0001537, HP:0012407, HP:0003487, HP:0001650, HP:0002341)

CaseHPOFreeText: Admitted to the hospital due to a 6-month history of progressive gait disturbance. Mental and motor development were normal. Past medical histories included Kawasaki disease at age 6 months. Deep tendon reflexes were mildly exaggerated in the upper extremities, and markedly exaggerated in the lower extremities with bilateral Babinski signs. Radiography of the chest demonstrated mild thoracic deformity and that of cervical spine demonstrated hypoplasia of vertebral body and spinous process. Brain MRI demonstrated enlarged perivascular space and small hyperintense lesions on fluid attenuated inversion recovery image. Cerebrospinal fluid (CSF) examinations demonstrated an elevated protein level (440 mg/dL; normal range 10–40 mg/dL), which would be resulted from CSF circulatory disturbance caused by severe spinal canal stenosis, without pleocytosis.

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CaseEnzymeAssay: The patient showed IDUA activity from peripheral leukocytes < 0.9 nmol/mg protein/h (normal range; 29.8–89.8 nmol/mg protein/h), and that of their mother showed 19.9 nmol/mg protein/h.

CaseUrineGAGs: Urine chemistry examination demonstrated increased excretion of uronic acid (63.1 mg/g creatinine; normal range 8.3–12.3 mg/g creatinine).

CaseERT: Yes, with laronidase

CaseBMT: N/A

Variant1: c.164dup (p.Leu56AlafsTer7) (c.252insC - in paper but nomenclature is not current)

Variant1ClinVarID: 855487

Variant1CAID: CA355945969

Variant2: c.1121C>A (p.Thr374Asn) (c.1209C>A - in paper but nomenclature is not current)

Variant2ClinVarID: 4078984

Variant2CAID: CA355963378

AdditionalVariants: N/A

ParentalGenotype: Mother: c.164dup; Father: c.1121C>A

PreviouslyPublished N/A

One assumption many social media sites make is that users can process a lot of fast-moving content and notifications at the same time. This might not be true for people with ADHD or anxiety, who can feel overwhelmed by constant alerts, autoplay videos, and infinite scrolling. For example, a user might open the app to check one message but get distracted by multiple notifications and lose focus. This can make the platform stressful instead of helpful. It shows that the design assumes a certain attention ability that not all users have.

Janie and Joe starks travel to the Eatonville. They rent a house and Joe buys land in the town. Janie’s relationship with Joe starks becomes more complex.

via Nils Behr at https://www.facebook.com/groups/TypewriterCollectors/posts/10163547121184678/

Small bottle used as applicator with a sponge inserted on the top covered by a small square of cloth which is rubberbanded on. This is then applied slowly to the typewriter ribbon on a winder.

Probably put this in qoutes or italicize to show it is happening on the screen.

Important -- bring in/merge updated content from https://valentinklotzbuecher.github.io/llm-uj-research-eval/

Meanwhile, link to that one for "the latest research"

doublecheck that these are NOT within the model context window

These numbers are being limited by Opus - we should do more evaluations in Opus and/or report the numbers just for the larger group.

This is because the self doubt and block is temporary and is a part of your state of mind at that moment.

That's probably a large reason for people who's career is an author with them needing to meet expectations and go through these stages often

This is probably the most common feeling for people when they are experiencing a writer's block.

I don't really get what the VP means by phenomenon, does it mean that they wanted to exploit it because it was something that was unusual?

Author response:

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

Reviewer #1 (Public review):

Weaknesses:

In my view, the presentation of the data is in some cases not ideal. The phrasing of some conclusions (e.g., group-attacks and wolf-pack-hunting by the bacteria) is in my opinion too strong based on the herein provided data.

We agree with your comment and have replaced the terms “Group-attacks” and “wolf-pack-hunting by “attacks” throughout the manuscript.

Reviewer #1 (Recommendations for the authors):

(1) Figure 2AB, please add the name of the statistical test and the number of replicates that the data is based on to the figure legend.

We thank Reviewer#1 for highlighting the need for more detail. We have revised the manuscript accordingly. The captions of figures 2, 3, 4 and S1 were revised to include the name of the statistical test and the number of replicates. Asterisks indicate significant differences in a multiple comparison test (One -way ANOVA with post hoc Tukey test),* P ≤ 0.05, ** P≤0.01, *** P≤ 0.001

(2) Figure 2C is this figure referred to in the text?

We apologize for this oversight. Figure 2C was replaced by new figures 2C and 2D and the old figure 2C is now referenced in the manuscript as Fig 3B1.

