???

I would really like to understand what this means exactly, and get some examples maybe.

How is transference connected to dialectic?

This seems to go along with our dialectic defintion

How are we using "dialectics" here? Is it in the sense that Vassar uses it? Are people creating conflict in order to draw more resources into their relationships with each other? That certainly happens sometimes.

I act out in order to get my parent's attention. This is a manufactured conflict in order to draw more resources in.

What does this mean?

Is it literally just what I was thinking before? ChatGPT seems to think so.

Preference falsification, desire for restricted or desired objects

The thing this brings to mind to me is the split-brain study.

i ddin't know

i didn't know 500 year old history in the Americas

wow didn't know

I didnt know

El diseño sistemático significa trabajar con un método claro: primero se planifica cómo organizar la información y luego se mejora poco a poco. No se trata de hacer todo de una vez, sino de construir y perfeccionar el sistema progresivamente.

Un programa es un sistema de información que integra usuarios y otros sistemas. En archivística y bibliotecología se refleja en plataformas digitales que gestionan documentos, catálogos o repositorios. Funciona en tres fases: Entrada (cargar documentos), Proceso (asignar metadatos) y Salida (documentos organizados para consulta). Conocer lo básico de programación facilita comunicarse con otras disciplinas y diseñar entornos claros, asegurando datos accesibles y comprensibles para usuarios y administradores.

No se trata de ser expertos de inmediato, sino de desarrollar una forma correcta de trabajar con la información desde el inicio, En las ciencias de la información, trabajar bien no significa solo usar herramientas, sino entender cómo funcionan y aplicar métodos organizados. Tanto en archivística como en bibliotecología, la clave está en planificar, estructurar y dar sentido a la información para que sea útil, accesible y de calidad.

El autor compara la programación con el arte para resaltar que el código también tiene una dimensión estética. Así como una pintura se aprecia por su técnica, un código limpio debe ser claro y fácil de entender. Del mismo modo, la gestión de la información puede verse como un arte: su estructura es la estética que permite que cualquier usuario navegue sin dificultad. Al final, la excelencia en la organización es lo que asegura la verdadera recuperación del conocimiento.

La programación no consiste solo en lograr que el programa funcione, sino en construirlo con una lógica clara desde el inicio. Para ello es útil, antes de escribir código, elaborar un diagrama de flujo que permita comprender el proceso, dividir un problema grande en partes más pequeñas (modularidad) y añadir comentarios que expliquen cada paso.

La verdadera dificultad en programación no está en lograr que el programa funcione, sino en que el estudiante comprenda la lógica detrás del código. Si modifica una sola línea y todo se rompe, sin saber cómo repararlo, es señal de que aún no domina los fundamentos ni los buenos hábitos de programación. La práctica profesional no consiste únicamente en resolver un rompecabezas, sino en escribir código que pueda ser entendido y modificado por otros o incluso por uno mismo meses después sin causar errores. En realidad, la programación comienza cuando el código ya funciona: es entonces cuando debemos refinarlo, probarlo y comprenderlo a fondo.

El texto amplía el concepto de programar: no se limita a escribir código, sino a dar instrucciones a herramientas digitales. En la actualidad, saber programar potencia las posibilidades de desarrollo profesional, ya que está presente en múltiples campos. Por ejemplo, un archivista que crea metadatos para describir documentos digitales, estructura una base de datos o aplica filtros avanzados, en cierta forma está programando. En este sentido, la programación fortalece la organización, el acceso y la recuperación de la información.

区块链技术是由多方共同记录和维护的一个分布式数据库，通过哈希索引形式一种链式结构，数据的记录和维护通过密码学技术来保证其完整性，使得任何一方难以篡改、抵赖、造假。基于区块链技术在数据要素安全流通交易，以区块链为基础建立安全可信的数融平台，将为数据要素自由流通保驾护航。 没懂他是什么玩意

hadoop,hbase,flink,spark，hive,dolphinscheduler，Impala、ClickHouse,hdfs

这里的bi服务是什么 这里的数仓集群有什么例子，有哪些工具 数仓的可视化分析平台又是用哪些工具 服务sla是什么意思 数仓dw是什么意思 这里面的埋点是什么意思 这里的etl是什么意思 离线数仓和实时数仓讲一下（全数据仓库） 这里的数据模型和数据治理说一下 etl里有kettle和flink-cdc 数据同步服务说一下 vpc防火墙，waf,是什么 单台缓存型服务器所能提供的NVMe存储容量 这什么意思

I appreciate these questions as it promotes connection and thinking about believe, beliefs reflect I think these questions must be always an anchor to our learning and work.

I found this source interesting because bulletin board systems feel like an early version of features that are still common today, especially threads and community discussion spaces. Even though the technology was much simpler, the basic social pattern is familiar: one person posts, others reply, and a conversation forms around shared interests. This source made me realize that many features we associate with modern social media actually have much older roots.

“related but distinct frameworks addressing the legacies of colonialism” this was another few words I didn’t know with its definition

What did they communicate about?

I wanna know why they’re deciding to do it that way and not a different way like did they try other ways? Addressing the problems critically are the best option

This is something I didn’t know and it means “the interconnected meaning in time”

I thought this was a very interesting thing so I said I wanted to annotate this because I really liked the way that they spoke on what this meaning was because I’ve never heard of it before, but I thought it was really interesting

I found this part interesting because it shows that the design of a platform affects how people interact. When personal webpages and communication tools were separate, online social life seems like it was more divided and maybe more intentional. Today, many platforms put everything together in one space, which makes interaction easier, but also creates more pressure to always be visible and active.

下面截图里有保函，这又是什么

什么是移动金币

这里的信用购机，买的是手机吗，还是什么

这下面截图的5g金币是什么业务 然后信用购，速兑通分成了这么多，他又是干嘛的 什么是电子凭证，电子保证

保理和再保理什么区别

这里的供应链金融是在干嘛的

这里业务域用来干嘛的啊

this shows the importance of citing sources and giving credit to others which is very important .

Helpful because I also think you should always add what you think

this explain that writers can choose when to write their conclusion depending on what works for them

Copying or using others peoples work without consent.

Ways on how to write a hook. Goes at the introduction paragraph.

Steps on making a paper with information and well written.

In general, this sample page was more useful than a bunch of greek text.

Let's not delete this yet.

Lidar sensors?

It helps me understand how to begin my paper and what I should include .

It helps me understand that an informative essay it meant to explain information and answer questions

It explains what an informative research report is and what it is supposed to do.

When it comes to writing your topic sentence don't forget to draw in your reader. However, don't overwhelm someone by trying to fit all 5 w's in the first sentence.

Important steps to make my paper interacting. a background will always make the audience interested.

Persuasive essays means trying to convence someone and the goal is to be informative.

Using these questions what who where when how and why will make our paper successful as were describing the topic from all their subjects.

Give out evidence and more detail about the topic itself. Finding resources and using its information.

Instruments 5

Worked?

Instruments 4

Instruments 3

Instruments 2

Timeline

Demographics 1

Timeline

Instruments 1

"nature does not make jumps"

Worked?

Instruments 2

Demographics

Game summary

Instruments 1

Game summary 1

Worked?

Instruments

Game summary 3

Game summary 1

Game summary 2

Mediation

Timeline

Very interesting result. If nucleation and stabilization are separable as suggested by your study, would you expect the older of the two basal bodies to have greater protofilament loss in these mutants? Do you see any ultrastructural differences between the two basal bodies in the same cell?

always cardiovert if unstable

less technical, more principle. add hyperlinks here too

not needed yet

here rather mention inventory, card hand, so on.

thise two points can be merged into a simple "tracking or stats" kind fo point

rather mention that attachment can be automated, or follow a mouse drag and drop type of behaviour, or VR hands, or 3rd person, etc.

rather mention that attachment can be automated using detection, and that there are many ways to support assisting drags and attachment

unecessary: simply state that attachment can be filtered, or resxtructed to conditions,including custom ones.

less technical: talk about drag origin

less technical: talk about outcome more than code

make it more approachable: users dont know what a pipeline is at that point: talk about movement modifiers

add that it's not only about transfer, but general spatial operation: movements,rotation, so on

Current difficult decision regarding when life "as a person" actually begins. switch from cellular experiment to human testing

Sequences of bans have halted and then restarted progress due to ethical concerns and faith.

Reference back to the Evans and Kaufman experiment isolating mouse embryonic cells.

Note

Here is a page note. Let's see how this works. I think this won't have the anchoring issues seen with annotations.

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

Learn more at Review Commons

Reply to the reviewers

Reviewer #1 (Evidence, reproducibility, and clarity (Required)):

Summary: In this manuscript, the authors examine how peripherin-2 (PRPH2) contributes to the localization of CNGβ1 within rod outer segment structures. PRPH2 and its homolog ROM1 are structural components of rod discs and are required for disc morphogenesis. In the absence of PRPH2, rod outer segments do not form, and various outer segment materials accumulate and are released as cilia-derived ectosomes. PRPH2 is thought to be transported through an unconventional secretory pathway, whereas cGMP-gated channels follow a conventional trafficking route. Although these components reach the outer segment through distinct pathways, PRPH2 is necessary for the proper delivery of CNGB1, a subunit of the cGMP-gated channel, to its correct destination. It was previously reported that a small fraction of PRPH2 reaches the outer segments through the conventional pathway when it forms a complex with Rom1 in mouse photoreceptors. Using Rom1 KO mice, the authors show that this conventionally trafficked PRPH2 fraction is not required for CNGB1 transport to the outer segment. Using various chimeric constructs, the authors verified that tetraspanin core of PRPH2, delivered to the OS, is sufficient to promote OS localization of CNGB1. Ct and Nt cytoplasmic regions of PRPH2 are dispensable for the role. Overall, the majority of the experiments are well-executed with statistical rigor, written in a way that others can reproduce, and support the major conclusion indicated in the title, "PRPH2 is essential for OS localization of CNGB1".

Major comments: I believe that the majority of the conclusions are well-supported in this manuscript. Below, I am listing the major points that may need additional experiments or clarifications: 1) CNGA1 subunit is transported to and enriched within ciliary exosomes or the outer segment in PRPH2 deficient mice (Figure 1). The reduced levels of CNGA1 and CNGB1 in rds-/- mice suggest limited stability of these proteins. Their diminished abundance is also influenced by decreased mRNA expression of the corresponding genes. These findings imply that CNGB1 may not be essential for outer segment delivery of cGMP-gated channels if CNGA1 alone contains adequate targeting information. Related to these points, it is unclear whether CNGB1 exhibits a trafficking defect or encounters other problems before leaving the endoplasmic reticulum. Such problems may involve deficiencies in folding, holo-channel assembly, or related quality control processes.

RESPONSE: We agree with this reviewer and have added additional data and interpretation to address this point. Our new data finds that in fact a low level of CNGB1 can reach ectosomes in rds-/- rods, which makes sense since we and others had observed CNGA1 was present and we know that channel assembly occurs in the ER. This suggests that the CNG channel can properly fold and assemble. Furthermore, overexpressing CNGB1 did not restore ciliary localization in Rds-/-, leading to our interpretation that in the absence of an outer segment membrane compartment, there is no place to deliver the CNG channel and it is subsequently degraded. Apart from perihperin’s binding partner, ROM1, this is unique to the CNG channel. CNG channel subunits are still significantly lower at P21 than other outer segment membrane proteins, such as ABCA4 (shown here), rhodopsin, and PCDH21(shown elsewhere).

