Here in the trending section, there is a good exhibition of the website being perceivable and understandable. There are sharp contrasting colors between the background and foreground to tell the user that the information in this section is more relevant towards their interests. Therefore, it would be easy to immediately understand what the most prominent news articles are for the user.

I think this would be the most robust section of the website, because it separates each of the news article in distinct themes. This can help improve the navigation for the website and ensure that readers know what kind of information that they are viewing. Anyone using assistive technology would know the overall theme of the articles that they are viewing.

This is a poor practice, as none of the videos have closed captioning to help users who may be deaf. There are also no transcriptions being provided for the users so that they would be able to understand the contents of the video. The videos on the website are not accessible to the deaf users who may wish to watch videos.

This section makes it easy for a screen reader to help direct the reader, as there are titles and a brief summary of what the article is about. This can help the reader immediately understand the content as it is navigable by a screen reader which many readers may rely on. There are also alt descriptions by examining the HTML code.

The most important headline is presented visually in a bold manner for the reader. The reader is able to gravitate immediately towards the headliner article and they are able to click into it for more detail. This is a good example of an operable interface, where the reader is also able to view a video as well.

[01:11:54 - 01:12:55] : Importance de partager des expériences pour développer une mixité sociale véritable et l’impact de cela sur l’empathie et la tolérance.

[00:43:00 - 00:45:00] : Discussion sur l’influence de l’environnement socio-économique sur les capacités d’apprentissage des enfants et l’importance de la diversité des expériences pour développer l’empathie et la compréhension sociale.

Résumé de la vidéo [00:00:00][^1^][1] - [00:24:32][^2^][2]:

Cette vidéo présente une conférence de Sama Karaki sur l'apprentissage pour l'apprentissage, et non pour la réussite. Elle aborde les neurosciences, la méritocratie, le talent, et comment ces concepts s'appliquent à l'éducation et au développement des compétences.

Points forts: + [00:00:00][^3^][3] Introduction de la conférence * Présentation de Sama Karaki, docteur en neurosciences * Discussion sur la méritocratie et le talent dans la société * Importance de développer des compétences à l'école + [00:02:13][^4^][4] Les neurosciences et l'éducation * Les neurosciences comme étude des bases biologiques des comportements * Importance du dialogue entre disciplines pour comprendre le talent et la méritocratie * Impact des inégalités sociales sur les capacités d'apprentissage + [00:10:01][^5^][5] Mythes et réalités sur le cerveau * Démystification de l'idée d'un cerveau latéralisé * Critique de la notion de traits fixes et mesurables comme la créativité ou l'empathie * Influence des attentes et perceptions des enseignants sur les élèves + [00:18:17][^6^][6] Intelligence et apprentissage * L'intelligence n'est pas un trait fixe ou héritable * Le cortex préfrontal et son rôle dans les fonctions exécutives * L'apprentissage comme processus coûteux nécessitant motivation et effort Résumé de la vidéo [00:24:34][^1^][1] - [00:48:09][^2^][2]:

Cette partie de la vidéo explore l'importance de la curiosité et du plaisir dans l'apprentissage, l'évolution de l'intelligence humaine, et les facteurs sociaux et culturels qui influencent l'intelligence. Elle souligne que l'intelligence n'est pas fixe et peut être façonnée par l'environnement et l'éducation.

Points forts: + [00:24:34][^3^][3] La curiosité dans l'apprentissage * La curiosité est essentielle pour rendre l'apprentissage agréable * L'apprentissage est comparé à des activités qui génèrent du plaisir malgré l'effort + [00:26:24][^4^][4] L'intelligence évolutive * L'intelligence n'est pas fixe et peut augmenter avec le temps * L'accès au raisonnement abstrait a augmenté l'intelligence au fil des générations + [00:31:34][^5^][5] Intelligence et culture * L'intelligence est influencée par des facteurs historiques et culturels * La perception de l'intelligence varie selon les époques et les cultures + [00:37:36][^6^][6] Intelligence et réussite sociale * L'intelligence ne prédit pas nécessairement la réussite sociale * Les inégalités socio-économiques peuvent affecter les résultats des tests d'intelligence Résumé de la vidéo [00:48:10][^1^][1] - [01:12:53][^2^][2]:

Cette vidéo explore le paradoxe de genre dans les domaines STEM et l'importance de l'apprentissage pour l'apprentissage, pas seulement pour réussir. Elle discute des différences de participation des femmes dans les pays égalitaires et non égalitaires, et comment les croyances et l'estime de soi influencent les choix de carrière.

Points forts: + [00:48:10][^3^][3] Le paradoxe de genre dans STEM * Moins de femmes dans les pays égalitaires * Plus grande égalité de genre, plus grand écart + [00:51:02][^4^][4] L'impact de l'estime de soi sur les choix de carrière * Les filles se voient moins brillantes à partir de 6 ans * L'importance de la mentalité de croissance + [00:54:00][^5^][5] L'intervention de Carol Dweck * Changement d'état d'esprit améliore les résultats * Apprendre pour apprendre, pas pour réussir + [01:04:00][^6^][6] L'importance de l'incertitude dans l'apprentissage * Valoriser la capacité à douter de soi * Résister à l'automatisme et promouvoir la métacognition Résumé de la vidéo [01:12:55][^1^][1] - [01:28:16][^2^][2]:

Cette partie de la vidéo aborde l'importance de l'apprentissage pour l'expérience personnelle plutôt que pour la réussite seule. Elle souligne la subjectivité de la perception de la réalité et la nécessité pour le système éducatif de mettre l'accent sur l'incertitude sociale et la construction collective de la connaissance.

Points forts: + [01:13:00][^3^][3] La perception subjective de la réalité * La réalité est liée à l'expérience personnelle * L'enseignement doit valoriser l'expérience subjective * Importance de reconnaître la subjectivité dans l'apprentissage + [01:14:00][^4^][4] L'illusion cognitive et la résistance à soi * Les automatismes influencent la perception * La tâche de l'enseignement est de former les bons automatismes * Nécessité de reconsidérer ce qui est appris face à la réalité + [01:17:20][^5^][5] Questions et réponses sur l'éducation * Discussion sur les stéréotypes de genre et l'éducation en Scandinavie * L'impact des expériences partagées sur l'empathie * Le rôle de l'école dans la formation de l'intuition et la déconstruction des préjugés + [01:25:43][^6^][6] Réflexions sur le talent et la méritocratie * Le défi de lutter contre une société méritocratique * La pression des parents et la conception individuelle du talent * L'importance de promouvoir l'égalité des chances dans l'éducation

The references show Riley, et al Part II twice. Need to add the reference for Part I here:

Minimum sample size for developing a multivariable prediction model: Part I - Continuous outcomes Richard D Riley 1 , Kym I E Snell 1 , Joie Ensor 1 , Danielle L Burke 1 , Frank E Harrell Jr 2 , Karel G M Moons 3 , Gary S Collins 4

PMID: 30347470 DOI: 10.1002/sim.7993

Exiting a session is called detaching

Being able to realize that not all is lost, that there are still some positive aspects of the dire situation that one could be in, allows one to move forward in hopes of protecting/obtaining those good qualities.

The rising of negative emotions inside of people can create an internal and/or external stagnation in terms of one's development/progress in life.

These feelings can be quite debilitating for some.

Answers the question of what quality one typically has that allows them to move forward through suffering.

This is something I was curious about as well while reading Victor Frankl's Man's Search For Meaning. While inside the concentration camps, he mentioned how some chose to "run into the wire" (commit suicide), and some had more motivation/drive to do what was asked of them and live another day. I was/am curious on what differentiates those who give up and those who don't.

Driving question of the article

You must set a restore scope, otherwise this login doesn't make sense.

Hurricanes need four main things to form: warm water, moist air, low wind, and a starting condition such as a thunderstorm.

This is very nice note for me

Proyecto "Anotación PFR", https://github.com/lmichan/PFR,

Tema/mesh/D012137/RespiratorySystem,

TipoDePrueba/mesh/D002000/ForcedSpirometry,

EtapaPrueba/Estandar,

PatronFuncional/Obstruccion,

PatronFuncional/PosibleRestriccion,

PatronFuncional/PosibleMixto,

PatronFuncional/Normal,

PatronFuncional/Broncodilatacion,

PatronFuncional/NoBroncodilatacion,

Enfermedad/mesh/D001249/Asthma,

Enfermedad/mesh/D029424/ChronicObstructivePulmonaryDisease,

Enfermedad/mesh/D003550/CysticFibrosis,

Enfermedad/mesh/D009468/NeuromuscularDisease,

Enfermedad/mesh/D001987/Bronchiectasis,

Enfermedad/mesh/D054990/IdiopathicPulmonaryFibrosis,

Enfermedad/mesh/D008175/LungNeoplasm,

Enfermedad/mesh/D001991/Bronchitis,

Enfermedad/mesh/mesh/D012130/RespiratoryHypersensitivity,

Enfermedad/mesh/D012829/Silicosis,

Proyecto "Anotación PFR", https://github.com/lmichan/PFR,

Tema/mesh/D012137/RespiratorySystem,

TipoDePrueba/mesh/D002000/ForcedSpirometry,

TipoDePrueba/mesh/D000089142/FractionalExhaledNitricOxideTesting,

TipoDePrueba/mesh/D001985/BronchialProvocationTests,

EtapaPrueba/Clinica,

PatronFuncional/Obstruccion,

PatronFuncional/PosibleRestriccion,

PatronFuncional/PosibleMixto,

PatronFuncional/Normal,

PatronFuncional/Broncodilatacion,

PatronFuncional/NoBroncodilatacion,

PatronFuncional/BajaInflamacionEosinofilica,

PatronFuncional/ModeradaInflamacionEosinofilica,

PatronFuncional/AltaInflamacionEosinofilica,

PatronFuncional/RetoBronquialPositivo,

PatronFuncional/RetoBronquialNegativo,

Enfermedad/mesh/D001249/Asthma,

Proyecto "Anotación PFR", https://github.com/lmichan/PFR,

Tema/mesh/D006266/HealthEducation,

TipoDePrueba/mesh/D002000/ForcedSpirometry,

EtapaPrueba/Interpretacion,

PatronFuncional/Obstruccion,

PatronFuncional/PosibleRestriccion,

PatronFuncional/PosibleMixto,

PatronFuncional/Normal,

PatronFuncional/Broncodilatacion,

PatronFuncional/NoBroncodilatacion,

Enfermedad/mesh/D001249/Asthma,

Enfermedad/mesh/D029424/ChronicObstructivePulmonaryDisease,

Proyecto "Anotación PFR", https://github.com/lmichan/PFR,

Tema/mesh/D006266/HealthEducation,

TipoDePrueba/mesh/D002000/ForcedSpirometry,

EtapaPrueba/Interpretacion,

PatronFuncional/Obstruccion,

PatronFuncional/PosibleRestriccion,

PatronFuncional/PosibleMixto,

PatronFuncional/Normal,

PatronFuncional/Broncodilatacion,

PatronFuncional/NoBroncodilatacion,

Enfermedad/mesh/D001249/Asthma,

Enfermedad/mesh/D029424/ChronicObstructivePulmonaryDisease,

This is another cool page note

This is a sick page note

Hmm! Sounds like a plan

Sounds cool

This is cool as well as sick!

follow up

WwbXR flips it on it's head

l1cs

91 9021766484

26/4 Aakar Building, Byramji Town Near Nelson Square Nagpur 440013

GST RETURN FILING

test

eLife assessment

This fundamental study provides insights into the interplay of endogenous orienting and the planning of goal-directed gaze shifts (saccades). Using an elegant experimental protocol and detailed analyses of the time course of saccadic choices, the authors provide compelling evidence for independent mechanisms that guide early, reflexive eye movements and later, voluntary gaze shifts. This work will be of interest to neuroscientists and psychologists working on vision and motor control and to those researching decision-making across disciplines.

Reviewer #1 (Public Review):

Summary:

The classical pro/antisaccade task has become a valuable diagnostic tool in neurology and psychiatry (Antoniades et al., 2013, Vision Res). Although it is well-established that antisaccades require substantially longer latencies than prosaccades, the exact attentional mechanisms underlying these differences are not yet fully elucidated. This study investigates the separate influences of exogenous and endogenous attention on saccade generation. These two mechanisms are often confounded in classical pro/antisaccade tasks. In the current study, the authors build on their previous work using an urgent choice task (Salinas et al., 2019, eLife) to time-resolve the influences of exogenous and endogenous factors on saccade execution. The key contribution of the current study is to show that, when controlling for exogenous capture, antisaccades continue to require longer processing times. This longer processing time may be explained by a coupling between endogenous attention and saccade motor plans.

Strengths:

In the classical pro/antisaccade task the direction of exogenous capture (caused by the presentation of the cue) is typically congruent with the direction of prosaccades and incongruent with antisaccades. A key strength of the current study is the introduction of different experimental conditions that control for the effects of exogenous capture on saccade generation. In particular, Experiments 3 and 4 provide strong evidence for two independent (exogenous and endogenous) mechanisms that guide saccadic choices, acting at different times. Differences in timing for pro and antisaccades during the endogenous phase were consistent and independent of whether the exogenous capture biased early saccades toward the correct prosaccade direction or toward the correct antisaccade directions.

As in previous studies by the same group (Salinas et al., 2019, eLife; Goldstein et al., 2023, eLife), the detailed analysis of the time course of goal-directed saccades allowed the authors to determine the exact, additional time of 30 ms that is necessary to generate a correct antisaccade versus prosaccade.

Overall, the manuscript is very well written, and the data are presented clearly.

Weaknesses:

The main research question could be defined more clearly. In the abstract and at some points throughout the manuscript, the authors indicate that the main purpose of the study was to assess whether the allocation of endogenous attention requires saccade planning [e.g., ll.3-5 or ll.247-248]. While the data show a coupling between endogenous attention and saccades, they do not point to a specific direction of this coupling (i.e., whether endogenous attention is necessary to successfully execute a saccade plan or whether a saccade plan necessarily accompanies endogenous attention).

Some of the analyses were performed only on subgroups of the participants. The reporting of these subgroup analyses is transparent and data from all participants are reported in the supplementary figures. Still, these subgroup analyses may make the data appear more consistent, compared to when data is considered across all participants. For instance, the exogenous capture in Experiments 1 and 2 appears much weaker in Figure 2 (subgroup) than Figure S3 (all participants). Moreover, because different subgroups were used for different analyses, it is often difficult to follow and evaluate the results. For instance, the tachometric curves in Figure 2 (see also Figure 3 and 4) show no motor bias towards the cue (i.e., performance was at ~50% for rPTs <75 ms). I assume that the subsequent analyses of the motor bias were based on a very different subgroup. In fact, based on Figure S2, it seems that the motor bias was predominantly seen in the unreliable participants. Therefore, I often found the figures that were based on data across all participants (Figures 7 and S3) more informative to evaluate the overall pattern of results.

Reviewer #2 (Public Review):

Goldstein et al. provide a thorough characterization of the interaction of attention and eye movement planning. These processes have been thought to be intertwined since at least the development of the Premotor Theory of Attention in 1987, and their relationship has been a continual source of debate and research for decades. Here, Goldstein et al. capitalize on their novel urgent saccade task to dissociate the effects of endogenous and exogenous attention on saccades towards and away from the cue. They find that attention and eye movements are, to some extent, linked to one another but that this link is transient and depends on the nature of the task. A primary strength of the work is that the researchers are able to carefully measure the timecourse of the interaction between attention and eye movements in various well-controlled experimental conditions. As a result, the behavioral interplay of two forms of attention (endogenous and exogenous) is illustrated at the level of tens of milliseconds as they interact with the planning and execution of saccades towards and away from the cued location. Overall, the results allow the authors to make meaningful claims about the time course of visual behavior, attention, and the potential neural mechanisms at a timescale relevant to everyday human behavior.

Reviewer #3 (Public Review):

Summary and overall evaluation:

Human vision is inherently limited so that only a small part of a visual scene can be perceived at a given moment. To address this limitation, the visual system has evolved a number of strategies and mechanisms that work in concert. First, humans move their eyes using saccadic eye movements. This allows us to place the high-resolution region in the center of the eye's retina (the fovea centralis) on objects of interest so that these are sampled with high acuity. Second, salient, conspicuous stimuli that appear abruptly and/or differ strongly from the other stimuli in the scene, seem to automatically attract ("exogenous") attention, so that a large share of the neuronal "resources" for visual processing is devoted to the stimuli, which improves the perception of the stimuli. Third, stimuli that are important for the current task and the current behavioral goals can be prioritized by attention mechanisms ("endogenous" attention), which also secures their allocated share of processing resources and helps them be perceived. It is well-established that eye movements are closely linked to the mechanisms of attention (for a review, see Carrasco, 2011, cited in the manuscript). However, it is still unclear what role voluntary, endogenous attention plays in the control of saccadic eye movements.

The present study used an experimental procedure involving time-pressure for responding, in order to uncover how the control of saccades by exogenous and endogenous attention unfolds over time. The findings of the study indicate that saccade planning was indeed influenced by the locus of endogenous attention, but that this influence was short-lasting and could be overcome quickly. Taken together, the present findings reveal new dynamics between endogenous attention and eye movement control, and lead the way for studying them using experiments under time pressure.

The results provided by the present study advance our understanding of vision, eye movements, and their control by brain mechanisms for attention. In addition, they demonstrate how tasks involving time pressure can be used to study the dynamics of cognitive processes. Therefore, the present study seems highly important not only for vision science, but also for psychology, (cognitive) neuroscience, and related research fields more generally.

Strengths:

The experiments of the study are performed with great care and rigor and the data is analyzed thoroughly and comprehensively. Overall, the results support the authors' conclusions, so I have only minor comments (see below). Taken together, the findings seem important for a wide community of researchers in vision science, psychology, and neuroscience.

Weaknesses (minor points):

(1) In this experimental paradigm, participants must decide where to saccade based on the color of the cue in the visual periphery (they should have made a prosaccade toward a green cue and an antisaccade away from a magenta cue). Thus, irrespective of whether the cue signaled that a prosaccade or an antisaccade was to be made, the identity of the cue was always essential for the task (as the authors explain on p. 5, lines 129-138). Also, the location where the cue appeared was blocked, and thus known to the participants in advance, so that endogenous attention could be directed to the cue at the beginning of a trial (e.g., p. 5, lines 129-132). These aspects of the experimental paradigm differ from the classic prosaccade/antisaccade paradigm (e.g. Antoniades et al., 2013, Vision Research). In the classic paradigm, the identity of the cues does not have to be distinguished to solve the task, since there is only one stimulus that should be looked at (prosaccade) or away from (antisaccade), and whether a prosaccade or antisaccade was required is constant across a block of trials. Thus, in contrast to the present paradigm, in the classic paradigm, the participants do not know where the cue is about to appear, but they know whether to perform a prosaccade or an antisaccade based on the location of the cue.

The present paradigm keeps the location of the cue constant in a block of trials by intention, because this ensures that endogenous attention is allocated to its location and is not overpowered by the exogenous capture of attention that would happen when a single stimulus appeared abruptly in the visual field. Thus, the reason for keeping the location of the cue constant seems convincing. However, I wondered what consequences the constant location would have for the task representations that persist across the task and govern how attention is allocated. In the classic paradigm, there is always a single stimulus that captures attention exogenously (as it appears abruptly). In a prosaccade block, participants can prioritize the visual transient caused by the stimulus, and follow it with a saccade to its coordinates. In an antisaccade block, following the transient with a saccade would always be wrong, so that participants could try to suppress the attention capture by the transient, and base their saccade on the coordinates of the opposite location. Thus, in prosaccade and antisaccade blocks, the task representations controlling how visual transients are processed to perform the task differ. In the present task, prosaccades and antisaccades cannot be distinguished by the visual transients. Thus, such a situation could favor endogenous attention and increase its influence on saccade planning, even though saccade planning under more naturalistic conditions would be dominated by visual transients. I suggest discussing how this (and vice versa the emphasis on visual transients in the classic paradigm) could affect the generality of the presented findings (e.g., how does this relate to the interpretation that saccade plans are obligatorily coupled to endogenous attention? See, Results, p. 10, lines 306-308, see also Deubel & Schneider, 1996, Vision Research).