(3) Movie 1, could the movie please also be provided as .mp4? I suggest including individual images across time in the main figure so that readers do not rely on opening a supplementary file for this key finding of the study.

In the revised manuscript, all the videos were converted to mp4 format and individual images across time were included in Figure 2C and 2D (Chronological snapshots of one attack) and in figure 3B1 (Chronological snapshots of the complete event), thereby improving the readability of the manuscript.

(4) Figure 3A2 (text l. 355), I am afraid I do not find this figure.

Fig. 3A2 which previously corresponded to Fig. 3B1, correspond now to Fig. 2C and Fig. 2D. This has been corrected in the revised version of the manuscript.

(5) Lines 356ff, I am afraid that I find it hard to follow what the authors refer to as the right cell or the left cell. I suggest either adding labels to the movies or providing individual images across multiple timepoints into the main figure that can be labelled and bring across the point.

Arrows have been added to videos 3–5 to clearly indicate the cells referred to in the text and facilitate tracking across time.

(6) In general, for all the microscopy, on how many cells have these phenomena been observed? What is n=x? Has this been quantified?

We thank the reviewer for pointing this out.

In caption of Fig. 3, the sentence “(A) Percentage of motile A. pacificum ACT03. (B) A. pacificum ACT03 attacked by V. atlanticus LGP32 and (C) A. pacificum ACT03 lysis after 0, 15, 30, 45 and 60 min of interaction. “was replaced by “(A) Cumulative percentage of motile A. pacificum ACT03 cells. (B) Cumulative number of cells attacked by V. atlanticus LGP32 and (C) Cumulative cell lysis after 0, 15, 30, 45 and 60 minutes of interaction.”. In Fig. 3 caption, the sentence “All percentages were determined based on a minimum of 2,000 cells of A. pacificum ACT03.” was also added.

In Fig. 4 caption, the sentence “All percentages were determined based on a minimum of 2,000 cells of A. pacificum ACT03.” was added.

In Fig. S1 caption, the sentence “All percentages were determined based on a minimum of 2,000 cells of A. pacificum ACT03.” was added.

(7) Figure S1A, does this figure show means plus/minus standard deviation? If yes, please add this to the figure legends.

In Fig. S1 caption, the sentence “Error bars represent the standard deviation of the mean of three independent experiments” was added.

How do the authors explain the big variation in the test condition and not in the control?

Regarding the higher variation observed in the test condition compared to the control, this may, on the one hand, reflect biological variability between independent batches of 60-h V. atlanticus cultures used to prepare the supernatants, and, on the other hand, a heterogeneity in the physiological status of independent algal batches (N = 3 ; 2 × 10^4 cells ; see Materials and Methods, Co-culture assay), which may not be perfectly synchronized . In contrast, the control condition consists of A. pacificum cultures incubated in fresh medium without bacterial supernatant, for which algal motility is highly reproducible and thus shows very little variation.

(8) Line 375, "The lysis phase corresponded to initial vesicle formation followed by the bursting of A. pacificum ACT03 cells (Movie 5) and was induced by the old-starved culture supernatant of V. atlanticus LGP32 (Fig. S1)." Is this reference to Figure S1 correct? S1 shows motility, doesn't it? I don't see how this data supports the statement made in this sentence.

We apologize for this unclear message.

"The lysis phase corresponded to initial vesicle formation followed by the bursting of A. pacificum ACT03 cells (Video 5) and was induced by the old-starved culture supernatant of V. atlanticus LGP32 (Fig. S1)." was replaced by "The lysis phase corresponded to initial vesicle formation followed by the bursting of A. pacificum ACT03 cells (Fig. 3C and 3C1).

And “We next tested whether this lytic effect was mediated by thermostable molecule (s) secreted by Vibrio. “was replaced by “We next tested whether this lytic effect was linked to Vibrio culture supernatant and mediated by thermostable molecule (s) secreted by Vibrio.

(9) Line 388ff, "Group attacks were observed on non-degraded A. pacificum ACT03 cells, but not on previously lysed cells." No reference to a figure is provided. I am afraid I don't see the data that this statement is based on.

As it is impossible to show a lack of attack, we just clarified the basis of our experiment.