2) CNGB1 overexpression in rds-/- mice does not result in outer segment localization of CNGB1 channels (Figure 2A). These findings do not clarify whether CNGB1 successfully transits through the Golgi apparatus or associates properly with CNGA1 subunits. Elevating expression levels alone would not compensate for problems in folding or assembly.

RESPONSE: We recognize that our previous submission lacked clarity on this point. Therefore, we have restructured the order of figures and provided additional controls to improve our manuscript. First, the fact that CNG channel is present at P21 and even increases over time suggests that in rds-/- rods channel processing (folding and assembly) is unaffected. Second, we recognize that channel stoichiometry is important for proper channel assembly, so we added a new supplementary figure that shows endogenous CNGA1 expression increases in rds-/- rods that are overexpressing myc-CNGB1 and FLAG-peripherin-2. This adds credence to our CNGB1 overexpression experiments and shows that CNGB1 being trapped is not due to inefficient channel assembly.

3) Claims related to Figure 6 (P45 rds-/-) need further evidence. It remains uncertain whether CNGA1 and CNGB1 are delivered to lamellar ciliary membranes or to a distinct plasma membrane compartment comparable to that observed in wild type rod outer segments, or whether they accumulate in ciliary ectosomes. Those lamellar structures could be a part of cone outer segments. The observed GARP signal may originate solely from soluble GARP proteins. It is also unclear if CNGA1 and ROM1 colocalize in P45 rds-/- mice. Clarifying these points would strengthen the conclusion that lamellar formation, rather than specific function of PRPH2, is sufficient for CNGB1 delivery to the cilium or outer segment plasma membrane.

RESPONSE: CNGA1/B1 are not expressed in cones, so the elevated outer segment localization observed at P45 must be coming from rods. In mouse retina, cones make up only 3% of the photoreceptor population. The SEM data clearly show that the lamellar ciliary protrusions are present on the majority of the photoreceptors. We now include CNGB1 staining from Rds-/- P45 sections that corroborate these data and show that CNGB1 is present at P45 and not P21 (Supplemental Figure 2).

Below are minor comments: 1) The study does not establish whether a direct interaction between PRPH2 and CNGB1 is required for CNGB1 delivery to rod outer segments. Prior work by the senior author (ref 13) suggests that this interaction is not essential, since the PRPH2 binding site within the GARP domain is distinct from outer segment transport signal of CNGB1. Including a discussion of the PRPH2-GARP (or CNGB1) interaction and its relevance to CNGB1 trafficking would help readers interpret the findings more fully.

RESPONSE: We have included this in our discussion.

2) The authors propose that the ROM1 core is sufficient for outer segment delivery of CNGB1 based on experiments with chimeric constructs. However, in Figure 1, ROM1 is present in the outer segments (or ciliary ectosomes) of rds-/- mice even though CNGB1 is not delivered to these structures.

RESPONSE: Our new data, including MS analysis and Western analysis from an enriched ectosome preparation, reveal that, along with ROM1, low levels of the CNG channel are delivered to ciliary ectosomes in Rds-/- mice. However, at this early timepoint photoreceptor cilia do not produce a membrane protrusion, which we observe is required to augment CNG delivery. We expressed a FLAG-ROM1 construct to try to drive earlier creation of these membrane protrusions, but this was unsuccessful, as we observed ROM1 was primarily localized to the inner segment. This suggests that overexpression of ROM1 did not increase ROM1 delivery to the cilia. Luckily, we were able to overcome this bottleneck with several of our chimeric ROM1/Prph2 constructs that did localize to the cilia and restore CNG localization. All of these new results have been included in the revised manuscript.

3) Line 80: "Theouter" A space shall be inserted between "The" and "outer".

RESPONSE: Done

**Referee cross-commenting**

Both reviewer #2 and reviewer #3 express views that align with mine. They clearly described the study's limitations, and their comments are highly valuable.

Reviewer #1 (Significance (Required)):

Prior studies showed that CNGB1 is not present in cilia-derived ectosomes of rds-/- mice, indicating that PRPH2 is necessary for ciliary or outer segment localization of CNGB1 in rods. Building on these earlier findings, I consider this study significant for the following reasons: 1) Using detailed analysis of different PRPH2 domains and chimeric constructs, it clarifies that PRPH2 core region, delivered to OSs, is essential and sufficient for OS localization of CNGB1. 2) PRPH2 and CNGB1 are thought to travel through different post-ER transport routes, with one pathway bypassing Golgi regions and the other passing through them. This study shows that CNGB1 depends on PRPH2, which suggests that these two routes may converge or interact at later stages and opens new directions for future investigation. 3) The study is relevant to basic scientists and biologists investigating how membrane structures acquire specialized functions in neurons, and its implications extend beyond photoreceptor biology.

Limitation of the study: I believe that clarifying these points will make the manuscript more significant. 1) Is it not clear, as mentioned above, how PRPH2 contributes to the delivery of CNGB1 to the OSs in the different secretory pathways.

RESPONSE: In the absence of ROM1, Prph2 only travels through the unconventional secretory pathway directly from the ER. By looking at CNG trafficking and localization in ROM1-/- mice, we rule out the possibility that the small portion of PRPH2/ROM1 complexes that traffic conventionally through the Golgi are required for channel localization (Figure 3). Further, our Rho-Prph2 chimera that includes the trafficking signal from Prprh2 did not rescue CNGB1 localization (Figure 4). These findings suggest that it is unlikely that these proteins engage during secretory transport to the outer segment.

2) The prior study using a fluorescence complementation approach (Ritter et al, 2011) suggests that PRPH2 and CNGB1 can associate within rod ISs, likely before their delivery to OSs. However, it remains unclear whether this interaction supports the potential cotransport of CNGB1 and PRPH2 or whether the authors view these proteins as being transported independently.

RESPONSE: As described above, our experiments rule out the notion that co-transport through the Golgi is driving CNG channel ciliary localization. We now note in our discussion that this data does not rule out the possibility of an earlier association between these proteins. However, the bulk of our data supports that any early interaction is not required for ciliary delivery.

3) At the end of the result section (Figure 6, rds-/- P45), the authors suggest that lamellar formation (evaginations?) is required for CNGB1 transport. However, CNGB1 is normally not seen in evaginations or lamellar structures, and thus the assumption is not consistent with prior findings.

RESPONSE: Absolutely, we agree that the CNG channel does not enter newly forming disc membranes, which has been shown by multiple groups. We included this in our discussion and have now added a clearer statement of our hypothesis: “Together, these data suggest that the partitioning of disc membranes from the plasma membrane by tetraspanin proteins is a key step for localizing the CNG channel and could play a role in segregating other proteins into the plasma membrane.”

Overall, the manuscript is insightful and has the potential to advance our field and related disciplines.

RESPONSE: Thanks!

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Cyclic nucleotide gated channels (CNG) localize to the plasma membrane of the rod photoreceptor outer segments, and are a key component of the phototransduction cascade. Understanding how outer segment proteins are trafficked and sequestered to the outer segments is an important field of investigation as it addresses both a fundamental aspect of cell biology and mechanism of disease, many of which have trafficking defects at the core of the pathogenic process. Using primarily IHC analysis of rodent models in combination with introduction of various expression constructs to the retina (through electroporation), this study finds that two rod outer segment structural proteins, peripheral-2 and ROM1, facilitate CNG channel localization to the outer segment.

While this conclusion is interesting, a major concern that tempers enthusiasm is that in peripherin-2 null photoreceptors, there are no outer bona fide segments. In lieu of outer segments, there are rudimentary membranous protrusions and vesicles distal to the connecting cilia where outer segments should be. So the basis for concluding that peripherin-2 is required for CNG localization to the outer segment seems a bit wobbly. It is understood that the authors assumed the membranous materials distal to cilia as proxy for outer segments in their analysis and narrative. This assumption may have some merits. However, it is well known that when outer segment morphogenesis is severely compromised, all normally outer segment-bound proteins are ectopically localized or largely absent due to increased degradation. This could be simply due to the loss of their destination compartment, among other things. It is not clear how the authors could distinguish between a direct causal relationship where loss of one protein leads to the mislocalization of another, from secondary outcomes due to loss of the outer segments. The last sentence of the Abstract is telling. "Interestingly, this notion is supported by endogenous staining of CNGB1, which reappears in aged Rds-/- rods that have produced ciliary membrane protrusions." So in aged mice CNGB1 did localize to the OS, but what changed? There was more OS like material to house the CNGB1 protein in the aged mice.

RESPONSE: We agree that the loss of the OS compartment is likely driving downregulation of all OS proteins and have included a statement as such in our manuscript. We also performed additional qRT-PCR analysis on ROM1 and ABCA4 to show global downregulation at the mRNA level – consistent with the notion that there are reduced outer segment proteins when morphogenesis is compromised. However, our Westerns and IHC (as well as published data) clearly find a specific decrease in the CNG channel at the protein level, suggesting that not all proteins behave similarly when the outer segment is not formed. We included additional discussion on this point as well. While not directly examined in our manuscript, previous reports have shown the reverse effect: some outer segment proteins (e.g. PCDH21, Prom1) are upregulated in rds-/- retinas (Rattner et al JBC 2004). Therefore, it is an oversimplification to state that all outer segment proteins behave the same when outer segments are not formed properly. Other models of outer segment dysmorphia (e.g. RhoKO, PCDH21KO, Prom1KO, or WASF3) localize the CNG channel properly. We have added this to the discussion and hope that by restructuring our manuscript, we clearly outline that we do think that membrane retention at the tip of the cilia is driving CNG channel localization and that molecularly the tetraspanin proteins play a role in organizing these membranes.

Reviewer #2 (Significance (Required)):

Trafficking of nascent proteins to the outer segment in support of its renewal is an important subject, which has significant impact in understanding the mechanisms of retinal degeneration. The conclusion from this study, that peripherin-2 and ROM1 have a direct role in supporting CNG subunit trafficking may well be meritorious. However the data presented are less than fully convincing, and specifically the question of a direct vs secondary effect needs to be better addressed.

RESPONSE: We appreciate this reviewer’s enthusiasm for investigating this process. The initial premise of our study was to investigate whether a direct effect of peripherin-2 on CNG delivery was possible, which was meritorious based on previously published data. However, we now find no direct trafficking link between CNG and peripherin-2; instead, our data largely find that CNG delivery is dependent on the presence of retained membranes at the ciliary tip – either through natural mechanisms or by driving “rudimentary” outer segment membrane lamination by overexpression of tetraspanin domains. We have restructured the manuscript to help guide the discussion.