(2) Discussion (p. 16, lines 472-475): The authors suppose that "It is as if the exogenous response was automatically followed by a motor bias in the opposite direction. Perhaps the oculomotor circuitry is such that an exogenous signal can rapidly trigger a saccade, but if it does not, then the corresponding motor plan is rapidly suppressed regardless of anything else.". I think this interesting point should be discussed in more detail. Could it also be that instead of suppression, other currently active motor plans were enhanced? Would this involve attention? Some attention models assume that attention works by distributing available (neuronal) processing resources (e.g., Desimone & Duncan, 1995, Annual Review of Neuroscience; Bundesen, 1990, Psychological Review; Bundesen et al., 2005, Psychological Review) so that the information receiving the largest share of resources results in perception and is used for action, but this happens without the active suppression of information.

(3) Methods, p. 19, lines 593-596: It is reported that saccades were scored based on their direction. I think more information should be provided to understand which eye movements entered the analysis. Was there a criterion for saccade amplitude? I think it would be very helpful to provide data on the distributions of saccade amplitudes or on their accuracy (e.g. average distance from target) or reliability (e.g. standard deviation of landing points). Also, it is reported that some data was excluded from the analysis, and I suggest reporting how much of the data was excluded. Was the exclusion of the data related to whether participants were "reliable" or "unreliable" performers?

(4) Results, p. 9, lines 262-266: Some data analyses are performed on a subset of participants that met certain performance criteria. The reasons for this data selection seem convincing (e.g. to ensure empirical curves were not flat, line 264). Nevertheless, I suggest to explain and justify this step in more detail. In addition, if not all participants achieved an acceptable performance and data quality, this could also speak to the experimental task and its difficulty. Thus, I suggest discussing the potential implications of this, in particular, how this could affect the studied mechanisms, and whether it could limit the presented findings to a special group within the studied population.

1. O Direito de ler não importa o quê- Cada pessoa deve ter a liberdade de ler qualquer tipo de livro que deseje, sem restrições ou censuras. Pois a leitura é uma atividade pessoal e privada e ninguém deve ser julgado ou impedido de ler um conteúdo que lhe interesse. Contribuindo para a liberdade de expressão, desenvolvimento intelectual e cultural de uma pessoa.
2. O Direito de amar os "Heróis" dos romances- As pessoas devem ser livres para admirar e apaixonar-se pelos personagens dos livros que leem. Amar esses personagens faz parte da experiência de leitura e permite que os leitores explorem emoções e a imaginação sem julgamentos ou limitações.

O Direito de Reler

Este direito permite ao leitor reviver momentos de prazer que teve na primeira leitura de um livro que gostou muito.

1. O direito de não acabar um livro Este direito é importante porque respeita a liberdade pessoal do leitor, evitando o desperdício de tempo com leituras não prazerosas e reconhece a diversidade de gostos literários.

2. O direito de ler não importa o quê Este direito é importante porque garante a liberdade de expressão, promove o acesso ao conhecimento, e encoraja a diversidade de perspetivas. Além disso, estimula o prazer pela leitura, contribuindo para o desenvolvimento pessoal e a sua identidade.

O Direito de Ler em Voz Alta

Na minha opinião, ler em voz alta é muito importante, porque podemos “saborear” as palavras de uma forma diferente do que se estivéssemos a ler “para dentro”.

O Direito de Ler em Voz Alta

Na minha opinião, ler em voz alta é muito importante, porque podemos “saborear” as palavras de uma forma diferente do que se estivéssemos a ler “para dentro”.

O Direito de Não Acabar um Livro

Na minha opinião, este direito do leitor é um dos mais importantes, pois, se não gostamos de um livro, devemos poder "abandoná-lo", para selecionarmos um de que verdadeiramente gostamos, não acham? E vocês, quais são os dois direitos que consideram fundamentais. Selecionem-nos e justifiquem as vossas opções.

мономорфизация как было сказано раньше - это когда комплилятор уже создаёт доп методы, из обобщенных типов. что по сути является статической диспетечеразией.

а есть еще динамическая - это когда уже во время программы, будет понятно, что использовать.

rust использует указатель, чтоб попробовать вызвать метода трейта у структуры, и если если его нету, то rust выдаст ошибку.

по сути. типаж-объект, это тот самый интерфейс. он даёт нам возможность, иметь не с опрелёленным типом. если бы мы использовали Where T: type. что объективности даст мономорфизацию. а с теми типами, которые реализуют в себе этот типаж. по сути, мы начинаем работать с типами не на прямую через тип, а через типаж-объект, что по сути может считатся эквавалентом интерфесов, либо абстракт класса.

Observation Facility

I guess it should be Phase 7v

in the side bar, these phases are in inverse alphabetical order, I assume they should be changed?

a stratum does not really "comprise" a phase perhaps "is included in"?

this, too, could be omitted -- same for the other rpages

perhaps best to leave out "A15" sionce it is already in the title above

just give link to fAB volume https://urkesh.org/pages/911-06.htm with reference especially to the courtyard

Het Nederlands Kwalificatieraamwerk (NLQF) is van grote waarde voor het non-formeel onderwijs. Hier zijn enkele redenen waarom:

1. Leven Lang Ontwikkelen (LLO): NLQF draagt bij aan Leven Lang Ontwikkelen (LLO) en het verbeteren van arbeidsmobiliteit¹. Het stelt mensen in staat om met een NLQF-niveau te laten zien wat ze waard zijn¹.

2. Transparantie: NLQF maakt de waarde en het niveau van non-formele kwalificaties transparanter en vergelijkbaar, zowel nationaal als internationaal¹.

3. Kwaliteitsborging: NLQF waarborgt de kwaliteit van examinering, leeruitkomsten en diploma's of certificaten¹.

4. Maatschappelijke erkenning: Voor private aanbieders en non-formeel onderwijs is het NLQF 'superbelangrijk', omdat inschaling hun aanbod maatschappelijke waarde geeft².

5. Groeiende acceptatie: In Nederland zijn inmiddels al 205 non-formele kwalificaties ingeschaald, en per jaar komen daar 60 inschalingen bij².

6. Arbeidsmarkt: Met NLQF kunnen werkgevers en werknemers/werkzoekenden beter beoordelen wat iemand al in huis heeft en wat hij nog kan bijleren².

Het is belangrijk om te weten dat hoewel NLQF een belangrijke rol speelt in het versterken van de waarde van non-formeel onderwijs, het geen invloed heeft op het recht om bepaalde titels of graden te voeren³.

Bron: Gesprek met Bing, 17-5-2024 (1) IN NL HEBBEN WE FORMEEL EN NON-FORMEEL ONDERWIJS (PRIVAAT). VOOR HET .... https://www.nlqf.nl/images/Daarom%20NLQF/Flyer%20Facts%20and%20figures.pdf. (2) 10 jaar NLQF - van niks naar één standaard voor alle kwalificaties. https://nlqf.nl/actueel/10-jaar-nlqf-van-niks-naar-een-standaard-voor-alle-kwalificaties. (3) NLQF. https://nlqf.nl/.

De inschaling in het Nederlands Kwalificatieraamwerk (NLQF) is een proces waarbij een kwalificatie wordt ingeschaald op een bepaald niveau van het NLQF¹. Hier zijn enkele belangrijke punten over de waarde van inschaling in het NLQF:

1. Procedure: Je dient een aanvraag in voor inschaling van een kwalificatie in het NLQF bij het Nationaal Coördinatiepunt Nederlands Kwalificatieraamwerk (NCP NLQF)¹. Het NCP NLQF beoordeelt of de aanvraag ontvankelijk is en stuurt de aanvraag door naar de experts van het NCP NLQF¹.

2. Best-fit methode: Bij de best-fit methode worden alle descriptoren bekeken en wordt vervolgens bepaald welk niveau het best bij het geheel van de kwalificatie past¹. Het kan namelijk zo zijn dat descriptoren van verschillende niveaus van toepassing zijn op een bepaalde kwalificatie¹.

3. Geldigheid: Zowel de validiteit van de aanbieder als de inschaling van de kwalificatie is 6 jaar geldig². Na 6 jaar vindt een herbeoordeling plaats².

4. Transparantie: De inschaling in het NLQF maakt de waarde van kwalificaties transparanter en vergelijkbaar, zowel nationaal als internationaal¹.

5. Geen instroomrechten: Aan het NLQF zijn geen instroomrechten verbonden. Het is aan de hogescholen zelf om hierin een besluit te nemen².

Het is belangrijk om te weten dat de inschaling in het NLQF geen invloed heeft op het recht om bepaalde titels of graden te voeren². Het voeren van titels en graden kan alleen op grond van de wet- en regelgeving waarin dit is geregeld².

Bron: Gesprek met Bing, 17-5-2024 (1) Proces inschaling - NLQF. https://nlqf.nl/inschaling. (2) Veelgestelde vragen na inschaling - NLQF. https://nlqf.nl/images/downloads/Toolbox/Veelgestelde_vragen_na_inschaling.pdf. (3) Inschaling - NLQF. https://nlqf.nl/inschaling-start. (4) undefined. https://aanvragen.nlqf.nl/.

How is the quality measured?

Author response:

[The following is the authors’ response to the current reviews.]

In response to Reviewer #2, we agree with the reviewer that it needs to be noted that not all forms of recognition are the same and have added the following: "However, we note that not all forms of recognition are the same; researchers may prefer to have their work featured instead of personal stories or critiques of the scientific environment."

[The following is the authors’ response to the previous reviews.]

We thank both reviewers for their detailed comments and insightful suggestions. Below we summarize our responses to each concern in addition to the edits within the manuscript.

We would also like to add a clarification to the eLife assessment, it states “This important bibliometric analysis shows that authors of scientific papers whose names suggest they are female or East Asian get quoted less often in news stories about their work.” We show that individuals with names predicted to be from women or East Asian name origins are less likely to be quoted or mentioned in Nature’s scientific news stories than expected by publication demographics. In this study, we did not compare the level of coverage of a scientific article by the demographics of the authors of the article.

Reviewer #1

The article is not so clearly structured, which makes it hard to follow. A better framing, contextualization, and conceptualization of their analysis would help the readers to better understand the results. There are some unclear definitions and wrong wording of key concepts.

We have adapted our wording in the text and added a more detailed discussion which hopefully makes the paper easier to comprehend. These changes are described in the context of your reviewer's suggestions and addressed in the next section.

Language use: Male/Female refers to sex, not to gender.

We have now updated the language throughout the text. Thank you for pointing this out.

Regional disparities are not the same as names' origin. While the first might relate to the academic origin of authors, inferred from their institutional belonging, the latter reflects the authors' inferred identity. Ethnic identities and the construction of prejudice against specific populations need proper contextualization.

We have added better contextualization in the manuscript and reworded the section in our results and discussion to clarify that we are analyzing disparities related to perceived ethnicity and not regions. We also added the following text to the results section “In our analysis, we use name origin as an estimate for the perceived ethnicity of a primary source by a journalist. Our prediction is not intended to assign ethnicity to an individual, but to be used broadly as a tool to quantify representational differences in a journalist's sociologically constructed perception of a primary source's ethnicity.” We also added the following text to our Discussion: “Our use of name origins is a proxy for a journalist's or referring scholarly peer’s potential perceptions of the ethnicity of a primary source as signaled by an individual's name. We do not intend to assign an identity to an individual, but to generate a broad metric to measure possible bias for particular ethnicities during journalists' primary source gathering.”

It would be helpful to have a clear definition of what are quotes, mentions, and citations. For me, it was not so clear and made understanding the results more difficult.

We added the following text to the results section Extracted Data Used for Analysis: “Quoted names are any names that were attached to a quote within the article. Mentioned names are any names that were stated within the article. Cited names are all author names of a scientific paper that was cited in the news article.”

The comparison against Nature published research articles is not perfect because journalists will also cover articles not published in Nature. If for example, the gender representation in the quoted articles is not the same between Nature journals and other journals, then this source of inequality would be missing (e.g. if the journalists are biased against women, but not as much when they published in Nature, because they are also biased towards Nature articles). Also, the gender representation among Nature authors could not be the same as in general. Nevertheless, this seems to be a fair benchmark, especially if the authors did not have access to other more comprehensive databases. But a statement of limitations including these potential issues would be good to have.

To add better context to the generalizability of our work, we added the following text to our discussion: “Furthermore, the news articles present on "www.nature.com" are intended for a very specific readership that may not be reflective of more broad scientific news outlets. In a separate analysis, we took a cursory look into a comparison with The Guardian and found similar disparities in gender and name origin. However, it is not clear which publications should be used as a comparator for science-related articles in The Guardian, and difficult to compare relative rates of representation. While other science news outlets may not have a direct comparator, it would be useful to take a broad comparison across multiple science news outlets to compare against one another. Our existing pipeline could be easily applied to other science news outlets and identify if there exists a consistent pattern of disparity regardless of the intended readership.”

"we select the highest probability origin for each name as the resultant assignment". Threshold based approaches for race/ethnicity name-based inference have been criticized by the literature as they might reproduce biases (see Kozlowski, D., Murray, D. S., Bell, A., Hulsey, W., Larivière, V., Monroe-White, T., & Sugimoto, C. R. (2022). Avoiding bias when inferring race using name-based approaches. Plos one, 17(3), e0264270.). The authors could use the full distribution of probabilities over names instead of selecting one. The formulae proposed (3-5) could be easily adapted to this change.

We thank the author for pointing this out. We have updated our analysis to use the probabilities instead of hard assignments. Figure 3 and formulae 3-5 have been updated. While we observe a slight shift in the calculated values, the overall trends are unchanged.

Is it possible to make an analysis that intersects both name origin and gender? I am not sure if the sample size would allow for this, but if some other dimensions were collapsed, it would be very important to show what happens at the intersection of these two dimensions of discrimination.

We agree that identifying any differences in quotation patterns at the intersection of gender and name origin would be very useful to identify. To address this, we added supplemental table 5. This table identifies the number of quotes per predicted name origin and gender over all years and article types. In this table, we don’t see a significant difference in gender distribution across predicted name origins.

Given a larger sample size, we would be able to better identify more subtle differences, but at this sample size, we cannot make more detailed inferences. Additionally, this also addresses a QC-issue, where predicted gender accuracy varies by name origin, specifically East Asian name origin. From our data, we don’t see a large difference in proportions across any name origin. We added the following text to the results section to incorporate this analysis:

“However, it should be noted that the error rate varies by name origin with the largest decrease in performance on names with an Asian origin [@doi:10.7717/peerj-cs.156;@doi:10.5195/jmla.2021.1252]

. In our analysis, we did not observe a large difference in names predicted to come from a man or woman between predicted East Asian and other name origins (Table 5). “

The use of vocabulary should be more homogeneous. For example, in page 13 the authors start to use the concepts of over/under enrichment, which appeared before in a title but was not used.

The text has been updated to remove all mentions of “over/under enrichment” with “over/under representation”

In the discussions section, it would be important to see as a statement of limitations the problems that automatic origin and gender inference have.

We thank the reviewer for this suggestion. We have added the following paragraph to our discussion.

Computational tools enabled us to automatically analyze thousands of articles to identify existing disparities by gender and name origin, but these tools are not without limitations. Our tools are unable to identify non-binary people and rely on gender predictors that are known to have region-specific biases, with the largest decrease in performance on names of an Asian origin [@doi:10.7717/peerj-cs.156;@doi:10.5195/jmla.2021.1252]. Furthermore, name origin is only a proxy for externally perceived racial or ethnic origins of a source or author and is not as accurate as self-identified race or ethnicity. Self-identification better captures the lived experience of an individual that computational estimates from a name can not capture. This is highlighted in our inability to distinguish between Black and White people from the US by their names. As the collection of demographic data by publication outlets grows, we believe this will enable a more fine-grained and accurate analysis of disparities in scientific journalism.

Figures 2a and 3a show that the affiliations of authors and their countries was going to be used in this analysis. Yet, this section is not present in the article. I would encourage the authors to add this to the analysis as it would show important patterns, and to intersect the dimensions of gender, name origin and country.

We were interested in using this analysis in our work, but unfortunately the sample size of cited works in each country was too small to make inferences. If this work was extended to larger scientific outlets to include larger corpora such as The Guardian or New York Times, we think one could be able to make more robust inferences. Since our work only focuses on Nature, we decided not to include this analysis. However, we do include a section in our discussion for future work.

“As a proxy for measuring possible geographical bias of a journalist, we attempted to identify if there was any geographical bias of cited authors. To do this, we identified the affiliation of each cited author and identified their affiliated country. Unfortunately, we could not robustly extract a large enough number of cited authors from different countries to make any conclusive statements. Expanding our work to other science journalism outlets could help identify possible ways in which geographic region, genders, and perceived ethnicity interact and affect scientific visibility of specific groups. While we are unable to identify that journalists have a specific geographical bias, having reporters explicitly focused on specific regional sources will broaden coverage of international opinions in science.”

It is not clear at that point what column dependence means.

The abstract has been updated to state, “Gender disparity in Nature quotes was dependent on the article type.”

Reviewer #2

We thank the reviewer for their very detailed and insightful suggestions regarding our analysis and the key caveats that needed better contextualization in our analysis. We went through each major point the reviewer brought up below and included any additional text that was needed.

In some cases, the manuscript lacks consistency in terminology, and uses word choice that is strange (e.g., "enrichment" and "depletion" when discussion representation).

We thank the review for pointing this out, we have removed all instances of depletion/enrichment for over/under-representation

Caveats to Claim 1. So while Claim 1 holds, it does not hold for all comparator sets and for all years. I don't think this is critical of the paper-the authors do discuss the trend in Claim 2-but interpretation of this claim should take care of these caveats, and readers should consider the important differences in first and last authorship.

We thank the reviewer for their detailed feedback on this section. We have added the missing contextualization of our results. In the results section, I changed the figure caption to: “Speakers predicted to be men are sometimes overrepresented in quotes, but this depends on the year and article type.” Added the following paragraph “When considering the relative proportion of authors and speakers predicted to be men, we only find a slight over-representation of men. This overrepresentation is dependent on the authorship position and the year. Before 2010, quotes predicted as from men are overrepresented in comparison to both first and last authors, but between 2010 and 2017 quotes predicted from men are only overrepresented in comparison for first authors. In 2020, we find a slight over-representation of quotes predicted to be from women relative to first and last authors, but still severely under-represented when considering the general population. The choice of comparison between first and last authors can reveal different aspects of the current state of academia. While this does not hold in all scientific fields, first authors are typically early career scientists and last authors are more senior scientists. It has also been shown that early career scientists tend to be more diverse than senior scientists [@doi:10.7554/eLife.60829; @doi:10.1096/fj.201800639]. Since we find that quotes are only slightly more likely to come from a last author, it is reasonable to compare the relative rate of predicted quotes from men to either authorship position. Comparison with last authorships may reveal more how gender bias currently exists whereas comparison with early career scientists may reveal bias in comparison to a future, more possibly diverse academic environment. We hope that increased representation and recognition of women in science, even beyond what is observed in authorship, can increase the proportion of women first and last authors such that it better reflects the general population.”

Generalizability to other contexts of science journalism:

We thank the reviewer for their feedback on the generalizability of our work. We have now added the following text to our discussion to provide the reader with a better context of our results: “To articles presented on "www.nature.com" are intended for a very specific readership that may not be reflective of more broad scientific news outlets. In a separate analysis, we took a cursory look into a comparison with The Guardian and found very similar disparities in gender and name origin. However, it is not clear which publications should be used as a comparator for science-related articles in The

Guardian, and difficult to compare relative rates of representation. While other science news outlets may not have a direct comparator, it would be useful to take a broad comparison across multiple science news outlets to compare against one another. Our existing pipeline could be easily applied to other science news outlets and identify if there exists a consistent pattern of disparity regardless of the intended readership. ”

Shallow discussion:

The authors highlight gender parity in career features, but why exactly is there gender parity in this format

We thank the reviewer for encouraging us to better contextualize our findings in the broader discourse. We have now added several sections to our Discussion. To address gender parity, we have added the following text: “This finding, coupled with the near equal number of articles written by journalists predicted to be men or women, argues for more diversity in topical coverage. "Career Feature" articles highlight current topics relevant to working scientists and frequently highlight systemic issues with the scientific environment. This column allows space for marginalized people to critique the current state of affairs in science or share their personal stories. This type of content encourages the journalist to seek out a diverse set of primary sources. Including more content that is not primarily focused on recent publications, but all topics surrounding the practice of science, can serve as an additional tool to rapidly achieve gender parity in journalistic recognition.”

Representation in quotations varies by first and last author, most certainly as a result of the academic division of labor in the life sciences. However, what does it say about the scientific quotation that it appears first authors are more often to be quoted? Does this mean that the division of labor is changing such that the first authors are the lead scientists? Or does it imply that senior authors are being skipped over, or giving away their chance to comment on a study to the first author?