“To this end, A. pacificum ACT03 in exponential growth phase was first exposed for 30 minutes to the supernatant of a 60-hour culture of V. atlanticus LGP32, which induced 25% lysis of A. pacificum ACT03 cells. Next, the corresponding V. atlanticus LGP32 cells were added. During exposure, attacks were observed only on undegraded A. pacificum ACT03 cells, but not on previously lysed cells” was replaced by “To this end, A. pacificum ACT03 in exponential growth phase was first exposed for 30 minutes to the supernatant of a 126-hour culture of V. atlanticus LGP32, which induced lysis of 70% of the A. pacificum ACT03 cells (Figures 3C and 3C1, arrow 2 and video 4). Next, cells of V. atlanticus LGP32 from a 60-hour culture, capable of attacking A. pacificum ACT03 cells (Fig. 3B), were added. For 1 hour of exposure, no attack was observed on the previously lysed algae.”

(10) Figure 4a, Based on the labeling of the figure, in particular the x-axis, it is not fully clear to me what I am looking at.

Figure 4A has been reworked and its legend modified. We hope that this graph is clearer now.

(11) Line 428, did the authors consider complementing the pvuD deletion mutant and testing for gain of function when providing the gene in trans?

We did not investigate pvuD in this study and did not construct a pvuD deletion mutant. We therefore assume that the recommendation refers to pvuB, which was the focus of our work. Unfortunately, we did not perform this experiment. However, several lines of evidence support the implication of PvuB and the vibrioferrin uptake system in this process: (i) the loss of attack behaviour is specific to the mutant in the vibrioferrin uptake pathway and (ii) our expression and proteomic data show a strong induction of vibrioferrin uptake components under starvation and iron-manipulated conditions, which correlate with the attack phenotype.

(12) Use of the term "group attack" in parentheses in the text, but in the section header and title. Is there really sufficient actual data to say that this is a "group attack"? What exactly are the indications for this being a behaviour of a group?

We agree with you. The terms “group attacks” and “wolf-pack hunting” were replaced by the more neutral term “attacks” throughout the manuscript.

(13) Table S1 and S2, those tables give a nice overview. Do the authors provide the raw data based on which they make a claim on "+" and "-" in the individual categories? I would prefer to see the actual data or at least have the possibility to look into this.

In the revised versions of Tables 1 and 2, we have improved the captions and clarified the meaning of each column in order to avoid any ambiguity between the results of this study and the bibliographic information.

Specifically regarding Table 2 :

We do not present any visuals of the interaction between Vibrio and Alexandrium because these species all look alike. Regarding the other algae species tested in interaction with Vibrio, phenomena other than lysis or cell attack have been observed and are the subject of specific laboratory studies.

(14) Line 456 "first study", line 40f "first evidence of a new mechanism". I suggest toning this down a bit and being clearer in the abstract about this being a working model that can be suggested based on individual bits of data.

We thank Reviewer #1 for this helpful suggestion.

In the summary:

“This is the first evidence of a new mechanism that could to be involved in regulating Alexandrium spp. blooms and giving Vibrio a competitive advantage in obtaining nutrients from the environment.” was replaced by “The interaction model we propose here suggests that Vibrio could play a role in regulating the proliferation of Alexandrium spp., giving it a competitive advantage in obtaining nutrients from the environment.”

In the discussion:

Considering predator as a free organism that feeds at the expense of another, this study is the first evidence of the capacity of some Vibrio to develop a predatory strategy against an alga. This behaviour differs from parasitism, because the survival of Vibrio is not exclusively dependent on algae in environment” was replaced by “Consider a predator as a free-living organism that kills its prey and feeds on it, this study provides data suggesting the ability of Vibrios to develop an original predator-like behaviour to kill and feed on algae.”

(15) Line 469 "Overall, these observations show that V. atlanticus LGP32 is able of wolf-pack hunting behaviour." I see the similarities. I feel that the term "show" is a bit too strong here, or I suggest referring to "wolf-pack-like behaviour".

The sentence “Overall, these observations show that V. atlanticus LGP32 is able of wolf-pack hunting attack behaviour” was replaced by “Overall, these observations suggest that V. atlanticus LGP32 can exhibit a predator-like behaviour”

Reviewer #2 (Public review):

As Weaknesses Reviewer #2 include:

(1) A lack of early, clear definitions for several important terms used in the paper, including 'predation', 'coordination' and 'coordinated action', 'group attack', and 'wolf-pack hunting', along with a corresponding lack of criteria for what evidence would warrant use of some of these labels. (For example, does mere simultaneity of attacks of an A. pacificum cell by many V. atlanticus cells constitute "coordination"? Or, as it seems to us, does coordination require some form of signalling between predator cells?)