The following quote underpins some of the reasoning in the study. Lines 139-144, "(Figure 2A). This localization pattern suggests that the CNGB1 subunit is trapped in the biosynthetic pathway. In contrast, when FLAG-tagged rhodopsin is overexpressed in Rds-/- rods it traffics properly to outer segment ectosomes (Figure 2B, (19)). We posit that without proper exit from the biosynthetic pathway, the endogenous CNGB1 protein is rapidly degraded to undetectable levels, which we circumvent through overexpression. These data suggest the localization defect of CNGB1 in Rds-/- rods is in the trafficking of CNGB1. " This in my view is an over- interpretation of limited data. The statement implies that rhodopsin and CNGB1 qualitatively differ in their fate but I would argue that both proteins are heavily degraded intracellularly except more of rhodopsin escaped to the "OS" and shows up in IHC. In many rhodopsin mutant transgenic mice, mutant rhodopsin appeared in OS even though intracellular degradation (gumming up the system) is a major factor in the disease process. The claim "rhodopsin trafficked properly to outer segment ectosomes" is not grounded in solid data.

RESPONSE: We do fundamentally agree that the endogenous CNG channel is heavily degraded, which we confirm by overexpressing an exogenous CNGB1-myc and finding it trapped in the biosynthetic pathway. As stated by the reviewer, this localization pattern is in contrast to what we and others have observed for endogenous rhodopsin, and now show for overexpressed FLAG-rhodopsin – that rhodopsin does traffic to the OS ectosomes. By comparing the localization of both endogenous and overexpressed constructs (using the same promoter), we feel that our conclusion is well supported. We appreciate that our wording of “rhodopsin trafficked properly to the outer segment” is misleading, as traffic of membrane proteins in Rds-/- rods is generally affected and not “proper”. Importantly, we follow up this “limited data” with additional experiments showing that at high expression levels, we are unable to drive CNGB1 localization to OS ectosomes unless we co-express with a tetraspanin domain.

A further minor comment is that the scope of the study appear limited, with no attempted experiments on how these proteins might interact to effect facilitation of trafficking.

RESPONSE: Our approach was to be agnostic to the outcome of our hypothesis that peripherin-2 was directly involved in CNG channel trafficking. The experiments we performed to test this (ROM1-/- analysis and Prph2 C-terminal chimeras) did not support a role for peripherin-2 in CNG trafficking. Instead, our data support a model in which membrane retention and organization at the ciliary tip drives CNG channel delivery. We feel that our approach was not limited.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

in the gene encoding tetraspanin protein peripherin 2 (Prph2), i.e., Rds-/-, examining the requirements for various portions of the Prph2 protein in the context of an assortment of chimeric constructs expressed via transfection into photoreceptor cells, to restore localization of the beta subunit of the cyclic nucleotide-gated channel (CNGbeta1) to photoreceptor outer segments (OS) (in a small number of experiments) or, in the majority of experiments, to do so for a recombinant tagged version of this protein also overexpressed by transfection.

The concluding sentences of the Discussion, which summarize the major conclusions are as follows: "Our data clearly show that localization of the CNG channel is dependent upon peripherin-2 after biosynthetic exit, further suggesting that the necessary action is at the ciliary base. Supporting evidence for this comes from analysis of Rhodopsin knockout outer segments which have internal disc-like structures and localize CNG channel properly. Therefore, in the absence of a fully elaborated outer segment, peripherin-2's ability to delineate a disc is sufficient to drive CNG channel delivery. Together, these data suggest that the partitioning of disc membranes from the plasma membrane by tetraspanin proteins is a key step for trafficking the CNG channel and could play a role in segregating other proteins into the plasma membrane.

The first sentence contains both reasonable conclusions and phrases whose meaning is unclear or not supported by the results presented. The statement: 'localization of the CNG channel is dependent upon peripherin-2 is supported by the data but, of course, has long been known from previous studies of Rds-/- mice. What is meant by "...after biosynthetic exit..." is unclear. If, by this term, apparently newly invented, the authors mean "after its synthesis of the protein is complete," the statement is accurate, but also a truism.

RESPONSE: The absence of CNGB1 was reported in previous studies, but the mechanism driving its absence has not been investigated. In our resubmission, we have added additional data that now shows CNGB1 is present at very low levels in Rds-/- ectosomes but remains undetectable by IHC, which is consistent with previous studies mentioned by the reviewer, but is also a novel finding. Importantly, we find specific downregulation of CNG channel subunits in Rds-/- retinas compared to ABCA4, supported by Western blot analysis (Figure 1), and we investigate the mechanism driving this result.

We appreciate the reviewer pointing out that “biosynthetic exit” is a niche term not broadly understood. We have removed this statement.

The statement, "the necessary action is at the ciliary base," is NOT supported by the data presented, as the effect of the "successful" Prph2 constructs on CNGbeta1 localization is primarily to increase its levels at the distal end of cilia and at the base of OS-related structures formed in response to the presence of the Prph2 constructs. The restoration of these membranes, which, as the authors note, has been previously reported, is overwhelmingly the biggest effect of these constructs, and it could be argued that the restored localization, rather than degradation, of CNGbeta1 is merely a downstream consequence of the formation of these structures, with perhaps, an element of stabilization of CNGbeta1 toward degradation from direct binding to Prph2, which has also been previously reported.

RESPONSE: We agree with the reviewer. Our interpretation of our data is that the presence of Prph2 (or its variants) at the distal end of the cilia localizes CNGB1, likely due to the formation of outer segment membrane structures. Previous to this work, there was a possibility that targeting information of Prph2 was required for CNGB1. That had never been explored. We definitively rule this possibility out when we express the C-terminal tail of Prph2, which is unable to rescue CNGB1 localization. Because the tetraspanin domain of Prph2 (or ROM1) can localize CNGB1, we do agree that the definition of an outer segment structure is the driving force for CNGB1 delivery – these are new findings. We’ve restructured and added additional discussion to the manuscript to clarify this point.

The next suggested conclusion is, "Therefore, in the absence of a fully elaborated outer segment, peripherin-2's ability to delineate a disc is sufficient to drive CNG channel delivery," is partly accurate and partly misleading. If the word "localization" were to replace the term, "delivery," concerning which there are no data (aside from those confirming that Prph2 and CNGbeta1 pass through distinct secretory pathways), this statement would be an accurate summary.

RESPONSE: We have updated to “localization”, but the fact that we confirm these two proteins do not traffic together through the Golgi would suggest that delivery is independent of trafficking.

The final sentence, "Together, these data suggest that the partitioning of disc membranes from the plasma membrane by tetraspanin proteins is a key step for trafficking the CNG channel and could play a role in segregating other proteins into the plasma membrane," sentence, would also be accurate if the word "localization," were to replace the term, "trafficking." The key point for these qualifications is that the experiments presented measure steady state levels of CNGbeta1 constructs at certain locations, which are determined not only by rates of trafficking, but also rates of synthesis and degradation, and the data presented confirm that total levels of CNGbeta1 are greatly diminished in the absence of functional Prph2, rendering any conclusions about the relative roles of trafficking kinetics and degradation kinetics speculative in nature.

RESPONSE: We agree and have revised.

Aside from these major conceptual issues, there is one overriding technical question: why are almost all the experiments presented carried out with a highly over-expressed engineered version of CNGb1 with a tag, which is clearly context far from the physiological one, as opposed to examining redistribution of the endogenous CNGbeta1, which is of much greater interest. In some results relegated to a Supplemental figure (Supp. Fig. 2), the authors clearly demonstrate that sufficient signal can be obtained from immunofluorescence staining the endogenous proteins for such experiments to be readily interpretable. If the concern was cross-reactivity with non-covalently attached GARP proteins, a few experiments showing that similar results are obtained for immunostaining of the endogenous protein or of the tagged construct would haver been sufficient, and the paper could have had more physiological relevance and impact.

RESPONSE: We agree that endogenous CNG staining is important and valuable, which is why we included it in our manuscript. We were able to confirm that overexpressed CNG recapitulated the endogenous staining. We proceeded with analyzing overexpressed, tagged CNG for the reasons stated by the reviewer. Yes, cross-reactivity with soluble GARP proteins was one consideration, as was the fact that the GARP antibody is a mouse monoclonal antibody. Increased IgG due to inflammation in the RDS-/- model can obscure the outer segment region in these retinas, confounding our quantification. The tagged versions of CNGB1 and corresponding quantification offered the most clarity and continuity for the reader; therefore, we relegate the endogenous staining to the supplement.

The remaining concerns are generally of less significance and mostly conceptual or quite minor technical concerns. Technically, the imaging data and their quantification are of good quality and analyzed with reasonable rigor.

RESPONSE: Thanks!

Abstract: "In this study, we investigate how peripherin-2 is engaged in CNG channel delivery to the outer segment. Might this not be more a question of how the absence of properly formed discs impacts the formation of outer segments with plasma membranes surrounding the disks? Is this really a question of "delivery" or "lack of address to make the delivery"?

RESPONSE: Our interpretation of this comment is that it boils down to semantics. Delivery is inclusive of both trafficking and localization, which we investigate in our manuscript.

Page 3, "fluorescence complementation between peripherin-2 and CNGb1 in the inner segment of transgenic Xenopus rods (23) ". The wording is unclear. It should be stated clearly that they are describing results of "bimolecular fluorescence complementation assays" of highly overexpressed recombinant proteins expressed from transgenes.

RESPONSE: We have revised.

Page 4, "...trapped in the biosynthetic pathway," It is unclear what the authors mean by this phrase. Obviously, "biosynthesis," i.e., translation is indeed complete, but biochemical pathways are not places. Is the intention to suggest that post-translational processing, such as addition and editing of carbohydrate chains or assembly with the alpha subunit has not been completed? If so, it would be better just to say so clearly. Or, is it meant to imply that it is physically "trapped" in the ER and/or Golgi apparatus? In any case the meaning should be made clear. Co-staining with ER and Golgi markers would have been very informative with respect to the compartments in which the highly overexpressed recombinant protein is trapped.

RESPONSE: We acknowledge that our phrasing here was indirect. We have revised. Co-staining with Calnexin (an ER-marker) was attempted, but proved to be uninformative.

It should also be noted that accumulation of highly overexpressed membrane proteins within internal membranes and membrane aggregates is a very commonly observed experimental phenomenon, and not restricted to the highly specialized trafficking routes in photoreceptors.

RESPONSE: We agree that exogenous expression of membrane proteins can lead to increased presence within internal membranes of the inner segment, which we routinely see in our experiments. Importantly, our analysis is restricted to the ability of these exogenously expressed proteins to reach the ciliary compartment in Rds mice. We also conduct these experiments in wild-type retinas to ensure that our constructs are expressed, and the proteins reach the ciliary outer segment under normal conditions.

Page 4, " peripherin-2 facilitates trafficking of the CNGb1 subunit to the outer segment " The data presented to this point do not demonstrate an enhancement of transport, but only of steady-state levels. There is nothing to rule out the possibility that some beta subunit is trafficked in Rds-/-, but is unstable to degradation in the region near the cilium when peripherin-2 and outer segments are not available. An increase in transport is certainly a possible explanation for the results, but should not be taken as an unambiguous conclusion.

RESPONSE: We have altered the description of these results to allow for more interpretation of our data, which show that CNGB1 delivery to the outer segment is reduced in Rds-/- mice and enhanced when peripherin-2 is re-expressed.

Page 4, " We confirmed that the fraction of peripherin-2 that traffics conventionally through the Golgi is indeed absent in Rom1-/- retinas and found that trafficking of the CNG channel via the conventional pathway is unaffected (Figure 3A) . This is one of the stronger and more interesting results in this manuscript, and tilts the argument against trafficking as being the mechanism for enhancement by overexpressed peripherin-2 of beta subunit levels in the distal region of the photoreceptor layer.

