This study looked at women in rural Wisconsin that were exposed to atrazine through well water.
Mcelroy, Jane A., Ronald E. Gangnon, Polly A. Newcomb, Marty S. Kanarek, Henry A. Anderson, Jim Vanden Brook, A. M. Y. Trentham-Dietz, and Patrick L. Remington. 2007. “Risk of Breast Cancer for Women Living in Rural Areas from Adult Exposure to Atrazine from Well Water in Wisconsin.” Journal of Exposure Science & Environmental Epidemiology 17 (2): 207–14.
runoff becomes part of slow violence when environmental exposure is linked to health risks.
Nixon, Rob. 2011. Slow Violence and the Environmentalism of the Poor. Cambridge, MA: Harvard University Press.
cancer risks, endocrine disruption, occupational exposure, and child health concerns over extended periods of time.
Hendryx, Michael, Jamison Conley, Evan Fedorko, Juhua Luo, and Matthew Armistead. 2012. “Permitted Water Pollution Discharges and Population Cancer and Non-Cancer Mortality: Toxicity Weights and Upstream Discharge Effects in US Rural-Urban Areas.” International Journal of Health Geographics 11 (1): 9. , Mcelroy, Jane A., Ronald E. Gangnon, Polly A. Newcomb, Marty S. Kanarek, Henry A. Anderson, Jim Vanden Brook, A. M. Y. Trentham-Dietz, and Patrick L. Remington. 2007. “Risk of Breast Cancer for Women Living in Rural Areas from Adult Exposure to Atrazine from Well Water in Wisconsin.” Journal of Exposure Science & Environmental Epidemiology 17 (2): 207–14. , New-Aaron, Moses, Zaeema Naveed, and Eleanor G. Rogan. 2021. “Estrogen Disrupting Pesticides in Nebraska Groundwater: Trends between Pesticide-Contaminated Water and Estrogen-Related Cancers in an Ecological Observational Study.” Water 13 (6): 790.
large region it is connected to via interconnected streams, rivers, groundwater, and wells.
Richards, R. Peter, David B. Baker, Nancy L. Creamer, John W. Kramer, David E. Ewing, Bruce J. Merryfield, and Larry K. Wallrabenstein. “Well Water Quality, Well Vulnerability, and Agricultural Contamination in the Midwestern United States.” Journal of Environmental Quality 25 (1996): 389–402
Once chemicals enter water systems, they will travel and in a manner that is difficult to trace as it is not damage that is visible to the naked eye
Richards, R. Peter, David B. Baker, Nancy L. Creamer, John W. Kramer, David E. Ewing, Bruce J. Merryfield, and Larry K. Wallrabenstein. “Well Water Quality, Well Vulnerability, and Agricultural Contamination in the Midwestern United States.” Journal of Environmental Quality 25 (1996): 389–402
ider regional water system
Bussan, Derek D., Clifford A. Ochs, Colin R. Jackson, Tarun Anumol, Shane A. Snyder, and James V. Cizdziel. 2017. “Concentrations of Select Dissolved Trace Elements and Anthropogenic Organic Compounds in the Mississippi River and Major Tributaries during the Summer of 2012 and 2013.” Environmental Monitoring and Assessment 189 (2)
production happens on a greater scale.
"Contaminants in the Mississippi River--Pesticides in the Mississippi River.” n.d. Pubs.usgs.gov.
protects crops and maintains high yield.
“Contaminants in the Mississippi River--Pesticides in the Mississippi River.” n.d. Pubs.usgs.gov.
water carries them off through agricultural runoff.
Richards, R. Peter, David B. Baker, Nancy L. Creamer, John W. Kramer, David E. Ewing, Bruce J. Merryfield, and Larry K. Wallrabenstein. “Well Water Quality, Well Vulnerability, and Agricultural Contamination in the Midwestern United States.” Journal of Environmental Quality 25 (1996): 389–402
Agricultural runoff is the result of water running over fields,
Richards, R. Peter, David B. Baker, Nancy L. Creamer, John W. Kramer, David E. Ewing, Bruce J. Merryfield, and Larry K. Wallrabenstein. “Well Water Quality, Well Vulnerability, and Agricultural Contamination in the Midwestern United States.” Journal of Environmental Quality 25 (1996): 389–402
Their rich literature was passed along from person to person and generation to generation through the oral tradition.
I think this is how Ebonics was developed into what the language is now.
Like jazz, which Ebonics has influenced along with hip-hop, blues, and other musical styles, however, Ebonics has its own particular take on the conventional American wisdom and it is a vital means for subverting that wisdom.
Ebonics will continue to be spoken and taught within the African American community.
Leon W. Todd, Jr., states, Ebonics is a politically correct term for dysfunctional speech. Legitimizing poor language habits will not help children find a job nor take control of their affairs later in life.
Scholars like Leon Todd may believe that SAE is the professional and correct English language that should be taught to students.
He notes that the current reality is that Standard American English (SAE) is the money language. Black English is identified with poverty and, therefore, holds a great stigma.
By making ebonics a second language this may diminish the poverty stigma that it encloses.
African American parents hold that their children suffer from linguistic problems deriving from structural differences in their spoken language just as Latino children do.
By African American children having the option of using Ebonics as a second language they have the opportunity to understand and comprehend Standard American English profoundly.
Teachers wish to use Ebonics to teach African American students so that they will perform better academically.
Educators using ebonics may allow student to significantly improve in their academics.
Ebonics is defined by Williams as a language spoken by African Americans that is not a variety of English but a separate language with its own mode of expression and rules of grammar.
Ebonics is spoken by African Americans to be able to express their form on the English language.
President Lincoln shook hands with me and said, “It is a good thing. What do you want?” I said, “I have a thousand men. We want to help fight to free our race. We want to know if you will take us in the service?” He said, “You have got good pluck. But I can’t take you now because you are contraband of war and not American citizens yet. But hold on to your society and there may be a chance for you.”
Why did Lincoln hesitate to enlist Singleton's regiment at first?
I took that five dollars and hired the A. M. E. Zion church at Newbern and commenced to recruit a regiment of colored men. I secured the thousand men and they appointed me as their colonel and I drilled them with cornstalks for guns.
Enslaved people didn't passively wait for the Union to free them. Singleton took his own money to recruit, organize, and drill 1,000 men with cornstalks. This proved that Black self organization created the forces the Union used.
This person you picked has something. A spark. A je ne sais quoi. That ‘something' is high agency.
Wow!
Table 5 shows that the AAE speakers and SAE speakers used nine of the complex syntax types in a similar manner. That is, all children, regardless of dialect, used simple infinitives with same subject, gerunds and participles, and the conjunctions and and because. The same number of children in each group used the let(s)/lemme and infinitive and wh-infinitive clause sentence types, but none of the children used tag questions or the conjunction since.
The similarity in performance of the two groups remained constant irrespective of the measurement unit. Thus, regardless of whether samples were analyzed by number of words per T-unit, number of clauses per T-unit, or number of words per clause, the performances of the groups were not statistically significantly different.
This study supports the findings of Craig and Washington (1994), who reported that children who spoke AAE used more complex sentence structures than children who spoke SAE.
In order to confirm this finding, further research with a larger sample needs to be done to determine if a group effect might exist for the words per T-unit and words per clause measures.
These results do not support Owen's (1995) finding that tasks that require a participant to give information will increase the words and clauses used per T-unit.
During the introductory questions phase, the children were asked six questions to which they responded based on their experiences and interests.
Table 1 presents mean raw scores and standard deviations for the performance of each participant group on the three speaking tasks.
Six samples were randomly selected and rescored by a certified speech--language pathologist using the complex syntax definitions outlined in Craig and Washingto
A Poisson regression was performed to determine if the AAE speakers and SAE speakers differed significantly in their production of specific complex sentence types.
There was no significant difference in the frequency of usage of the conjunction but (p = .0346) and the unmarked infinitive (p = .0994).
To determine whether a child spoke SAE or AAE, we employed the criteria used by Terrell and Terrell
To be included in the study, the Anglo American children had to speak SAE and the African American children had to use AAE as their native dialects
Specifically, this study attempted to answer several questions:
One such factor is the type of conversational task that is used to elicit the language sample.
Some tasks could be more difficult then others.
using criterion-referenced tests with no psychometric norms.
suggested that standardized tests should not be used to assess the language and cognitive skills of nonmainstream English speakers.
Should not be used to determine eligibility base off language ability.
"the groups were remarkably similar"
Johnson's hypothesis was found to be partially correct.
he stated that "Advantaged children will have higher scores on test measures of language than disadvantaged children, but there will be no differences between these groups on the spontaneous speech measures"
sed the T-unit to compare the language samples of 40 African American and Anglo American Head Start students age 4 years
(Quisenberry, 1975), achievement levels (Calvert, 1971), and grade levels (Klecan-Aker & Lopez, 1985). The results of these studies have shown that language samples collected from children of higher socioeconomic levels, higher achievement levels, and higher grade levels contained longer T-units.
compared the number of T-units used by children of different socioeconomic levels
defined the T-unit as a grammatical sentence containing one main clause and any attached or embedded clause or nonclausal structure.
family members and familiar peers can be enlisted to help obtain the sample, and the sample can be collected in an environment that is comfortable and familiar to the speaker of a nonstandard English dialect.
Help make the environment feel more comfortable and open
culturally appropriate materials and topics can be incorporated into the sampling procedure
indicated that language samples are valuable.
Can be useful in a natural environment
problem with language samples is that they are not always reliable.
collecting language samples in naturalistic settings
researchers have suggested two possible solutions: that existing tests be renormed on different ethnic minority groups or that test scores be adjusted for dialect-sensitive items
biased against nonmainstream English speakers
in order for children to qualify for federally funded special education services, they must take standardized, norm-referenced tests.
Must understand english or use translating devices to understand.
The process of assessing the speech and language skills of children who are culturally and linguistically different from the mainstream population,
Published in Database https://doi.org/10.1093/database/baag042
8 §4.1 / §4.8 Umbral normativo Las fuentes mencionan el umbral de 100 clientes (NCG 502) y, en minutas, referencias a 100 empleados/colaboradores. No queda claro si son gatillos distintos o una confusión de las minutas. Documento de orientación / matriz Minutas de cumplimiento Confirmar si 100 clientes y 100 empleados/colaboradores son umbrales distintos de la NCG 502 o un error de las minutas.