The term “Coordinate” was replaced by “simultaneous” throughout the manuscript

The terms “Group attack” and “wolf pack hunting” were replaced by “attack” throughout the manuscript

(2) Absence of controls for cell density in the test for starvation effects on predatory behaviour; unclear how the length of incubation affects the density of V. atlanticus cells.

We thank the reviewer for pointing this out.

Cells density experiment was already performed (cf. Fig. 4A).

The sentence. ”All percentages were determined based on a minimum of 2,000 cells of A. pacificum ACT03.“ was added in captions of Fig. 3, Fig. 4 and Fig S1

(3) Lack of clarity in some of the methodological descriptions

The Methodology has been checked and some improvements have been made.

Reviewer #2 (Recommendations for the authors):

(A) Title

(1) Could 'induces' be better than 'promotes'?

We agree with Reviewer #2. The initial title, “Starvation of the bacterium Vibrio atlanticus promotes lightning group-attacks on the dinoflagellate Alexandrium pacificum”, was replaced by “Starvation of the bacterium Vibrio atlanticus induces simultaneous attacks on the dinoflagellate Alexandrium pacificum”.

(B) Abstract

(1) Perhaps define pycosphere in the abstract - many readers might not know this word.

We have revised the abstract to define the term phycosphere and added the sentence “This occurs in the microenvironment surrounding phytoplankton cells, the phycosphere. An interface rich in nutrients and organic molecules exuded by the cell.”

(2) Perhaps "on dinoflagellates".

We thank Reviewer #2 for this suggestion. We have revised the abstract by replacing “on the dinoflagellates species” with “on dinoflagellates”.

(3) Line 33 - The word 'prey' is used without a claim of predation having yet been made; only killing has been claimed so far.

We agree and have replaced the word “prey” by “algae” in the abstract.

(4) Line 34 - It is unclear whether the description refers to the 'attack stage' or to 'wolf-pack attack' in general. The sentence is written in such a way that it seems to refer to 'wolf-pack attack'. However, this would seem to be incorrect, with the description being specific to V. atlanticus.

To avoid this ambiguity, we have removed the sentence “resembles the ‘wolf-pack attack’ strategy” from the abstract.

(5) Line 35 - Should there be a 'consumption phase'?

We agree with the reviewer #2, “degradation” was replaced by “consumption”.

(6) If predation is claimed later in the manuscript (which it is), it should be explicitly claimed in the abstract.

We thank Reviewer #2 for this helpful suggestion.

We have revised the abstract. The sentence “Results showed that Vibrio atlanticus was able to coordinate lightning group attacks then kill the dinoflagellate Alexandrium pacificum ACT03” was replaced by “The results showed that Vibrio atlanticus was capable of attacking and killing the dinoflagellate Alexandrium pacificum ACT03”.

(C) Main text

(1) Line 54 - Perhaps "Among HAB-causing organisms...".

We agree with the reviewer’s suggestion and have revised the wording.

(2) Line 56 - "that, together with..., form the "Alexandrium tamarense" complex".

We agree with the reviewer’s suggestion and have revised the sentence.

(3) Line 57 - What this "complex" is and its significance should be explained.

“Among them, Alexandrium pacificum is a flagellated eukaryotic unicellular organism that together with Alexandrium tamarense and Alexandrium fundyense form the "Alexandrium tamarense" complex (Hadjadji et al., 2020)” was replaced by

“Among them, Alexandrium pacificum is a flagellated eukaryotic unicellular organism that together with Alexandrium tamarense and Alexandrium fundyense form the "Alexandrium tamarense" complex, responsible for paralytic shellfish poisoning worldwide (Hadjadji et al., 2020)”

(4) Line 58 - What is a Rephy survey?

We clarified this point, “by rephy survey” was replaced by “by the French phytoplankton observation and monitoring network (Rephy)”

(5) Line 59 - 'resulting in' instead of 'resulting of'.

We agree with the reviewer and have replaced “resulting of” with “resulting in”.

(6) Line 65 - It seems that ', influencing the time of appearance of blooms' would be more correct than the current phrasing. The current phrasing is unclear regarding the relation between species, tolerance range, and the time of appearance of blooms.

To address this point, “Depending on the phytoplankton species, the tolerance range of physicochemical parameters is different and influences the time of appearance of blooms” was replaced by “Depending on the species of phytoplankton, tolerance to physicochemical parameters varies, which influences when blooms occur.”

(7) Line 76 - Run-on sentence which should probably be split after the reference to Wang et al., 2020.

We agree with the reviewer and have split the sentence.

(8) Line 89 - What are these observations?

This sentence was reformulated.