We thank the reviewer for asking bringing up these important questions. We have added better context to our first author analysis in our discussion. We have included the following two sections to address this. Also, we want to state that we find last authors to be slightly more quoted than first authors, as depicted in Fig. 2d., with first author quotation percentage largely appearing below the red line. We included this text in a response above and include it again here for convenience.

“Before 2010, quotes predicted as from men are overrepresented in comparison to both first and last authors, but between 2010 and 2017 quotes predicted from men are only overrepresented in comparison for first authors. In 2020, we find a slight over-representation of quotes predicted to be from women relative to first and last authors, but still severely under-represented when considering the general population. The choice of comparison between first and last authors can reveal different aspects of the current state of academia. While this does not hold in all scientific fields, first authors are typically early career scientists and last authors are more senior scientists. It has also been shown that early career scientists tend to be more diverse than senior scientists [@doi:10.7554/eLife.60829; @doi:10.1096/fj.201800639]. Since we find that quotes are only slightly more likely to come from a last author, it is reasonable to compare the relative rate of predicted quotes from men to either authorship position. Comparison with last authorships may reveal more how gender bias currently exists whereas comparison with early career scientists may reveal bias in comparison to a future, more possibly diverse academic environment. We hope that increased representation and recognition of women in science, even beyond what is observed in authorship, can increase the proportion of women first and last authors such that it better reflects the general population.”

“In our analysis, we also find that there are more first authors with predicted East Asian name origin than last authors. This is in contrast to predicted Celtic/English and European name origins.

Furthermore, we see that the amount of first author people with predicted East Asian name origins is increasing at a much faster rate than quotes are increasing. If this mismatched rate of representation continues, this could lead to an increasingly large erasure of early career scientists with East Asian name origins. As noted before, focusing on increasing engagement with early career scientists can help to reduce the growing disparity of public visibility of scientists with East Asian name origins.”

What might be the downstream impacts on the public stemming from the under-representation of scientists with East Asian names? According to Figure 3d, not only are East Asian names under-represented in quotations, but they are becoming more under-represented over time as they appear as authors in a greater number of Nature publications; Those with European names are proportionately represented in quotations given their share of authors in Nature. Why might this be, especially seeing as Anglo names are heavily over-represented?

To address this point, we have added the following text to our discussion: “In our analysis, we also find that there are more first authors with predicted East Asian name origin than last authors. This is in contrast to predicted Celtic/English and European name origins. Furthermore, the amount of first author people with predicted East Asian name origins is increasing at a much faster rate than quotes are increasing. If this mismatched rate of representation continues, this could lead to an increasingly large erasure of early career scientists with East Asian name origins. As noted before, focusing on increasing engagement with early career scientists can help to reduce the growing disparity of public visibility of scientists with East Asian name origins.”

I am very confused by Figure 1B. It mixes the counts of News-related items with (non-Springer) research articles in a single stacked bar plot which makes determining the quantity of either difficult. I would advise splitting them out

Figure 1B has been updated, and the News and Research articles have been separated.

When querying the first 2000 or so results from the SpringerNature API, are the authors certain that they are getting a random sample of papers?

These papers were the first 200 English language "Journal" papers returned by the Springer Nature API for each month, resulting in 2400 papers per year from 2005 through 2020. These papers are the first 200 papers published each month by a Springer Nature journal, which may not be completely random, but we believe to be a reasonably representative sample. Furthermore, the Springer Nature comparator set is being used as an additional comparator to the complete set of all Nature research papers used in our analyses.

In all figures: the authors use capital letters to indicate panels in the caption, but lowercase letters in the figure itself and in the main text. This should be made consistent.

This has been updated.

In all figures: the authors should make the caption letter bold in the figure captions, which makes it much easier to find descriptions of specific panels

This has been updated.

In the section "coreNLP": the authors mention "co-reference resolution" but without really remarking why it is being used. This is an issue throughout the methods-the authors describe what method they are using but either they don't mention why they are using that method until later, or else not at all.

We have added better reasoning behind our coreNLP selected methods: “We used the standard set of annotaters: tokenize, ssplit, pos, lemma, ner, parse, coref, and additionally the quote annotator. These perform text tokenization, sentence splitting, part of speech recognition, lemmatization, named entity recoginition, division of sentences into constituent phrases, co-reference resolution, and identification of quoted entities, respectively. We used the "statistical" algorithm to perform coreference resolution for speed. Each of these aspects is required to identify the names of quoted or mentioned speakers and identify any of their associated pronouns. All results were output to json format for further downstream processing.”

We included a better description of scrapy: “Scrapy is a tool that applies user-defined rules to follow hyperlinks on webpages and return the information contained on each webpage.

We used Scrapy to extract all web pages containing news articles and extract the text.”

We also included our motivation for bootstrapping: “We used the boostrap method to construct confidence intervals for each of our calculated statistics.”

In the section "Name Formatting for Gender Prediction in Quotes or Mentions", genderizeR is mentioned before an introduction to the tool

We added the following text to provide context: “Even though genderizeR, the computational method used to predict the name's gender, only uses the first name to make the gender prediction, identifying the full name gives us greater confidence that we correctly identified the first name. “

In the section "Name Formatting for Gender Prediction of Authors", you state that you exclude papers with only one author. How many papers is this? I assume few, in Nature, but if not I can imagine gender differences based on who writes first-authored papers.

We find that the number excluded is roughly 7% of all papers, which is consistent across Nature and Springer Nature (1113/15013 for cited springer articles, 2899/42155 for random springer articles, 955/12459 for nature authors). We have added the following text to the manuscript for better context: “Roughly 7% of all papers were estimated to be by a single author and removed from this analysis.: 1113/15013 for cited Springer articles, 2899/42155 for random Springer articles, 955/12459 for Nature research articles.”

In "Name Origin Analysis", for the in-text reference to Equation 3: include the prefix "Eq." or similar to mark this as referencing the equation and not something else

This has been updated.

The use of the word "enrichment" in reference to the representation of East Asian authors is strange and does not fit the colloquial definition of the term. I suggest just using a simpler term like "representation" instead

Similarly, the authors use the word "depletion" to reflect the lower rate of quotes to scientists with East-Asian names, but I feel a simpler word would be more appropriate.

We thank the reviewer for this suggestion, all instances of “enrichment/depletion” have been replaced with “over/under representation”

The authors claim in Figure 2d that there is a steady increase in the rate of first author citations, however, this graph is not convincing. It appears to show much more noise than anything resembling a steady change.

We have reworded our figure description to state that there is a consistent bias towards quoting last authors. Our figure description now states: “Panel d shows a consistent but slight bias towards quoting the last author of a cited article than the first author over time.”

Supplemental Figures 1b and 1c do not seem to be mentioned in the main text, and I struggle to see their relevance.

We thank the reviewer for identifying this error; these subpanels have been removed.

ipns agregore cache

De vier doelstellingen van de minister om gelijkheid in het (voortgezet) onderwijs te verbeteren

• Duidelijke sectoridentiteitsen: De minister benadrukt het belang van duidelijke sectoridentiteiten in het onderwijs om aan te sluiten bij het doel en de maatschappelijke missie van elke sector. Deze duidelijkheid helpt studenten het aanbod van elke sector te begrijpen .

• Gelijke kansen voor groei: Het doel is ervoor te zorgen dat, ondanks de verschillen in de manier waarop mbo-, hbo- en wo-sectoren studenten opleiden, ze allemaal als gelijk worden beschouwd. Deze gelijkheid biedt iedereen de mogelijkheid om zich tot het beste van zichzelf te ontwikkelen .

• Hiërarchische labels vermijden: De minister pleit ervoor om hiërarchische labels zoals „middelbaar” en „hoger” in onderwijsterminologie te vermijden. Integendeel, het bevorderen van het gebruik van neutrale termen zoals mbo, hbo en wo bevordert gelijkheid en voorkomt stigmatisering op basis van opleidingsniveaus .

• Naadloze studententransities: Het verbeteren van de afstemming van onderwijsprogramma's en studentenondersteuning tussen mbo-, hbo- en wo-sectoren is cruciaal om ontmoediging en uitval van studenten tijdens transities te voorkomen. Door deze verbindingen te verbeteren, kan de voortgang en het succes van studenten soepeler verlopen .

**Door de minister geïdentificeerde problemen en knelpunten **

• Beperkte sectormobiliteit: De minister benadrukt dat er weliswaar een toename is van havo- en vwo-studenten die naar het mbo gaan, maar dat de meeste studenten de neiging hebben om door te gaan in de sector die overeenkomt met hun hoogst behaalde niveau. Dit gebrek aan sectormobiliteit kan studenten hinderen die mogelijk beter gedijen in een andere onderwijssector .

• Transition Challenges: De overgang tussen mbo-, hbo- en wo-sectoren brengt uitdagingen met zich mee vanwege onvoldoende afstemming in onderwijsprogramma's en studentenondersteunende diensten. Dit gebrek aan samenhang kan leiden tot ontmoediging en uitval van studenten, met een aanzienlijk uitvalpercentage van 19% voor mbo-studenten die overstappen naar het hbo .

• Hiërarchische percepties: De minister erkent het voortbestaan van een hiërarchische maatschappelijke visie die praktische beroepen en onderwijs devalueert. Deze achterhaalde perceptie komt voort uit een negentiende-eeuws hiërarchisch maatschappelijk model, dat barrières opwerpt voor de gelijke erkenning van alle vormen van onderwijs en werk .

ipns cached

.

What stops disclosee to not send the "Admit"? Thus being repudiable to "I haven't received the promised and agreed upon info".

eLife assessment:

This paper characterises a novel gene (Spar), and presenting valuable findings in the field of insect biology and behaviour. The experiments are well designed, with attention to detail, showcasing the potential of the Drosophila melanogaster model and the use of online resources. The mixed approach presents a convincing argument for a genetic interaction between Alk and Spar.

Reviewer #2 (Public Review):

This manuscript illustrates the power of "combined" research, incorporating a range of tools, both old and new to answer a question. This thorough approach identifies a novel target in a well-established signalling pathway and characterises a new player in Drosophila CNS development.

Largely, the experiments are carried out with precision, meeting the aims of the project, and setting new targets for future research in the field. It was particularly refreshing to see the use of multi-omics data integration and Targeted DamID (TaDa) findings to triage scRNA-seq data. Some of the TaDa methodology was unorthodox, however, this does not affect the main finding of the study. The authors (in the revised manuscript) have appropriately justified their TaDa approaches and mentioned the caveats in the main text.

Their discovery of Spar as a neuropeptide precursor downstream of Alk is novel, as well as its ability to regulate activity and circadian clock function in the fly. Spar was just one of the downstream factors identified from this study, therefore, the potential impact goes beyond this one Alk downstream effector.

Reviewer #3 (Public Review):

Summary:

The receptor tyrosine kinase Anaplastic Lymphoma Kinase (ALK) in humans is nervous system expressed and plays an important role as an oncogene. A number of groups have been studying ALK signalling in flies to gain mechanistic insight into its various roles. In flies, ALK plays a critical role in development, particularly embryonic development and axon targeting. In addition, ALK was also shown to regulate adult functions including sleep and memory. In this manuscript, Sukumar et al., used a suite of molecular techniques to identify downstream targets of ALK signalling. They first used targeted DamID, a technique that involves a DNA methylase to RNA polymerase II, so that GATC sites in close proximity to PolII binding sites are marked. They performed these experiments in wild type and ALK loss of function mutants (using an Alk dominant negative ALkDN), to identify Alk responsive loci. Comparing these loci with a larval single cell RNAseq dataset identified neuroendocrine cells as an important site of Alk action. They further combined these TaDa hits with data from RNA seq in Alk Loss and Gain of Function manipulations to identify a single novel target of Alk signalling - a neuropeptide precursor they named Sparkly (Spar) for its expression pattern. They generated a mutant allele of Spar, raised an antibody against Spar, and characterised its expression pattern and mutant behavioural phenotypes including defects in sleep and circadian function.

Strengths:

The molecular biology experiments using TaDa and RNAseq were elegant and very convincing. The authors identified a novel gene they named Spar. They also generated a mutant allele of Spar (using CrisprCas technology) and raised an antibody against Spar. These experiments are lovely, and the reagents will be useful to the community. The paper is also well written, and the figures are very nicely laid out making the manuscript a pleasure to read.

Weaknesses:

The manuscript has improved very substantially in revision. The authors have clearly taken the comments on board in good faith.

Editors' note: The authors have satisfactorily addressed the concerns raised in the previous rounds of review. These were related to the unconventional analysis of the TaDa data, the addition of other means of down regulated gene function, and the nature of analyses of behavioural data.

Author response:

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

Point-by-point response to concerns raised by reviewer #3:

The manuscript has improved very substantially in revision. The authors have clearly taken the comments on board in good faith. Yet, some small concerns remain around the behavioural analysis.

In Fig. 8H and H' average sleep/day is ~100. Is this minutes of sleep? 100 min/day is far too low, is it a typo?

The numbers for sleep bouts are also too low to me e.g. in Fig 9 number of sleep bouts avg around 4.

In their response to reviewers the authors say these errors were fixed, yet the figures appear not to have been changed. Perhaps the old figures were left in inadvertently?

Indeed this correction was somehow missed and we thank the reviewer for noticing this. We have now corrected Fig 8H-H’ and Fig 9D.  

The circadian anticipatory activity analyses could also be improved. The standard in the field is to perform eduction analyses and quantify anticipatory activity e.g. using the method of Harrisingh et al. (PMID: 18003827). This typically computed as the ratio of activity in the 3hrs preceding light transition to activity in the 6hrs preceding light transition.

In their response to reviewers, the authors have revised their anticipation analyses by quantifying the mean activity in the 6 hrs preceding light transition. However, in the method of Harrisingh et al., anticipation is the ratio of activity in the 3hrs preceding light transition to activity in the 6hrs preceding light transition. Simply computing the activity in the 6hrs preceding light transition does not give a measure of anticipation, determining the ratio is key.

We acknowledge the importance of obtaining accurate results in our analysis, therefore we have re-evaluated the anticipation activity by measuring the ratio of the mean activity in the 3h preceding light transition over the activity in the 6h preceding light transition. We have reported the data as percentages in Fig 8F-G and modified the figure legends accordingly.

this is duplicated in the "Deposition" page, and may be eliminated here

Reviewer #1 (Public Review):

Olszyński and colleagues present data showing variability from canonical "aversive calls", typically described as long 22 kHz calls rodents emit in aversive situations. Similarly long but higher-frequency (44 kHz) calls are presented as a distinct call type, including analyses both of their acoustic properties and animals' responses to hearing playback of these calls. While this work adds an intriguing and important reminder, namely that animal behavior is often more variable and complex than perhaps we would like it to be, there is some caution warranted in the interpretation of these data.

The exclusive use of males is a major concern lacking adequate justification and should be disclosed in the title and abstract to ensure readers are aware of this limitation. With several reported sex differences in rat vocal behaviors this means caution should be exercised when generalizing from these findings. The occurrence of an estrus cycle in typical female rats is not justification for their exclusion. Note also that male rodents experience great variability in hormonal states as well, distinguishing between individuals and within individuals across time. The study of endocrinological influences on behavior can be separated from the study of said behavior itself, across all sexes. Similarly, concerns about needing to increase the number of animals when including all sexes are usually unwarranted (see Shansky [2019] and Phillips et al. [2023]).

Regarding the analysis where calls were sorted using DBSCAN based on peak frequency and duration, my comment on the originally reviewed version stands. It seems that the calls are sorted by an (unbiased) algorithm into categories based on their frequency and duration, and because 44kHz calls differ by definition on frequency and duration the fact that the algorithm sorts them as a distinct category is not evidence that they are "new calls [that] form a separate, distinct group". I appreciate that the authors have softened their language regarding the novelty and distinctness of these calls, but the manuscript contains several instances where claims of novelty and specificity (e.g. the subtitle on line 193) is emphasized beyond what the data justifies.

The behavioral response to call playback is intriguing, although again more in line with the hypothesis that these are not a distinct type of call but merely represent expected variation in vocalization parameters. Across the board animals respond rather similarly to hearing 22 kHz calls as they do to hearing 44 kHz calls, with occasional shifts of 44 kHz call responses to an intermediate between appetitive and aversive calls. This does raise interesting questions about how, ethologically, animals may interpret such variation and integrate this interpretation in their responses. However, the categorical approach employed here does not address these questions fully.

I appreciate the amendment in discussing the idea of arousal being the key determinant for the increased emission of 44kHz, and the addition of other factors. Some of the items in this list, such as annoyance/anger and disgust/boredom, don't really seem to fit the data. I'm not sure I find the idea that rats become annoyed or disgusted during fear conditioning to be a particularly compelling argument. As such the list appears to be a collection of emotion-related words, with unclear potential associations with the 44kHz calls.

Later in the Discussion the authors argue that the 44kHz aversive calls signal an increased intensity of a negative valence emotional state. It is not clear how the presented arguments actually support this. For example, what does the elongation of fear conditioning to 10 trials have to do with increased negative emotionality? Is there data supporting this relationship between duration and emotion, outside anthropomorphism? Each of the 6 arguments presented seems quite distant from being able to support this conclusion.

In sum, rather than describing the 44kHz long calls as a new call type, it may be more accurate to say that sometimes aversive calls can occur at frequencies above 22 kHz. Individual and situational variability in vocalization parameters seems to be expected, much more so than all members of a species strictly adhering to extremely non-variable behavioral outputs.

[Editors' note: The reviewer agrees that the additional analysis has ruled out the possibility that the calls are due to fatigue.]

The Guardian: Donald Trump hat Big-Oil Managern angeboten, klimapolitische Maßnahmen der Biden-Administration rückgängig zu machen, wenn sie seinen Wahlkampf mit einer Milliarde Dollar unterstützen. Einer Studie des Guardian zufolge können die Ölkonzerne von Trump vor allem 110 Milliaren Dollar Subventionen (u.a. Steuererleichterungen für neue fossile Projekte) erwarten, die die Biden-Regierung abschaffen will. Hintergrundartikel zu Lobbyisten im US-Ölgeschäft und aktuellen Konflikten<br /> https://www.theguardian.com/us-news/article/2024/may/16/donald-trump-big-oil-executives-alleged-deal-explained

this behavior beetween peer, how them will talk to each oher

we created PeerId by publicKey

WirtschaftsWoche: Hintergrundbericht zur geplanten Fusion der Petrochemie-Sparten von OMV und Adnoc. Die Adnoc will mit #Bourouge einen Petrochemie-Großkonzern aufbauen, der dann statt Treibstoffe scheinbar saubere Produkte wie Kunstdünger und Plastik verkauft und auf Recycling und Kreislaufwirtschaft ausgerichtet ist. Dazu ist man auch an einer Übernahme von Covestro und Wintershall interessiert. Der Konzern soll zur Hälfte der OMV gehören. https://www.wiwo.de/unternehmen/industrie/chemieindustrie-was-hinter-dem-megageschaeft-zwischen-omv-und-adnoc-steckt/29560900.html

Author response:

eLife assessment 

This important study provides evidence for a combination of the latest generation of Oxford Nanopore Technology long reads with state-of-the art variant callers enabling bacterial variant discovery at accuracy that matches or exceeds the current "gold standard" with short reads. The evidence supporting the claims of the authors is convincing, although the inclusion of a larger number of reference genomes would further strengthen the study. The work will be of interest to anyone performing sequencing for outbreak investigations, bacterial epidemiology, or similar studies. 

We thank the editor and reviewers for the accurate summary and positive assessment. We address the comment about increasing the number of reference genomes in the response to reviewer 2.

Public Reviews: 

Reviewer #1 (Public Review): 

Summary: 

The authors assess the accuracy of short variant calling (SNPs and indels) in bacterial genomes using Oxford Nanopore reads generated on R10.4 flow cells from a very similar genome (99.5% ANI), examining the impact of variant caller choice (three traditional variant callers: bcftools, freebayes, and longshot, and three deep learning based variant callers: clair3, deep variant, and nano caller), base calling model (fast, hac and sup) and read depth (using both simplex and duplex reads). 

Strengths: 

Given the stated goal (analysis of variant calling for reads drawn from genomes very similar to the reference), the analysis is largely complete and results are compelling. The authors make the code and data used in their analysis available for re-use using current best practices (a computational workflow and data archived in INSDC databases or Zenodo as appropriate). 