Respecto a este punto, debemos informar una novedad muy favorable que se generó durante esta semana. Tras una consulta realizada por Camila Bernat con abogados y expertos, se logró redefinir el criterio para el límite de 100 clientes al que estábamos a punto de llegar.
Nuevos Criterios de Exclusión Se concluyó legal y operativamente que, del cálculo total, se deben excluir dos grupos de la cartera:
Clientes a los cuales no se les ha presentado una propuesta comercial en los últimos tres meses.
Clientes que cumplen con la definición de "Cliente Calificado".
Al aplicar estas exclusiones, nuestra cifra activa se redujo drásticamente, pasando de 96 (cercanoa los 100) a aproximadamente 20 clientes. En consecuencia, la probabilidad de tener inconvenientes o bloqueos por alcanzar este límite durante los próximos años queda descartada, lo cual elimina este tema de nuestra lista de preocupaciones inminentes.
Finalmente, respecto a la mención de "100 empleados o colaboradores", consideramos que debe tratarse de un error. Actualmente, no tenemos ninguna referencia, métrica ni aspecto dentro del proyecto que enlace nuestras operaciones a un límite o grupo de 100 colaboradores.
7 §4.2 / §4.7 Presión de CPU y FIFO El feedback de revisión define FIFO como algoritmo de posiciones vigentes (la rentabilidad se deriva después). La documentación vincula la lógica FIFO a picos de CPU en ventanas matutina y nocturna. Comentario de revisión Documento consolidado de arquitectura Confirmar qué proceso genera la presión de CPU observada si FIFO no calcula rentabilidad en sí.
En nuestro sistema, el FIFO es un algoritmo diseñado para determinar la cartera vigente de cada cliente. Su función principal es filtrar las transacciones, aislando únicamente aquellas que afectan las posiciones actuales y descartando las que ya no tienen impacto operativo. A partir de este resultado depurado, se procede a calcular las rentabilidades, tal como se menciona correctamente en el punto 7.
La documentación vincula correctamente la ejecución de la lógica FIFO a picos en el consumo de CPU. Esto se explica por la forma en que está construido a nivel de base de datos:
El cálculo se realiza utilizando cursores dentro de SQL Server a través de procedimientos almacenados (stored procedures).
Al tener que iterar y procesar un gran volumen de información histórica (aplicando el FIFO por cada activo y por cada cliente), el consumo de CPU y memoria se dispara. Cabe destacar que el impacto es variable, ya que cuando se realizan ejecuciones parciales el consumo de recursos es menor.
El estrés sobre el sistema se hace especialmente evidente debido a los horarios en los que debe operar:
Proceso nocturno: Se encarga de actualizar las carteras de los clientes procesando la información de varias semanas hacia atrás.
Proceso matutino: Aproximadamente a las 6:00 a.m., inmediatamente después de la carga de precios, el algoritmo debe ejecutarse nuevamente para actualizar las posiciones del último día vigente. Es precisamente durante este bloque de la mañana cuando se registran las mayores elevaciones en el uso de la CPU.
6 §3.11 Cronología Passport El feedback de revisión sitúa el nacimiento de Passport en 2012; documentación previa indica 2008. Comentario de revisión Documento consolidado / orientación inicial Confirmar el año de puesta en producción de Passport.
Respecto al origen y los años de implementación del sistema Passport, es importante aclarar que su desarrollo histórico se divide en dos grandes etapas:
Passport V1 (Aprox. 2007 - 2008): Corresponde a la versión original que dio inicio al sistema.
Passport V2 (2012 - Actualidad): Es la versión vigente que opera hoy en día. Durante este hito, se consolidaron múltiples sistemas anteriores y se crearon nuevos módulos para robustecer la plataforma.
La diferencia arquitectónica más importante entre ambas versiones fue la construcción de un Data Mart durante el desarrollo de la V2 en 2012. Este repositorio de datos es el encargado de alimentar la información que se visualiza actualmente en el Factsheet.
Debido a esta evolución tecnológica conjunta, hoy en día existen diferentes versiones de ambos componentes, las cuales están estrictamente vinculadas entre sí:
El Factsheet V1 corresponde y está asociado a la versión original (Passport V1).
El Factsheet 2 (el actual) corresponde y se alimenta del Datamart construido para la versión vigente (Passport V2).
5 §3.4.1 / §3.8 Flujos CMF y AFP El feedback de revisión indica CMF → LocalVisionDB y AFP → FLUJOSAFP. FLUJOSAFP no figura en el levantamiento documental; LocalVisionGlobal aparece en fuentes previas con un rol distinto o ambiguo en estos flujos. Comentario de revisión Documento consolidado de arquitectura Confirmar destinos definitivos de CMF y AFP, el rol de LocalVisionGlobal y registrar FLUJOSAFP en el inventario de bases si corresponde.
Para comprender bien en qué consiste este punto, es fundamental distinguir entre los dos tipos de información que manejamos y sus respectivas fuentes:
Información de la CMF: Corresponde a los datos relacionados con los activos locales. Esta información se almacena en la base de datos principal LocalVisionDB y requiere una actualización diaria.
Flujos de Inversión de las AFP: Esta información se extrae de plataformas gubernamentales y se utiliza exclusivamente para la confección de un reporte mensual. Dicho documento detalla el monto actual que las AFP invierten en fondos específicos, así como su variación mes a mes. Se almacena en la base de datos FLUJOSAFP.
Es importante destacar que la gestión de ambas fuentes se realiza de manera totalmente independiente y convergen en bases de datos distintas. Dado que el seguimiento de los flujos de las AFP es un proceso secundario o de menor impacto, es muy probable que por esta razón no haya sido detallado en los reportes anteriores.
Finalmente, cabe mencionar que la base de datos local denominada "LocalVisionGlobal" no tiene un uso intensivo. Su propósito es netamente para fines de configuración del sistema, almacenar algunos datos de clientes, y no está destinada al almacenamiento de información operativa o histórica.
4 Roadmap / fases PETI La Matriz Ejecutiva prioriza PETI-10, PETI-11 y PETI-01 como Crítico / Fase 1. El Documento Único de Orientación Inicial presenta una secuencia distinta (automatización en Fase 2, hardware en Fase 4) y, además, su propio texto narrativo no coincide con su tabla de fases. Matriz Ejecutiva Documento Único de Orientación Inicial Confirmar la secuencia de fases PETI vigente y la prioridad actual de PETI-10, PETI-11, PETI-13 y PETI-01.
Para complementar la información, es importante considerar solamente la estructura de fases indicada en la matriz ejecutiva que fue entregada en el Data Room. A continuación, detallamos el estado actual y la justificación de cada iniciativa:
PETI 1 (En curso): Actualmente nos encontramos en la etapa de cotización del equipamiento. Este avance ha sido posible gracias a la asesoría que nos entregaron para definir cuál debería ser nuestra plataforma tecnológica óptima.
PETI 10 y PETI 11 (Fase 1): Ambas iniciativas se mantienen en la primera fase. Si bien son problemáticas que ya estamos gestionando y resolviendo en el día a día, todavía queda un trabajo significativo por completar para darles cierre.
PETI 13 (Fase 1): También conserva su prioridad inicial. Esto responde a la necesidad apremiante de simplificar los flujos de ingresos, actualizaciones y cuadraturas, ya que nuestro sistema actual ha demostrado ser ineficiente en estas áreas.
En línea con los desafíos levantados (especialmente por el PETI 13), el escenario ideal sería la implementación de un nuevo sistema integral que nos entregue las herramientas necesarias para gestionar de manera mucho más eficiente todos nuestros procesos internos.
3 §4.3 Cuadratura Vision vs. custodio El feedback de revisión describe la cuadratura como semiautomatizada (procesos que toman archivos de Gletir e Interactive Brokers y generan la cuadratura; Operaciones revisa y completa), con prioridad baja-media y frecuencia mensual. Documentación previa menciona un informe semanal automático. Comentario de revisión Documento consolidado / orientación inicial Confirmar el grado real de automatización y la frecuencia operativa vigente (mensual vs. semanal).
Creemos que existe una confusión respecto a la información presentada entre ambas entregas, generada principalmente al mezclar dos instancias que tienen propósitos y frecuencias distintas. Es fundamental distinguir entre el informe de estado y el proceso de ejecución:
1. Mail semanal: Estado de las cuadraturas La documentación que menciona un informe semanal automático se refiere exclusivamente a un correo de monitoreo. Este reporte semanal tiene como único objetivo mostrar el estado actual de las cuadraturas. Es una herramienta informativa que permite visualizar la situación semana a semana, pero no es el momento en el que se ejecutan las correcciones.
2. Proceso mensual: Ejecución de la cuadratura Por otro lado, el proceso de cuadratura y corrección formal es de frecuencia en el mejor de los casos mensual. Este procedimiento existe, funciona bastante bien en la actualidad y cumple con lo requerido. Se ejecuta una vez al mes, obteniendo los archivos de los bancos internacionales para cargar la información de manera semiautomatizada a través de un cargador implementado en el sistema.
En resumen, lo que se debe informar con claridad es que no hay que confundir el proceso operativo de cuadratura (que es mensual y semiautomatizado) con el correo semanal (que es puramente un reporte informativo sobre el estado de dichas cuadraturas).
2 §4.3 Reproceso por inconsistencias Lakpa La Matriz Ejecutiva (PETI-11) califica la carga de actividad Lakpa como Crítico / Fase 1, con nota "debe resolver la dependencia actual de personas para la interpretación de cartolas". El feedback de revisión indica que la prioridad "ahora es media" dado que existe una automatización. Matriz Ejecutiva PETI-11 Comentario de revisión Confirmar si la automatización implementada resuelve el problema estructural o si sigue siendo prioritario.
Es fundamental entender la diferencia y el alcance central de ambas iniciativas:
PETI 10: Se centra en el problema de la duplicidad operativa. Aborda los errores y diferencias generados al tener que ingresar la misma información dos veces (en los bancos o corredoras y, paralelamente, en nuestro sistema).