“Based on observations from the natural environment showing a potent relationship between Vibrio and Alexandrium algae bloom events, this study aim to determine in vitro, the main factors implicated in this relationship” was replaced by ”This study aims to describe observations made in the natural environment between Vibrio bacteria and Alexandrium algal blooms, and to determine in vitro the main factors involved in this relationship.”

(9) Line 94 - This is the first clear reference to a predator-prey interaction, and it is stated as if it's established. Is it not a central goal of the study to demonstrate that predation is even happening?

Based on the title and abstract, I would have expected the major claims of the paper highlighted in the abstract to be:

(i) that predation of algae by bacteria occurs in this system,

(ii) there is a social component of predation,

(iii) claims about what induces this predatory behaviour.

The summary has been amended accordingly, and the term “predation” has been removed, along with all sentences referring to it.

(10) Line 99 - What does n.d. mean?

This point was addressed in the revised version.

(11) Line 97 section - specify qPCR.

This point was clarified in the revised version.

(12) Line 139 - Mentioning the oligonucleotides in this part of the methods seems out of place. Would this not fit better in the section on Gene expression analysis?

This sentence was discarded from this paragraph.

(13) Line 147 - Where did the co-cultured phytoplankton species come from?

To answer this point, reference to Table 2 was added

(14) Line 149 - Is it known if the phytoplankton strains had all grown to the same density after 24 hours?

The doubling time of dinoflagellates in laboratory culture is between 5 and 7 days. During the duration of the experiments, the dinoflagellate concentration did not change significantly.

The sentence “(doubling time between 5 and 7 days)” was added

(15) Line 150 - Was the density of the Vibrio cultures at the different incubation times measured? Density might play an important role in predation, and so it would be important to control for density in these assays.

The concentrations of live vibrio in each individual culture were not actually measured. However, the role of vibrio density in attacks was measured and is shown in Figure 4A and observed in Fig 2B.

(16) Line 153 - How long was the co-incubation?

The incubation times were added in the revised version.

(17) Line 158 - What is mean by "independent experiments", more exactly?

To clarify this point, “Data are the means of three independent experiments” was replaced by “The data come from three independent experiments using independent phytoplankton cultures and independent bacterial cultures.”

(18) Line 161 - Perhaps give the source information about the Vibrio strain at its first mention.

A reference has been added in the revised preprint.

(19) Line 163 - line 141 refer to multiple non-axenic species, whereas here "the algal strain" is referred to.

And

(20) Line 164 - language phrasing throughout the manuscript could use some polishing, e.g., "this means that additional bacteria...".

To address this comment, “As the algal strain used in the study is not axenic, means that additional bacteria, other than the V. atlanticus LGP32, are potentially present in the experiments.” was replaced by “As the A. pacificum ACT03 strain (table 2) used in the study is not axenic, there is potential for bacteria other than V. atlanticus LGP32 to be present in the experiments.”

(21) Line 208 - Why were both magnitude and p-value criteria used rather than just p-values?

In the present proteomic approach each experimental condition was measured six times, and the average (mean) value was used to reduce random noise. Then we selected differences that had to be large enough to matter biologically, this is a central criterion and at least a 2-fold change was considered to focus exclusively on biologically relevant differences, which allowed us to control for the effect size. However, the differences also had to be statistically significant, we applied a statistical confidence at P < 0.01, to be sure that there is less than a 1% chance the result happened randomly. In the present proteomic approach each experimental condition was measured six times, and the average (mean) value was used to reduce random noise.

Then we selected differences that had to be large enough to matter biologically, this is a central criteria and at least a 2-fold change was considered to focus exclusively on biologically relevant differences, which allowed us to control for the effect size. However, the differences also had to be statistically significant, we applied a statistical confidence at P < 0.01, to be sure that there is less than a 1% chance the result happened randomly. We considered that using both criteria makes the results meaningful and trustworthy, not just a small or random fluctuation.

(22) Line 270 - Were these three replicate experiments also "independent"; if yes, in what sense?

“All experiments were conducted in triplicate” was replaced by “The experiments were performed using biological triplicates, each of which was analyzed in triplicate.”

(23) Line 296 - Perhaps "the temperature-sensitivity (or resistance) of" rather than "the nature of".

The modification was made in the new manuscript.

(24) Line 307 - The sentence mentions only one influential period that was removed from the dataset, but the word 'whenever' suggests multiple occurrences.

We agree, “whenever” was replaced by “because”.

(25) Line 325 - line 327 - The rationale behind the first part of the following sentence isn't clear to me, and what is meant by the second part is also not clear.