Weaknesses: 

While the medaka variant caller is now deprecated for diploid calling, it is still widely used for haploid variant calling and should at least be mentioned (even if the mention is only to explain its exclusion from the analysis). 

We agree that this would be an informative addition to the study and will add it to the benchmarking.

Appraisal: 

The experiments the authors engaged in are well structured and the results are convincing. I expect that these results will be incorporated into "best practice" bacterial variant calling workflows in the future. 

Thank you for the positive appraisal.

Reviewer #2 (Public Review): 

Summary: 

Hall et al describe the superiority of ONT sequencing and deep learning-based variant callers to deliver higher SNP and Indel accuracy compared to previous gold-standard Illumina short-read sequencing. Furthermore, they provide recommendations for read sequencing depth and computational requirements when performing variant calling. 

Strengths: 

The study describes compelling data showing ONT superiority when using deep learning-based variant callers, such as Clair3, compared to Illumina sequencing. This challenges the paradigm that Illumina sequencing is the gold standard for variant calling in bacterial genomes. The authors provide evidence that homopolymeric regions, a systematic and problematic issue with ONT data, are no longer a concern in ONT sequencing. 

Weaknesses: 

(1) The inclusion of a larger number of reference genomes would have strengthened the study to accommodate larger variability (a limitation mentioned by the authors). 

Our strategic selection of 14 genomes—spanning a variety of bacterial genera and species, diverse GC content, and both gram-negative and gram-positive species (including M. tuberculosis, which is neither)—was designed to robustly address potential variability in our results. Moreover, all our genome assemblies underwent rigorous manual inspection as the quality of the true genome sequences is the foundation this research is built upon. Given this, the fundamental conclusions regarding the accuracy of variant calls would likely remain unchanged with the addition of more genomes.  However, we do acknowledge that a substantially larger sample size, which is beyond the scope of this study, would enable more fine-grained analysis of species differences in error rates.

(2) In Figure 2, there are clearly one or two samples that perform worse than others in all combinations (are always below the box plots). No information about species-specific variant calls is provided by the authors but one would like to know if those are recurrently associated with one or two species. Species-specific recommendations could also help the scientific community to choose the best sequencing/variant calling approaches.

Thank you for highlighting this observation. The precision, recall, and F1 scores for each sample and condition can be found in Supplementary Table S4. We will investigate the samples that consistently perform below expectation to determine if this is associated with specific species, which may necessitate tailored recommendations for those species. Additionally, we will produce a species-segregated version of Figure 2 for a clearer interpretation and will place it in the supplementary materials.

(3) The authors support that a read depth of 10x is sufficient to achieve variant calls that match or exceed Illumina sequencing. However, the standard here should be the optimal discriminatory power for clinical and public health utility (namely outbreak analysis). In such scenarios, the highest discriminatory power is always desirable and as such an F1 score, Recall and Precision that is as close to 100% as possible should be maintained (which changes the minimum read sequencing depth to at least 25x, which is the inflection point).

We agree that the highest discriminatory power is always desirable for clinical or public health applications. In which case, 25x is probably a better minimum recommendation. However, we are also aware that there are resource-limited settings where parity with Illumina is sufficient. In these cases, 10x depth from ONT would provide sufficient data.

The manuscript currently emphasises the latter scenario, but we will revise the text to clearly recommend 25x depth as a conservative aim in settings where resources are not a constraint, ensuring the highest possible discriminatory power for applications like outbreak analysis.

(4) The sequencing of the samples was not performed with the same Illumina and ONT method/equipment, which could have introduced specific equipment/preparation artefacts that were not considered in the study. See for example https://academic.oup.com/nargab/article/3/1/lqab019/6193612. 

To our knowledge, there is no evidence that sequencing on different ONT machines or barcoding kits leads to a difference in read characteristics or accuracy. To ensure consistency and minimise potential variability, we used the same ONT flowcells for all samples and performed basecalling on the same Nvidia A100 GPU. We will update the methods to emphasise this.

For Illumina and ONT, the exact machines used for which samples will be added as a supplementary table. We will also add a comment about possible Illumina error rate differences in the ‘Limitations’ section of the Discussion.

In summary, while there may be specific equipment or preparation artifacts to consider, we took steps to minimise these effects and maintain consistency across our sequencing methods.

Reviewer #3 (Public Review): 

Hall et al. benchmarked different variant calling methods on Nanopore reads of bacterial samples and compared the performance of Nanopore to short reads produced with Illumina sequencing. To establish a common ground for comparison, the authors first generated a variant truth set for each sample and then projected this set to the reference sequence of the sample to obtain a mutated reference. Subsequently, Hall et al. called SNPs and small indels using commonly used deep learning and conventional variant callers and compared the precision and accuracy from reads produced with simplex and duplex Nanopore sequencing to Illumina data. The authors did not investigate large structural variation, which is a major limitation of the current manuscript. It will be very interesting to see a follow-up study covering this much more challenging type of variation. 

We fully agree that investigating structural variations (SVs) would be a very interesting and important follow-up. Identifying and generating ground truth SVs is a nontrivial task and we feel it deserves its own space and study. We hope to explore this in the future.

In their comprehensive comparison of SNPs and small indels, the authors observed superior performance of deep learning over conventional variant callers when Nanopore reads were basecalled with the most accurate (but also computationally very expensive) model, even exceeding Illumina in some cases. Not surprisingly, Nanopore underperformed compared to Illumina when basecalled with the fastest (but computationally much less demanding) method with the lowest accuracy. The authors then investigated the surprisingly higher performance of Nanopore data in some cases and identified lower recall with Illumina short read data, particularly from repetitive regions and regions with high variant density, as the driver. Combining the most accurate Nanopore basecalling method with a deep learning variant caller resulted in low error rates in homopolymer regions, similar to Illumina data. This is remarkable, as homopolymer regions are (or, were) traditionally challenging for Nanopore sequencing. 

Lastly, Hall et al. provided useful information on the required Nanopore read depth, which is surprisingly low, and the computational resources for variant calling with deep learning callers. With that, the authors established a new state-of-the-art for Nanopore-only variant, calling on bacterial sequencing data. Most likely these findings will be transferred to other organisms as well or at least provide a proof-of-concept that can be built upon. 

As the authors mention multiple times throughout the manuscript, Nanopore can provide sequencing data in nearly real-time and in remote regions, therefore opening up a ton of new possibilities, for example for infectious disease surveillance. 

However, the high-performing variant calling method as established in this study requires the computationally very expensive sup and/or duplex Nanopore basecalling, whereas the least computationally demanding method underperforms. Here, the manuscript would greatly benefit from extending the last section on computational requirements, as the authors determine the resources for the variant calling but do not cover the entire picture. This could even be misleading for less experienced researchers who want to perform bacterial sequencing at high performance but with low resources. The authors mention it in the discussion but do not make clear enough that the described computational resources are probably largely insufficient to perform the high-accuracy basecalling required. 

We have provided runtime benchmarks for basecalling in Supplementary Figure S16 and detailed these times in Supplementary Table S7. In addition, we state in the Results section (P10 L228-230) “Though we do note that if the person performing the variant calling has received the raw (pod5) ONT data, basecalling also needs to be accounted for, as depending on how much sequencing was done, this step can also be resource-intensive.”

Even with super-accuracy basecalling considered, our analysis shows that variant calling remains the most resource-intensive step for Clair3, DeepVariant, FreeBayes, and NanoCaller. Therefore, the statement “the described computational resources are probably largely insufficient to perform the high-accuracy basecalling required”, is incorrect. However, we will endeavour to make the basecalling component and considerations more prominent in the Results and Discussion.

eLife assessment

This important study provides evidence for a combination of the latest generation of Oxford Nanopore Technology long reads with state-of-the art variant callers enabling bacterial variant discovery at accuracy that matches or exceeds the current "gold standard" with short reads. The evidence supporting the claims of the authors is convincing, although the inclusion of a larger number of reference genomes would further strengthen the study. The work will be of interest to anyone performing sequencing for outbreak investigations, bacterial epidemiology, or similar studies.

Reviewer #1 (Public Review):

Summary:

The authors assess the accuracy of short variant calling (SNPs and indels) in bacterial genomes using Oxford Nanopore reads generated on R10.4 flow cells from a very similar genome (99.5% ANI), examining the impact of variant caller choice (three traditional variant callers: bcftools, freebayes, and longshot, and three deep learning based variant callers: clair3, deep variant, and nano caller), base calling model (fast, hac and sup) and read depth (using both simplex and duplex reads).

Strengths:

Given the stated goal (analysis of variant calling for reads drawn from genomes very similar to the reference), the analysis is largely complete and results are compelling. The authors make the code and data used in their analysis available for re-use using current best practices (a computational workflow and data archived in INSDC databases or Zenodo as appropriate).

Weaknesses:

While the medaka variant caller is now deprecated for diploid calling, it is still widely used for haploid variant calling and should at least be mentioned (even if the mention is only to explain its exclusion from the analysis).

Appraisal:

The experiments the authors engaged in are well structured and the results are convincing. I expect that these results will be incorporated into "best practice" bacterial variant calling workflows in the future.

Reviewer #2 (Public Review):

Summary:

Hall et al describe the superiority of ONT sequencing and deep learning-based variant callers to deliver higher SNP and Indel accuracy compared to previous gold-standard Illumina short-read sequencing. Furthermore, they provide recommendations for read sequencing depth and computational requirements when performing variant calling.

Strengths:

The study describes compelling data showing ONT superiority when using deep learning-based variant callers, such as Clair3, compared to Illumina sequencing. This challenges the paradigm that Illumina sequencing is the gold standard for variant calling in bacterial genomes. The authors provide evidence that homopolymeric regions, a systematic and problematic issue with ONT data, are no longer a concern in ONT sequencing.

Weaknesses:

(1) The inclusion of a larger number of reference genomes would have strengthened the study to accommodate larger variability (a limitation mentioned by the authors).

(2) In Figure 2, there are clearly one or two samples that perform worse than others in all combinations (are always below the box plots). No information about species-specific variant calls is provided by the authors but one would like to know if those are recurrently associated with one or two species. Species-specific recommendations could also help the scientific community to choose the best sequencing/variant calling approaches.

(3) The authors support that a read depth of 10x is sufficient to achieve variant calls that match or exceed Illumina sequencing. However, the standard here should be the optimal discriminatory power for clinical and public health utility (namely outbreak analysis). In such scenarios, the highest discriminatory power is always desirable and as such an F1 score, Recall and Precision that is as close to 100% as possible should be maintained (which changes the minimum read sequencing depth to at least 25x, which is the inflection point).

(4) The sequencing of the samples was not performed with the same Illumina and ONT method/equipment, which could have introduced specific equipment/preparation artefacts that were not considered in the study. See for example https://academic.oup.com/nargab/article/3/1/lqab019/6193612.

Reviewer #3 (Public Review):

Hall et al. benchmarked different variant calling methods on Nanopore reads of bacterial samples and compared the performance of Nanopore to short reads produced with Illumina sequencing. To establish a common ground for comparison, the authors first generated a variant truth set for each sample and then projected this set to the reference sequence of the sample to obtain a mutated reference. Subsequently, Hall et al. called SNPs and small indels using commonly used deep learning and conventional variant callers and compared the precision and accuracy from reads produced with simplex and duplex Nanopore sequencing to Illumina data. The authors did not investigate large structural variation, which is a major limitation of the current manuscript. It will be very interesting to see a follow-up study covering this much more challenging type of variation.

In their comprehensive comparison of SNPs and small indels, the authors observed superior performance of deep learning over conventional variant callers when Nanopore reads were basecalled with the most accurate (but also computationally very expensive) model, even exceeding Illumina in some cases. Not surprisingly, Nanopore underperformed compared to Illumina when basecalled with the fastest (but computationally much less demanding) method with the lowest accuracy. The authors then investigated the surprisingly higher performance of Nanopore data in some cases and identified lower recall with Illumina short read data, particularly from repetitive regions and regions with high variant density, as the driver. Combining the most accurate Nanopore basecalling method with a deep learning variant caller resulted in low error rates in homopolymer regions, similar to Illumina data. This is remarkable, as homopolymer regions are (or, were) traditionally challenging for Nanopore sequencing.

Lastly, Hall et al. provided useful information on the required Nanopore read depth, which is surprisingly low, and the computational resources for variant calling with deep learning callers. With that, the authors established a new state-of-the-art for Nanopore-only variant, calling on bacterial sequencing data. Most likely these findings will be transferred to other organisms as well or at least provide a proof-of-concept that can be built upon.

As the authors mention multiple times throughout the manuscript, Nanopore can provide sequencing data in nearly real-time and in remote regions, therefore opening up a ton of new possibilities, for example for infectious disease surveillance.

However, the high-performing variant calling method as established in this study requires the computationally very expensive sup and/or duplex Nanopore basecalling, whereas the least computationally demanding method underperforms. Here, the manuscript would greatly benefit from extending the last section on computational requirements, as the authors determine the resources for the variant calling but do not cover the entire picture. This could even be misleading for less experienced researchers who want to perform bacterial sequencing at high performance but with low resources. The authors mention it in the discussion but do not make clear enough that the described computational resources are probably largely insufficient to perform the high-accuracy basecalling required.

Kleine Zeitung: In einem Hintergrundgespräch bestätigt Edith Hlawati, die Chefin der österreichischen Staatsholding Öbag, dass die Fusion der Petrochemietöchter von OMV und Adnoc weiter von beiden Seiten angestrebt wird. Hlawati war federführend beim aktuellen Syndikatsvertrag der Öbag mit dem Ölkonzern des autoritär regierten und mit Russland kooperierenden Abu Dhabi. https://www.kleinezeitung.at/wirtschaft/18467296/ich-bin-optimistisch-rueckt-omv-adnoc-deal-naeher

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)):

I have trialled the package on my lab's data and it works as advertised. It was straightforward to use and did not require any special training. I am confident this is a tool that will be approachable even to users with limited computational experience. The use of artificial data to validate the approach - and to provide clear limits on applicability - is particularly helpful.

The main limitation of the tool is that it requires the user to manually select regions. This somewhat limits the generalisability and is also more subjective - users can easily choose "nice" regions that better match with their hypothesis, rather than quantifying the data in an unbiased manner. However, given the inherent challenges in quantifying biological data, such problems are not easily circumventable.

*

* I have some comments to clarify the manuscript:

1. A "straightforward installation" is mentioned. Given this is a Method paper, the means of installation should be clearly laid out.*

__This sentence is now modified. In the revised manuscript we now describe how to install the toolset and we give the link to the toolset website if further information is needed. __On this website, we provide a full video tutorial and a user manual. The user manual is provided as a supplementary material of the manuscript.

* It would be helpful if there was an option to generate an output with the regions analysed (i.e., a JPG image with the data and the drawn line(s) on top). There are two reasons for this: i) A major problem with user-driven quantification is accidental double counting of regions (e.g., a user quantifies a part of an image and then later quantifies the same region). ii) Allows other users to independently verify measurements at a later time.*

We agree that it is helpful to save the analyzed regions. To answer this comment and the other two reviewers' comments pointing at a similar feature, we have now included an automatic saving of the regions of interest. The user will be able to reopen saved regions of interest using a new function we included in the new version of PatternJ.

* 3. Related to the above point, it is highlighted that each time point would need to be analysed separately (line 361-362). It seems like it should be relatively straightforward to allow a function where the analysis line can be mapped onto the next time point. The user could then adjust slightly for changes in position, but still be starting from near the previous timepoint. Given how prevalent timelapse imaging is, this seems like (or something similar) a clear benefit to add to the software.*

We agree that the analysis of time series images can be a useful addition. We have added the analysis of time-lapse series in the new version of PatternJ. The principles behind the analysis of time-lapse series and an example of such analysis are provided in Figure 1 - figure supplement 3 and Figure 5, with accompanying text lines 140-153 and 360-372. The analysis includes a semi-automated selection of regions of interest, which will make the analysis of such sequences more straightforward than having to draw a selection on each image of the series. The user is required to draw at least two regions of interest in two different frames, and the algorithm will automatically generate regions of interest in frames in which selections were not drawn. The algorithm generates the analysis immediately after selections are drawn by the user, which includes the tracking of the reference channel.

* Line 134-135. The level of accuracy of the searching should be clarified here. This is discussed later in the manuscript, but it would be helpful to give readers an idea at this point what level of tolerance the software has to noise and aperiodicity.

*

We agree with the reviewer that a clarification of this part of the algorithm will help the user better understand the manuscript.__ We have modified the sentence to clarify the range of search used and the resulting limits in aperiodicity (now lines 176-181). __Regarding the tolerance to noise, it is difficult to estimate it a priori from the choice made at the algorithm stage, so we prefer to leave it to the validation part of the manuscript. We hope this solution satisfies the reviewer and future users.

*

**Referees cross-commenting**

I think the other reviewer comments are very pertinent. The authors have a fair bit to do, but they are reasonable requests. So, they should be encouraged to do the revisions fully so that the final software tool is as useful as possible.

Reviewer #1 (Significance (Required)):

Developing software tools for quantifying biological data that are approachable for a wide range of users remains a longstanding challenge. This challenge is due to: (1) the inherent problem of variability in biological systems; (2) the complexity of defining clearly quantifiable measurables; and (3) the broad spread of computational skills amongst likely users of such software.

In this work, Blin et al., develop a simple plugin for ImageJ designed to quickly and easily quantify regular repeating units within biological systems - e.g., muscle fibre structure. They clearly and fairly discuss existing tools, with their pros and cons. The motivation for PatternJ is properly justified (which is sadly not always the case with such software tools).

Overall, the paper is well written and accessible. The tool has limitations but it is clearly useful and easy to use. Therefore, this work is publishable with only minor corrections.

*We thank the reviewer for the positive evaluation of PatternJ and for pointing out its accessibility to the users.

*

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

# Summary

The authors present an ImageJ Macro GUI tool set for the quantification of one-dimensional repeated patterns that are commonly occurring in microscopy images of muscles.

# Major comments

In our view the article and also software could be improved in terms of defining the scope of its applicability and user-ship. In many parts the article and software suggest that general biological patterns can be analysed, but then in other parts very specific muscle actin wordings are used. We are pointing this out in the "Minor comments" sections below. We feel that the authors could improve their work by making a clear choice here. One option would be to clearly limit the scope of the tool to the analysis of actin structures in muscles. In this case we would recommend to also rename the tool, e.g. MusclePatternJ. The other option would be to make the tool about the generic analysis of one-dimensional patterns, maybe calling the tool LinePatternJ. In the latter case we would recommend to remove all actin specific wordings from the macro tool set and also the article should be in parts slightly re-written.

*

We agree with the reviewer that our initial manuscript used a mix of general and muscle-oriented vocabulary, which could make the use of PatternJ confusing especially outside of the muscle field. To make PatternJ useful for the largest community, we corrected the manuscript and the PatternJ toolset to provide the general vocabulary needed to make it understandable for every biologist. We modified the manuscript accordingly.

* # Minor/detailed comments

# Software

We recommend considering the following suggestions for improving the software.

## File and folder selection dialogs

In general, clicking on many of the buttons just opens up a file-browser dialog without any further information. For novel users it is not clear what the tool expects one to select here. It would be very good if the software could be rewritten such that there are always clear instructions displayed about which file or folder one should open for the different buttons.*

We experienced with the current version of macOS that the file-browser dialog does not display any message; we suspect this is the issue raised by the reviewer. This is a known issue of Fiji on Mac and all applications on Mac since 2016. We provided guidelines in the user manual and on the tutorial video to correct this issue by changing a parameter in Fiji. Given the issues the reviewer had accessing the material on the PatternJ website, which we apologize for, we understand the issue raised. We added an extra warning on the PatternJ website to point at this problem and its solution. Additionally, we have limited the file-browser dialog appearance to what we thought was strictly necessary. Thus, the user will experience fewer prompts, speeding up the analysis.