PETI 11: Se enfoca en la brecha de funcionalidades e información. Aborda las discrepancias que existen entre los sistemas y lo almacenado en nuestro sistema de carteras locales frente a lo que tenemos en el sistema de carteras internacionales.
El PETI 11 no es un elemento aislado, sino que se conecta directamente con otros puntos levantados:
Con el PETI 10: Ambos comparten el objetivo final de eliminar manualidad y mejorar la calidad de la información que se ingresa a nuestra plataforma, beneficiando directamente al sistema de carteras locales.
Con el PETI 06: Comparten una problemática idéntica, pero a nivel de propuestas. En ambos casos se evidencia que el sistema de carteras internacionales está automatizado, mientras que el de carteras locales sigue operando de forma manual.
Respecto a la diferencia entre lo indicado en la matriz ejecutiva original y el feedback posterior (donde el PETI 11 baja a prioridad media), la justificación es muy similar a la expuesta en el primer punto de esta tabla. Se recomienda mantener la criticidad en nivel crítico. Sigue siendo una necesidad apremiante nivelar nuestra tecnología para que el sistema de carteras locales alcance el mismo estándar y nivel de automatización que ya posee el sistema de carteras internacionales. Que aparezca Lakpa es solo un indicador de que es una parte de un problema mayor.
1 §4.3 Doble ingreso operación La Matriz Ejecutiva (PETI-10) califica la automatización de blotters como Crítico / Fase 1, con nota explícita de "eliminar duplicidad de ingreso de datos". El feedback de revisión indica que "no está el gran problema y tampoco genera grandes riesgos". Matriz Ejecutiva PETI-10 Comentario de revisión Confirmar con Vision cuál es la prioridad vigente y si la visión ha cambiado respecto a la Matriz Ejecutiva.
El objetivo principal del PETI 10 es eliminar el ingreso manual y la duplicidad de datos en la empresa. Aunque se menciona el Blotter por ser nuestro sistema principal para esta tarea, el problema abarca también otras vías de ingreso. La dificultad central radica en la duplicidad del trabajo: actualmente, el equipo comercial u operativo debe registrar una operación en la plataforma del banco o corredora y, posteriormente, volver a ingresarla en nuestro sistema interno.
El feedback reciente, alineado con los comentarios de Camila Bernat, destaca que ya estamos ejecutando iniciativas concretas para solucionar este problema:
Automatización con Lakpa: Tenemos un sistema de scraping activo que extrae datos de esta fuente y los carga en nuestro sistema de manera automática todos los días.
Integración con bancos internacionales: Utilizamos scripts en Python para procesar archivos de instituciones como Pershing e Interactive Brokers, lo que nos permite cargar información y realizar cuadraturas. Además, estamos gestionando la implementación de una API para automatizar completamente este flujo diario.
Las diferencias con las matrices originales se explican por el contexto de aquel momento: cuando se elaboraron, no contábamos con estas soluciones y existía una gran preocupación sobre cómo manejar las cargas de datos no estructurados. Hoy, el problema es mucho más manejable gracias a los avances mencionados.
A pesar de estas mejoras, se recomienda mantener el nivel de criticidad original, ya que sigue siendo un tema contingente. Si bien el escenario ideal sería eliminar por completo el uso del blotter y el ingreso manual, en la práctica siempre existirán situaciones excepcionales o límites que requerirán intervención nuestra.
A documented elicitation limitation.
Make this is a folding box
displayed submittable slider defaults, so some responses may be anchored or untouched.
Clarify this - the language here doesn't make it clear what you're talking about, and can't we just skip the ones where they did not move the slider?
Direct validation of the linear WELLBY against better-specified alternatives — per Benjamin, not yet done.
How would this be conducted - give at least a hint, perhaps in a tooltip?
would answer the question a
which is 'the question' ?
What would change this answer
"The answer" given is diffuse, hedged, and multipart. Which part of the answer would change because of these things?
evidence
Again, whenever you cite evidence, you should link or refer directly to it.
Do if you have budget or control an instrument
This discussion seems to ignore another point we have been covering in this conversation, namely the potential failure of cardinality of this scale. Shouldn't this also be mentioned?
Flag explicitly when a conclusion depends on the location of the neutral point.
This seems great, but it comes with not much explanation. This isn't well joined up.
Use directly measured life satisfaction wherever a trial reports it, in preference to a converted mental-health measure.
I'm missing a justification of this. Was this justified above, or is it a recommendation coming directly from one of the authors/participants?
nd publish sensitivity bounds
Give a little more hint as to how someone would publish sensitivity bounds or perhaps a link we're referencing explaining how to do this.
1. The decision this is about
This needs more background. You give a brief explanation of the relevance of the question, although more links would be helpful, but you haven't explained the context in a way that makes this page work by itself. What is this individual page meaning to do? How is it based on evidence coming out of our Pivotal question our evaluation and our workshop?
Ratios move even when coefficients do not
That's a confusing overstatement. For the ratios to move, the coefficients also have to move. I believe it's just that they're much more sensitive.
instruments
What do you mean by "instruments" here? I guess you're talking about the set of questions used to measure well-being, etc., such as the Cantrell ladder or a set of depression and anxiety questions? But I really don't understand the implication here. Yes, I see they overlap and they are not completely nested, but what's the relevance of that? You need to complete the argument, even if it means using more space, which you can be judicious with, using tool tips and folds.
SDs inherit the population you measured them in A standard deviation is a property of a sample, not of a person.
I don't completely understand what the implication of "a property of a sample, not a person" is. What's the relevance of that?
Mechanically, comparisons will tend to favour interventions run in more heterogeneous populations.
I don't see this, so obviously. Will these interventions not be more noisy in such populations, in line with the greater heterogeneity? But also, I don't even understand directionally what you're talking about here. If the population is homogenous, then there will be less underlying variation. If some intervention has a sort of unit-level effect, it will be recorded as being more impactful in more homogenous populations, not more heterogeneous populations. Consider this more carefully. Explain it better, including possible tool tips and folds for making the explanation complete, and also source this to and link a page or paper that makes this point explicitly.
2. Bottom line
You can, to the extent that this is indeed justified, keep this formulation without putting in all the references I mentioned below, as long as you give a major caveat and signpost that these are explained in sections below - which I wouldput hyperlinks to.
The reason for the SD step is data, not theory.
We write this in a more normal way, not as "not this but that."
income-doubling to lives-saved, lives-saved to DALYs — then backing out the remaining si
I don't completely see how this relates to the SD-SD method?
its conversion
"Arrived at it's WELLBY/DALY conversion"
Founders Pledge raised these questions with us through our Pivotal Questions programme;
Hyperlink this backgtound here.
3. What this is based on
Good to know what your sources are for this, but it's not linked narrowly enough that I can understand what is attributable to what: - what's attributable to evidence - what's attributable to careful arguments - what's only one person's claim, etc.
cheap fixes are known
That's a bold claim. I'm not sure how "cheap" these would be in practice. I think you should hedge this claim and provide evidence for it.
ny single global conversion factor therefore systematically mis-weights domains
I don't see how the "therefore" follows here. This is underexplained.
Disability weights top out around 0.3, while depression and anxiety show losses above a full life-satisfaction point.
Same comments here - you need to give sources for everything, but also I don't quite understand what it is you're talking about here. Ok, it's kind of understandable, but it needs a little bit better explanation. What are the "conditions" we're talking about, etc.?
The likely error is directional, not symmetric. Compression and ceiling effects both push toward overstating the wellbeing gains from mental-health interventions relative to physical ones. A conversion factor that is wrong in a known direction should not be treated as noise.
What's the epistemic basis for this - is this your own thinking? Is this something that was explicitly claimed in the workshop, reflecting a peer-reviewed paper etc? Again, we need to reference these things.
han a near-1:1 point estimate implies
You're not providing context here. Where is this near one-to-one point estimate that you're talking about coming from? (I know, but you should reference it and link it, etc., perhaps with tool tips with embedded hyperlinks, and.or folding boxes
Do not describe it as established.
Do not describe what? What is "it" referring to here?
and
and --> but
The SD-to-SD step is defensible as a working assumption, and it is nobody's preferred method — including its originators'. Keep using it; report it as an assumption; run sensitivity analysis. Do not describe it as established.
This is a recommendation without any evidence presented. It's not reasoned transparently. You need to justify this or explain that it will be justified below and link that.
The question funders ask us is not "is subjective wellbeing valid." It is: can I keep using this step, and how wrong might it make my ranking?
This is your AI. Not this, but that slop. Make it more like my own language and avoid using this bold all the time.
Founders Pledge's moral weights use it. Happier Lives Institute's cost-effectiveness work generates it. Anyone reading either inherits it.
Link citations/references to this, please.
Almost every such conversion in current use
We only have two listed. That seems like an overstatement to say "almost every"
one standard deviation of improvement on a depression or anxiety instrument as one standard deviation of improvement in life satisfaction
avoid this excess bolding
means
"that often means" -- not always
Violência Institucional
Vitimização Secundária (Revitimização) Resposta Legislativa no Brasil
No cenário jurídico brasileiro, o debate sobre as teorias da revitimização impulsionou importantes marcos de proteção:
Lei nº 13.431/2017 (Depoimento Especial): Criou salvaguardas para que crianças e adolescentes vítimas de violência sejam ouvidos em ambiente acolhedor, por profissionais especializados, evitando a reiteração da oitiva.
Lei nº 14.245/2021 (Lei Mariana Ferrer): Alterou o Código de Processo Penal para vedar expressamente a utilização de linguagem ou material que ofenda a dignidade da vítima ou questione sua vida pregressa durante audiências de instrução.