To clarify this point, “This result is consistent with the difficulty that Vibrio has in growing at temperatures below 20°C and with the complex interacting factors driving bloom dynamics (Laanaia et al., 2013)” was replaced by “This result is consistent with the difficulty Vibrio has in growing at temperatures below 20°C and with the many environmental factors that influence the dynamics of algae proliferation (Laanaia et al., 2013)."

(26) Line 327 - line 328 - Hard to interpret; does this refer to living algal cells, or all algal cells, living and degraded?

To improve clarity, “Interestingly, in spring 2015, the mean densities of all Alexandrium cells and of free-living Vibrio were positively correlated” was replaced by “Interestingly, in spring 2015, the mean densities of Alexandrium cells (living and degraded) and of free-living Vibrio were positively correlated”

(27) Figure 2 - These results strongly point to predation, but why the Vibrio population would already be elevated in the co-culture treatment relative to the control immediately after inoculation (0 hrs) is not clear.

The experiments were not conducted at the same time, and the first value on the graphs corresponds to the concentration of vibrio determined after 1 hour of exposure/incubation and not at time 0. Figures 2A and 2B have been modified accordingly, and substantial changes have been made to the relevant section of the results.

(28) Line 348 - There's no mention of Figure 2C in the main text, or of the statistical test associated with it in the Figure 2 legend.

To address this comment, Figure 2C has now been cited in the main text, and the statistical analysis method has been added to the Figure 2 caption.

(29) Line 352 - Text descriptions of videos are not easy to connect with the video content. Label the file names the same as how they are referred to in the text.

We agree with you, the sentence “Epifluorescence microscopy observation of GFP-labelled V. atlanticus LGP32 (previously grown in Zobell medium) in interaction showed that A. pacificum ACT03 cells that had lost their motility were attacked individually by V. atlanticus LGP32 before being lysed (Fig, 2C and Video 1). “was rephrased and replaced by “Epifluorescence microscopy observation of GFP-labelled V. atlanticus LGP32 (previously grow in Zobell medium) in interaction showed that V. atlanticus LGP32 simultaneously attacks A. pacificum ACT03 cells (Fig, 2C and Video 1).”

(30) Movie 1 could be cut to remove uninteresting footage at the start. What indicates lysis? Is the deformation of the cells an indication of lysis?

To respond to this comment, Video 1 has been shortened and in the caption, “degraded” was replaced by “lysed”

(31) Line 353 - Video could be zoomed in more on a few typical attacks to remove visual noise.

A chronological overview of an attack has been added to Figure 2 corresponding to Figure 2D, and a chronological overview of the overall event has been added to Figure 3 corresponding to Figure 3B1.

(32) Line 355 - There does not seem to be a Figure 3A2.

To address this point, the Fig. 2 and Fig. 3 has been revised for more clarity. See above

(33) Figure 3 - Can the authors fully exclude an effect of bacterial density as distinct from an effect of growth/starvation phase? It would be helpful to determine bacterial viable population densities at 12, 36, 60, and 126 hrs of incubation in Zobell medium, and to control for density in testing for effects on algae.

Information on Vibrio densities incubated in Zobell medium for 12, 36, 60, and 126 hours has been now included in the results section “Attack of A. pacificum ACT03 is activated by V. atlanticus LGP32 starvation.”

(34) Line 363 - It is unclear how the degradation of the flagella is apparent from movie 3. It would be helpful to have a comparison with healthy flagella.

Alexandrium cells with intact flagella move so quickly that it is impossible for us to follow them and film their flagella with the tools at our disposal.

For greater clarity, arrows have been added to videos 3, 4 and 5.

(35) Line 364 - Sudden change from referring to the recording as 'video' instead of movie. What is meant by erratic swimming? The cell does not seem to move much.

To address this comment, “Movie” was replaced by “Video” throughout the manuscript and “erratic swimming” was replaced by “irregular swimming”

(36) Line 365 - How did you observe the detachment of the flagellum?

The detachment of the flagellum can be observed using a confocal microscope. This process was filmed and presented in Video 3. Arrows have been added to the video to clearly indicate the flagellum detachment.

(37) Line 368 - Perhaps this is due to it not being clear regarding which movie is meant, but there is no clear attack visible in movie 4.

To make this clearer, arrows have been added to the video 4 to indicate attached cells.