RESPONSE: We agree.

Page 5, " Our finding that secretory trafficking of peripherin-2 and CNGb1 is distinct . Clumsy syntax- needs to be rewritten for clarity.

RESPONSE: Revised

Page 5, "two previously characterized fusion proteins... have been shown to localize to the outer segment and build a rudimentary membrane structure (19) " This previous result, which is critical to interpretation of the results in this manuscript, should be introduced early, before any experimental results using related constructs are presented, in order to avoid confusion.

RESPONSE: Prior to these experiments, we used only full-length peripherin-2, rhodopsin, or CNGB1. This paragraph is the first introduction of any chimeric protein, and we explain these two constructs thoroughly. We believe this satisfies this reviewer’s request.

Page 5, " We confirmed these data by staining for endogenous CNGb1 in Rds-/- rods electroporated with each construct (Supplemental Figure 2B,C) " This is the most informative result in this manuscript with regard to the ability of these constructs to restore proper localization of CNGB1- it is not clear that the overexpression constructs for CNGB1 present any advantage beyond stronger signal and they may not be assumed, a priori, to be faithfully reporting on interactions of Prph2 with endogenous CNGB1, which is the biologically significant question. A big problem with Supp. Fig. 2 is that there is no real control, i.e., one without any Prph2 construct electroporated. Even the Rho-Prph2CT construct has some ROS-related structures and some CNGB1 localized to the one shown at higher magnification. The Prph2-RhoCT construct seems to lead to a substantial increase in endogenous CNGB1 in inner segment membranes. This looks like a phenomenon that is potentially very interesting, although it doesn't fit with any of the models put forth in the manuscript.

RESPONSE: We agree that endogenous staining (shown in Supplemental Figure 3 of our revised manuscript) is informative, but it was technically challenging. Once we verified that our overexpression system recapitulated results for endogenous CNGB1, we went forward with the epitope-tagged CNGB1, which was clearer when quantifying CNGB1 localization to rudimentary outer segments.

Our electroporation method provides an excellent internal control, as all of the non-electroporated cells show no endogenous CNGB1 localization without peripherin expression (Sup Fig 3A).

Page 5, " cytosolic N- and C-termini of peripherin-2 are dispensable for CNGb1 outer segment localization " No- if you could simply remove them and get proper localization, that would show they are "dispensable." In these experiments they are always replaced with the corresponding region of some other protein that is localized to OS, or in one case, with 3 copies of the FLAG tag at the N-terminus. There are also clear differences in the efficacy of the different "successful" constructs, but these results and their implications are not really discussed.

RESPONSE: We make this statement in the context of these termini being dispensable to CNGB1 localization, not to peripherin-2’s stability, function, or localization. A complete truncation of either domain results in a non-functioning protein. Our supplemental data shows reduced expression with a truncated N-terminus, preventing analysis (Sup Fig 5C). The 3X-FLAG has no known function in the cell, and we believe it serves as a proxy for removing the N-terminus altogether. Removing the C-terminus would prevent proper outer segment targeting, which is key to determining how peripherin-2 impacts CNGB1 ciliary delivery. Replacing this C-terminus with an outer segment targeting domain from another protein is an established method of investigation.

Page 6, " We then wanted to determine whether the ROM1 tetraspanin region was sufficient to facilitate CNGb1 delivery by further replacing ROM1's cytoplasmic N-terminus with that of peripherin-2 (Prph2NT/CT-ROM1) . " This experiment obviously does NOT test "sufficiency" of the TM segments, as the construct has the termini replaced with the corresponding regions of Prph2, which might functionally substitute for the missing ROM1 regions.

RESPONSE: Our previous results had already ruled out a role for these termini in CNGB1 localization.

Page 6, " We show a dramatic increase in GARP staining in the aged Rds-/- retinal sections " The age dependence of this phenomenon is quite interesting and puzzling. Any thoughts on the mechanism?

RESPONSE: We agree that this natural process is very interesting. We have restructured the order of our figures and provided additional controls to support this finding. We have added this to the discussion and hope that by restructuring our manuscript, we clearly outline that we do think that membrane retention at the tip of the cilia is driving CNG channel localization and that molecularly the tetraspanin proteins play a role in organizing these membranes.

Page 6, " Although CNGα1, known to form homotetramers, can localize to the extracellular vesicles released into the outer segment area. " Not a sentence.

RESPONSE: Revised

Page 6, " Our data now shows that the population of peripherin-2 in complex with ROM1 that travels through the conventional trafficking pathway does not play a role in CNGb1 localization to the outer segment. " This is an oddly accurate, albeit somewhat contradictory sentence. Yes, you have failed to answer the question you claim this work was designed to address. Apart from this negative result, nothing is learned about trafficking, per se, from the experiments in this manuscript.

RESPONSE: Please see our response to the reviewer’s comment above that clarifies our thinking regarding our results on trafficking.

Page 7, " anticipated " Hopefully, the authors mean to say, "hypothesized," here.

RESPONSE: Revised

**Referee cross-commenting**

My impression from reading the reviewers' comments is that there is general agreement on both the strengths and the limitations of this work. In my opinion, the issues raised by the reviewers could be addressed by editing the manuscript to be more circumspect in drawing definite conclusions from data that are not fully conclusive, without necessarily adding new experiments.

Reviewer #3 (Significance (Required)):

This study addresses a problem of great interest in the photoreceptor field and in cell biology more generally of trafficking and localization of specialized membrane proteins to specialized ciliary membranes. The strengths are technical quality of data with good controls, in most cases. The limitations are largely conceptual in nature and derive from the rather simplistic approach to the experimental design, as described above. The rather dated, "mix and match" approach based on chimeric construct with pieces of sequences removed and replaced at will does not properly account for the conclusion reached many times from many experiments, including some this manuscript, that the "roles" of stretches of amino acid sequence depend exquisitely on the multidimensional context in which they are tested, not simply on their position in the linear sequence. The paper presents interesting and convincing results with respect to functional requirements for formation disc-like membranes, but very little with respect to 'trafficking."

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Referee #3

Evidence, reproducibility and clarity

in the gene encoding tetraspanin protein peripherin 2 (Prph2), i.e., Rds-/-, examining the requirements for various portions of the Prph2 protein in the context of an assortment of chimeric constructs expressed via transfection into photoreceptor cells, to restore localization of the beta subunit of the cyclic nucleotide-gated channel (CNGbeta1) to photoreceptor outer segments (OS) (in a small number of experiments) or, in the majority of experiments, to do so for a recombinant tagged version of this protein also overexpressed by transfection.

The concluding sentences of the Discussion, which summarize the major conclusions are as follows: "Our data clearly show that localization of the CNG channel is dependent upon peripherin-2 after biosynthetic exit, further suggesting that the necessary action is at the ciliary base. Supporting evidence for this comes from analysis of Rhodopsin knockout outer segments which have internal disc-like structures and localize CNG channel properly. Therefore, in the absence of a fully elaborated outer segment, peripherin-2's ability to delineate a disc is sufficient to drive CNG channel delivery. Together, these data suggest that the partitioning of disc membranes from the plasma membrane by tetraspanin proteins is a key step for trafficking the CNG channel and could play a role in segregating other proteins into the plasma membrane.

The first sentence contains both reasonable conclusions and phrases whose meaning is unclear or not supported by the results presented. The statement: 'localization of the CNG channel is dependent upon peripherin-2 is supported by the data but, of course, has long been known from previous studies of Rds-/- mice. What is meant by "...after biosynthetic exit..." is unclear. If, by this term, apparently newly invented, the authors mean "after its synthesis of the protein is complete," the statement is accurate, but also a truism. The statement, "the necessary action is at the ciliary base," is NOT supported by the data presented, as the effect of the "successful" Prph2 constructs on CNGbeta1 localization is primarily to increase its levels at the distal end of cilia and at the base of OS-related structures formed in response to the presence of the Prph2 constructs. The restoration of these membranes, which, as the authors note, has been previously reported, is overwhelmingly the biggest effect of these constructs, and it could be argued that the restored localization, rather than degradation, of CNGbeta1 is merely a downstream consequence of the formation of these structures, with perhaps, an element of stabilization of CNGbeta1 toward degradation from direct binding to Prph2, which has also been previously reported.

The next suggested conclusion is, "Therefore, in the absence of a fully elaborated outer segment, peripherin-2's ability to delineate a disc is sufficient to drive CNG channel delivery," is partly accurate and partly misleading. If the word "localization" were to replace the term, "delivery," concerning which there are no data (aside from those confirming that Prph2 and CNGbeta1 pass through distinct secretory pathways), this statement would be an accurate summary. The final sentence, "Together, these data suggest that the partitioning of disc membranes from the plasma membrane by tetraspanin proteins is a key step for trafficking the CNG channel and could play a role in segregating other proteins into the plasma membrane," sentence, would also be accurate if the word "localization," were to replace the term, "trafficking." The key point for these qualifications is that the experiments presented measure steady state levels of CNGbeta1 constructs at certain locations, which are determined not only by rates of trafficking, but also rates of synthesis and degradation, and the data presented confirm that total levels of CNGbeta1 are greatly diminished in the absence of functional Prph2, rendering any conclusions about the relative roles of trafficking kinetics and degradation kinetics speculative in nature.

Aside from these major conceptual issues, there is one overriding technical question: why are almost all the experiments presented carried out with a highly over-expressed engineered version of CNGb1 with a tag, which is clearly context far from the physiological one, as opposed to examining redistribution of the endogenous CNGbeta1, which is of much greater interest. In some results relegated to a Supplemental figure (Supp. Fig. 2), the authors clearly demonstrate that sufficient signal can be obtained from immunofluorescence staining the endogenous proteins for such experiments to be readily interpretable. If the concern was cross-reactivity with non-covalently attached GARP proteins, a few experiments showing that similar results are obtained for immunostaining of the endogenous protein or of the tagged construct would haver been sufficient, and the paper could have had more physiological relevance and impact.

The remaining concerns are generally of less significance and mostly conceptual or quite minor technical concerns. Technically, the imaging data and their quantification are of good quality and analyzed with reasonable rigor.

Abstract: "In this study, we investigate how peripherin-2 is engaged in CNG channel delivery to the outer segment. Might this not be more a question of how the absence of properly formed discs impacts the formation of outer segments with plasma membranes surrounding the disks? Is this really a question of "delivery" or "lack of address to make the delivery"?

Page 3, "fluorescence complementation between peripherin-2 and CNG1 in the inner segment of transgenic Xenopus rods (23) ". The wording is unclear. It should be stated clearly that they are describing results of "bimolecular fluorescence complementation assays" of highly overexpressed recombinant proteins expressed from transgenes.