Lei nº 14.321/2022 (Violência Institucional): Tipificou o crime de violência institucional na Lei de Abuso de Autoridade (Art. 15-A), punindo o agente público que submeter a vítima de infração penal ou a testemunha a procedimentos desnecessários, repetitivos ou hostis que a façam reviver a situação de violência.
supporting K-12 schools to implement coherent instruction and colleges and universities to holistically meet the needs of their students is among the hardest, most necessary work in education
Gates Foundation: "holistically meet the needs of their students" which makes me think about - Persistence - Quality (getting the learning they came to get) - Relevance (WIL) - Advising + support services - Community (belonging, mentors, friends) - Purpose + Meaningfulness (it's what learners want and it's what motivates people)
Connecticut’s policy has the explicit intention of strengthening the parent-child relationship (Dutcher 2021). The state requires that at least two persons visit an incarcerated person and one must be the incarcerated person’s child (under age 18). The second person may be the incarcerated person’s spouse or parent and/or the child’s legal guardian. If an incarcerated person does not have a minor child, they cannot participate in the program.
I appreciate this information as it clarifies why incarcerated individuals cannot participate in the program if they do not have a minor child.
Sources : California Department of Corrections and Rehabilitation (n.d.), Dutcher (2021)
Very insightful information. I think students will appreciate this table. It will generate considerable discussion.
"Personal Wiki" just a Knowledge Graph in disguise?
Yes
WE called it 'wikiNizr
as in wikilike Organizer of all yOur individual and collaorative mutual learning
building knowledge that compounds -- and the graph layer is what makes it scale.
yes but what's in a name, sat's in a link, Are te critical questions
Cyc was right with atomic formulas for capturing trails
But instead of trying to create standard, create homiconic ways to create extend revicsse notations that drives the creation of aormulas
stop using tokens to "discover" relationships and start using them to reason over them.
especially because realationshis of real interest and importance tend to be not smal workd graphs bt 10 or even 10s of hops complex long trais simply composed
In fact learning advances when large separte graphs are joined in meaninful ways
10k+
using mndgraph and IPFS there are no efficiencey bottleneck
In fact the entire grah that connects people documents named interest itsef is interplanetary
shared ed by the participant individals, they have a complete record of all their interactions and own creation wit story and provenance and all is just Out There limitless scaling yet eerything 1 engaes with one has acces to the entire complete graph
By the judicious use of pining services and infrastucture like Peergos availability can be ensured
consistency is guaranteed by the way informatio is echanged
changes are always made only a given machine nrpwser that is actively i use at the moment and as one withes devices, and all the information created on a evice ar published on IPfsor Peergos or anu other drive soution emplued
Oriinally Minddive as piloted using a round trip nbetween goolde Docs and at the time wik\nizer and its Mind#graph
links
wih TeailMakrs notation yu can ut anything into the lines
The inks themselves are cyc like atomic formulas with a siple selfdocumented syntax and working 2NOTATION PEODESSO which iteslf is extensibel and ever coevolvable
Since in Indywiki the Maker App for the IndyWeb all concens are integral and omnioptional, so you will never be locked into any conceptsion
The whole thing is designed with making radical cahnges easy and cheap.
This applies to the very means that the notation orchestrates
Since the interpay between authors rely exhanes of all that is needed to change one thing to another named version
future proof forward compatiility can be ensured/
Personal Wiki just a Knowledge Graph in disguise?
It is a learning graph
freed from the limitation of knowledge graps as currently envisaged which are but propositional trails
the great thing about the origina ida of the wiki that it is by nature always in complete and informal
git repo of markdown files
can do better tha git, Not only willthis be dcentralied and local first
but communicae bakc using diffs
heavy bookkeeping
By construction and the imliate order imposed by using the traiMaks notation would result in transparent automatic two way linking ofpages
as pages themseles are first class interplanetary accesibnle information onjects
and they are class free and any typing qualifying, costraints are named and constructed by users or euse existing trailmakrs metadesin open soure and open sauce constructs, that are incorporated into the sapce of everyne who acceses them from known sources wit ful provenance of the the origin and co-evokutian of ak etalevel ideas
which themselves are autopoieti and homoiconic, in the sene that pages formulating what a givn network of trailmakrs mean or able to produce are themselves created and organized around trailmarked pages which in turn can ink and run javascript snippets that are themselves art of a subnetwork and can be hooked in
le good answers back as new pages
Any LLM Wiki Page, like any other page on te eb is amenable to be Indywikifyication.
Agreat anitdote to enshittification
Or indeed could e etended to support deep interplay
given that the Markdown fies that are organied by an LLM wiki are, if I understand correctly, are local first
But even if tthey would not be suitable two way dta exchange mechanism could be developed and once develoed addd to the etensible Indywiki
markdown pages
by construction can be turned into HTML pages as a uniersal document exchange format Turn them or any named part into a wiki page and
and turn them into trailmarked structured pages
LLM reads
sted of using LLMs so AI assisted web searhes for significant salient searh terms
and use these comination of words as part of new wiki pages to be assoiated with Searhes
annotate innotate link new wiki pages as needed
and weave your own learning trails
ones that will never faded and since they are incorporated into omni present personally attributed wiki pages people can readily reference and link to them and engage in conversations with the attributed author
take a copy of any wiki page into their own work spaces back links to the source pages for these personal copies are maintined and bot parties have verifiale provenance of sharing and a way to engage in conversations around the immediate neighbourhood te focal page and even create new shared wiki paes as starting points to named threaded conversations
The wiki is multiplayer and every page can be a starting point for a community of colaborators
Zero durable memory. No accumulation.
Each individual human actor through these interation become part of a network of collaborators or just author audience networks
everything is saved on IP#fs so it is permanent, unstopable, unenclosable
as access is only provided through interpersonal pper to peer recorded channes with full history of all proposed changes and additional elaorations
When you add a new source
Instead of leaving it up to the LLM annotate and Innotate them using trailmaks named wiki pages link tem along named links that use TailMakrs atomic formulas to make explicit the intent prpsoe role of theassociation at least going one way, and rely on automatically generated and maintained back links, which as approriate an be again named intentionally
a structured, interlinked collection of markdown files.
Flip THAT
give people the ability to create WYSIWYN
What you See IS What You Need to do with ideas to be captured
resources s.o be made sense of
using a Mark In notations that can be used to name and formulate the intentional/meaningful structure implicatd by the text 1 reads or write!
Personally archie complete web pages of selected parts
highlight any contiguos block of text and associate wiki pages name them with TrailMarks notation and blaze trails across the content ths created contextualize them and and capture them in a two way linked intentionall named blocks that can not only e highresolution addressable but transcludable and outcludable in theor own named wiki pages fragments
Have the ability to designate neighbourhood of trails and generate virtual hyper documents and or Wiki Pages
LLM incrementally builds and maintains a persistent wiki
M maintains a wiki
the LLM is rediscovering knowledge from scratch on every question. There's no accumulation
LLM rediscovering from scratch
no accumulation
document-to-knowledge graph system
That was the round trip from Google Docs creted using TrailMarks Mrk-in intentional notation back in the late wikiNizer Days ~ 2014
using MindGraph that handled Proporisional Trail very well, but it was clear that what is required as a sweet spot was more like what Vannevar Bush's wrote about in "As We May Think" the new professions of TrailBlazers would be able to make sense of associative trails as they were to organie their learnings in form taat was ready to share, and perhaps co=laborate.
That is what IndyWiki Brings to any web page on the Web
involvement and belief.
I recommend individual paragraphs for "involvement and belief" to maintain consistency.
The team forfeited its season after it came to light that seven players were actually ineligible to play due to their low grades (NBC 5 Chicago 2014). The Chicago Sun-Times conducted a broader follow-up investigation in 2016. The newspaper’s investigative team found that the Chicago School District did not discipline coaches for failing to submit incomplete, inaccurate, or no documentation about the academic performance of their student athletes (Chicago Sun-Times 2016). In this lax environment it is easy to see how a coach could find it more beneficial to not follow or enforce policies. The potential reward would include maintaining or improving their reputation and employability as a winning coach and their school’s status in a sport.
Great example. I think this will resonate with students.
No había habido otra opción para la tendencia revolucionaria del peronismo que Montoneros. Si ellos succionaron todas las organizaciones de base entre el 73 y el 75, ¿para qué iban a cambiar la política si todo el mundo quería militar en Montoneros? Había una vieja bronca en el pueblo peronista. Que a Aramburu había que ajusticiarlo, hasta el más pacífico lo pensaba. Y Montoneros funda su origen sobre ese hecho. En el origen estaba la concepción que la guerra era lo único que podía vencer al enemigo, y en el final también.
Key point
tracer-bullet
Substack writers, you need a website!
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
Note to readers on BioRxiv: In this response to reviewer comments, we have drafted some display items to illustrate the preliminary analyses that we have performed. If the reader sees a reference to a display item and wishes to view it in its original format, please see the original revision response PDF file. Our response begins below:
Manuscript number: RC-2026-03518
Corresponding author(s): Tijana, Ivanovic
General Statements
We first provide the general statements from Reviewers #1, 2, and 3 to give a holistic view of their impressions of the paper. Afterwards, we describe our plan to address their comments and the structure of this document.
Reviewer #1(Evidence, reproducibility and clarity (Required)): Planitzer et al. have developed a clever flow cytometry assay to monitor hemagglutinin (HA)-mediated membrane fusion. This novel approach provides several advantages over bulk and single-particle fusion assays, including dramatically higher throughput and detection of lipid mixing under low efficiency conditions. As such, on purely technical grounds, this manuscript provides a significant advance to the field. The authors then apply their new approach to probe the role of HA-sialic acid binding in the membrane fusion reaction. This reflects an important open question in the field, which, as the authors point out, has been elusive. They conclude that sialic acid binding promotes efficient membrane fusion, probably by increasing the likelihood of the fusion peptide engaging the target membrane after acid-induced conformational changes. Overall, the manuscript is very well written; the data are rigorously analyzed and nicely presented; and the results are important and informative. My comments are, for the most part, minor.
__Reviewer #1 (Significance (Required)): Planitzer et al. have developed a clever flow cytometry assay to monitor hemagglutinin (HA)-mediated membrane fusion. This novel approach provides several advantages over bulk and single-particle fusion assays, including dramatically higher throughput and detection of lipid mixing under low efficiency conditions. As such, on purely technical grounds, this manuscript provides a significant advance to the field. The authors then apply their new approach to probe the role of HA-sialic acid binding in the membrane fusion reaction. This reflects an important open question in the field, which, as the authors point out, has been elusive. They conclude that sialic acid binding promotes efficient membrane fusion, probably by increasing the likelihood of the fusion peptide engaging the target membrane after acid-induced conformational changes. Overall, the manuscript is very well written; the data are rigorously analyzed and nicely presented; and the results are important and informative.