And the sentence in the caption of the video 4 “Vibrio, filmed under a confocal microscope, attacks in groups one immobilized Alexandrium cell then moves on to attack — still as a group — another cell without touching the other whole cells, suggesting active communication between Vibrio cells” was rewritten and replaced by “This video, recorded under a confocal microscope, shows Vibrios simultaneously attacking a first immobilized Alexandrium cell, then moving on to attack a second cell without ever targeting the other cells present, suggesting active communication between the Vibrio bacteria.”

(38) Line 369 - It seems the peak attach % was reached at 45 minutes, not 15-30 minutes.

Sorry for the confusion. In fig. 3 for more clarity, the sentence “(A) Percentage of A. pacificum ACT03 motile cells. (B) cells attacked by V. atlanticus LGP32 and (C) cells lysis after 0, 15, 30, 45 and 60 min of interaction” was replaced by “(A) Cumulative percentage of motile A. pacificum ACT03 cells. (B) Cumulative number of cells attacked by V. atlanticus LGP32 and (C) Cumulative cell lysis after 0, 15, 30, 45 and 60 minutes of interaction.”

(39) Line 382 - "clearly show role of nutrient limitation", see comment re controlling for any role of bacterial density.

To address this point, information’s on Vibrio densities were added in the manuscript. See cf comment 33.

(40) Line 385 - line 386 - Phrasing unclear.

We have revised the text accordingly, “To this aim, A. pacificum ACT03 in exponential growth phase was first exposed for 30 min to supernatant from 60 hours starved V. atlanticus LGP32 Zobell media that induced 25% lysis of A. pacificum ACT03 cells and next to the corresponding V. atlanticus LGP32 cells. Group attacks were observed on non-degraded A. pacificum ACT03 cells, but not on lysed cells.“ was replaced by “To this end, A. pacificum ACT03 in exponential growth phase was first exposed for 30 minutes to the supernatant of a 126-hour culture of V. atlanticus LGP32, which induced lysis of 70% of the A. pacificum ACT03 cells (Figures 3C and 3C1, arrow 2 and video 4). Next, cells of V. atlanticus LGP32 from a 60-hour culture, capable of attacking A. pacificum ACT03 cells (Fig. 3B), were added. For 1 hour of exposure, no attack was observed on the previously lysed algae.”

(41) Line 413 - Is this the only pathway for quorum sensing in V. atlanticus?

Indeed, the last two sentences of this paragraph are unclear.

To address this point:

“By targeted mutagenesis of key genes involved in QS pathways ΔluxM (HAI-1 production), ΔluxS (AI-2 production) and ΔluxR (high-density QS master regulator) did not lead to any change in the attack behaviour of V. atlanticus LGP32 (Fig. 4C).” was replaced by “Targeted mutagenesis of key genes involved in two of the three known QS pathways in vibrios (Fig. S3), ΔluxM (HAI-1 production), ΔluxS (AI-2 production), and ΔluxR (main high-density QS regulator), did not result in any changes in the attack behavior of V. atlanticus LGP32 (Fig. 4C).”

And “Taken together these results showed that attack by V. atlanticus LGP32 is not link to QS.” was replaced by. “Combined with the absence of overexpression of the CqsS gene (inducible by CAI-1) involved in the last known QS pathway in Vibrio (Fig. S3), these results indicated that the attack by V. atlanticus LGP32 is most likely unrelated to QS.”

(42) The references to tropism aren't clear.

You're right, there's no reason to use the term tropism here. We have removed it.

(43) Line 439 - Why was H3BO4 used as a control for the addition of FeCl3?

For clarity, the sentence “Boron being known to be a regulator or capable of being transported by vibrioferrin (Romano et al., 2013; Weerasinghe et al., 2013), we tested its potential involvement in the interaction but no effect was evidenced here.” was replaced by “Given that boron is known for its role in regulating a global bacterial cellular response to phytoplankton and to bind to vibrioferrin (Romano et al., 2013; Weerasinghe et al., 2013), we tested its potential involvement in simultaneous vibrio attacks. Compared to the Zobell control, no effect on the number of attacks was observed”

(44) Line 441 - line 449 - Should explicitly say in text that no attacks were observed for any species other than the Alexandrium and Gymnodinium species.

We agree and have explicitly stated in the text that no attacks were observed for any species other than Alexandrium and Gymnodinium.

(45) Line 454 - line 455 - The last part of this sentence seems a strange statement, since

(i) it has long been know that predatory bacteria can eat a wide range of eukaryotes, ii) one of the cited papers (Perez et al) actually highlights a case of bacterial predation on algae, and iii) in the next paragraph the authors themselves highlight Streptomyces predation of algae.