Page 4, "...trapped in the biosynthetic pathway," It is unclear what the authors mean by this phrase. Obviously, "biosynthesis," i.e., translation is indeed complete, but biochemical pathways are not places. Is the intention to suggest that post-translational processing, such as addition and editing of carbohydrate chains or assembly with the alpha subunit has not been completed? If so, it would be better just to say so clearly. Or, is it meant to imply that it is physically "trapped" in the ER and/or Golgi apparatus? In any case the meaning should be made clear. Co-staining with ER and Golgi markers would have been very informative with respect to the compartments in which the highly overexpressed recombinant protein is trapped. It should also be noted that accumulation of highly overexpressed membrane proteins within internal membranes and membrane aggregates is a very commonly observed experimental phenomenon, and not restricted to the highly specialized trafficking routes in photoreceptors.

Page 4, " peripherin-2 facilitates trafficking of the CNG1 subunit to the outer segment " The data presented to this point do not demonstrate an enhancement of transport, but only of steady-state levels. There is nothing to rule out the possibility that some beta subunit is trafficked in Rds-/-, but is unstable to degradation in the region near the cilium when peripherin-2 and outer segments are not available. An increase in transport is certainly a possible explanation for the results, but should not be taken as an unambiguous conclusion.

Page 4, " We confirmed that the fraction of peripherin-2 that traffics conventionally through the Golgi is indeed absent in Rom1-/- retinas and found that trafficking of the CNG channel via the conventional pathway is unaffected (Figure 3A) . This is one of the stronger and more interesting results in this manuscript, and tilts the argument against trafficking as being the mechanism for enhancement by overexpressed peripherin-2 of beta subunit levels in the distal region of the photoreceptor layer.

Page 5, " Our finding that secretory trafficking of peripherin-2 and CNG1 is distinct . Clumsy syntax- needs to be rewritten for clarity.

Page 5, "two previously characterized fusion proteins... have been shown to localize to the outer segment and build a rudimentary membrane structure (19) " This previous result, which is critical to interpretation of the results in this manuscript, should be introduced early, before any experimental results using related constructs are presented, in order to avoid confusion.

Page 5, " We confirmed these data by staining for endogenous CNG1 in Rds-/- rods electroporated with each construct (Supplemental Figure 2B,C) " This is the most informative result in this manuscript with regard to the ability of these constructs to restore proper localization of CNGB1- it is not clear that the overexpression constructs for CNGB1 present any advantage beyond stronger signal and they may not be assumed, a priori, to be faithfully reporting on interactions of Prph2 with endogenous CNGB1, which is the biologically significant question. A big problem with Supp. Fig. 2 is that there is no real control, i.e., one without any Prph2 construct electroporated. Even the Rho-Prph2CT construct has some ROS-related structures and some CNGB1 localized to the one shown at higher magnification. The Prph2-RhoCT construct seems to lead to a substantial increase in endogenous CNGB1 in inner segment membranes. This looks like a phenomenon that is potentially very interesting, although it doesn't fit with any of the models put forth in the manuscript.

Page 5, " cytosolic N- and C-termini of peripherin-2 are dispensable for CNG1 outer segment localization " No- if you could simply remove them and get proper localization, that would show they are "dispensable." In these experiments they are always replaced with the corresponding region of some other protein that is localized to OS, or in one case, with 3 copies of the FLAG tag at the N-terminus. There are also clear differences in the efficacy of the different "successful" constructs, but these results and their implications are not really discussed.

Page 6, " We then wanted to determine whether the ROM1 tetraspanin region was sufficient to facilitate CNG1 delivery by further replacing ROM1's cytoplasmic N-terminus with that of peripherin-2 (Prph2NT/CT-ROM1) . " This experiment obviously does NOT test "sufficiency" of the TM segments, as the construct has the termini replaced with the corresponding regions of Prph2, which might functionally substitute for the missing ROM1 regions.

Page 6, " We show a dramatic increase in GARP staining in the aged Rds-/- retinal sections " The age dependence of this phenomenon is quite interesting and puzzling. Any thoughts on the mechanism?

Page 6, " Although CNGα1, known to form homotetramers, can localize to the extracellular vesicles released into the outer segment area. " Not a sentence.

Page 6, " Our data now shows that the population of peripherin-2 in complex with ROM1 that travels through the conventional trafficking pathway does not play a role in CNG1 localization to the outer segment. " This is an oddly accurate, albeit somewhat contradictory sentence. Yes, you have failed to answer the question you claim this work was designed to address. Apart from this negative result, nothing is learned about trafficking, per se, from the experiments in this manuscript.

Page 7, " anticipated " Hopefully, the authors mean to say, "hypothesized," here.

Referee cross-commenting

My impression from reading the reviewers' comments is that there is general agreement on both the strengths and the limitations of this work. In my opinion, the issues raised by the reviewers could be addressed by editing the manuscript to be more circumspect in drawing definite conclusions from data that are not fully conclusive, without necessarily adding new experiments.

Significance

This study addresses a problem of great interest in the photoreceptor field and in cell biology more generally of trafficking and localization of specialized membrane proteins to specialized ciliary membranes. The strengths are technical quality of data with good controls, in most cases. The limitations are largely conceptual in nature and derive from the rather simplistic approach to the experimental design, as described above. The rather dated, "mix and match" approach based on chimeric construct with pieces of sequences removed and replaced at will does not properly account for the conclusion reached many times from many experiments, including some this manuscript, that the "roles" of stretches of amino acid sequence depend exquisitely on the multidimensional context in which they are tested, not simply on their position in the linear sequence. The paper presents interesting and convincing results with respect to functional requirements for formation disc-like membranes, but very little with respect to 'trafficking."

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Referee #2

Evidence, reproducibility and clarity

Cyclic nucleotide gated channels (CNG) localize to the plasma membrane of the rod photoreceptor outer segments, and are a key component of the phototransduction cascade. Understanding how outer segment proteins are trafficked and sequestered to the outer segments is an important field of investigation as it addresses both a fundamental aspect of cell biology and mechanism of disease, many of which have trafficking defects at the core of the pathogenic process. Using primarily IHC analysis of rodent models in combination with introduction of various expression constructs to the retina (through electroporation), this study finds that two rod outer segment structural proteins, peripheral-2 and ROM1, facilitate CNG channel localization to the outer segment.

While this conclusion is interesting, a major concern that tempers enthusiasm is that in peripherin-2 null photoreceptors, there are no outer bona fide segments. In lieu of outer segments, there are rudimentary membranous protrusions and vesicles distal to the connecting cilia where outer segments should be. So the basis for concluding that peripherin-2 is required for CNG localization to the outer segment seems a bit wobbly. It is understood that the authors assumed the membranous materials distal to cilia as proxy for outer segments in their analysis and narrative. This assumption may have some merits. However, it is well known that when outer segment morphogenesis is severely compromised, all normally outer segment bound proteins are ectopically localized or largely absent due to increased degradation. This could be simply due to the loss of their destination compartment, among other things. It is not clear how the authors could distinguish between a direct causal relationship where loss of one protein leads to the mislocalization of another, from secondary outcomes due to loss of the outer segments. The last sentence of the Abstract is telling. "Interestingly, this notion is supported by endogenous staining of CNGB1, which reappears in aged Rds-/- rods that have produced ciliary membrane protrusions." So in aged mice CNGB1 did localize to the OS, but what changed? There was more OS like material to house the CNGB1 protein in the aged mice.

Significance

Trafficking of nascent proteins to the outer segment in support of its renewal is an important subject, which has significant impact in understanding the mechanisms of retinal degeneration. The conclusion from this study, that peripherin-2 and ROM1 have a direct role in supporting CNG subunit trafficking may well be meritorious. However the data presented are less than fully convincing, and specifically the question of a direct vs secondary effect needs to be better addressed.

The following quote underpins some of the reasoning in the study. Lines 139-144, "(Figure 2A). This localization pattern suggests that the CNGB1 subunit is trapped in the biosynthetic pathway. Incontrast, when FLAG-tagged rhodopsin is overexpressed in Rds-/- rods it traffics properly to outer segment ectosomes (Figure 2B, (19)). We posit that without proper exit from thebiosynthetic pathway, the endogenous CNGB1 protein is rapidly degraded to undetectablelevels, which we circumvent through overexpression. These data suggest the localization defect of CNGB1 in Rds-/- rods is in the trafficking of CNGB1. " This in my view is an over- interpretation of limited data. The statement implies that rhodopsin and CNGB1 qualitatively differ in their fate but I would argue that both proteins are heavily degraded intracellularly except more of rhodopsin escaped to the "OS" and shows up in IHC. In many rhodopsin mutant transgenic mice, mutant rhodopsin appeared in OS even though intracellular degradation (gumming up the system) is a major factor in the disease process. The claim "rhodopsin trafficked properly to outer segment ectosomes" is not grounded in solid data.

A further minor comment is that the scope of the study appear limited, with no attempted experiments on how these proteins might interact to effect facilitation of trafficking.

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Referee #1

Evidence, reproducibility and clarity

Summary: In this manuscript, the authors examine how peripherin-2 (PRPH2) contributes to the localization of CNGβ1 within rod outer segment structures. PRPH2 and its homolog ROM1 are structural components of rod discs and are required for disc morphogenesis. In the absence of PRPH2, rod outer segments do not form, and various outer segment materials accumulate and are released as cilia-derived ectosomes. PRPH2 is thought to be transported through an unconventional secretory pathway, whereas cGMP-gated channels follow a conventional trafficking route. Although these components reach the outer segment through distinct pathways, PRPH2 is necessary for the proper delivery of CNGB1, a subunit of the cGMP-gated channel, to its correct destination.

It was previously reported that a small fraction of PRPH2 reaches the outer segments through the conventional pathway when it forms a complex with Rom1 in mouse photoreceptors. Using Rom1 KO mice, the authors show that this conventionally trafficked PRPH2 fraction is not required for CNGB1 transport to the outer segment. Using various chimeric constructs, the authors verified that tetraspanin core of PRPH2, delivered to the OS, is sufficient to promote OS localization of CNGB1. Ct and Nt cytoplasmic regions of PRPH2 are dispensable for the role. Overall, the majority of the experiments are well-executed with statistical rigor, written in a way that others can reproduce, and support the major conclusion indicated in the title, "PRPH2 is essential for OS localization of CNGB1".

Major comments: I believe that the majority of the conclusions are well-supported in this manuscript. Below, I am listing the major points that may need additional experiments or clarifications:

1) CNGA1 subunit is transported to and enriched within ciliary exosomes or the outer segment in PRPH2 deficient mice (Figure 1). The reduced levels of CNGA1 and CNGB1 in rds-/- mice suggest limited stability of these proteins. Their diminished abundance is also influenced by decreased mRNA expression of the corresponding genes. These findings imply that CNGB1 may not be essential for outer segment delivery of cGMP-gated channels if CNGA1 alone contains adequate targeting information. Related to these points, it is unclear whether CNGB1 exhibits a trafficking defect or encounters other problems before leaving the endoplasmic reticulum. Such problems may involve deficiencies in folding, holo-channel assembly, or related quality control processes.

2) CNGB1 overexpression in rds-/- mice does not result in outer segment localization of CNGB1 channels (Figure 2A). These findings do not clarify whether CNGB1 successfully transits through the Golgi apparatus or associates properly with CNGA1 subunits. Elevating expression levels alone would not compensate for problems in folding or assembly.