*

## Extract button

The tool asks one to specify things like whether selections are drawn "M-line-to-M-line"; for users that are not experts in muscle morphology this is not understandable. It would be great to find more generally applicable formulations. *

We agree that this muscle-oriented vocabulary can make the use of PatternJ confusing. We have now corrected the user interface to provide both general and muscle-specific vocabulary ("center-to-center or edge-to-edge (M-line-to-M-line or Z-disc-to-Z-disc)").*

## Manual selection accuracy

The 1st step of the analysis is always to start from a user hand-drawn profile across intensity patterns in the image. However, this step can cause inaccuracy that varies with the shape and curve of the line profile drawn. If not strictly perpendicular to for example the M line patterns, the distance between intensity peaks will be different. This will be more problematic when dealing with non-straight and parallelly poised features in the image. If the structure is bended with a curve, the line drawn over it also needs to reproduce this curve, to precisely capture the intensity pattern. I found this limits the reproducibility and easy-usability of the software.*

We understand the concern of the reviewer. On curved selections this will be an issue that is difficult to solve, especially on "S" curved or more complex selections. The user will have to be very careful in these situations. On non-curved samples, the issue may be concerning at first sight, but the errors go with the inverse of cosine and are therefore rather low. For example, if the user creates a selection off by 5 degrees, which is visually obvious, lengths will be affected by an increase of only 0.38%. The point raised by the reviewer is important to discuss, and we therefore added a paragraph to comment on the choice of selection (lines 94-98) and a supplementary figure to help make it clear (Figure 1 - figure supplement 1).*

### Reproducibility

Since the line profile drawn on the image is the first step and very essential to the entire process, it should be considered to save together with the analysis result. For example, as ImageJ ROI or ROIset files that can be re-imported, correctly positioned, and visualized in the measured images. This would greatly improve the reproducibility of the proposed workflow. In the manuscript, only the extracted features are being saved (because the save button is also just asking for a folder containing images, so I cannot verify its functionality). *

We agree that this is a very useful and important feature. We have added ROI automatic saving. Additionally, we now provide a simplified import function of all ROIs generated with PatternJ and the automated extraction and analysis of the list of ROIs. This can be done from ROIs generated previously in PatternJ or with ROIs generated from other ImageJ/Fiji algorithms. These new features are described in the manuscript in lines 120-121 and 130-132.

*

## ? button

It would be great if that button would open up some usage instructions.

*

We agree with the reviewer that the "?" button can be used in a better way. We have replaced this button with a Help menu, including a simple tutorial showing a series of images detailing the steps to follow by the user, a link to the user website, and a link to our video tutorial.

* ## Easy improvement of workflow

I would suggest a reasonable expansion of the current workflow, by fitting and displaying 2D lines to the band or line structure in the image, that form the "patterns" the author aims to address. Thus, it extracts geometry models from the image, and the inter-line distance, and even the curve formed by these sets of lines can be further analyzed and studied. These fitted 2D lines can be also well integrated into ImageJ as Line ROI, and thus be saved, imported back, and checked or being further modified. I think this can largely increase the usefulness and reproducibility of the software.

*

We hope that we understood this comment correctly. We had sent a clarification request to the editor, but unfortunately did not receive an answer within the requested 4 weeks of this revision. We understood the following: instead of using our 1D approach, in which we extract positions from a profile, the reviewer suggests extracting the positions of features not as a single point, but as a series of coordinates defining its shape. If this is the case, this is a major modification of the tool that is beyond the scope of PatternJ. We believe that keeping our tool simple, makes it robust. This is the major strength of PatternJ. Local fitting will not use line average for instance, which would make the tool less reliable.

* # Manuscript

We recommend considering the following suggestions for improving the manuscript. Abstract: The abstract suggests that general patterns can be quantified, however the actual tool quantifies specific subtypes of one-dimensional patterns. We recommend adapting the abstract accordingly.

*

We modified the abstract to make this point clearer.

* Line 58: Gray-level co-occurrence matrix (GLCM) based feature extraction and analysis approach is not mentioned nor compared. At least there's a relatively recent study on Sarcomeres structure based on GLCM feature extraction: https://github.com/steinjm/SotaTool with publication: *https://doi.org/10.1002/cpz1.462

• *

We thank the reviewer for making us aware of this publication. We cite it now and have added it to our comparison of available approaches.

* Line 75: "...these simple geometrical features will address most quantitative needs..." We feel that this may be an overstatement, e.g. we can imagine that there should be many relevant two-dimensional patterns in biology?!*

We have modified this sentence to avoid potential confusion (lines 76-77).

• *

• Line 83: "After a straightforward installation by the user, ...". We think it would be convenient to add the installation steps at this place into the manuscript. *

__This sentence is now modified. We now mention how to install the toolset and we provide the link to the toolset website, if further information is needed (lines 86-88). __On the website, we provide a full video tutorial and a user manual.

* Line 87: "Multicolor images will give a graph with one profile per color." The 'Multicolor images' here should be more precisely stated as "multi-channel" images. Multi-color images could be confused with RGB images which will be treated as 8-bit gray value (type conversion first) images by profile plot in ImageJ. *

We agree with the reviewer that this could create some confusion. We modified "multicolor" to "multi-channel".

* Line 92: "...such as individual bands, blocks, or sarcomeric actin...". While bands and blocks are generic pattern terms, the biological term "sarcomeric actin" does not seem to fit in this list. Could a more generic wording be found, such as "block with spike"? *

We agree with the reviewer that "sarcomeric actin" alone will not be clear to all readers. We modified the text to "block with a central band, as often observed in the muscle field for sarcomeric actin" (lines 103-104). The toolset was modified accordingly.

* Line 95: "the algorithm defines one pattern by having the features of highest intensity in its centre". Could this be rephrased? We did not understand what that exactly means.*

We agree with the reviewer that this was not clear. We rewrote this paragraph (lines 101-114) and provided a supplementary figure to illustrate these definitions (Figure 1 - figure supplement 2).

* Line 124 - 147: This part the only description of the algorithm behind the feature extraction and analysis, but not clearly stated. Many details are missing or assumed known by the reader. For example, how it achieved sub-pixel resolution results is not clear. One can only assume that by fitting Gaussian to the band, the center position (peak) thus can be calculated from continuous curves other than pixels. *

Note that the two sentences introducing this description are "Automated feature extraction is the core of the tool. The algorithm takes multiple steps to achieve this (Fig. S2):". We were hoping this statement was clear, but the reviewer may refer to something else. We agree that the description of some of the details of the steps was too quick. We have now expanded the description where needed.

* Line 407: We think the availability of both the tool and the code could be improved. For Fiji tools it is common practice to create an Update Site and to make the code available on GitHub. In addition, downloading the example file (https://drive.google.com/file/d/1eMazyQJlisWPwmozvyb8VPVbfAgaH7Hz/view?usp=drive_link) required a Google login and access request, which is not very convenient; in fact, we asked for access but it was denied. It would be important for the download to be easier, e.g. from GitHub or Zenodo.

*

We are sorry for issues encountered when downloading the tool and additional material. We thank the reviewer for pointing out these issues that limited the accessibility of our tool. We simplified the downloading procedure on the website, which does not go through the google drive interface nor requires a google account. Additionally, for the coder community the code, user manual and examples are now available from GitHub at github.com/PierreMangeol/PatternJ, and are provided as supplementary material with the manuscript. To our knowledge, update sites work for plugins but not for macro toolsets. Having experience sharing our codes with non-specialists, a classical website with a tutorial video is more accessible than more coder-oriented websites, which deter many users.

* Reviewer #2 (Significance (Required)):

The strength of this study is that a tool for the analysis of one-dimensional repeated patterns occurring in muscle fibres is made available in the accessible open-source platform ImageJ/Fiji. In the introduction to the article the authors provide an extensive review of comparable existing tools. Their new tool fills a gap in terms of providing an easy-to-use software for users without computational skills that enables the analysis of muscle sarcomere patterns. We feel that if the below mentioned limitations could be addressed the tool could indeed be valuable to life scientists interested in muscle patterning without computational skills.

In our view there are a few limitations, including the accessibility of example data and tutorials at sites.google.com/view/patternj, which we had trouble to access. In addition, we think that the workflow in Fiji, which currently requires pressing several buttons in the correct order, could be further simplified and streamlined by adopting some "wizard" approach, where the user is guided through the steps.

*As answered above, the links on the PatternJ website are now corrected. Regarding the workflow, we now provide a Help menu with:

1. __a basic set of instructions to use the tool, __
2. a direct link to the tutorial video in the PatternJ toolset
3. a direct link to the website on which both the tutorial video and a detailed user manual can be found. We hope this addresses the issues raised by this reviewer.

*Another limitation is the reproducibility of the analysis; here we recommend enabling IJ Macro recording as well as saving of the drawn line ROIs. For more detailed suggestions for improvements please see the above sections of our review. *

We agree that saving ROIs is very useful. It is now implemented in PatternJ.

We are not sure what this reviewer means by "enabling IJ Macro recording". The ImageJ Macro Recorder is indeed very useful, but to our knowledge, it is limited to built-in functions. Our code is open and we hope this will be sufficient for advanced users to modify the code and make it fit their needs.*

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

Summary In this manuscript, the authors present a new toolset for the analysis of repetitive patterns in biological images named PatternJ. One of the main advantages of this new tool over existing ones is that it is simple to install and run and does not require any coding skills whatsoever, since it runs on the ImageJ GUI. Another advantage is that it does not only provide the mean length of the pattern unit but also the subpixel localization of each unit and the distributions of lengths and that it does not require GPU processing to run, unlike other existing tools. The major disadvantage of the PatternJ is that it requires heavy, although very simple, user input in both the selection of the region to be analyzed and in the analysis steps. Another limitation is that, at least in its current version, PatternJ is not suitable for time-lapse imaging. The authors clearly explain the algorithm used by the tool to find the localization of pattern features and they thoroughly test the limits of their tool in conditions of varying SNR, periodicity and band intensity. Finally, they also show the performance of PatternJ across several biological models such as different kinds of muscle cells, neurons and fish embryonic somites, as well as different imaging modalities such as brightfield, fluorescence confocal microscopy, STORM and even electron microscopy.

This manuscript is clearly written, and both the section and the figures are well organized and tell a cohesive story. By testing PatternJ, I can attest to its ease of installation and use. Overall, I consider that PatternJ is a useful tool for the analysis of patterned microscopy images and this article is fit for publication. However, i do have some minor suggestions and questions that I would like the authors to address, as I consider they could improve this manuscript and the tool:

*We are grateful to this reviewer for this very positive assessment of PatternJ and of our manuscript.

* Minor Suggestions: In the methodology section is missing a more detailed description about how the metric plotted was obtained: as normalized intensity or precision in pixels. *

We agree with the reviewer that a more detailed description of the metric plotted was missing. We added this information in the method part and added information in the Figure captions where more details could help to clarify the value displayed.

* The validation is based mostly on the SNR and patterns. They should include a dataset of real data to validate the algorithm in three of the standard patterns tested. *

We validated our tool using computer-generated images, in which we know with certainty the localization of patterns. This allowed us to automatically analyze 30 000 images, and with varying settings, we sometimes analyzed 10 times the same image, leading to about 150 000 selections analyzed. From these analyses, we can provide with confidence an unbiased assessment of the tool precision and the tool capacity to extract patterns. We already provided examples of various biological data images in Figures 4-6, showing all possible features that can be extracted with PatternJ. In these examples, we can claim by eye that PatternJ extracts patterns efficiently, but we cannot know how precise these extractions are because of the nature of biological data: "real" positions of features are unknown in biological data. Such validation will be limited to assessing whether a pattern was found or not, which we believe we already provided with the examples in Figures 4-6.

* The video tutorial available in the PatternJ website is very useful, maybe it would be worth it to include it as supplemental material for this manuscript, if the journal allows it. *

As the video tutorial may have been missed by other reviewers, we agree it is important to make it more prominent to users. We have now added a Help menu in the toolset that opens the tutorial video. Having the video as supplementary material could indeed be a useful addition if the size of the video is compatible with the journal limits.

* An example image is provided to test the macro. However, it would be useful to provide further example images for each of the three possible standard patterns suggested: Block, actin sarcomere or individual band.*

We agree this can help users. We now provide another multi-channel example image on the PatternJ website including blocks and a pattern made of a linear intensity gradient that can be extracted with our simpler "single pattern" algorithm, which were missing in the first example. Additionally, we provide an example to be used with our new time-lapse analysis.

* Access to both the manual and the sample images in the PatternJ website should be made publicly available. Right now they both sit in a private Drive account. *

As mentioned above, we apologize for access issues that occurred during the review process. These files can now be downloaded directly on the website without any sort of authentication. Additionally, these files are now also available on GitHub.

* Some common errors are not properly handled by the macro and could be confusing for the user: When there is no selection and one tries to run a Check or Extraction: "Selection required in line 307 (called from line 14). profile=getProfile( ;". A simple "a line selection is required" message would be useful there. When "band" or "block" is selected for a channel in the "Set parameters" window, yet a 0 value is entered into the corresponding "Number of bands or blocks" section, one gets this error when trying to Extract: "Empty array in line 842 (called from line 113). if ( ( subloc . length == 1 ) & ( subloc [ 0 == 0) ) {". This error is not too rare, since the "Number of bands or blocks" section is populated with a 0 after choosing "sarcomeric actin" (after accepting the settings) and stays that way when one changes back to "blocks" or "bands".*

We thank the reviewer for pointing out these bugs. These bugs are now corrected in the revised version.

* The fact that every time one clicks on the most used buttons, the getDirectory window appears is not only quite annoying but also, ultimately a waste of time. Isn't it possible to choose the directory in which to store the files only once, from the "Set parameters" window?*

We have now found a solution to avoid this step. The user is only prompted to provide the image folder when pressing the "Set parameter" button. We kept the prompt for directory only when the user selects the time-lapse analysis or the analysis of multiple ROIs. The main reason is that it is very easy for the analysis to end up in the wrong folder otherwise.

* The authors state that the outputs of the workflow are "user friendly text files". However, some of them lack descriptive headers (like the localisations and profiles) or even file names (like colors.txt). If there is something lacking in the manuscript, it is a brief description of all the output files generated during the workflow.*

PatternJ generates multiple files, several of which are internal to the toolset. They are needed to keep track of which analyses were done, and which colors were used in the images, amongst others. From the user part, only the files obtained after the analysis All_localizations.channel_X.txt and sarcomere_lengths.txt are useful. To improve the user experience, we now moved all internal files to a folder named "internal", which we think will clarify which outputs are useful for further analysis, and which ones are not. We thank the reviewer for raising this point and we now mention it in our Tutorial.

I don't really see the point in saving the localizations from the "Extraction" step, they are even named "temp".

We thank the reviewer for this comment, this was indeed not necessary. We modified PatternJ to delete these files after they are used.

* In the same line, I DO see the point of saving the profiles and localizations from the "Extract & Save" step, but I think they should be deleted during the "Analysis" step, since all their information is then grouped in a single file, with descriptive headers. This deleting could be optional and set in the "Set parameters" window.*

We understand the point raised by the reviewer. However, the analysis depends on the reference channel picked, which is asked for when starting an analysis, and can be augmented with additional selections. If a user chooses to modify the reference channel or to add a new profile to the analysis, deleting all these files would mean that the user will have to start over again, which we believe will create frustration. An optional deletion at the analysis step is simple to implement, but it could create problems for users who do not understand what it means practically.

* Moreover, I think it would be useful to also save the linear roi used for the "Extract & Save" step, and eventually combine them during the "Analysis step" into a single roi set file so that future re-analysis could be made on the same regions. This could be an optional feature set from the "Set parameters" window. *

We agree with the reviewer that saving ROIs is very useful. ROIs are now saved into a single file each time the user extracts and saves positions from a selection. Additionally, the user can re-use previous ROIs and analyze an image or image series in a single step.

* In the "PatternJ workflow" section of the manuscript, the authors state that after the "Extract & Save" step "(...) steps 1, 2, 4, and 5 can be repeated on other selections (...)". However, technically, only steps 1 and 5 are really necessary (alternatively 1, 4 and 5 if the user is unsure of the quality of the patterning). If a user follows this to the letter, I think it can lead to wasted time.

*

We agree with the reviewer and have corrected the manuscript accordingly (line 119-120).

• *

*I believe that the "Version Information" button, although important, has potential to be more useful if used as a "Help" button for the toolset. There could be links to useful sources like the manuscript or the PatternJ website but also some tips like "whenever possible, use a higher linewidth for your line selection" *

We agree with the reviewer as pointed out in our previous answers to the other reviewers. This button is now replaced by a Help menu, including a simple tutorial in a series of images detailing the steps to follow, a link to the user website, and a link to our video tutorial.

* It would be interesting to mention to what extent does the orientation of the line selection in relation to the patterned structure (i.e. perfectly parallel vs more diagonal) affect pattern length variability?*

As answered to reviewer 1, we understand this concern, which needs to be clarified for readers. The issue may be concerning at first sight, but the errors grow only with the inverse of cosine and are therefore rather low. For example, if the user creates a selection off by 3 degrees, which is visually obvious, lengths will be affected by an increase of only 0.14%. The point raised by the reviewer is important to discuss, and we therefore have added a comment on the choice of selection (lines 94-98) as well as a supplementary figure (Figure 1 - figure supplement 1).

* When "the algorithm uses the peak of highest intensity as a starting point and then searches for peak intensity values one spatial period away on each side of this starting point" (line 133-135), does that search have a range? If so, what is the range? *

We agree that this information is useful to share with the reader. The range is one pattern size. We have modified the sentence to clarify the range of search used and the resulting limits in aperiodicity (now lines 176-181).

* Line 144 states that the parameters of the fit are saved and given to the user, yet I could not find such information in the outputs. *

The parameters of the fits are saved for blocks. We have now clarified this point by modifying the manuscript (lines 186-198) and modifying Figure 1 - figure supplement 5. We realized we made an error in the description of how edges of "block with middle band" are extracted. This is now corrected.

* In line 286, authors finish by saying "More complex patterns from electron microscopy images may also be used with PatternJ.". Since this statement is not backed by evidence in the manuscript, I suggest deleting it (or at the very least, providing some examples of what more complex patterns the authors refer to). *

This sentence is now deleted.

* In the TEM image of the fly wing muscle in fig. 4 there is a subtle but clearly visible white stripe pattern in the original image. Since that pattern consists of 'dips', rather than 'peaks' in the profile of the inverted image, they do not get analyzed. I think it is worth mentioning that if the image of interest contains both "bright" and "dark" patterns, then the analysis should be performed in both the original and the inverted images because the nature of the algorithm does not allow it to detect "dark" patterns. *

We agree with the reviewer's comment. We now mention this point in lines 337-339.

* In line 283, the authors mention using background correction. They should explicit what method of background correction they used. If they used ImageJ's "subtract background' tool, then specify the radius.*

We now describe this step in the method section.

*

Reviewer #3 (Significance (Required)):

• Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field. Being a software paper, the advance proposed by the authors is technical in nature. The novelty and significance of this tool is that it offers quick and simple pattern analysis at the single unit level to a broad audience, since it runs on the ImageJ GUI and does not require any programming knowledge. Moreover, all the modules and steps are well described in the paper, which allows easy going through the analysis.
• Place the work in the context of the existing literature (provide references, where appropriate). The authors themselves provide a good and thorough comparison of their tool with other existing ones, both in terms of ease of use and on the type of information extracted by each method. While PatternJ is not necessarily superior in all aspects, it succeeds at providing precise single pattern unit measurements in a user-friendly manner.
• State what audience might be interested in and influenced by the reported findings. Most researchers working with microscopy images of muscle cells or fibers or any other patterned sample and interested in analyzing changes in that pattern in response to perturbations, time, development, etc. could use this tool to obtain useful, and otherwise laborious, information. *

We thank the reviewer for these enthusiastic comments about how straightforward for biologists it is to use PatternJ and its broad applicability in the bio community.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #3

Evidence, reproducibility and clarity

Summary

In this manuscript, the authors present a new toolset for the analysis of repetitive patterns in biological images named PatternJ. One of the main advantages of this new tool over existing ones is that it is simple to install and run and does not require any coding skills whatsoever, since it runs on the ImageJ GUI. Another advantage is that it does not only provide the mean length of the pattern unit but also the subpixel localization of each unit and the distributions of lengths and that it does not require GPU processing to run, unlike other existing tools. The major disadvantage of the PatternJ is that it requires heavy, although very simple, user input in both the selection of the region to be analyzed and in the analysis steps. Another limitation is that, at least in its current version, PatternJ is not suitable for time-lapse imaging.

The authors clearly explain the algorithm used by the tool to find the localization of pattern features and they thoroughly test the limits of their tool in conditions of varying SNR, periodicity and band intensity. Finally, they also show the performance of PatternJ across several biological models such as different kinds of muscle cells, neurons and fish embryonic somites, as well as different imaging modalities such as brightfield, fluorescence confocal microscopy, STORM and even electron microscopy.