__
Reviewer #2 (Evidence, reproducibility and clarity (Required)): This manuscript by Planitzer et al. investigates how NA activity, HA receptor binding avidity, and the identity and density of sialic acid receptors within the target membrane influence membrane fusion. This is an important question, as functional balance between HA and NA drives the antigenic evolution of seasonal influenza viruses and contributes to the pandemic potential of zoonotic viruses. As discussed in this manuscript, prior studies of how sialic acid receptors contribute to fusion following initial viral attachment are relatively few, and these have frequently reached different conclusions. Many of these discrepancies are likely attributable to details of the experimental system used to address the question, highlighting the importance of approaches in which the receptor identity and density are precisely controlled. Within this context, the sensitive and quantitative method the authors have developed to measure the efficiency and kinetics of virion fusion using flow cytometry ('spCALM') stands out as an important contribution of this manuscript. Using this approach, the authors convincingly demonstrate that engagement of HA with sialic acid receptors during the fusion process promotes efficient fusion, and that conditions that disrupt this engagement (low receptor densities combined with NA activity or receptor-HA mismatch) lead to reduced fusion efficiency by reducing the probability of fusion peptide insertion. Overall, the methods and findings of this paper are a strong contribution to the field and will be of interest to researchers studying viral membrane fusion as well as HA-NA balance. The manuscript is well-written, and the figures are very clear. The supporting figures provide additional context that is valuable. Addressing the comments below would strengthen the manuscript further.
Reviewer #2 (Significance (Required)): This study establishes a powerful experimental approach to quantitatively dissect fusion between influenza viruses and target membranes across a wide range of experimental conditions. This approach represents a significant methodological advance and its high throughput allows the authors to quantify rare events. Additionally, the manuscript presents convincing data showing that, within the regimes tested here, sustained receptor engagement promotes membrane fusion by increasing the likelihood of fusion peptide insertion. The observation that this is reduced by NA activity at low receptor densities carries implications for HA-NA functional balance.
While the specific findings may be restricted to the range of experimental conditions tested here, the results will be of interest to researchers studying virus-membrane fusion and functional balance between the influenza virus glycoproteins.
__Reviewer #3 (Evidence, reproducibility and clarity (Required)): Summary: The manuscript by Plamitzer et al. investigates influenza A virus hemagglutinin (HA)-mediated membrane fusion using bulk flow cytometry analysis. In the assay, they use red blood cells and liposomes carrying defined, variable densities of sialic acid residues. They used H3N2 and H1N1 model viruses with spherical morphology. The liposomes were produced using double-stranded DNA to attach sialic acid. Similar studies have been done using DNA-tethers to study membrane fusion between liposomes and influenza virions (PMID: 35143209, PMID: 27410740). Some previous studies have shown that reduced HA binding does not influence membrane fusion, while others have shown that binding increases membrane fusion. This work aims to reconcile prior conflicting observations. Based on their results, they conclude that HA-receptor binding avidity and the extent of neuraminidase (NA)-mediated receptor cleavage directly influence lipid mixing, which is used here as a proxy for membrane fusion and allows monitoring of the first step of fusion, namely the hemifusion intermediate. The results of the test whether receptor binding modulates the HA extended intermediate should be interpreted with more caution. The manuscript would benefit from more data on the characterisation of liposome carrying dsDNA sialic acid receptors and from using the TIRF SBP method to validate the flow cytometry.
__
__Reviewer #3 (Significance (Required)):
__
__Significance: The study attempts to answer an important question and is of interest to the membrane fusion field and the virology field. It provides flow cytometry to study membrane fusion and a method for DNA-based sialic acid receptors that can be displayed at control density.
Limitations and Advances: 1) Flow cytometry is a high throughput methods which allows to analyze many conditions. Recently published methods on flow virometry are promising methods that allow to study viron morphology. However, it might not be suitable to address specific mechanistic questions of membrane fusion to the molecular details presented in the model and discussed in the discussion. Overall, TIRF microscopy and SBP (Fig S6) present a much more accurate method than flow cytometry as it allows to study individaul events and provides additional information on virus size, fluorescence signal etc. In addition, single-molecule FRET methods could be used to monitor HA conformational changes at varying concentrations of sialic acid receptors. Previous studies have already shown that binding to sialic acid allosterically regulates HA2 conformational dynamics (PMID: 29961575). 2) A limitation is that only lipid mixing is monitored and no full fusion. Hence, one cannot conclude that all lipid mixing here leads to a full fusion pore opening. This should be addressed in the manuscript or at least noted as a limitation noted in the manuscript as a limitation.__
We thank the reviewers for their careful evaluation of our manuscript and for their thoughtful and constructive comments. We are encouraged that Reviewers 1 and 2 recognized both the methodological advances afforded by the spCALM assay and the importance of the biological insights into the role of HA-receptor interactions in influenza membrane fusion. We also appreciate the critical perspective of Reviewer 3, whose comments prompted us to more carefully articulate the capabilities and limitations of the assay, clarify aspects of its implementation that may not be immediately apparent to readers more familiar with conventional bulk fusion assays or supported planar bilayer/TIRF approaches, and consider alternative mechanistic interpretations of our findings.
Overall, we believe the reviewers have identified several opportunities to substantially strengthen the manuscript. Briefly, prompted by Reviewer 1 and Reviewer 3, we have developed improved analyses of virion recovery, binding, and liposome occupancy that refine our assessment of virion attachment, reveal previously unrecognized nonspecific particle losses, and will provide more rigorous quality-control metrics throughout the study. These new analyses also strengthen the inference that receptor interactions similarly influence both virion attachment and the lipid-mixing efficiency of bound virions, although the two processes exhibit distinct quantitative dependencies across multiple experiments. In response to Reviewer 2, we will extend the experimental parameter space by attempting measurements at higher receptor densities and with longer DNA receptor linkers. If these experiments cannot be completed successfully, we will better define the experimental regimes over which our conclusions are expected to apply and discuss the corresponding limitations. In response to Reviewer 3, we will revise the manuscript to more clearly describe the single-particle nature and unique advantages of the spCALM assay, explicitly discuss alternative mechanistic interpretations of the receptor-density dependence, attempt experiments to distinguish between competing mechanistic models, and evaluate the effects of soluble receptor on binding and lipid mixing to further probe the role of receptor engagement during membrane fusion.
In summary, we believe all reviewer comments can be satisfactorily addressed. The revised manuscript will include new analyses, expanded discussion, clarification of the experimental methodology, and even new experiments where feasible. Together, these revisions will strengthen both the technical presentation of the spCALM assay and the mechanistic conclusions regarding the role of receptor engagement in regulating influenza virus lipid mixing.
We have organized our responses to reviewer comments according to the Revision Plan template, separating planned revisions (Section 2), revisions that are already complete (Section 3), and revisions which we prefer not to carry out (Section 4). Within each section, we present our responses in order from reviewer #1 to reviewer #3 in the original order that they appear in the compiled reviews document. We have included line numbers in this revision plan to indicate where text revisions can be found in the (partially) revised manuscript and have tracked those changes in Microsoft Word. We also cite several publications in our responses and have included a list of references at the end of this revision plan.
Description of the planned revisions
Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.
Reviewer #1: “In the spCALM assay, the authors indicate that labeled virions were incubated with cells or liposomes at a ratio of "0.15 virions per target". How were the numbers of virions determined for these experiments? The Methods refer to MOIs in units of PFU/cell but do not indicate how the number of physical particles were determined.”
Thank you for pointing out this omission. We have for now extended our description of the “Viruses and reagents” subsection of Materials and Methods in lines 457-463 to refer to our previous study describing how physical virus particle and vesicle counts were determined.
However, since this is a relatively new method, in the revision we will add supplemental panels to Figure S1 to show how we count free virions and assess whether they are monodisperse and uniform in both size (mostly spherical) and R18-dye incorporation by flow virometry. Then, these figures will also be referenced in the Methods.
Reviewer #1: “The authors indicate that the MEDI antibody "increased binding across all tested conditions." But this was not mentioned again. By what mechanism do they expect the antibody enhances virus binding?”
We thank Reviewer #1 for prompting us to reevaluate this unexpected observation. Upon additional analysis, we found that our original interpretation was incorrect. Rather than increasing receptor-mediated attachment, MEDI increases the recovery of virions in our assay, including both free and liposome-attached particles, while the total liposome concentration remains unchanged (Display Item 1A,B). Consistent with this interpretation, the fraction of virions bound to liposomes is not meaningfully changed by the presence of MEDI (Display Item 1E). We therefore infer that MEDI reduces nonspecific losses of virions, likely through decreased adsorption to plastic surfaces, rather than enhancing receptor-mediated binding. We have added a mention of this point in lines 314-317 and will incorporate these additional analyses in the manuscript revision.
Display Item 1 caption: MEDI reduces virion losses but does not improve virion attachment. In the experiment presented in Fig 5C and S15, MEDI8852-Fab (MEDI) increased the fraction of liposomes with bound virions because it reduced particle losses. We extracted total virion counts (A), total liposome counts (B), the ratio of total recovered virions to liposomes (C), the percentage of liposomes with bound virions (D), and the percentage of virions bound to liposomes (E) before pH drop. MEDI increased total virion counts (A) but did not affect total liposome counts (B). This increased the actual virion-to-liposome ratio in MEDI relative to no-MEDI condition (C). By instead analyzing attachment efficiency as the percentage of recovered virions which are attached to liposomes (note the liposomes are always in access) (E), we control for differential particle loss with or without MEDI and find no meaningful difference in virion attachment efficiency in the presence or absence of MEDI, as expected. Error bars indicate standard error of the mean.