To make this clearer, « Among predators, predatory bacteria are found in a wide variety of environments, and like bacteriophages and predatory protists, they have been reported to prey exclusively on other bacteria » was replaced by “Among predators, predatory bacteria are found in a wide variety of environments and, like bacteriophages and predatory protists, feed primarily on other bacteria, although a few cases of predation on microbial eukaryotes have also been reported.”

(46) Line 455 - Better to clarify the authors' definition of a predator at the start of the paper. The offered definition seems more like a definition of 'consumer' than 'predator', as the latter normally involves both the killing and consumption of other organisms, not just consumption with some kind of "expense".

To address this comment:

- “predator behaviour” was replaced by “predator-like behaviour”

- and “Considering predator as a free organism that feeds at the expense of another, this study is the first evidence of the capacity of some Vibrio to develop a predatory strategy against an alga. This behaviour differs from parasitism, because the survival of Vibrio is not exclusively dependent on algae in environment” was replaced by “Consider a predator as a free-living organism that kills its prey and feeds on it, this study provides data suggesting the ability of Vibrios to develop an original predator-like behaviour to kill and feed on algae.”

(47) Line 457 - Don't see the benefit of trying to distinguish from parasitism here, especially since parasitism can be facultative, whereas the authors' phrasing suggests that it is always obligate.

You are right, this sentence has been deleted.

(48) Line 463 - line 464 - The authors should clearly explain exactly what detailed aspects of Myxococcus and Lysobacter predation they think the "attack stage" of V. atlanticus resembles.

Accordingly, “The second stage, the ‘attack stage’ corresponding to physical contact between Vibrio and Alexandrium resembles the ‘wolf-pack attack’ strategy described for Myxococcus xanthus and Lysobacter regardless of the prey species used, M. xanthus must be in close proximity to prey cells in order to induce their lysis and to benefit from their biomass (Martin, 2002; Perez et al., 2014)” was replaced by “The second stage, the ‘attack stage’ corresponding to the physical contact between Vibrios and Alexandrium, is similar to the strategy used by Myxococcus xanthus and Lysobacter. These bacteria must be in close proximity to their prey in order to cause lysis and utilize their biomass, regardless of the prey's species (Martin, 2002; Genovesi et al., 2013; Perez et al., 2016; Zhang et al., 2020)”

(49) Line 466 - line 467 - The comparison to bacteria clustering around lysed cells is surprising since the authors show that V. atlanticus does not attack already lysed cells.

The sentence was rephrased, “This phenomenon is comparable to that of bacteria clustering around lysed ciliate cells “was replaced by “Visually, this phenomenon resembles bacteria clustering around lysed ciliate cells.”

(50) Line 469 - Missing is a statement of exactly what criteria constitute "wolf-pack hunting behaviour" and exactly how V. atlanticus meets those criteria.

To address this point, “wolf-pack hunting behaviour” was replaced by “predator-like behaviour”

'Able of' should be corrected to 'Capable of'.

We agree and have reworded the sentence.

(51) Line 470 - Consider starting a new paragraph for the material on quorum sensing.

Accordingly, we have separated the section concerning QS pathway from the section concerning iron pathway.

(52) As part of their discussion on the role of iron uptake, can the authors comment on any relationship between starvation and iron uptake, and in particular the observations that, while general nutrient deprivation induces attacks, supplementation with a specific nutrient (iron) also induces attacks (Figure 4D)? Do bacteria starved for general growth substrates take up more iron than growing bacteria?

To respond to this comment, “Future study could demonstrate further the role of vibrioferrin in group attack, by adding iron-saturated vibrioferrin to algae-Vibrio co-cultures.” was replaced by “Interestingly, if a general nutrient deficiency causes attacks, iron supplementation increases the number of attacks (Figure 4D), suggesting the importance of iron absorption in the attack behavior. Future studies should determine whether nutrient deficiency increases the iron absorption capacity of Vibrios and whether this plays a major role in the attack mechanism.”

(53) Line 486 - Of what is boron known to be a regulator?

To respond to this comment, “Given that boron is known for its regulatory properties and for being transportable by vibrioferrin“ was replaced by “Given that boron is known for its role in regulating a global bacterial cellular response to phytoplankton and to bind to vibrioferrin”.

Yamamoto Paper Writing Pads: Tomoegawa Tomoe River vs. Sanzen Tomoe River S<br /> by The Well Appointed Desk<br /> accessed on 2026-04-28T09:51:08

Comparison of paper from the No. 7 machine, the No. 9 machine at Tomoegawa and the newer Sanzen S method.