This manuscript is clearly written, and both the section and the figures are well organized and tell a cohesive story. By testing PatternJ, I can attest to its ease of installation and use. Overall, I consider that PatternJ is a useful tool for the analysis of patterned microscopy images and this article is fit for publication. However, i do have some minor suggestions and questions that I would like the authors to address, as I consider they could improve this manuscript and the tool:

Minor Suggestions:

In the methodology section is missing a more detailed description about how the metric plotted was obtained: as normalized intensity or precision in pixels. The validation is based mostly on the SNR and patterns. They should include a dataset of real data to validate the algorithm in three of the standard patterns tested. The video tutorial available in the PatternJ website is very useful, maybe it would be worth it to include it as supplemental material for this manuscript, if the journal allows it. An example image is provided to test the macro. However, it would be useful to provide further example images for each of the three possible standard patterns suggested: Block, actin sarcomere or individual band. Access to both the manual and the sample images in the PatternJ website should be made publicly available. Right now they both sit in a private Drive account. Some common errors are not properly handled by the macro and could be confusing for the user: When there is no selection and one tries to run a Check or Extraction: "Selection required in line 307 (called from line 14). profile=getProfile( <)>;". A simple "a line selection is required" message would be useful there. When "band" or "block" is selected for a channel in the "Set parameters" window, yet a 0 value is entered into the corresponding "Number of bands or blocks" section, one gets this error when trying to Extract: "Empty array in line 842 (called from line 113). if ( ( subloc . length == 1 ) & ( subloc [ 0 <]> == 0) ) {". This error is not too rare, since the "Number of bands or blocks" section is populated with a 0 after choosing "sarcomeric actin" (after accepting the settings) and stays that way when one changes back to "blocks" or "bands".<br /> The fact that every time one clicks on the most used buttons, the getDirectory window appears is not only quite annoying but also, ultimately a waste of time. Isn't it possible to choose the directory in which to store the files only once, from the "Set parameters" window? The authors state that the outputs of the workflow are "user friendly text files". However, some of them lack descriptive headers (like the localisations and profiles) or even file names (like colors.txt). If there is something lacking in the manuscript, it is a brief description of all the output files generated during the workflow. I don't really see the point in saving the localizations from the "Extraction" step, they are even named "temp". In the same line, I DO see the point of saving the profiles and localizations from the "Extract & Save" step, but I think they should be deleted during the "Analysis" step, since all their information is then grouped in a single file, with descriptive headers. This deleting could be optional and set in the "Set parameters" window. Moreover, I think it would be useful to also save the linear roi used for the "Extract & Save" step, and eventually combine them during the "Analysis step" into a single roi set file so that future re-analysis could be made on the same regions. This could be an optional feature set from the "Set parameters" window. In the "PatternJ workflow" section of the manuscript, the authors state that after the "Extract & Save" step "(...) steps 1, 2, 4, and 5 can be repeated on other selections (...)". However, technically, only steps 1 and 5 are really necessary (alternatively 1, 4 and 5 if the user is unsure of the quality of the patterning). If a user follows this to the letter, I think it can lead to wasted time. I believe that the "Version Information" button, although important, has potential to be more useful if used as a "Help" button for the toolset. There could be links to useful sources like the manuscript or the PatternJ website but also some tips like "whenever possible, use a higher linewidth for your line selection" It would be interesting to mention to what extent does the orientation of the line selection in relation to the patterned structure (i.e. perfectly parallel vs more diagonal) affect pattern length variability? When "the algorithm uses the peak of highest intensity as a starting point and then searches for peak intensity values one spatial period away on each side of this starting point" (line 133-135), does that search have a range? If so, what is the range? Line 144 states that the parameters of the fit are saved and given to the user, yet I could not find such information in the outputs. In line 286, authors finish by saying "More complex patterns from electron microscopy images may also be used with PatternJ.". Since this statement is not backed by evidence in the manuscript, I suggest deleting it (or at the very least, providing some examples of what more complex patterns the authors refer to). In the TEM image of the fly wing muscle in fig. 4 there is a subtle but clearly visible white stripe pattern in the original image. Since that pattern consists of 'dips', rather than 'peaks' in the profile of the inverted image, they do not get analyzed. I think it is worth mentioning that if the image of interest contains both "bright" and "dark" patterns, then the analysis should be performed in both the original and the inverted images because the nature of the algorithm does not allow it to detect "dark" patterns. In line 283, the authors mention using background correction. They should explicit what method of background correction they used. If they used ImageJ's "subtract background' tool, then specify the radius.

Significance

• Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field. Being a software paper, the advance proposed by the authors is technical in nature. The novelty and significance of this tool is that it offers quick and simple pattern analysis at the single unit level to a broad audience, since it runs on the ImageJ GUI and does not require any programming knowledge. Moreover, all the modules and steps are well described in the paper, which allows easy going through the analysis.
• Place the work in the context of the existing literature (provide references, where appropriate). The authors themselves provide a good and thorough comparison of their tool with other existing ones, both in terms of ease of use and on the type of information extracted by each method. While PatternJ is not necessarily superior in all aspects, it succeeds at providing precise single pattern unit measurements in a user-friendly manner.
• State what audience might be interested in and influenced by the reported findings. Most researchers working with microscopy images of muscle cells or fibers or any other patterned sample and interested in analyzing changes in that pattern in response to perturbations, time, development, etc. could use this tool to obtain useful, and otherwise laborious, information.
• Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate. I am a biologist with extensive experience in confocal microscopy and image analysis using classical machine vision tools, particularly using ImageJ and CellProfiler.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

Summary

The authors present an ImageJ Macro GUI tool set for the quantification of one-dimensional repeated patterns that are commonly occurring in microscopy images of muscles.

Major comments

In our view the article and also software could be improved in terms of defining the scope of its applicability and user-ship. In many parts the article and software suggest that general biological patterns can be analysed, but then in other parts very specific muscle actin wordings are used. We are pointing this out in the "Minor comments" sections below. We feel that the authors could improve their work by making a clear choice here. One option would be to clearly limit the scope of the tool to the analysis of actin structures in muscles. In this case we would recommend to also rename the tool, e.g. MusclePatternJ. The other option would be to make the tool about the generic analysis of one-dimensional patterns, maybe calling the tool LinePatternJ. In the latter case we would recommend to remove all actin specific wordings from the macro tool set and also the article should be in parts slightly re-written.

Minor/detailed comments

Software

We recommend considering the following suggestions for improving the software.

File and folder selection dialogs

In general, clicking on many of the buttons just opens up a file-browser dialog without any further information. For novel users it is not clear what the tool expects one to select here. It would be very good if the software could be rewritten such that there are always clear instructions displayed about which file or folder one should open for the different buttons.

Extract button

The tool asks one to specify things like whether selections are drawn "M-line-to-M-line"; for users that are not experts in muscle morphology this is not understandable. It would be great to find more generally applicable formulations.

Manual selection accuracy

The 1st step of the analysis is always to start from a user hand-drawn profile across intensity patterns in the image. However, this step can cause inaccuracy that varies with the shape and curve of the line profile drawn. If not strictly perpendicular to for example the M line patterns, the distance between intensity peaks will be different. This will be more problematic when dealing with non-straight and parallelly poised features in the image. If the structure is bended with a curve, the line drawn over it also needs to reproduce this curve, to precisely capture the intensity pattern. I found this limits the reproducibility and easy-usability of the software.

Reproducibility

Since the line profile drawn on the image is the first step and very essential to the entire process, it should be considered to save together with the analysis result. For example, as ImageJ ROI or ROIset files that can be re-imported, correctly positioned, and visualized in the measured images. This would greatly improve the reproducibility of the proposed workflow. In the manuscript, only the extracted features are being saved (because the save button is also just asking for a folder containing images, so I cannot verify its functionality).

? button

It would be great if that button would open up some usage instructions.

Easy improvement of workflow

I would suggest a reasonable expansion of the current workflow, by fitting and displaying 2D lines to the band or line structure in the image, that form the "patterns" the author aims to address. Thus, it extracts geometry models from the image, and the inter-line distance, and even the curve formed by these sets of lines can be further analyzed and studied. These fitted 2D lines can be also well integrated into ImageJ as Line ROI, and thus be saved, imported back, and checked or being further modified. I think this can largely increase the usefulness and reproducibility of the software.

Manuscript

We recommend considering the following suggestions for improving the manuscript. Abstract: The abstract suggests that general patterns can be quantified, however the actual tool quantifies specific subtypes of one-dimensional patterns. We recommend adapting the abstract accordingly.

Line 58: Gray-level co-occurrence matrix (GLCM) based feature extraction and analysis approach is not mentioned nor compared. At least there's a relatively recent study on Sarcomeres structure based on GLCM feature extraction: https://github.com/steinjm/SotaTool with publication: https://doi.org/10.1002/cpz1.462

Line 75: "...these simple geometrical features will address most quantitative needs..." We feel that this may be an overstatement, e.g. we can imagine that there should be many relevant two-dimensional patterns in biology?!

Line 83: "After a straightforward installation by the user, ...". We think it would be convenient to add the installation steps at this place into the manuscript.

Line 87: "Multicolor images will give a graph with one profile per color." The 'Multicolor images' here should be more precisely stated as "multi-channel" images. Multi-color images could be confused with RGB images which will be treated as 8-bit gray value (type conversion first) images by profile plot in ImageJ.

Line 92: "...such as individual bands, blocks, or sarcomeric actin...". While bands and blocks are generic pattern terms, the biological term "sarcomeric actin" does not seem to fit in this list. Could a more generic wording be found, such as "block with spike"?

Line 95: "the algorithm defines one pattern by having the features of highest intensity in its centre". Could this be rephrased? We did not understand what that exactly means.

Line 124 - 147: This part the only description of the algorithm behind the feature extraction and analysis, but not clearly stated. Many details are missing or assumed known by the reader. For example, how it achieved sub-pixel resolution results is not clear. One can only assume that by fitting Gaussian to the band, the center position (peak) thus can be calculated from continuous curves other than pixels.

Line 407: We think the availability of both the tool and the code could be improved. For Fiji tools it is common practice to create an Update Site and to make the code available on GitHub. In addition, downloading the example file (https://drive.google.com/file/d/1eMazyQJlisWPwmozvyb8VPVbfAgaH7Hz/view?usp=drive_link) required a Google login and access request, which is not very convenient; in fact, we asked for access but it was denied. It would be important for the download to be easier, e.g. from GitHub or Zenodo.

Significance

The strength of this study is that a tool for the analysis of one-dimensional repeated patterns occurring in muscle fibres is made available in the accessible open-source platform ImageJ/Fiji. In the introduction to the article the authors provide an extensive review of comparable existing tools. Their new tool fills a gap in terms of providing an easy-to-use software for users without computational skills that enables the analysis of muscle sarcomere patterns. We feel that if the below mentioned limitations could be addressed the tool could indeed be valuable to life scientists interested in muscle patterning without computational skills.

In our view there are a few limitations, including the accessibility of example data and tutorials at sites.google.com/view/patternj, which we had trouble to access. In addition, we think that the workflow in Fiji, which currently requires pressing several buttons in the correct order, could be further simplified and streamlined by adopting some "wizard" approach, where the user is guided through the steps. Another limitation is the reproducibility of the analysis; here we recommend enabling IJ Macro recording as well as saving of the drawn line ROIs. For more detailed suggestions for improvements please see the above sections of our review.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #1

Evidence, reproducibility and clarity

I have trialled the package on my lab's data and it works as advertised. It was straightforward to use and did not require any special training. I am confident this is a tool that will be approachable even to users with limited computational experience. The use of artificial data to validate the approach - and to provide clear limits on applicability - is particularly helpful.

The main limitation of the tool is that it requires the user to manually select regions. This somewhat limits the generalisability and is also more subjective - users can easily choose "nice" regions that better match with their hypothesis, rather than quantifying the data in an unbiased manner. However, given the inherent challenges in quantifying biological data, such problems are not easily circumventable.

I have some comments to clarify the manuscript:

1. A "straightforward installation" is mentioned. Given this is a Method paper, the means of installation should be clearly laid out.
2. It would be helpful if there was an option to generate an output with the regions analysed (i.e., a JPG image with the data and the drawn line(s) on top). There are two reasons for this: i) A major problem with user-driven quantification is accidental double counting of regions (e.g., a user quantifies a part of an image and then later quantifies the same region). ii) Allows other users to independently verify measurements at a later time.
3. Related to the above point, it is highlighted that each time point would need to be analysed separately (line 361-362). It seems like it should be relatively straightforward to allow a function where the analysis line can be mapped onto the next time point. The user could then adjust slightly for changes in position, but still be starting from near the previous timepoint. Given how prevalent timelapse imaging is, this seems like (or something similar) a clear benefit to add to the software.
4. Line 134-135. The level of accuracy of the searching should be clarified here. This is discussed later in the manuscript, but it would be helpful to give readers an idea at this point what level of tolerance the software has to noise and aperiodicity.

Referees cross-commenting

I think the other reviewer comments are very pertinent. The authors have a fair bit to do, but they are reasonable requests. So, they should be encouraged to do the revisions fully so that the final software tool is as useful as possible.

Significance

Developing software tools for quantifying biological data that are approachable for a wide range of users remains a longstanding challenge. This challenge is due to: (1) the inherent problem of variability in biological systems; (2) the complexity of defining clearly quantifiable measurables; and (3) the broad spread of computational skills amongst likely users of such software.

In this work, Blin et al., develop a simple plugin for ImageJ designed to quickly and easily quantify regular repeating units within biological systems - e.g., muscle fibre structure. They clearly and fairly discuss existing tools, with their pros and cons. The motivation for PatternJ is properly justified (which is sadly not always the case with such software tools).

Overall, the paper is well written and accessible. The tool has limitations but it is clearly useful and easy to use. Therefore, this work is publishable with only minor corrections.

eLife assessment

The study, from the group that pioneered migrasome, describes a novel vaccine platform derived from this newly discovered organelle. Using these cleverly engineered migrasomes – that behave like natural migrasomes – as a novel vaccine platform has the potential to overcome obstacles such as cold chain issues for vaccines like messenger RNA. Although the findings are important with practical implications for the vaccine technology, and the evidence, based on appropriate and validated methodology is convincing and is in line with current state-of-the-art, there are some critical issues that need to be addressed. These include a head-to-head comparison with proven vaccine platforms, for example, a SARS-CoV-2 mRNA vaccine or an adjuvanted recombinant spike protein.

non-nieuws in de sterrenkunde, maar de vragen die Schilling oproept te stellen gaan eigenlijk over elk dossier. Wel 'steeds meer x' roepen maar geen echte vergelijking (het is nog altijd minder dan 10 jr geleden) of duiding geven (bijv dat x gewoon met bevolking meegroeit), geen relatie met impact, geen relatie met blijvende waarde etc. cf [[Crap detection is civic duty 2018010073052]]

Die Zeit: Interview mit Leonore Gewessler zur Abhängigkeit Österreichs von russischem Gas. Es wird deutlich, dass der Umstieg auf andere Lieferanten bisher an der OMV und ihren Verbündeten wie der Wirtschaftskammer und der Industriellenvereinigung scheitert. https://www.zeit.de/2024/22/gasimporte-russland-oesterreich-leonore-gewessler-gazprom/komplettansicht

我选择都要。使用Emacs 29的--init-directory参数可以同时使用两份配置，这样一份doom emacs 用于上手实践，一份vanilla用于自己折腾理解，甚至还可以将doom emacs 复制一份用做测（ 版本，折腾完毕后才把配置同步到日常使用版本中

Emacs的话，可以做到哪里不对改那里。前提是需要具备一些elisp的知识

Reviewer #1 (Public Review):

Summary:

Winged seeds or ovules from the Devonian are crucial to understanding the origin and early evolutionary history of wind dispersal strategy. Based on exceptionally well-preserved fossil specimens, the present manuscript documented a new fossil plant taxon (new genus and new species) from the Famennian Series of Upper Devonian in eastern China and demonstrated that three-winged seeds are more adapted to wind dispersal than one-, two- and four-winged seeds by using mathematical analysis.

Strengths:

The manuscript is well organised and well presented, with superb illustrations. The methods used in the manuscript are appropriate.

Weaknesses:

I would only like to suggest moving the "Mathematical analysis of wind dispersal of ovules with 1-4 wings" section from the supplementary information to the main text, leaving the supplementary figures as supplementary materials.

eLife assessment

This useful manuscript describes the second earliest known winged ovule without a capule in the Famennian of Late Devonian. Using solid mathematical analysis, the authors demonstrate that three-winged seeds are more adapted to wind dispersal than one-, two- and four-winged seeds. The manuscript will help the scientific community to understand the origin and early evolutionary history of wind dispersal strategy of early land plants.

Reviewer #2 (Public Review):

Summary:

This manuscript described the second earliest known winged ovule without a capule in the Famennian of Late Devonian. Using Mathematical analysis, the authors suggest that the integuments of the earliest ovules without a cupule, as in the new taxon and Guazia, evolved functions in wind dispersal.

Strengths:

The new ovule taxon's morphological part is convincing. It provides additional evidence for the earliest winged ovules, and the mathematical analysis helps to understand their function.

Weaknesses:

The discussion should be enhanced to clarify the significance of this finding. What is the new advance compared with the Guazia finding? The authors can illustrate the character transformations using a simplified cladogram. The present version of the main text looks flat.

The Guardian: Die Parteien, die die neue niederländische Rechtsregierung unterstützen, haben sich auf ein Arbeitsprogramm geeinigt. Wichtige klimapolitische Maßnahmen werden zurückgenommen. So soll die Höchstgeschwindigkeit auf Autobahnen wieder 130 km/h betragen; Agrardiesel wird wieder subventioniert. Bei der Installation von Wärmepumpen verzichtet man auf Zielwerte. Es sollen 4 Atomkraftwerke gebaut werden. Was die Regierung durchsetzen kann, ist noch unklar. https://www.theguardian.com/world/article/2024/may/16/new-dutch-coalition-aims-to-reintroduce-80mph-limit-in-cull-of-climate-goals

eLife assessment

This important study reports the deep evolutionary conservation of a core genetic program regulating spermatogenesis in flies, mice, and humans. The data presented are supportive of the main conclusion and generally convincing. This work will be of interest to evolutionary and reproductive biologists.

Reviewer #1 (Public Review):

Summary:

By combining an analysis of the evolutionary age of the genes expressed in male germ cells, a study of genes associated with spermatocyte protein-protein interaction networks and functional experiments in Drosophila, Brattig-Correia and colleagues provide evidence for an ancient origin of the genetic program underlying metazoan spermatogenesis. This leads to identifying a relatively small core set of functional interactions between deeply conserved gene expression regulators, whose impairment is then shown to be associated with cases of human male infertility.

Strengths:

In my opinion, the work is important for three different reasons. First, it shows that, even though reproductive genes can evolve rapidly and male germ cells display a significant level of transcriptional noise, it is still possible to obtain convincing evidence that a conserved core of functionally interacting genes lies at the basis of the male germ transcriptome. Second, it reports an experimental strategy that could also be applied to gene networks involved in different biological problems. Third, the authors make a compelling case that, due to its effects on human spermatogenesis, disruption of the male germ cell orthoBackbone can be exploited to identify new genetic causes of infertility.

Weaknesses:

The main strength of the general approach followed by the authors is, inevitably, also a weakness. This is because a study rooted in comparative biology is unlikely to identify newly emerged genes that may adopt key roles in processes such as species-specific gamete recognition. Additionally, using a TPM >1 threshold for protein-coding transcripts may exclude genes, such as those encoding proteins required for gamete fusion, which are thought to be expressed at a very low level. Although these considerations raise the possibility that the chosen approach may miss information that, depending on the species, could be potentially highly functionally important, this by no means reduces its value in identifying genes belonging to the conserved genetic program of spermatogenesis.

Reviewer #2 (Public Review):

Summary:

This is a tour de force study that aims to understand the genetic basis of male germ cell development across three animal species (human, mouse, and flies) by performing a genetic program conservation analysis (using phylostratigraphy and network science) with a special emphasis on genes that peak or decline during mitosis-to-meiosis. This analysis, in agreement with previous findings, reveals that several genes active during and before meiosis are deeply conserved across species, suggesting ancient regulatory mechanisms. To identify critical genes in germ cell development, the investigators integrated clinical genetics data, performing gene knockdown and knockout experiments in both mice and flies. Specifically, over 900 conserved genes were investigated in flies, with three of these genes further studied in mice. Of the 900 genes in flies, ~250 RNAi knockdowns had fertility phenotypes. The fertility phenotypes for the fly data can be viewed using the following browser link: https://pages.igc.pt/meionav. The scope of target gene validation is impressive. Below are a few minor comments.