While investigating this effect, we also identified an error in our description of a subset of the experimental conditions. Specifically, the experiments shown in Figs. 5A and 5B were performed using 0.5 virions per liposome rather than the previously stated ratio of 0.15. This higher input was intentionally used to increase the number of bound virion-liposome pairs under conditions of very low lipid-mixing efficiency. We will correct the manuscript to accurately report these experimental conditions. Importantly, this did not increase the number of virions bound per liposome (see our response to reviewer 3 #2, Display Item 3).
Finally, these analyses prompted us to systematically evaluate particle recovery and liposome binding in other experiments in the study (Display Item 2, Display Item 6). We found that low-input virions are subject to nonspecific adsorption losses, explaining why the observed fraction of bound liposomes is often lower than expected from the nominal input ratio (Display Item 2). We will incorporate these new particle recovery and liposome binding analyses into the revised manuscript and extend this systematic evaluation across all experiments.
Display Item 2 caption: Particle recovery and attachment analysis for the experiment presented in Fig. 4B. From events detected before pH drop, we derive particle recovery metrics, including total virion counts (A), total liposome counts (B), and total virion-to-liposome ratio (C), as well as attachment metrics, including the fraction of liposomes with bound virions (D), the fraction of total virions attached to liposomes (E).
Reviewer #2: “Related to my first point, a tradeoff in the approach described here is that the observations may be specific to the regime that is being tested. Any effort to expand the range of conditions tested would therefore be valuable. For example, the authors discuss prior work at higher receptor densities (line 364); it seems that this higher-density regime could be evaluated here for direct comparison. Similarly, it is interesting that the authors do not observe differences when they use 12nt vs 24nt for the display of sialic acid on DNA tethers. I would imagine that sustained receptor binding could be detrimental to fusion peptide insertion only if it occurs at too large of a distance from the target membrane. It seems likely that the distances involved here (~4nm vs. 8nm) may not be sufficient to observe this, but testing larger DNA anchors seems feasible. If not, additional discussion of contexts where the findings described here may no longer be applicable would strengthen the manuscript.”
Thank you for this valuable suggestion. We agree that extending the range of receptor densities and spacer lengths would strengthen this manuscript by more directly comparing our result with previous studies and by testing our proposed mechanism at the high end of receptor densities. We therefore plan to extend our analysis to receptor incorporations of at least 5 mol% and to compare lipid mixing using receptor mimetics displayed on 12 (+4.08 nm), 24 (+8.16 nm), and 32 (+10.88 nm)-nucleotide oligo linkers.
If these experiments cannot be completed successfully within the revision, we will expand the Discussion to more clearly define the range of conditions over which we expect our conclusions to apply and where they may not.
Reviewer #3: “1) The conclusions are drawn from a flow cytometry, which allows to analyse samples in bulk. On one hand, it allows analysis of a large number of fusion events; on the other hand, it cannot resolve individual events or the relationship between binding and membrane fusion in individual binding states. The flow cytometry method used in the manuscript should be validated against single-particle fusion events in microscopy-based assays, which would also allow investigation of both the binding area and fusion (as done in the Fig S6). Here, authors could evaluate the effect of neutralizing antibody, NA inhibitor or the presence of soluble sialic acid pretreated virions at receptor concentrations higher than 16%.”
Thank you for revealing this potential point of confusion. Fundamentally, flow cytometry does measure individual particles which are sampled from a particle suspension; from this, we derive the distribution of liposome-bound virus particles that have undergone R18 dye dequenching within discrete time intervals. It is true that we do not track a single virion from binding to fusion, but we instead measure the distribution of single virions (in virion-liposome pairs) in sampled time bins. In other words, this is not a single-particle tracking experiment (which SPB/TIRF is), but it is nonetheless a single-particle experiment well suited to derive single-particle kinetics and especially efficiencies under attenuation. To address this reviewer’s comment, we will improve our description of this assay to make sure this distinction is clearly made in writing.
We have developed this novel assay after much experimentation using SPB/TIRF, our key area of expertise (Ivanovic, Choi et al. 2013, Li, Li et al. 2021, Li, Li et al. 2022) and realizing that this more standard approach is critically limited. Some of these limitations can be gleaned from the presented results as follows. Based on the decreases in particle attachment that we observed on supported planar bilayers with 16% receptor saturation with TIRF microscopy in Fig. S6, it would be impractical to evaluate differential effects on lipid mixing from receptor binding when receptor saturation is further reduced. In the spCALM experiment presented in Fig. 4B, which tests lipid mixing by the same virus strain used in the TIRF experiment presented in Fig. S6, the 6’SL receptor did not reduce lipid mixing efficiency until receptor saturation was decreased to values lower than 8%. Therefore, we are unable to assess the relevant receptor saturation regimes using TIRF microscopy; we expect that other treatments which influence receptor binding will likewise restrict virion attachment too severely to be practical. Finally, diffraction-limited optical microscopy methods do not provide any more information on virion-target membrane contact area than our flow cytometry-based method.
We presented several layers of validation of our assay and summarize them in order. Attachment of PR8 virions to erythrocyte membranes is inhibited by the neutralizing antibody SbH36-26 in the expected concentration range (Fig. 2C, (Partlow, Jaeggi-Wong et al. 2025)). As in SPB/TIRF, PR8 has lower baseline efficiency than X31 in spCALM (Fig. S2, (Otterstrom, Brandenburg et al. 2014)). As is well established including by SPB/TIRF, fusion kinetics and efficiency are pH-dependent in spCALM (Fig. 3E, (Floyd, Ragains et al. 2008, Ivanovic, Choi et al. 2013)). As in SPB/TIRF, Gamma cumulative distribution fitting of lipid mixing vs. time trajectories in spCALM revealed 3-4 underlying steps (Fig. S8, (Floyd, Ragains et al. 2008, Ivanovic, Choi et al. 2013, Otterstrom, Brandenburg et al. 2014, Ivanovic and Harrison 2015, Li, Li et al. 2021, Li, Li et al. 2022).
Reviewer #3: “2) In particular, authors do not provide data showing that flow cytometry can quantify time-resolved distributions of lipid mixing states among single virions bound to individual cells or vesicles, as claimed in the introduction (Page 3, line 85). Flow cytometry can not distinguish a single virion bound to a 1µm vesicle from multiple virions bound to a 1µm vesicle. Liposomes often can bind to multiple virions, as shown by previous cryo-EM studies. Since authors use liposomes extruded via a 1µm filter, it is expected that these liposomes will be heterogeneous in size and shape, and thus single liposomes can bind a variable amount of viruses, leading to a variable amount of dequnching. In addition, this will lead to aggregation - analogous to a hemagglutination assay, which will complicate the interpretation of flow cytometry results.”
We thank Reviewer #3 for raising this point – in response, we have performed additional analyses which, as we demonstrate in what follows, formalize our claims. Since flow virometry can readily distinguish single virions from pairs or groups of associated virions by virion fluorescence intensity (bioRxiv: https://doi.org/10.64898/2026.06.15.729605), we decided to analyze fluorescence distributions of free and liposome-associated virions in our experiments to probe whether multi-virion binding to single liposomes occurs with notable frequency. Liposomes with more than one bound virion would, just like clumped virions do, shift upward in fluorescence intensity. We illustrate here with the results of this analysis for the experiment shown in Fig. 5A,B, where virion-liposome input was the highest (0.5 vs. 0.15 in the remaining experiments; also see our response regarding the MEDI8852 experiment to Reviewer 1). We used virus-only and liposome-only controls to define cytometry gates for unbound virions and virion-free liposomes. We extracted the R18 fluorescence distributions for liposomes with and without bound virions, and for unbound virions in the same sample (Display Item 3). The fluorescence distributions for virions and liposome-virion bound complexes strongly overlap and are both considerably higher than the distribution for liposomes without virions. This is consistent with liposomes not appreciably binding more than one virion. We will extend this analysis to the remaining samples in this experiment and to other experiments and present it as a supplementary figure in the revised manuscript. This analysis furthermore excludes the existence of aggregates such as in a hemagglutination assay.
Display Item 3 caption: Liposomes do not bind multiple particles. (A) Representative sample from the experiment presented in Fig. 5A,B showing that liposome-virion complexes have nearly overlapping R18 fluorescence distribution to unbound virions, and both have higher intensity than liposomes without bound virions. (B) Violin plots showing the distribution of median R18 fluorescence distributions for the specified populations representing all samples from the experiment presented in Fig. 5A,B.
Reviewer #3: 3) Liposomes with variying concentration of receptors should be characterized by cryo-EM as a quality check. It is not clear what the size distribution of liposomes and multilamellarity is. A fraction of small liposomes can skew the analysis as they would not be gated in the flow cytometry. In addition, cryo-ET could be used to analyze the contact zone area at different concentrations of DNA-linked sialic acid receptors.
We agree that the size distribution of the liposome preparations used in our study is likely heterogeneous to some extent and that an analysis of the efficiency of lipid mixing for liposomes of different sizes is therefore useful. We thank this reviewer for suggesting we explore this further. In spCALM, liposomes are detected with a range of DiD membrane dye fluorescence and 488-nm side-scatter intensities. For the experiment presented in Fig. 4B, we separately gated subsets of liposomes with low DiD fluorescence and side-scatter intensity, which would comprise smaller liposomes on average, and of liposomes with high DiD fluorescence and side-scatter, which would comprise larger liposomes (Display Item 4A). We compared lipid-mixing efficiency of bound virion-liposomes pairs between the small and large liposome subpopulations to the total liposome population. Differences in efficiency across liposome subpopulations within each replicate were small relative to differences across replicates (Display Item 4B). The smallest ~7-9% of liposomes fused slightly more efficiently at very low or no receptor saturation whereas the largest ~1-3% liposomes tended to fuse somewhat less efficiently overall. The results of the experiment with respect to the effect of receptor density, NA-inhibition, and receptor type are thus practically indistinguishable between the liposome size subpopulations (Display Item 4C). From this, we infer that size heterogeneity in our liposome preparations neither explains nor confounds our interpretations of the regulatory effect of receptor binding on lipid mixing. We will present this analysis as a supplementary figure in the revised manuscript.