3) Claims related to Figure 6 (P45 rds-/-) need further evidence. It remains uncertain whether CNGA1 and CNGB1 are delivered to lamellar ciliary membranes or to a distinct plasma membrane compartment comparable to that observed in wild type rod outer segments, or whether they accumulate in ciliary ectosomes. Those lamellar structures could be a part of cone outer segments. The observed GARP signal may originate solely from soluble GARP proteins. It is also unclear if CNGA1 and ROM1 colocalize in P45 rds-/- mice. Clarifying these points would strengthen the conclusion that lamellar formation, rather than specific function of PRPH2, is sufficient for CNGB1 delivery to the cilium or outer segment plasma membrane.

Below are minor comments:

1. The study does not establish whether a direct interaction between PRPH2 and CNGB1 is required for CNGB1 delivery to rod outer segments. Prior work by the senior author (ref 13) suggests that this interaction is not essential, since the PRPH2 binding site within the GARP domain is distinct from outer segment transport signal of CNGB1. Including a discussion of the PRPH2-GARP (or CNGB1) interaction and its relevance to CNGB1 trafficking would help readers interpret the findings more fully.
2. The authors propose that the ROM1 core is sufficient for outer segment delivery of CNGB1 based on experiments with chimeric constructs. However, in Figure 1, ROM1 is present in the outer segments (or ciliary ectosomes) of rds-/- mice even though CNGB1 is not delivered to these structures.
3. Line 80: "Theouter" A space shall be inserted between "The" and "outer".

Referee cross-commenting

Both reviewer #2 and reviewer #3 express views that align with mine. They clearly described the study's limitations, and their comments are highly valuable.

Significance

Prior studies showed that CNGB1 is not present in cilia-derived ectosomes of rds-/- mice, indicating that PRPH2 is necessary for ciliary or outer segment localization of CNGB1 in rods. Building on these earlier findings, I consider this study significant for the following reasons:

1) Using detailed analysis of different PRPH2 domains and chimeric constructs, it clarifies that PRPH2 core region, delivered to OSs, is essential and sufficient for OS localization of CNGB1.

2) PRPH2 and CNGB1 are thought to travel through different post-ER transport routes, with one pathway bypassing Golgi regions and the other passing through them. This study shows that CNGB1 depends on PRPH2, which suggests that these two routes may converge or interact at later stages and opens new directions for future investigation.

3) The study is relevant to basic scientists and biologists investigating how membrane structures acquire specialized functions in neurons, and its implications extend beyond photoreceptor biology.

Limitation of the study:

I believe that clarifying these points will make the manuscript more significant.

1) Is it not clear, as mentioned above, how PRPH2 contributes to the delivery of CNGB1 to the OSs in the different secretory pathways.

2) The prior study using a fluorescence complementation approach (Ritter et al, 2011) suggests that PRPH2 and CNGB1 can associate within rod ISs, likely before their delivery to OSs. However, it remains unclear whether this interaction supports the potential cotransport of CNGB1 and PRPH2 or whether the authors view these proteins as being transported independently.

3) At the end of the result section (Figure 6, rds-/- P45), the authors suggest that lamellar formation (evaginations?) is required for CNGB1 transport. However, CNGB1 is normally not seen in evaginations or lamellar structures, and thus the assumption is not consistent with prior findings.

Overall, the manuscript is insightful and has the potential to advance our field and related disciplines.

Decreto nº 9.830/2019

Responsabilização na hipótese de dolo ou erro grosseiro

Art. 12. O agente público somente poderá ser responsabilizado por suas decisões ou opiniões técnicas se agir ou se omitir com dolo, direto ou eventual, ou cometer erro grosseiro, no desempenho de suas funções.

• § 1º Considera-se erro grosseiro aquele manifesto, evidente e inescusável praticado com culpa grave, caracterizado por ação ou omissão com elevado grau de negligência, imprudência ou imperícia.

• § 2º Não será configurado dolo ou erro grosseiro do agente público se não restar comprovada, nos autos do processo de responsabilização, situação ou circunstância fática capaz de caracterizar o dolo ou o erro grosseiro.

• § 3º O mero nexo de causalidade entre a conduta e o resultado danoso não implica responsabilização, exceto se comprovado o dolo ou o erro grosseiro do agente público.

• § 4º A complexidade da matéria e das atribuições exercidas pelo agente público serão consideradas em eventual responsabilização do agente público.

• § 5º O montante do dano ao erário, ainda que expressivo, não poderá, por si só, ser elemento para caracterizar o erro grosseiro ou o dolo.

• § 6º A responsabilização pela opinião técnica não se estende de forma automática ao decisor que a adotou como fundamento de decidir e somente se configurará se estiverem presentes elementos suficientes para o decisor aferir o dolo ou o erro grosseiro da opinião técnica ou se houver conluio entre os agentes.

• § 7º No exercício do poder hierárquico, só responderá por culpa in vigilando aquele cuja omissão caracterizar erro grosseiro ou dolo.

• § 8º O disposto neste artigo não exime o agente público de atuar de forma diligente e eficiente no cumprimento dos seus deveres constitucionais e legais.

Acórdão 1525/2025 Primeira Câmara (Tomada de Contas Especial, Relator Ministro Jhonatan de Jesus)

Responsabilidade. Culpa. Erro grosseiro. Omissão no dever de prestar contas. Débito. Multa.

• A omissão no dever de prestar contas constitui grave inobservância do dever de cuidado no trato com a coisa pública, revelando a existência de culpa grave, uma vez que se distancia do que seria esperado de um administrador médio, o que caracteriza erro grosseiro a que alude o art. 28 do Decreto-lei 4.657/1942 (Lindb), incluído pela Lei 13.655/2018, legitimando a condenação em débito do responsável e a aplicação da sanção pecuniária prevista no art. 57 da Lei 8.443/1992.

Acórdão 755/2025 Plenário (Tomada de Contas Especial, Relator Ministro Jhonatan de Jesus)

Responsabilidade. Culpa. Erro grosseiro. Conduta. Avaliação. Sanção.

• Para fins do exercício do poder sancionatório do TCU, o erro grosseiro a que alude o art. 28 do Decreto-lei 4.657/1942 (Lindb) fica configurado quando a conduta do agente público se distancia acentuadamente daquela que seria esperada do administrador médio, parâmetro que retrata o dever de cuidado objetivo esperado de um gestor comum, capaz e prudente.

Acórdão 1089/2025 - Plenário (Tomada de Contas Especial, Relator Ministro Benjamin Zymler)

Responsabilidade. Culpa. Parecerista. Fundamentação. Parecer jurídico.

• Os pareceres jurídicos desprovidos de fundamentação adequada, favoráveis a contratações manifestamente ilegais ou que deixem de considerar jurisprudência pacificada do TCU podem ensejar a responsabilização do seu autor, se o ato concorrer para eventual irregularidade praticada pela autoridade que nele se embasou.

Acórdão 2467/2025 - Segunda Câmara (Tomada de Contas Especial, Relator Ministro-Substituto Marcos Bemquerer)

Responsabilidade. Culpa. Erro grosseiro. Convênio. Execução física. Plano de trabalho.

• Para fins do exercício do poder sancionatório do TCU, considera-se erro grosseiro (art. 28 do Decreto-lei 4.657/1942 - Lindb) a execução do objeto conveniado em desacordo com o plano de trabalho aprovado pelo concedente, sem qualquer justificativa.

Acórdão 5284/2025 - Segunda Câmara (Recurso de Reconsideração, Relator Ministro Augusto Nardes)

Responsabilidade. Débito. Culpa. Dolo. Lei de Introdução às Normas do Direito Brasileiro. Erro grosseiro.

• A regra prevista no art. 28 do Decreto-lei 4.657/1942 (Lindb), que estabelece que o agente público só responderá pessoalmente por suas decisões ou opiniões técnicas em caso de dolo ou erro grosseiro, <u>não se aplica à responsabilidade financeira por dano ao erário</u>. O dever de indenizar prejuízos aos cofres públicos permanece sujeito à comprovação de dolo ou culpa, sem qualquer gradação, tendo em vista o tratamento constitucional dado à matéria (art. 37, § 6º, da Constituição Federal).

OBS.: Muito embora o regulamento equipare culpa grave a erro grosseiro, a literalidade da lei fala que o agente público será responsabilizado na hipótese de <u>dolo</u> ou <u>erro grosseiro</u>.

Synthèse de Mission d’Information : Le Journalisme d'Investigation Indépendant à l'Ère Numérique

Résumé Exécutif

Ce document synthétise les auditions menées par la mission d’information du Sénat sur les « zones grises de l’information », réunissant les représentants de trois médias d'investigation de premier plan : Disclose, Mediapart et Off-Investigation.

Le point central de cette rencontre est la redéfinition du journalisme de temps long face à la précarité de l'information numérique.

Les intervenants rejettent fermement l'appellation de « zone grise », arguant que leur pratique est ancrée dans une rigueur factuelle et juridique stricte (Loi de 1881).

Leurs modèles économiques, qu'ils soient basés sur l'abonnement, le don ou le mécénat, visent à s'affranchir de la dépendance aux régies publicitaires, aux aides publiques et à l'influence des grands groupes industriels.

Les principaux défis identifiés incluent la concentration des médias entre les mains de quelques milliardaires, la prolifération des « procédures bâillons » visant à épuiser financièrement les rédactions, et une dépendance paradoxale aux plateformes (YouTube, Facebook) qui, tout en offrant une audience massive, imposent une censure algorithmique opaque sur des sujets d'intérêt public.

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1. Identité et Modèles Économiques : L'Impératif de l'Indépendance

Les trois structures représentées partagent une mission commune : produire une information d'intérêt général exclusive, tout en expérimentant des modèles financiers alternatifs.

Comparaison des Modèles de Presse Indépendante

| Média | Statut Juridique | Modèle de Financement | Accessibilité des Contenus | | --- | --- | --- | --- | | Mediapart | Société par actions (fonds de dotation) | 100 % Abonnements (270 000 abonnés) | Payant (Mur payant / Paywall) | | Disclose | Association loi 1901 (But non lucratif) | Dons de lecteurs et fondations philanthropiques | Gratuit (Accès libre / Open Access) | | Off-Investigation | Structure indépendante | Campagnes de dons annuelles et abonnements | Mixte (YouTube gratuit / Site payant) |

Rejet des Financements Traditionnels

• Indépendance vis-à-vis des milliardaires : Les intervenants dénoncent la concentration des médias (95 % des marques d'information en France) aux mains d'industriels dont l'information n'est pas le métier.

• Refus des aides publiques : Mediapart et Disclose n'utilisent pas les aides à la presse.

Ils critiquent l'opacité et le manque de conditionnalité de ces aides, soulignant que des groupes milliardaires (ex: LVMH/Bernard Arnault) en sont les principaux bénéficiaires.

• Absence de publicité : Ce choix protège les rédactions contre l'influence des annonceurs et la « course au clic » qui dégrade la qualité de l'information.

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2. L'Enquête comme Outil de Contre-Pouvoir et Impact Public

Le journalisme d'investigation se distingue par sa capacité à générer un impact concret sur la société et le cadre législatif.

Exemples d'Impacts Documentés par Disclose

• Ventes d'armes (Made in France) : Révélations sur l'utilisation d'armes françaises contre des civils au Yémen, menant à la création d'une délégation parlementaire de contrôle.