(1) In Supplemental Figure 2, it is notable that enterocyte transcriptomes are predominantly composed of younger genes, contrasting with the genetic age profile observed in brain and muscle cells. This difference is an intriguing observation and it would be curious to hear the author's comments.

(2) Regarding the document, the figures provided only include supplemental data; none of the main text figures are in the full PDF.

(3) Lastly, it would be great to section and stain mouse testis to classify the different stages of arrest during meiosis for each of the mouse mutants in order to compare more precisely to flies.

This paper serves as a vital resource, emphasizing that only through the analysis of hundreds of genes can we prioritize essential genes for germ cell development. its remarkable that about 60% of conserved genes have no apparent phenotype during germ cell development.

Strengths:

The high-throughput screening was conducted on a conserved network of 920 genes expressed during the mitosis-to-meiosis transition. Approximately 250 of these genes were associated with fertility phenotypes. Notably, mutations in 5 of the 250 genes have been identified in human male infertility patients. Furthermore, 3 of these genes were modeled in mice, where they were also linked to infertility. This study establishes a crucial groundwork for future investigations into germ cell development genes, aiming to delineate their essential roles and functions.

Weaknesses:

The fertility phenotyping in this study is limited, yet dissecting the mechanistic roles of these proteins falls beyond its scope. Nevertheless, this work serves as an invaluable resource for further exploration of specific genes of interest.

eLife assessment

This important study reports the developmental dynamics and molecular markers of the rete ovarii during ovarian development. However, the data supporting the main conclusions remain incomplete. This study will be of interest to developmental and reproductive biologists.

Reviewer #1 (Public Review):

Summary:

The manuscript by Anbarcia et al. re-evaluates the function of the enigmatic Rete Ovarii (RO), a structure that forms in close association with the mammalian ovary. The RO has generally been considered a functionless structure in the adult ovary. This manuscript follows up on a previous study from the lab that analyzed ovarian morphogenesis using high-resolution microscopy (McKey et al., 2022). The present study adds finer details to RO development and possible function by (1) identifying new markers for OR sub-regions (e.g. GFR1a labels the connecting rete) suggesting that the sub-regions are functionally distinct, (2) showing that the OR sub-regions are connected by a luminal system that allows transport of material from the extra-ovarian rete (EOR) to the inter-ovarian rete (IOG), (3) identifies proteins that are secreted into the OR lumen and that may regulate ovarian homeostasis, and finally, (4) better defines how the vasculature, nervous, and immune system integrates with the OR.

Strengths:

The data is beautifully presented and convincing. They show that the RO is composed of three distinct domains that have unique gene expression signatures and thus likely are functionally distinct.

Weaknesses:

It is not always clear what the novel findings are that this manuscript is presenting. It appears to be largely similar to the analysis done by McKey et al. (2022) but with more time points and molecular markers. The novelty of the present study's findings needs to be better articulated.

Reviewer #2 (Public Review):

A large number of ovarian experiments have been conducted - especially in morphological and molecular biology studies - specifically removing the ovarian membrane. This experiment is a good supplement to existing knowledge and plays an important role in early ovarian development and the regulation of ovarian homeostasis during the estrous cycle. There are also innovations in research ideas and methods, which will meet the requirements of experimental design and provide inspiration for other researchers.

This reviewer did not identify any major issues with the article. However, the following points could be further clarified:

(1) Is there any comparative data on the proteomics of RO and rete testis in early development? With some molecular markers also derived from rete testis, it would be better to provide the data or references.

(2) Although the size of RO and its components is quite small and difficult to operate, the researchers in this article had already been able to perform intracavitary injection of EOR and extract EOR or CR for mass spectrometry analysis. Therefore, can EOR, CR, or IOR be damaged or removed, providing further strong evidence of ovarian development function?

(3) Although IOR is shown on the schematic diagram, it cannot be observed in the immunohistochemistry pictures in Figure 1 and Figure 3. The authors should provide a detailed explanation.

Reviewer #3 (Public Review):

Summary:

The rete ovarii (RO) has long been disregarded as a non-functional structure within the ovary. In their study, Anbarci and colleagues have delineated the markers and developmental dynamics of three distinct regions of the RO - the intraovarian rete (IOR), the extraovarian rete (EOR), and the connecting rete (CR). Notably focusing on the EOR, the authors presented evidence illustrating that the EOR forms a convoluted tubular structure culminating in a dilated tip. Intriguingly, microinjections into this tip revealed luminal flow towards the ovary containing potentially secreted functional proteins. Additionally, the EOR cells exhibit associations with vasculature, macrophages, and neuronal projections, proposing the notion that the RO may play a functional role in ovarian development during critical ovariogenesis stages. By identifying marker genes within the RO, the authors have also suggested that the RO could serve as a potential structure linking the ovary with the neuronal system.

Strengths:

Overall, the reviewer commends the authors for their systematic research on the RO, shedding light on this overlooked structure in developing ovaries. Furthermore, the authors have proposed a series of hypotheses that are both captivating and scientifically significant, with the potential to reshape our understanding of ovarian development through future investigations.

Weaknesses:

There is a lack of conclusive data supporting many conclusions in the manuscript. Therefore, the paper's overall conclusions should be moderated until functional validations are conducted.

You could also cite my paper :p https://scholar.google.com/citations?view_op=view_citation&hl=en&user=KCi_988AAAAJ&citation_for_view=KCi_988AAAAJ:zYLM7Y9cAGgC

Maybe mention https://arxiv.org/abs/1805.11593

I'd talk a bit more about why it works on some games, why it doesn't on others. Overall performance is not that different to DQN.

If you're going to talk about this, move it to the DQN section and make a small subsection on DQN enhancements (basically the things from the Rainbow paper). Don't spend much space since this isn't core.

Make this a subheading under DQN (maybe also explain target networks quickly)

interesting

eLife assessment

The authors combined human genetic analysis with zebrafish experiments to produce evidence that alleles that impair the function of EPHA4 cause idiopathic scoliosis (IS), a common spinal deformity. The significance of the findings is important because the cellular and molecular mechanisms that contribute to IS remain poorly understood. The human genetic data are quite convincing whereas the zebrafish data, although supportive, are incomplete.

Joint Public Review:

Summary:

Idiopathic scoliosis (IS) is a common spinal deformity. Various studies have linked genes to IS, but underlying mechanisms are unclear such that we still lack understanding of the causes of IS. The current manuscript analyzes IS patient populations and identifies EPHA4 as a novel associated gene, finding three rare variants in EPHA4 from three patients (one disrupting splicing and two missense variants) as well as a large deletion (encompassing EPHA4) in a Waardenburg syndrome patient with scoliosis. EPHA4 is a member of the Eph receptor family. Drawing on data from zebrafish experiments, the authors argue that EPHA4 loss of function disrupts the central pattern generator (CPG) function necessary for motor coordination.

Strengths:

The main strength of this manuscript is the human genetic data, which provides convincing evidence linking EPHA4 variants to IS. The loss of function experiments in zebrafish strongly support the conclusion that EPHA4 variants that reduce function lead to IS.

Weaknesses:

The conclusion that disruption of CPG function causes spinal curves in the zebrafish model is not well supported. The authors' final model is that a disrupted CPG leads to asymmetric mechanical loading on the spine and, over time, the development of curves. This is a reasonable idea, but currently not strongly backed up by data in the manuscript. Potentially, the impaired larval movements simply coincide with, but do not cause, juvenile-onset scoliosis. Support for the authors' conclusion would require independent methods of disrupting CPG function and determining if this is accompanied by spine curvature. At a minimum, the language of the manuscript could be toned down, with the CPG defects put forward as a potential explanation for scoliosis in the discussion rather than as something this manuscript has "shown". An additional weakness of the manuscript is that the zebrafish genetic tools are not sufficiently validated to provide full confidence in the data and conclusions.

great

地方

萨达

eLife assessment

This work is important because it attempts to elucidate how immune cells migrate across the blood brain barrier. The authors developed a convincing framework to visualize, recognize and track the movement of different immune cells across primary human and mouse brain microvascular endothelial cells without the need for fluorescence-based imaging using microfluidic devices. The data gathered are solid, and this work will be of interest to the cancer biology, immunology and medical therapeutics fields.

Reviewer #1 (Public Review):

Summary:

It is evident that studying leukocyte extravasation in vitro is a challenge. One needs to include physiological flow, culture cells and isolate primary immune cells. Timing is of utmost importance and a reproducible setup essential. Extra challenges are met when extravasation kinetics in different vascular beds is required, e.g., across the blood-brain barrier. In this study, the authors describe a reliable and reproducible method to analyze leukocyte TEM under physiological flow conditions, including this analysis. That the software can also detect reverse TEM is a plus.

Strengths:

It is quite a challenge to get this assay reproducible and stable, in particular as there is flow included. Also for the analysis, there is currently no clear software analysis program, and many labs have their own methods. This paper gives the opportunity to unify the data and results obtained with this assay under label-free conditions. This should eventually lead to more solid and reproducible results.

Also, the comparison between manual and software analysis is appreciated.

Weaknesses:

The authors stress that it can be done in BBB models, but I would argue that it is much more broadly applicable. This is not necessarily a weakness of the study but more an opportunity to strengthen the method. So I would encourage the authors to rewrite some parts and make it more broadly applicable.

Reviewer #2 (Public Review):

Summary:

This paper develops an under-flow migration tracker to evaluate all the steps of the extravasation cascade of immune cells across the BBB. The algorithm is useful and has important applications.

Strengths:

Algorithm is almost as accurate as manual tracking and importantly saves time for researchers.

Weaknesses:

Applicability can be questioned because the device used is 2D and physiological biology is in 3D. Comparisons to other automated tools was not performed by the authors.

Reviewer #3 (Public Review):

Summary:

The authors aimed to establish a faster and more efficient method of tracking steps of T-cell extravasation across the blood brain barrier. The authors developed a framework to visualize, recognize and track the movement of different immune cells across primary human and mouse brain microvascular endothelial cells without the need for fluorescence-based imaging. The authors succinctly describe the basic requirements for tracking in the introduction followed by an in-depth account of the execution.

Weaknesses and Strengths:

Materials & methods and results:

(1) The methods section also lacks details of the microfluidic device that the authors talk about in the paper. Under physiological sheer stress, the T-cells detach from the pMBMEC monolayer, and are hence unable to be detected; however, this observation requires an explanation pertaining to the reason of occurrence and potential solutions to circumvent it to ensure physiologically relevant experimental parameters.

(2) The author describes a method for debris exclusion using UFMTrack that eliminates objects of <30 pixels in size from analysis based on a mean pixel size of 400 for T lymphocytes. However, this mean pixel size appears to stem from in-vitro activated CD8 T cells, which rapidly grow and proliferate upon stimulation. In line with this, activated lymphocytes exhibit increased cytoplasmic area, making them appear less dense or "brighter" by phase microscopy compared to naïve lymphocytes, which are relatively compact and subsequently appear dimmer. Given this, it is not clear whether UFMTrack is sufficiently trained to identify naïve human lymphocytes in circulating blood, nor smaller, murine lymphocytes. Analysis of each lymphocyte subtype in terms of pixel size and intensity would be beneficial to strengthen the claim that UFMTrack can identify each of these populations. Additionally, demonstrating that UFMTrack can correctly characterize the behavior of naïve versus activated lymphocytes isolated from murine and human sources would strengthen the claim that UFMTrack can be broadly applied to study lymphocyte dynamics in diverse models without additional training

(3) Average precision was compared to the analysis of UFMTrack but it is unclear how average precision was calculated. This information should have been included in the methods section

(4) CD4 and CD8 T cells exhibit distinct biology and interaction kinetics driven in part by their MHC molecule affinity and distinct receptor expression profiles. Thus, it is unclear why two distinct mechanisms of endothelial cell activation are needed to see differences between the populations.

(5) The BMECs are barrier tissues but were cultured on µdishes in this study. To study the transmigration of T-cells across the endothelium, the model would have been more relevant on a semi-permeable membrane instead of a closed surface.

(6) Methods are provided for the isolation and expansion of human effector and memory CD4+ T cells. However, there is no mention of specific CD4+ T cell populations used for analysis with UFMTrack, nor a clear breakdown of tracking efficiency for each subpopulation. Further, there is no similar method for the isolation of CD8+ T cell compartments. A clear breakdown of the performance efficiency of UFMTrack with each cell population investigated in this study would provide greater insight into the software's performance with regard to tracking the behavior and movement of distinct immune populations.

(7) The results section is quite extensive and discusses details of establishment of the framework while highlighting both the pros and cons of the different aspects of the process, for example the limitation of the two models, 2D and 2D+T were highlighted well. However, the results section includes details which may be more fitting in the methods section.

(8) A few statements in the results section lacked literary support, which was not provided in the discussion either, such as support for increased variance of T-cell instantaneous speed on stimulated vs non-stimulated pMBMECs. Another example is the enhancement of cytokine stimulation directed T-cell movement on the pMBMECs that the authors observed but failed to relay the physiological relevance of it. The authors don't provide enough references for developments in the field prior to their work which form the basis and need for this technology.

(9) The rationale for use of OT-1 and 2D2-derived murine lymphocytes is unclear here. The OT-1 model has been generated to study antigen-specific CD8+ T cell responses, while the 2D2 model has been generated to recapitulate CD4 T cell-specific myelin oligodendrocyte glycoprotein (MOG) responses.

Figures and text:

(1) There are certain discrepancies and misarrangement of figures and text. For example, discussion of the effect of sheer flow on T cell attachment as part of the introduction in figure 1 and then mentioning it in the text again in the results section as part of figure 4 is repetitive.

(2) Section IV, subsection 1 of the results section, refers to 'data acquisition section above' in line 279, however the said section is part of materials and methods which is provided towards the end of the manuscript.

(3) There are figures in the manuscript that have not been referenced in the results section, for example, figure 3A and B. Figure 1 hasn't been addressed until subsection 7 of materials and methods

(4) A lack of significance but an observed trend of increased variance of T cell instantaneous speed is reported in line 296-298; however, the graph (figure 4G) shows a significant change in instantaneous speed between non-stimulated and TNFα-stimulated systems. This is misleading to the readers.

(5) The authors talk about three beginner experimentors testing the manual T cell tracking process but figure 5 only showcases data from two experimentors without stating the reason for excluding experimentor 1.

Discussion:

(1) While the discussion captures the major takeaways from the paper, it lacks relevant supporting references to relate the observation to physiological conditions and applicability.

(2) The discussion lacks connection to the results since the figures were not referenced while discussing an observed trend

(3) The authors briefly looked into mouse and human BMECs and their individual interaction with T-cells, but don't discuss the differences between the two, if any, that challenged their framework.

(4) Even though though the imaging tool relies on difference in appearance for detection, the authors talk about lack of feasibility in detecting transmigration of BMDMs due to their significantly different appearance. The statement lacks a problem solving approach to discuss how and why this was the case.

Relevance to the field:

Utilizing the framework provided by the authors, the application can be adapted and/or utilized for visualizing a range of different cell types, provided they are different in appearance. However, this would require extensive changes to the script and won't be adaptable in its current form.

eLife assessment

This fundamental study provides a modeling regime that provides new insight into the energy-preservation parameters among schooling fish. The strength of the evidence supporting observations such as distilled dynamics between leading and lagging schooling fish which are derived from emergent properties is convincing. Overall, the study provides exciting insights into energetic coupling with respect to group swimming dynamics. Some potential improvements to strengthen the study include clarification regarding degrees of freedom and parameter ranges in the model.

Reviewer #1 (Public Review):

Summary:

The study seeks to establish accurate computational models to explore the role of hydrodynamic interactions on energy savings and spatial patterns in fish schools. Specifically, the authors consider a system of (one degree-of-freedom) flapping airfoils that passively position themselves with respect to the streamwise direction, while oscillating at the same frequency and amplitude, with a given phase lag and at a constant cross-stream distance. By parametrically varying the phase lag and the cross-stream distance, they systematically explore the stability and energy costs of emergent configurations. Computational findings are leveraged to distill insights into universal relationships and clarify the role of the wake of the leading foil.

Strengths:

(1) The use of multiple computational models (computational fluid dynamics, CFD, for full Navier-Stokes equations and computationally efficient inviscid vortex sheet, VS, model) offers an extra degree of reliability of the observed findings and backing to the use of simplified models for future research in more complex settings.

(2) The systematic assessment of the stability and energy savings in multiple configurations of pairs and larger ensembles of flapping foils is an important addition to the literature.

(3) The discovery of a linear phase-distance relationship in the formation attained by pairs of flapping foils is a significant contribution, which helps compare different experimental observations in the literature.

(4) The observation of a critical size effect for in-line formations of larger, above which cohesion and energetic benefits are lost at once, is a new discovery in the field.

Weaknesses:

(1) The extent to which observations on one-degree-of-freedom flapping foils could translate to real fish schools is presently unclear so some of the conclusions on live fish schools are likely to be overstated and would benefit from some more biological framing.

(2) The analysis of non-reciprocal coupling is not as novel as the rest of the study and potentially not as convincing due to the chosen linear metric of interaction (that is, the flow agreement).

Overall, this is a rigorous effort on a critical topic: findings of the research can offer important insight into the hydrodynamics of fish schooling, stimulating interdisciplinary research at the interface of computational fluid mechanics and biology.

Reviewer #2 (Public Review):

The document "Mapping spatial patterns to energetic benefits in groups of flow-coupled swimmers" by Heydari et al. uses several types of simulations and models to address aspects of stability of position and power consumption in few-body groups of pitching foils. I think the work has the potential to be a valuable and timely contribution to an important subject area. The supporting evidence is largely quite convincing, though some details could raise questions, and there is room for improvement in the presentation. My recommendations are focused on clarifying the presentation and perhaps spurring the authors to assess additional aspects:

(1) Why do the authors choose to set the swimmers free only in the propulsion direction? I can understand constraining all the positions/orientations for investigating the resulting forces and power, and I can also understand the value of allowing the bodies to be fully free in x, y, and their orientation angle to see if possible configurations spontaneously emerge from the flow interactions. But why constrain some degrees of freedom and not others? What's the motivation, and what's the relevance to animals, which are fully free?

(2) The model description in Eq. (1) and the surrounding text is confusing. Aren't the authors computing forces via CFD or the VS method and then simply driving the propulsive dynamics according to the net horizontal force? It seems then irrelevant to decompose things into thrust and drag, and it seems irrelevant to claim that the thrust comes from pressure and the drag from viscous effects. The latter claim may in fact be incorrect since the body has a shape and the normal and tangential components of the surface stress along the body may be complex.

(3) The parameter taudiss in the VS simulations takes on unusual values such as 2.45T, making it seem like this value is somehow very special, and perhaps 2.44 or 2.46 would lead to significantly different results. If the value is special, the authors should discuss and assess it. Otherwise, I recommend picking a round value, like 2 or 3, which would avoid distraction.

(4) Some of the COT plots/information were difficult to interpret because the correspondence of beneficial with the mathematical sign was changing. For example, DeltaCOT as introduced on p. 5 is such that negative indicates bad energetics as compared to a solo swimmer. But elsewhere, lower or more negative COT is good in terms of savings. Given the many plots, large amounts of data, and many quantities being assessed, the paper needs a highly uniform presentation to aid the reader.

(5) I didn't understand the value of the "flow agreement parameter," and I didn't understand the authors' interpretation of its significance. Firstly, it would help if this and all other quantities were given explicit definitions as complete equations (including normalization). As I understand it, the quantity indicates the match of the flow velocity at some location with the flapping velocity of a "ghost swimmer" at that location. This does not seem to be exactly relevant to the equilibrium locations. In particular, if the match were perfect, then the swimmer would generate no relative flow and thus no thrust, meaning such a location could not be an equilibrium. So, some degree of mismatch seems necessary. I believe such a mismatch is indeed present, but the plots such as those in Figure 4 may disguise the effect. The color bar is saturated to the point of essentially being three tones (blue, white, red), so we cannot see that the observed equilibria are likely between the max and min values of this parameter.

(6) More generally, and related to the above, I am favorable towards the authors' attempts to find approximate flow metrics that could be used to predict the equilibrium positions and their stability, but I think the reasoning needs to be more solid. It seems the authors are seeking a parameter that can indicate equilibrium and another that can indicate stability. Can they clearly lay out the motivation behind any proposed metrics, and clearly present complete equations for their definitions? Further, is there a related power metric that can be appropriately defined and which proves to be useful?