Display Item 4 caption: Liposome size heterogeneity does not produce meaningful differences in lipid-mixing efficiency. (A) We re-analyzed the experiment presented in Fig. 4B by separately analyzing lipid mixing for liposomes with low (Small liposomes) or high (Big liposomes) 488-nm side-scatter (SSC-A) and DiD membrane fluorescence (APC-A). (B) Lipid mixing efficiency for liposomes displaying 3’SL or 6’SL receptors at the indicated range of saturations in the presence or absence of 100 nM NAI; analyses with each liposome size subpopulation are connected by lines for each replicate. (C) For each liposome size subpopulation, logistic curves with a shared Hill slope were fit to the combined data from each condition, with shaded 95% confidence intervals. The overall effect of receptor saturation, receptor type, and NA-inhibition is not meaningfully different between the liposome size subpopulations.
Reviewer 3: “4) Data on receptor binding promote HA insertion is not cleanly explained and not experimentally justified. Increased binding of virion and liposomes, will potentially lead to an extended binding zone, which could give rise to multiple fusion pores, which could accelerate dequenching and the R18 fluorescence burst. Previous cryo-ET work has shown that interaction with liposomes can engage one to seven HA glycoproteins when 5 mol% gangliosides are present in 200 nm liposomes (PMID: 27572837). This should be considered in the model and the manuscript would largely benefit from having a resolution analysis of the liposomes at varying concentrations of the DNA-sialic acid and the binding area with a virus.”
We thank reviewer #3 for raising the alternative hypothesis that receptor binding could deform the target membrane, thereby increasing the virion-target contact area and the number of HAs contained within. We agree that this is a plausible mechanism that merits consideration. Our current data do not distinguish between this possibility and the hypothesis that receptor binding promotes HA fusion peptide insertion, and we will revise the interpretation of Figure 5A, B results and the Discussion to more explicitly acknowledge these alternative interpretations.
To discriminate between these two mechanistic models, we will evaluate whether receptor density differentially affects lipid mixing of spherical and filamentous virions. We hypothesize that if receptor binding promotes lipid mixing by increasing membrane wrapping and thereby expanding the virion-target contact area, this effect would be more pronounced for spherical virions than for filaments. For a spherical particle, relatively small increases in the extent of membrane wrapping produce comparatively large increases in the buried contact area and, consequently, in the number of HA molecules within the virion-target contact zone. In contrast, for a filament, comparable increases in membrane wrapping produce substantially smaller relative changes in buried contact area. Moreover, membrane wrapping around a sphere is expected to incur greater membrane deformation costs than wrapping around a filament, such that increasing receptor density may produce a relatively sharp transition to extensive wrapping for spherical virions, whereas the corresponding response for filaments would be expected to be more gradual. Thus, if receptor density primarily regulates lipid mixing by modulating the extent of membrane wrapping, we would expect the receptor-density dependence to be steeper for spherical virions than for filaments.
We note that this experiment presents technical challenges. Filamentous virions require larger target membranes to generate sufficient R18 dequenching to resolve unfused and lipid-mixed virion-target pairs, and we therefore anticipate using erythrocyte ghosts rather than liposomes as targets. Receptor density on erythrocyte ghosts would be modulated with bacterial neuraminidase under varying conditions. If these experiments can be completed in time for the revision, we will include them in the revised manuscript. Regardless, we will revise our discussion to explicitly consider membrane deformation as an alternative mechanism underlying the receptor-density dependence observed in Fig. 5AB.
Can strong binding induce liposome membrane rupture?”
To test if liposomes are rupturing or aggregating, we examined the total number of detected liposomes before pH drop for the experiment presented in Fig. 4B (Display Item 2B). The total liposome count decreased somewhat at high receptor saturation for liposomes bearing 3’SL but not 6’SL. This effect was associated with a small increase in the fraction of brighter liposomes for 3’SL but not 6’SL liposomes (Display Item 5). From this, we infer that this apparent loss of liposomes is due to aggregation and not rupture. Importantly, the effect on total liposome counts was relatively small and only present for 3’SL-bearing liposomes, for which efficient lipid mixing required greater receptor density than for 6’SL-bearing liposomes. We therefore conclude that this putative aggregation effect is inconsequential for our lipid mixing results.
Display Item 5 caption: Mild liposome aggregation at high receptor saturation. Representative scatter plots of DiD membrane fluorescence (y-axis) and 488-nm side-scatter (x-axis) before pH drop for liposomes decorated with 0, 16, or 100% saturation with either 3’SL (top) or 6’SL (bottom) receptor mimetics. Gates for overall liposomes, small liposomes, and large liposomes are provided to illustrate the emergence of a larger subpopulation for 3’SL 100%.
and
“6) As in 3D space, viruses can also bind several vesicles; membrane fusion would lead to an increase in the size of the vesicles. Can this be detected by flow cytometry? If not, what is the benefit of the flow cytometer over the classical fluorimeter, which has been routinely used to monitor membrane fusion in bulk?”
As we demonstrate in Display Item 3 and Fig. 2A-B, virions bound to vesicles are distinguishable from vesicles without virions and virion-vesicle pairs that have undergone lipid mixing are distinguishable from pairs which have not. Flow cytometry thus offers a fundamental advantage over bulk fluorimetry because it measures membrane fusion on individual virion-liposome pairs rather than as an ensemble average. Each detected event reports whether a virion is bound to a liposome and whether that virion has undergone lipid mixing. This allows us to determine the probability of lipid mixing among bound virions while independently quantifying binding efficiency. In contrast, bulk fluorimetry reports only the summed fluorescence of the entire sample, making it impossible to distinguish changes in membrane fusion efficiency from changes in the number of virion-target interactions.
“Also, please comment on the sensitivity of the instrument and required volumes, as well as how acid is added. How was the final pH of the mixture determined so precisely?”
We thank the reviewer for pointing out that this description was ambiguous. The reported pH values correspond to the fusion-triggering buffer rather than the final reaction mixture, and we have revised the text to make this explicit on lines 116-118, 189, 194, 299, 737-738, 754, 769, 773, 786-787, 801-802, 807-808. Because the neutral pH virion-liposome mixture is diluted approximately 10-fold into the acidic triggering buffer, the final reaction pH is expected to be very close to that of the triggering buffer. We will experimentally verify this and report the result in the revised manuscript.
Reviewer #3: “7) How efficient is the membrane fusion compared to total disassembly included by a detergent? Normalisation to total dequnching should be used. “
We thank Reviewer #3 for this comment. We believe there may be a misunderstanding regarding the readout of the spCALM assay. Unlike conventional bulk fluorimetry experiments, spCALM does not quantify the magnitude of ensemble R18 fluorescence dequenching. Instead, it analyzes individual virion-liposome pairs and classifies each virion as either quenched or dequenched within discrete sampling intervals. Thus, the question addressed by spCALM is, "What fraction of virion-liposome pairs have undergone lipid mixing?", whereas bulk fluorimetry asks, "How much total R18 dequenching has occurred?" Lipid-mixing efficiency is therefore determined from the fraction of individual virions occupying the dequenched population and is independent of the absolute fluorescence intensity of the R18 signal. Consequently, normalization to detergent-induced total dequenching, which is appropriate for bulk fluorescence assays, is not applicable to the single-particle measurements reported here because complete lipid mixing is intrinsically represented by complete occupancy of the dequenched state. We will revise the manuscript to make this distinction clearer and avoid potential confusion for readers more familiar with ensemble fusion assays.
Reviewer #3: “8) HA/NA functional balance is not only constrained by the distribution of sialic species but also by the ratio of the HA/NA and by the NA spatial distribution, which is clustered only at one end and hence it cannot interfere in the binding zone. This is not discussed in the manuscript or introduced.”
We agree that the spatial organization of HA and NA is an important consideration when interpreting HA/NA functional balance. However, the polarized distribution of NA observed on mature virions might not persist under the low-pH conditions of membrane fusion; polarized NA organization on the virion surface is maintained by the interactions with the underlying M1 matrix which restrict NA mobility (Vahey and Fletcher 2019), and acidification is known to dissociate the underlying M1 matrix layer (Calder, Wasilewski et al. 2010). We had discussed in the original submission that in the context of its polarized distribution, NA might limit receptor availability before the virion-target contact interface is established or, alternatively, it might become mobile at the pH of fusion and contribute from within the interface (lines 400-408). In the revision, we will ensure that the concept of HA/NA polarization is clearly introduced so that it is not overlooked.
Reviewer #3: “9) Conclusions in the manuscript could be strengthened by providing data on binding and fusion using viruses that were pre-treated with different amounts of soluble sialic acid prior to the fusion kinetics measurement.”
We thank Reviewer #3 for this interesting suggestion. While soluble receptor (kd for monomeric binding is ~1 - 4 mM) might not effectively compete with the multivalent receptor displays on the target membrane, this experiment is relatively simple to implement, and a positive outcome has a potential to further hone the mechanistic interpretations of our findings. We will perform experiments measuring virion binding and lipid mixing in the presence of varying concentrations of soluble sialyllactose and include the results in the revised manuscript. Depending on the outcome, these experiments may help distinguish whether receptor binding primarily acts through membrane-associated interactions or by directly modulating HA conformation or function. We will discuss the implications of these results for the proposed mechanism by which receptor binding promotes lipid mixing. A negative result (i.e. no effect from the soluble receptors) will be difficult to interpret and will be simply reported as such.
Reviewer #3: “1) Figure 2 lacks information on time fo the three plots. A time stemp could be also included to the video.”
We will revise Fig. 2 and Movies 1 and 2 to include information on time.
Reviewer #3: “2) It is not clear why the model is presented as figure 1 and the density of spikes does not reflect the density on the viruses - it is not drawn to the scale, which in this study might be important.”
We thank Reviewer #3 for this comment. The purpose of Figure 1 is to provide a schematic illustration of the local conformational changes associated with membrane fusion rather than to accurately represent the surface density or number of HA molecules on influenza virions. We recognize that this was not sufficiently clear in the original figure. To avoid any confusion, we will explicitly indicate in the figure that the illustration is not drawn to scale. We will also revise the legend to clarify that the virion-target contact patch for a representative particle in our experiments is expected to contain on the order of 100 HA molecules, and that the simplified depiction is intended solely to illustrate the key molecular events within the virion-target interface, specifically the fusion cluster and ways HA might be rendered nonparticipating. Depicting the full complement of HA molecules expected within the contact patch would substantially obscure the illustration of the fusion cluster in Fig. 1B and of inhibitor or antibody binding and nonproductive HA refolding in Fig. 1C.