• Environnement : Enquêtes sur la pollution de Lactalis et la contamination massive aux PFAS (polluants éternels), déclenchant des plaintes de communes et de citoyens.

• Santé et Histoire : L'enquête « Toxique » sur les essais nucléaires en Polynésie a abouti à une proposition de loi pour l'indemnisation des victimes, votée à l'unanimité par les députés.

• Libertés publiques : Documentation de l'usage illégal de la reconnaissance faciale par la police, provoquant des contrôles de la CNIL et des suspensions de logiciels (Briefcam).

La Notion de « Droit de Savoir »

Pour Mediapart, la mission démocratique consiste à éclairer les citoyens sur les agissements de ceux qui gouvernent ou détiennent une puissance économique.

Cette approche se veut « hors camp », ne servant que l'intérêt général et le respect de la Constitution.

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3. Les Obstacles au Journalisme d'Investigation

Les intervenants ont identifié plusieurs menaces systémiques qui pèsent sur la liberté d'informer.

La Censure et la Concentration

• Censure industrielle : Des cas de censure directe par Vincent Bolloré (Canal+) ont été cités par Disclose et Off-Investigation (enquête sur le Crédit Mutuel).

• Hystérisation du débat : Off-Investigation dénonce une stratégie de certains médias (CNews) visant à « diviser pour régner » et à créer des tensions sociales.

Les Pressions Judiciaires (Procédures Bâillons)

Les médias indépendants sont confrontés à une multiplication de poursuites (diffamation, violation du secret de la défense nationale) dont le but est l'épuisement financier :

• Coûts élevés : Même en cas de victoire, les frais d'avocats restent à la charge du média.

• Gardes à vue et perquisitions : Mention du cas d'Ariane Lavrilleux (Disclose) placée 40 heures en garde à vue pour ses enquêtes sur l'Égypte.

• Revendication : Appel à la transposition rapide de la directive européenne contre les procédures bâillons.

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4. La Relation Ambivalente avec les Plateformes Numériques

Bien que les réseaux sociaux permettent de contourner les barrières de diffusion traditionnelles, ils imposent de nouvelles contraintes.

Opportunités et Dépendance

• L'Autoroute YouTube : Pour Off-Investigation, YouTube est indispensable pour toucher une audience massive (plusieurs millions de vues pour un documentaire) là où les chaînes de télévision traditionnelles (M6, TF1, France TV) opposent souvent des refus.

• Dîme numérique : Les plateformes captent une part importante des revenus (environ 40-45 % pour YouTube).

Censure Algorithmique et Valeurs Américaines

Les plateformes imposent des règles morales et politiques qui interfèrent avec l'information :

• Déréférencement : YouTube a « tué » la portée de documentaires montrant des violences policières (Gilets Jaunes) ou traitant de violences sexuelles, au nom de la « pudeur » ou de la sensibilité des contenus.

• Opacité : Les médias déplorent l'absence d'interlocuteurs directs pour contester ces décisions arbitraires de modération.

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5. Régulation, Éthique et Intelligence Artificielle

Défense de la Loi de 1881

Les intervenants s'opposent fermement à toute nouvelle forme de labellisation ou de régulation par des autorités administratives (type Arcom).

Ils considèrent que :

• Le juge indépendant est le seul régulateur légitime de la presse.

• La loi de 1881 est un socle suffisant et protecteur.

• Toute instance de labellisation supplémentaire risquerait d'être politisée ou corporatiste.

L'Intelligence Artificielle (IA)

L'IA est perçue comme une menace pour le pluralisme si elle est utilisée pour générer des contenus de masse (« sites de désinformation »).

Cependant, elle est jugée incapable de remplacer l'investigation car :

• Elle ne peut pas produire d'informations exclusives.- Elle ne possède pas le « facteur humain » nécessaire pour instaurer une confiance avec les sources et les victimes.

• Les médias présents certifient n'utiliser aucune IA générative pour leurs enquêtes.

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Conclusions et Recommandations des Intervenants

Pour renforcer la fiabilité de l'information et protéger la presse indépendante, les recommandations suivantes ont été formulées :

• Transposer la directive européenne contre les procédures bâillons.

• Renforcer le secret des sources et l'indépendance des rédactions face aux actionnaires (droit d'agrément).

• Limiter les seuils de concentration des médias.

• Conditionner les aides à la presse à des critères de transparence et d'indépendance éditoriale.

• Soutenir l'éducation aux médias pour restaurer le lien de confiance avec les jeunes générations.

I personally believe that the person who made the post is the person who ran it. I don't think that the bot made the code but is closer to a tool that can be used like a shovel. I also think that the person who made the post is simply the person who created the tool to make the post.

so ein "zufall", jetzt wo praktisch alle gemeinden dringend geld brauchen, weil immer weniger steuereinnahmen und immer mehr ausgaben für sozialhilfe...

Another thing to add about Taoism is the idea of Yin and Yang. This idea is that the world is composed of opposing forces that must exist in harmony. There is also a belief that the concept of good or bad does not exist.

Demographics and Instruments 4

Instruments 3

Instruments 1

Instruments 2

Timeline 2

Game Summary

separa en una sección titulada referencias.

Revisa en gh-pages de github: https://mara-dl.github.io/Tesis_Maestria/

habrá que profundizar en este resultado en capítulos previos

usa formato de algortmo de latex. bastaria con cambiar el tipo de letra a currier

link de enlace

que tenga link de enlace

Note de Synthèse : Analyse des Mécanismes et du Traitement Judiciaire des Violences Intrafamiliales

Résumé Exécutif

Ce document analyse les dynamiques systémiques des violences intrafamiliales telles qu'observées lors des audiences judiciaires.

Les points clés révèlent que la violence n'est pas un incident isolé mais un système de domination fondé sur le contrôle coercitif et un sentiment d'appropriation de l'autre (« tu m'appartiens »).

Les auteurs de violences utilisent des mécanismes de défense récurrents : minimisation, déni, inversion de la culpabilité et décrédibilisation de la victime.

Le passage à l'acte ultime, le féminicide, survient souvent lorsque la victime tente de s'extraire de ce contrôle (séparation, grossesse).

Face à cet héritage historique et culturel de domination masculine, l'institution judiciaire évolue vers une approche plus spécialisée.

Le rôle du juge est désormais de décrypter ces mécanismes, de nommer précisément les faits et de corriger les inégalités systémiques pour interrompre le cycle intergénérationnel de la violence.

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I. La Logique de Domination et le Contrôle Coercitif

Les violences intrafamiliales s'inscrivent dans un schéma de comportement structuré visant à instaurer un climat de captivité au sein du foyer.

• Le sentiment de propriété : L'auteur considère la victime comme un objet lui appartenant.

Cette logique se traduit par des expressions telles que « je t'aime, je vais te tuer, tu m'appartiens ».

• Le contrôle coercitif : Ce mécanisme consiste en une micro-régulation de la vie quotidienne de la victime.

Il inclut :

• La surveillance des communications (lectures de SMS, contrôle des réseaux sociaux).

• La restriction des mouvements et des sorties.

• Le contrôle des relations sociales.

• Le climat de peur : L'usage de menaces de mort (« Je te crève », « Je vous crève tous ») vise à maintenir l'entourage dans un état de soumission et de terreur constante.

II. Rhétorique et Mécanismes de Défense des Auteurs

L'analyse des audiences met en lumière des stratégies discursives systématiques employées par les prévenus pour échapper à leur responsabilité.

1. La minimisation et l'euphémisation

Les auteurs présentent souvent les actes de violence comme des accidents ou des erreurs de calcul :

• Utilisation de termes comme « manque de peau » ou « mal calculé la distance » pour justifier un coup de tête ou une dégradation matérielle.

• Substitution de termes violents par des mots atténuants (ex: parler d'un crachat qu'on « amène » comme s'il s'agissait d'un cadeau).

• Distinction entre être « colérique » et être « violent ».

2. L'inversion de la culpabilité

Les auteurs tentent de justifier leurs actes par le comportement de la victime :

• La violence est présentée comme une réponse à des « provocations ».

• L'argument du « il n'y a pas de fumée sans feu » est utilisé pour rejeter la responsabilité sur la victime.

• L'auteur se présente parfois comme la véritable victime, poussée à bout.

3. La décrédibilisation de la victime

Une tactique fréquente consiste à faire passer la victime pour « folle », « menteuse » ou « hystérique » afin d'invalider sa parole devant le tribunal.

III. Le Cycle de la Violence et les Facteurs de Risque

La violence intrafamiliale suit une trajectoire de progressivité qui peut mener au féminicide.

| Stade | Caractéristiques | | --- | --- | | Enfermement | Mise en place silencieuse du contrôle coercitif, parfois sans violence physique préalable. | | Escalade | Augmentation graduelle de la gravité des sanctions : menaces, puis violences physiques. | | Passage à l'acte | Souvent déclenché par une rupture du contrôle (annonce du divorce, séparation). |

Facteurs aggravants du risque de féminicide :

• La volonté de départ : Quand la victime s'échappe, l'homme peut préférer « briser son jouet » plutôt que de perdre le contrôle.

• La grossesse : Perçue comme une intrusion dans la relation fusionnelle au profit de l'enfant, menaçant l'exclusivité de la possession.

• La jalousie : La découverte d'une tierce personne, même potentielle, déclenche une réaction de destruction.

IV. Dimensions Sociétales et Culturelles

Le document souligne que ces violences ne sont pas des faits isolés mais s'adossent à une structure historique.

• Héritage juridique : Jusqu'au XIXe siècle (Code Napoléon), l'homme avait un « droit de correction » légitime sur sa femme et ses enfants.

Ce reliquat historique influence encore les consciences actuelles.

• Responsabilité de la culture populaire : La fiction (séries, films) participe souvent à une « culture du féminicide » en banalisant les corps de femmes violentées ou en romantisant le crime sous l'appellation de « crime passionnel ».

• Réalité du crime : Le document insiste sur le fait qu'« on ne tue pas par amour ».

Le terme « passionnel » occulte la réalité criminelle de l'acte.

• Transmission intergénérationnelle : Les enfants témoins de violences sont marqués.

Si les garçons ont tendance à reproduire le schéma d'agresseur, les filles ont tendance à reproduire un schéma de victimation.

V. L'Office du Juge : Vers une Justice de Qualité

Le rôle des magistrats évolue pour mieux répondre aux enjeux des violences de genre.

• Décryptage des mécanismes : Le juge doit être capable d'identifier le contrôle coercitif et de ne pas se laisser abuser par la rhétorique de l'auteur.

• Manifestation de la vérité : Il s'agit de renommer les faits avec précision et de remettre la culpabilité du côté de l'auteur, indépendamment du comportement de la victime.

• Analyse systémique : Le juge doit se poser trois questions fondamentales pour corriger les inégalités de genre :

  • Mon jugement est-il le fruit d'une inégalité systémique ?

• Ma façon de parler à la victime peut-elle aggraver cette inégalité (ex: lui demander pourquoi elle n'est pas partie) ?

• Ma décision peut-elle corriger une inégalité systémique ?

VI. Ressources et Dispositifs d'Aide

Il est impératif de ne pas rester isolé face à ces situations. Des outils concrets existent pour les victimes et les témoins :

• 3919 : Numéro national de référence pour les violences faites aux femmes (anonyme et gratuit).