(7) Why do the authors not carry out CFD simulations on the larger groups? Some explanations should be given, or some corresponding CFD simulations should be carried out. It would be interesting if CFD simulations were done and included, especially for the in-line case of many swimmers. This is because the results seem to be quite nuanced and dependent on many-body effects beyond nearest-neighbor interactions. It would certainly be comforting to see something similar happen in CFD.

(8) Related to the above, the authors should discuss seemingly significant differences in their results for long in-line formations as compared to the CFD work of Peng et al. [48]. That work showed apparently stable groups for numbers of swimmers quite larger than that studied here. Why such a qualitatively different result, and how should we interpret these differences regarding the more general issue of the stability of tandem groups?

(9) The authors seem to have all the tools needed to address the general question about how dynamically stable configurations relate to those that are energetically optimal. Are stable solutions optimal, or not? This would seem to have very important implications for animal groups, and the work addresses closely related topics but seems to miss the opportunity to give a definitive answer to this big question.

(10) Time-delay particle model: This model seems to construct a simplified wake flow. But does the constructed flow satisfy basic properties that we demand of any flow, such as being divergence-free? If not, then the formulation may be troublesome.

Not sure if I should use trailmarks and listicle here or not? I will choose to use it.

gendered syntax - I understand, but I also pointed out that the evolutionary nature of a language's syntax gives it unique gender characteristics. - I gave the example of my own mother tongue of Cantonese which is syntactically more gender neutral instead of English, which is patriarchal: - Cantonese (play the audio at the following links) - person - https://www.cantoneseclass101.com/cantonese-dictionary/ - man - https://www.cantoneseclass101.com/cantonese-dictionary/ - woman - https://www.cantoneseclass101.com/cantonese-dictionary/ - In the Cantonese language, the suffix (Yan) means person, - It is then modified by the respective female and male prefix - Noi (female) - Nam (male) - This gives us gender neutral syntax, as opposed to English where we have patriarchal gender syntax, where the suffix is male and the female is constructed as a secondary concatenation using the male syntactical suffix - male - FEmale - man - WOman - HUman - HUmanITY - men - WOmen - The English language gives syntactical primacy to the male gender, while a language such as Cantonese does not - What the psychological effects are, I'm not sure of. For within the Cantonese language, there is as much patriarchism as any other culture. It is not a particularly feminine culture. - And the gender neutrality does not even take into account of the more recent transgender category.

to - Cantonese syntax - person - man - woman - https://hyp.is/3wgg0BQOEe-uRQ-kpQf8Eg/www.cantoneseclass101.com/cantonese-dictionary/ - With English, we have to read between the lines and project the author's salience landscape because it's not explicit in the syntax.

PROBLEM - This page does not generate a unique URL for each of the onpage search results returned. - Can Indyweb create unique CID for this?

This quote stood out to me because I believe that no one should have to hide who they are and how they learn. Faculty at schools are supposed to be the ones who embrace and encourage students, so I find it very saddening that people who are neurodivergent are treated differently by the faculty, in any sort of way that makes them uncomfortable. This relates to equity as even if a student needs to LEARN differently than others, it does not mean in any way that they should be TREATED differently. Being in the healthcare field, and taking harder STEM classes than I am used to, I have noticed that every single student learns differently. I think that it is important that faculty understands that we all have differences, and different ways of learning. And are able to help us in those ways, to where everyone succeeds. -Presley Labell

from - Indyweb Alpha team convo - https://hyp.is/tAQQkhQOEe-mQ5djqIdORg/ipfs.indy0.net/ipfs/QmU7UtYmmd1nXB4dsRLmAdrquPe7eVao6XxRV5ES4q6vkc/messages.html

Here, Sue Klebold introduces the Pentadic element of scene to her speech, being the aftermath of the Columbine shooting. A scene in which she continues to paint throughout her speech.

The scene of this speech is also the third (yet ongoing) event in her narrative arc, which she continues to explain and explore through the end of her speech.

Klebold's ends her presentation emphasizing the need for her audience to continue to care for others despite the possibility of tragedy, leaving the audience on an empowered note.

Klebold invokes logos yet again using statistics about suicide attempts.

This statement deconstructs the idea that if someone is loved or well cared for, they will not go to drastic measures to end their (or others') lives. In video diaries published after the massacre, Dylan is known for saying that his parents treated him well.

Here, Klebold refers back to the criticism of how continually accessible lethal weapons are to obtain that she began towards the beginning of her speech. This also introduces another element of the pentad, Agency, or the means or tools used by the agent for the act.

This anecdote provides a very poignant piece of insight on Sue's experience in the aftermath of the massacre. This allows the audience to see through a more vulnerable window of Sue's life and connect with her more as a speaker.

The statement "Dylan could not have loved me if her could do something as horrible as he did." invokes pathos both in the woman who was speaking to her at the time, as well as her audience.

Klebold discusses suicide as a decision that is made when not in a coherent mental state. She believes that the decision to commit suicide is something that comes from a lack of rationality, despite the meticulous planning that went into the massacre for months before it occurred.

Another insertion of logos into her presentation, she brings in rough estimates to tell of how often people with mental illness are hardly ever recognized or supported until they go through a behavioral crisis of their own. The word choice of labelling the massacre as a murder-suicide rather than a mass shooting emphasizes the suicidality that Dylan was experiencing, in comparison to his friend Eric, who is described to be more involved in te murder aspect.

Here, Klebold uses the element of logos, or the logical appeal to her audience based in Neo-Aristotelian rhetoric. She introduces (albeit rough) statistics about mental health that attempts destigmatize thoughts surrounding mental illness.

Again invoking pathos in her speech, she emphasizes her lack of knowledge in her son's situation, which many parents can often relate to. On the other hand, people who have experienced self-harming or suicidal ideation can empathize with the tendency to hide these elements of their lives from loved ones, and can therefore understand her reasoning for not having known prior to his death, despite there being physical evidence of video and written diaries discovered and publicized after the massacre.

With this declaration, Sue Klebold introduces another element of the pentad, that being Purpose. She deciphers that the purpose of the act of the Columbine massacre, at least on Dylan's part, was mainly to die, rather than to kill.

In Bartol & Bartol's 2017 publication Criminal Behavior: a Psychological Approach, the two suggest that there is no singular profile for a mass shooter, though there are many similar factors that come into play when analyzing them as a collective, explaining that it is often argued that most school shooters have a history of social rejection, along with psychological problems, an interest in weapons, and/or a fascination with death. (p.311-314)

This exposition introduces the fact that she is in fact Dylan Klebold's mother. This evokes the element of ethos, giving her audience reason to find her credible in talking about the situation, giving context for the rest of the speech. This point is the start of events in her narrative arc, as well as pinpoints the Act and Agentsof her Pentadic rhetoric structure.

Pentadic criticism was created by scholar Kenneth Burke, which consists of "using the five basic elements of a drama—act, agent, agency, scene and purpose," as defined by Sonja K. Foss' book Rhetorical Criticism: Exploration and Practice. In this case, the Act being the Columbine massacre, and the Agents being Dylan Klebold and Eric Harris.

Klebold acknowledges the lives taken by the two boys, as well as those who's lives were permanently physically altered. At 6:42 of this video, Columbine survivor Sean Graves tells of how he lost his ability to walk due to the injuries he sustained from the shooting.

Her son's suicide, in her narrative arc, is Klebold's second event. Though it could be included as a part of the first event specified (the Columbine massacre), she makes an effort to separate it from the massacre as a whole.

This statement is fueled with intention to evoke pathos in the audience, attempting to relate to other parents in the audience and speaking on how often parents of teenagers cannot truly understand their child’s life outside of their home.

Klebold later continues this argument to promote awareness of mental health crises and criticize the ease of which it was for her son to obtain lethal weapons.

An ironically contradictory statement to make after stating that "...he was a completely different person from the one I knew."

Klebold continues to evoke pathos in her speech, showing the audience her vulnerability via expression of guilt for her son's actions.

With these opening lines, Sue Klebold easily evokes the element of pathos within her audience. Pathos is an element of Neo-Aristotelian rhetoric that appeals to the emotions of the audience. Being the mother of one of the Columbine shooters, it may have been more difficult if not impossible for the audience to attempt to sympathize or empathize with her if she had not opened with the element of pathos. The word choice of "darkness" and her son's brief farewell foreshadows a tragedy that she could not have seen coming.

Pro: BlogTo does a great job in ensuring that all their videos has a headline regarding the subject of their content as well as captioning all of their dialog. All captions are always timed accurately and legible.

CON: BlogTO fails to include alternate text on their images. As every blog post includes one if not more images that introduce or highlight subjects within the article, providing alternate text will allow images to properly communicate information for those that are unable to visually see the context of the images.

CON: The use of red font maybe distinguishable to the average person as an indicator of a embedded text-link, however this can be quite difficult for those that are colourblind (especially with red-type) as the text will look all the same. The use of more contrasting colours or by implementing the typical defaulted link underline, this could allow for a more accessible user experience.

PRO-ish but mainly CON: Although you are able to go through the navigation menu by clicking tab on the keyboard (the slight pro), the whole navigation system of the blogTO site is tucked away behind a small button that is only able to be hovered over with a mouse, making it unaccessible for those that need to use a keyboard alone. Due to it being hidden, it is also unknown how many tabs or its context there are within the navigation.

CON: Despite taking into consideration accessibility through close captioning, their videos fail to allow access to any user controls such as slowing down/speeding up the video, rewind/fast-forward or any custom preferences like the option to use automated cc's, descriptions or transcripts. Due to the quick nature of these videos that are originally made to fit apps like TikTok, not being able to slow down the video or even scroll through the timeframe makes watching the content extremely difficult for those that get easily overwhelmed or need more time to process so much information at once.

Cela dépend de la configuration clavier physique vs clavier entré dans les préférences, ensuite de l'ordinateur. Moi j'ai un clavier del qui montre les deux formes de balises et je n'ai qu'à les utiliser avec "Alt" et cela uniquement oarce que j'ai installé la configuration clavier "FRA (cdn multi)". Si je l'avais laissé en ENG d'origine la touche "Alt" est inutile; j'appuie direct. J'aurais modéré ce propos au moins signalant que cela dépend de nos réglages.

This situation highlights a broader issue within professional spheres—namely, the failure to provide inclusive and considerate support to individuals with disabilities and their families. Rather than offering constructive advice and assistance, the instructor's response seems to suggest a lack of willingness to engage with the complexities of the child's needs.

It's deeply disheartening to hear about the lack of empathy and understanding displayed by the professional who was supposed to offer guidance and reassurance to the author regarding her child. To be told that the professional "doesn't know how to deal" with a child who is described as "oppositional" and "developmentally delayed" feels like a profound betrayal of trust and a dismissal of the challenges the author and her daughter face.

Ultimately, this paper serves as a powerful call to action, urging educators and policymakers to reevaluate their approaches to supporting students with disabilities and to prioritize the creation of educational spaces that celebrate diversity, promote equity, and empower every student to reach their full potential.

This paper calls for a critical review of current educational practices to identify and address systemic barriers that hinder the inclusion and success of students with disabilities. It advocates for the implementation of strategies and interventions that foster a more supportive and inclusive learning environment for all students, regardless of their abilities or differences.

By shedding light on the limitations and implications of the "child with special needs" label, the document emphasizes the need for a fundamental shift in how we perceive and support students with disabilities. It underscores the importance of recognizing each child as an individual with unique strengths, interests, and abilities, rather than reducing them to a single label.

Moreover, isolation can hinder academic performance. Without a supportive peer group, students may struggle to engage fully in their studies, participate in class, or seek help when needed. The absence of a social support network can make school a daunting and unwelcoming place, further impacting a student's ability to succeed.

Belonging to a group helps students feel recognized and valued for who they are, which is essential for their self-esteem and mental well-being. Peer groups offer companionship, understanding, and shared experiences, which are crucial for personal development and resilience. These groups can provide support in times of stress, offer diverse perspectives, and encourage positive social behaviors, all of which are beneficial for a student's overall growth.

Finding a group is extremely important for students because it provides a sense of belonging, acceptance, and acknowledgment, contributing significantly to a safer and more supportive environment. During formative years, especially in educational settings, being part of a group offers emotional security and validation, helping students navigate the complexities of social and academic life.

A critical issue within our education system is the widespread misunderstanding and misapplication of laws and policies related to bullying and sexual harassment. It's deeply concerning that many teachers do not fully grasp the distinction between bullying and sexual harassment, particularly regarding peer-based harassment. This lack of understanding can lead to a failure to recognize and appropriately address instances of gender-based harassment, thereby perpetuating a hostile learning environment for students.

When educators are not equipped with the proper knowledge and training to differentiate between these forms of harassment, they cannot effectively intervene or support affected students. This oversight can allow harmful behaviors to continue unchecked, creating a school culture where students feel unsafe and unsupported. It's crucial for schools to provide comprehensive training for teachers and staff on the nuances of bullying and sexual harassment, emphasizing the specific dynamics of gender-based harassment.

Addressing this issue is not just about compliance with laws and policies; it's about creating an educational environment where all students can learn and thrive without fear of harassment. By ensuring that educators understand and can effectively apply anti-bullying and anti-harassment policies, we can move towards a more inclusive and respectful school culture. This shift is essential for fostering the well-being and academic success of all students, particularly those who are most vulnerable to gender-based harassment.

The fact that LGBTQ students who endure severe harassment are more likely to plan for higher education speaks volumes about their determination and resilience. Nevertheless, the detrimental effects on their academic performance and attendance cannot be overlooked. Schools must recognize the critical role they play in creating a safe and inclusive environment where all students, regardless of their sexual orientation or gender identity, can thrive.

The research findings also reveal the remarkable resilience of LGBTQ students. Despite facing increased harassment, those who are open about their identities often exhibit higher self-esteem. This illustrates the profound importance of authenticity and the strength that comes from being true to oneself, even in the face of adversity. However, the everyday experiences of isolation and harassment still take a significant toll, leading to lower grades and more absences among these students.

This passage poignantly underscores the personal toll that everyday acts of homophobia and transphobia can inflict on LGBTQ students. It's heartbreaking to consider that these students, who are merely trying to navigate the complexities of school life, often face isolation and harassment. These negative experiences can severely impact their attendance and educational aspirations, highlighting the urgent need for schools to be more supportive and understanding.

This behavior not only fosters unnecessary hatred but also deepens societal divides. Our fear of external perceptions drives this negative behavior, which ultimately harms everyone involved. It's crucial to challenge these attitudes and promote a culture of respect and acceptance, ensuring that no student feels targeted or unsafe because of their identity.

The 2019 GLSEN survey found that 60% of LGBTQ students had been sexually harassed in the past year. This statistic highlights a pervasive issue: the public often perceives LGBTQ students as weak or unable to defend themselves, which bullies exploit to target them. Such harassment is never acceptable, regardless of a person's sexual orientation or gender identity.

I strongly agree with this statement. Increasing awareness and education on LGBTQ topics empowers students to educate themselves and others, promoting a more inclusive and respectful school culture. It's particularly important for students who are exploring their identities but lack a safe space to discuss these issues. By integrating LGBTQ representation and discussions into the curriculum, schools can provide the support and visibility these students need, encouraging acceptance and understanding among all students.

Ignoring the issue of sexuality in school curricula means neglecting to provide LGBTQ students with essential representations of themselves, which are crucial for their self-understanding and development. This neglect also fails to offer examples of ways to counter bias and promote respect among peers who may initially be unwilling to accept LGBTQ individuals. Addressing sexuality in education is vital for fostering an inclusive environment where all students can see themselves reflected and valued.

This is a crucial statement because it reminds us that gender bias is a multifaceted issue affecting both genders. While much attention is rightly given to how gender roles belittle women, it is equally important to recognize that men can also feel misjudged and pressured by these roles. Addressing gender bias comprehensively requires acknowledging and addressing its impact on all individuals, regardless of gender.

The analysis points out that despite some progress towards more flexible gender norms, many schools continue to uphold outdated practices such as gender sorting and conservative sex education. These practices perpetuate traditional notions of gender and significantly affect students' well-being. For example, sex education programs focusing on abstinence until marriage often exclude LGBTQ students and fail to address crucial issues like pregnancy and reproductive responsibility, disproportionately affecting young women.

This passage effectively highlights the pervasive issue of gender bias, emphasizing that it impacts not only women but also men, especially young men in educational settings. Societal expectations of masculinity often compel young men to assert dominance through aggressive behaviors like taunting, driven by the fear of being perceived as weak or non-heterosexual. These behaviors reflect deeply ingrained stereotypes about masculinity and strength, underscoring the restrictive nature of traditional gender norms.

Thorne's work highlights the significant role educational institutions play in sorting and labeling individuals based on gender and sexuality. However, she also points out that within these institutions, elementary school students often challenge and reinterpret gender norms. This indicates that gender, as a social construct, can be viewed as a flexible and open-to-interpretation category. By embracing and encouraging this perspective, schools can become spaces where traditional gender norms are questioned, allowing for a more inclusive and supportive environment that reduces bullying and harassment.

The misinterpretation of the link between gender nonconformity and sexual minority status by peers, leading to increased harassment of transgender students, underscores the urgent need for better education and awareness in schools. This is a critical point, as understanding normative identities is essential for fostering an environment where each student's unique identity is respected. By enhancing educational strategies to improve connection and relatability among students, schools can effectively break down barriers and create a more equitable learning environment for all.

In Hispanic households, gender norms tend to be particularly rigid. Hispanic men are often pressured to embody a form of masculinity that can border on toxic, while women are expected to adhere strictly to traditional femininity. Deviating from these norms can lead to harsh judgments and being perceived as a disgrace to the family. This cultural context highlights the broader societal pressures that reinforce rigid gender roles, making the journey towards acceptance and self-expression even more challenging for LGBTQ individuals. Recognizing and addressing these entrenched norms is essential for fostering a more inclusive and understanding society.

Gender and sexuality are fundamental organizing categories in schools and society, deeply influencing everyone's experiences, including those who challenge these norms. Unfortunately, gender nonconformity and sexual minority status are often conflated by peers, leading to heightened harassment of transgender youth. This conflation underscores the interconnectedness of gender and sexuality, as LGBTQ students navigate their identities through both lenses, complicating attempts to separate these concepts.

This highlights a significant yet often overlooked point: the social construct of gender is so deeply ingrained that we unconsciously perpetuate gendered practices. Recognizing and addressing these behaviors, especially in our interactions with young children, is crucial for promoting gradual social change and deconstructing entrenched gender norms. By consciously avoiding gendered distinctions in educational and social settings, we can help cultivate a more inclusive and equitable mindset from an early age.

Reflecting on my elementary school experience, I recall how teachers often divided us into boys versus girls for games, creating an immediate sense of tension and competition to prove which gender was superior. These early experiences undoubtedly influenced me, fostering a competitive spirit and a strong sense of camaraderie among girls. Growing up surrounded by females, I became a staunch advocate for feminism and the importance of girls supporting girls.

The perspective that young children naturally perceive gender in a simplified way and are not deeply affected by gender differentiations in early education is an important one. Indeed, at very young ages, children are more.

Possible baselines: 1. re-ranking using QE 2. add noise to QE 3. Can we hill climb directly on QE? 4. All the baselines but with xIoD's demonstration examples

what happens when there's no difficult word?

QE score is used during the process, so this is not trustworthy.

but how are x and y decided?

how?

1. 更好的附件东方红郡地方规划局
2. 红烧鸡块
3. 收到会尽快

J'espérais carrément commencer par moi qui a besoin d'un site sur mesure qui se détache du lot. Je pense changer de métier, c'est pourquoi je suis ici mais je débarque du métier, justement, de la photo... J'avoue que je vais constemment doubler mon regard afin de faire mon propre site en parallèle à moins que ce ne soit tout simplement pas possible dans le cadre du cours. Ce serait dommage... pourquoi ne pas nous lancer carrément sur notre propre création pour nous-mêmes par le biais de ces exercies. Nous sommes censés savoir le faire à la fin du cours, non ?

The tile of the article is written in a larger font. This makes the tile perceivable to users; the information of the text is presentable. The title is also understandable; it lets the reader know that article is about Modi's political popularity in the span of 10 years.

intro survey

syllabus

welcoming everyone to achieve their full potential

connective tissue between students, engagement and rigor is an interesting reason for not using AI graders etc all the time, which are getting enticed to do.

humanizing online learning

You need tap the pencil icon next to "Single Posts" and enter editing mode and then find Options button at top-right.