• 119 : Numéro dédié à la protection de l'enfance.

• Services de police et gendarmerie : Accessibles 24h/24 pour le dépôt de plainte, constituant une réponse concrète et immédiate.

• Lieux d'écoute : Espaces de conseils pour ceux qui craignent d'être victimes ou qui souhaitent anticiper un processus judiciaire perçu comme impressionnant.

Worked? 2

Instruments 2 and Worked?

Demographics 1

Demographics 2

Mediation 3 and Instruments 1

Mediation 1

Mediation 2

Game summary

Teamwork science. People experimenting with or photographing things they are interested in and uploading that for other people to look at.

Relating observed natural processes or phenomena through a scientific lens (ex: erosion in canyons, halos around the sun, lunar eclipse)

Units, terms, and other scientific concepts are talked about in the news (ex: health regimens, weather courses) so it is expected to know a bit about it

Ability to recognize chemicals and reason whether or not to buy the product

Understanding of natural orders that can be taken advantage of in ones environment

organizations like "moms for nuclear"

climate change, ocean acidification, certain chemicals recognized as carcinogens

Game summary 2

Instruments 7

Timeline 2

Demographics 3

Worked? 2

Instruments 6 and Worked?

Instruments 5

Game summary

Instruments 4

Mediation 2

Mediation 3

Demographics 2

Demographics 1

Instruments 4

Instruments 3

Timeline 3

Demographics 1 and Mediation 1

Instruments 2, Mediation 2, and Timeline 2

Timeline 1

Trait blanc again.

No a-conceptuality.

It also seems to ape the royal road to the unconcious line in Freud.

Life as idealisation.

In other words, dislocation.

But the reverse is also true.

Nice.

Breaching immediately contaminates.

Great sumnmary of the quasi-concept.

Yes.

Yes queen, give him nothing.

This is a pretty good summary of espacement, but it doesn't seem to cover the grammatisation.

A bit of an oversimplification. The exterorior is more often than not doubled into a pure exterior and a perverted exterior-- and what about the Khora?

The author claims that trade in Mecca fails to provide a sufficient explanation of the emergence of Islam. This questions previous historians who focused on economic influences. It implies that there are other political or social explanations that should be taken into account.

The authors used a clinical prediction model to estimate the individual risk of developing breast cancer, but it is questionable if models can do this.

A recently updated description of these models reads (1): An individuals’ risk of an event as estimated by a prediction model refers to the probability of that event in the subgroup of individuals with the same predictor values. Truly individual risks do not exist because of the ‘reference class problem’: an individual can belong to an infinite number of subgroups. By choosing a different set of predictors to condition on, the reference class changes, and so does the risk. It may seem paradoxical that using prediction models for decision making can benefit outcomes at a population level without having trustworthy risk estimates for individuals from that population. ... Clinical utility at the population level, for example in terms of cost-effectiveness, is sufficient to warrant the use of a decision strategy based on a model. We may need to accept that two models may have the same clinical utility yet very different risk estimates for the same individual, often to the extent that model A recommends treatment initiation, but model B does not.

Another concluded that (2): clinical prediction models cannot, do not, and need not estimate individual risk.

Since there is no evidence individual risk exist (3) and clinical prediction models don't estimate individual risk, these models are better understood to be risk stratifying a population. They separate a population into groups differing in risk, not individuals differing in risk, which can then be used to devise policies that efficiently allocate surveillance or preventive measures. This may make sense for the general population, but is unlikely to be useful for BRCA1 and BRCA2 pathogenic variant carriers. If predictors are added to a model, the risk distribution often broadens (detected by measures of discrimination) which may allow even more efficient allocations. This was nicely shown by the figure depicting the broader risk distribution resulting from addition of the polygenic risk score to age alone.

Discordance in predictions for an individual across models is well documented and has been called "the multiverse of madness (4), having been observed with breast cancer risk models (5) and polygenic risk models (6). This does not impact their use for population risk stratification, but should preclude their use for individual risk estimation.

Holmberg and Parascandola critiqued the use of breast cancer risk models, including the presentation of numbers as personalized risks (7): But we would argue that the more general problem here is with the promise of individualised risk information, which implies a sense of authority and certainty about the individual. Framing risk estimates as individualised may be misleading as it implies a high level of specificity for the individual. The use of individualised risk estimates suggests that a risk model says something personal about a specific individual rather than an entire group.

Models including more or better predictors or using machine learning may provide greater discrimination but will not evade the reference class problem. So, this problem will persist and geneticists using clinical prediction models need to understand this counterargument to the conventional narrative on individual risk. To date, the reference class problem has been a well-kept secret in medicine with only 10 results in PubMed. A new points to consider statement from the ACMG (8) does caution that risk estimates cannot be directly applied to an individual and that discordant risks may be estimated. Nevertheless, personalized or individualized risk estimates remain the goal (with future model improvements expected to make this possible) and the discordance is not flagged, as it usually is, as a reason to question the use of these numbers. There no discussion of the reference class problem and its implication that individual risks don't exist and that the continuum of risk applies to groups, not individuals, so models provide population risk stratification suitable for policy development but not for counseling.

As geneticists considering counseling based on a clinical prediction model need to know about the reference class problem and its implications, this merits inclusion in the discussion of limitations when the research is eventually published. Ralph Stern Division of Cardiovascular Medicine Department of Internal Medicine University of Michigan

1. Barreñada L, Steyerberg EW, Timmerman D, Thomassen D, Wynants L, Van Calster B. The fundamental problem of risk prediction for individuals: health AI, uncertainty, and personalized medicine. arXiv. Preprint posted online 2025. doi:10.48550/ARXIV.2506.17141

2. Stern RH. Accuracy of Preoperative Risk Assessment: Individuals versus Groups. Am J Med. 2025;138(6):926-927. doi:10.1016/j.amjmed.2025.02.007

3. Dawid P. On individual risk. Synthese. 2017;194(9):3445-3474. doi:10.1007/s11229-015-0953-4

4. Riley RD, Pate A, Dhiman P, Archer L, Martin GP, Collins GS. Clinical prediction models and the multiverse of madness. BMC Med. 2023;21(1):502. doi:10.1186/s12916-023-03212-y

5. Paige JS, Lee CI, Wang PC, et al. Variability Among Breast Cancer Risk Classification Models When Applied at the Level of the Individual Woman. J Gen Intern Med. 2023;38(11):2584-2592. doi:10.1007/s11606-023-08043-4

6. Clifton L, Collister JA, Liu X, Littlejohns TJ, Hunter DJ. Assessing agreement between different polygenic risk scores in the UK Biobank. Sci Rep. 2022;12(1):12812. doi:10.1038/s41598-022-17012-6

7. Holmberg C, Parascandola M. Individualised risk estimation and the nature of prevention. Health, Risk & Society. 2010;12(5):441-452. doi:10.1080/13698575.2010.508835 1.

8. Pal T, Christopher J, Astiazaran-Symonds E, et al. Consideration of inherited cancer risk on a continuum: An international and multidisciplinary perspective: A points to consider statement of the American College of Medical Genetics and Genomics (ACMG). Genet Med. 2026;28(3):101659. doi:10.1016/j.gim.2025.101659

Any

Sp and punc

Spent

Comme une très grande part de la tradition de l’ethnographie

Privilégiée sur quel plan ? Symbolique ? Epistémique ? Matériel ?

Quels biais exactement ?

Il n'y a pas là de différence avec la sociologie compréhensive et l'éthnographie

Une remarque comme ça : Donc cela implique-t-il de prendre connaissance des connaissances empiriques disponibles sur son objet pour savoir ce qui en était su précédemment ?

Est-ce que cela ne correspond pas à la démarche de la sociologie compréhensive ?

Une remarque comme ça : … S’il revient aux acteurs de changer, pourquoi ne pas co-construire les sujets de recherche avec eux ? Un peu étrange alors non ?

Du coup ne devrais-je pas être située plus bas sur l’axe épistémologique ?

Vous pouvez aussi préciser que paradoxalement je n’envisage pas nécessairement de transformer la pratique du terrain étudié mais d’avoir un impact sur les autres expériences (voir aussi « Travailler « à partir de » et « sur » les justifications des enquêtés. Un retour d’expérience. », in Terrains philosophiques, dir. Alison Bouffet, Théophile Lavault, Pauline Vermeren, Presses Universitaires de Dijon., 2025)

Il me semble dès lors qu’il pourrait être utile pour chaque type de situer les autres auteurs qui en font partie, par exemple en les mentionnant brièvement à la fin de chaque partie. Car si on dit qu’il y a des travaux «pionniers» d’une approche, c’est que d’autres ont suivis (même s’ils ne savaient pas qu’ils reprenaient une approche déjà existante)

ou en tous cas s’envisage comme complémentaire ?

Du coup un angle mort important de votre classification est la question de l’appui non pas sur les méthodes des sciences sociales, mais bien des données des sciences sociales: il y a des philosophes qui mobilisent les données déjà existante (ce qu’on sait déjà) en sociologie, en histoire, etc. sur leur objet ; et d’autres non.

Ég gerði smá nafnavillu þegar ég vann "about me" þar á að vera Þýsk kamilla (Matricaria chamomilla) ekki kamilla offincialis, ég kann ekki að laga:(

hahaha þolinmæði :)greinin er komin hjá mér:)

hæhæ ég þarf hjálp hér, þegar ég birti færsluna þá bara hverfur hún?? kærleikakveðja

The conversion process for PDF files submitted to medRxiv can fail to recognize block quotations. The "Full-Text" version will be hard to read when block quotations look indistinguishable from the surrounding text. Please consider the PDF version. We regret the inconvenience.

I found it interesting the problems he identifies as sourced in division. For example, divided churches cannot practice discipline.

Campbell not only recognizes brethren among the denominations, he expects them to rise up in cooperation with this evangelical, ecumenical movement

A concise statement of the overall purpose

Campbell sees a remarkable degree of unity as already the case among all the churches of Christianity. They agree on doctrines and ordinances. What prohibits their visible unity?

complex interaction of increased visceral afferent sensitivity, gastric delayed emptying or impaired accommodation to food or psychosocial stressors. Or Peptic ulcer disease is present in 5–15% and GERD is present in up to 20% of patients with dyspepsia, even without significant heartburn

还有一种设计是

"System initialized. User profile and session protocols have been synchronized. I am ready to assist based on the defined skills and constraints."

/💻/asus/🧊/me/📓/2026/04/10

in English

n'hésite pas à mentionner mon article ! ;)

Mais il y'a bien un humain qui gère ses "influenceurs" virtuels? pourquoi elle ne pourrait pas faire d'erreur ?

Est‑ce que cette impression de proximité fonctionne de la même manière avec un avatar virtuel qu’avec une célébrité?

Hotel in Sigmaringen, across from the castle

Ajoute des mots clés pour qu'on puisse trouver cet article plus facilement.

DOI: 10.7554/eLife.88466

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @maulamb

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.7554/eLife.88466

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @maulamb

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.7554/eLife.88466

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @maulamb

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.7554/eLife.88466

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @maulamb

SciCrunch record: RRID:SCR_006457

What is this?