Reviewer #3: “3) The manuscript structure could be improved, although it is understandable that the final format depends on the journal. Some information on methods is present in the main text, and some in the extended methods in the supplementary data. This makes the manuscript harder to review. Authors could consider redistributing some supplementary information to the main data, and consider publishing the first method and its validation, and another manuscript on biology analysis.”
We will certainly rearrange the structure of the methods to accommodate the guidelines of the eventual journal of publication. We aimed to structure our main figures to convey the most essential findings of the work but are open to promoting supplementary content to main figures if it can be accommodated within journal guidelines.
Reviewer #3: “Overall, TIRF microscopy and SBP (Fig S6) present a much more accurate method than flow cytometry as it allows to study individaul events and provides additional information on virus size, fluorescence signal etc. In addition, single-molecule FRET methods could be used to monitor HA conformational changes at varying concentrations of sialic acid receptors. Previous studies have already shown that binding to sialic acid allosterically regulates HA2 conformational dynamics (PMID: 29961575).”
We respectfully disagree that TIRF microscopy of supported planar bilayers is inherently more accurate than flow cytometry in the ways described in this comment. As we detail in other responses in this document (see our responses to “Reviewer #3: “2) In particular, authors do not provide data showing that…” __and “6) As in 3D space, viruses can also bind several vesicles; …”), flow cytometry in the form of spCALM permits analysis of lipid mixing deriving from fluorescence measurements of single, membrane-attached virions. For analysis of virus particles with sizes below the diffraction limit of visible light, which are the sizes that we study here, flow cytometry provides as much size information per particle as TIRF microscopy. Please see our response to reviewer 3 (“1) The conclusions are drawn from a flow cytometry, …”__) regarding the suitability of SPB/TIRF for the current mechanistic questions.
We agree that single-molecule FRET has provided important insight into HA conformational dynamics, including the observation that receptor binding shifts HA toward downstream conformational intermediates (Das, Govindan et al. 2018). However, smFRET addresses a fundamentally different mechanistic question than the present study. smFRET reports on the conformational dynamics of individual labeled HA molecules, whereas our experiments measure the functional outcome of the collective action of many HAs during membrane fusion. Moreover, interpretation of smFRET measurements relies on site-specific fluorophore labeling within conformationally sensitive regions of HA, an experimental strategy that may itself influence the native conformational landscape. Thus, while highly complementary, smFRET cannot directly establish how receptor binding alters the probability of productive lipid mixing.
Implementing and validating an smFRET assay for our virus-membrane fusion system would require substantial development of a highly specialized methodology and is therefore beyond the scope of the present study. Instead, we will expand the Discussion to place our findings in the context of previous smFRET studies. Specifically, we will discuss that, although previous work indicates that receptor binding increases occupancy of later HA conformational intermediates, our data support a different mechanistic interpretation: receptor engagement does not primarily drive HA further along its conformational pathway toward HA extension but instead alters the probability that receptor-bound HAs proceed to membrane insertion following low-pH triggering. In sum, smFRET provides an indication that receptor binding influences conformational HA dynamics whereas our study evaluates the effect of receptor binding on lipid mixing by virions. Both approaches are important, but neither can fully recapitulate the value of the other, nor are they necessarily in conflict.
__ __
3. Description of the revisions that have already been incorporated in the transferred manuscript
Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.
Reviewer #1: “A couple points in the text, the authors use the term "distributional dynamics". This term is not defined and could use clarification.”
We agree that this wording was unclear and have revised our wording for clarity on lines 105-106, 204, 801, and 807.
Reviewer #1: “Why have the authors used this technique using lipid-ssDNA and receptor-DNA to display sialic acid on the liposomes? In principle, this allows control over the distance between the sialic acid and the membrane. But in the spCALM assay the length of the spacer DNA (12 or 24 nt) had not effect. So, why then not use the simpler approach of incorporating sialylated lipids (e.g. GD1a, GD1b) in the membrane?”
We thank this reviewer for the opportunity to clarify two additional key advantages of our approach. Our system allows us to display glycans of precisely defined structure, with either α2,3 or α2,6 sialic acid linkage, on identical carbohydrate-lipid conjugates, which ensures identical chemistry and display geometry with only the intended differences. This is not possible with gangliosides familiar to us including GD1a (displays a terminal alpha2,3-linked sialic acid) and GD1b (contains only internal sialic acids, alpha2,8-linked in tandem). Furthermore, to alter receptor density using gangliosides, one must alter membrane lipid composition, while our system allows tuning of receptor density without changing lipid composition.
We have added a description of these key advantages in lines 161-163. We have furthermore restructured the introductory paragraph in the Results section “Programmable glycan-receptor displays for membrane-fusion studies” to first emphasize programmability of the receptor type, then tunability of density without altering lipid or display chemistry, and the spacer component is now mentioned last (as this, indeed, turned out inconsequential for lipid mixing under our current assay conditions).
__Reviewer #2: “____While the precise control of receptor identity and density is a strength of this experimental approach, more discussion of how the conditions tested here compare to physiological conditions would strengthen the manuscript. The receptor densities tested here are We agree that placing the tested receptor densities into a physiological context is valuable. Estimates of the density of influenza receptors in the endosomal compartments where membrane fusion takes place are not available, so we instead compare our system with the plasma membrane of human erythrocytes in the Discussion section, lines 361-372. The maximum receptor density used in our experiments is approximately 15-fold lower than the total sialic acid content of the human erythrocyte glycocalyx. However, this comparison almost certainly overestimates the density of physiologically relevant influenza receptors because it includes all sialic acid regardless of glycosidic linkage, underlying glycan structure, or accessibility. Consequently, the density of functional influenza receptors may be substantially closer to the receptor densities explored in our chemically defined liposome system. Moreover, the receptor composition and density within the endosomal lumen remain unknown and might differ considerably from those of the erythrocyte surface. Finally, receptor density may vary widely across potential cell targets in complex systems of natural infections.
Our aim was to identify the mechanisms by which receptor binding regulates HA-mediated lipid mixing using a molecularly defined receptor display. This approach allowed us to systematically vary receptor density over a controlled range while avoiding the compositional complexity of native membranes, thereby enabling mechanistic interpretation of receptor density-dependent effects.
Reviewer #2: Regarding the experiments in Figure 4 and S13: what is the anticipated phenotype for the A227T variant? It appears to have the opposite effect of the avidity-increasing E246G mutation, restoring WT avidity in the double mutant (line 245), and forming larger plaques in MDCK cells when introduced alone (in contrast to the smaller plaques formed by E246G). However, in spCALM measurements (Fig 4C), it behaves similarly to WT. The authors mention that this result is surprising (line 259), but more context for this observation would be helpful. Similarly, it is interesting that all of the variants tested exhibit higher peak fusion efficiency than the WT (50-75% vs. 25%). I would be interested how the authors interpret this result.
We agree that these observations merit additional discussion. We have revised the manuscript to more carefully interpret the plaque assay results, acknowledging that plaque size reflects the combined effects of virus attachment, entry, membrane fusion, replication, and release, and therefore cannot be directly attributed to altered receptor binding or fusion alone (lines 249-256).
Our reanalysis of the spCALM attachment data (prompted by the Reviewer 1 comment regarding MEDI8852-Fab’s effects on virion attachment) provides additional context for interpreting HA mutants. The attachment phenotypes closely parallel the lipid-mixing results: E246G exhibits the greatest increase in virion binding, A227T alone behaves similarly to WT (maybe slightly reduced binding), and the A227T mutation partially or fully reverses the enhanced binding conferred by E246G (see Display Item 6 below). We have incorporated these observations into the revised Results (lines 256-265) and expanded our interpretations to emphasize that receptor binding and lipid mixing exhibit similar qualitative trends but distinct quantitative dependencies on receptor density (lines 265-271).
Display Item 6 caption: Trends in virion attachment efficiency mirror those of lipid-mixing efficiency, but attachment is sensitive to receptor density at greater values than for lipid mixing. (A) The percentage of total recovered virions which are attached to liposomes. (B) Efficiency of lipid mixing among bound virion-liposome pairs, replotted from Fig. 4C for ease of comparison.
Finally, we agree that the increased maximal lipid-mixing efficiencies observed for all three HA variants relative to WT are intriguing. At present, we do not know the molecular basis for this effect, and we do not believe it can be reliably inferred from the available data. We will therefore discuss this as an interesting observation that warrants future investigation while emphasizing that it does not affect the principal conclusion of the study that HA-receptor interactions regulate lipid-mixing efficiency through receptor avidity.
Reviewer #2: “Line 49: "3-4 neighboring HA2 molecules" should be clarified to indicate that this means 3-4 trimers (as stated a few lines later).”
Thank you, we have clarified the wording in this clause on line 49.
__Reviewer #2: “Line 278: "presence of absence" should read "presence or absence".” __
We thank Reviewer #2 for pointing this out; we found and revised a total of three instances of this typographical error in the main manuscript (lines 289, 309, 769) and one in the supplementary appendix (Fig. S9 legend).
Reviewer #2: “Line 296: I am surprised that 450nM MEDI8852 is a sub-neutralizing dose; this seems higher than what has been reported elsewhere, although it may be a specific feature of the virus used here. Can the authors clarify how this was determined, i.e., is this based on an infection assay or a lipid mixing assay?”
Our intended meaning is that the tested dose of MEDI8852-Fab reduces lipid-mixing efficiency and does not fully inhibit it. We agree that this was unclear and have clarified our language on lines 308-309. As an aside, we note here that in vitro membrane fusion experiments seem to be less susceptible to inhibition by MEDI-Fab than infection experiments (i.e. neutralization), as was reported by our lab in 2021 (Li, Li et al. 2021).
__Reviewer #3: __
__“5) How does 60% receptor saturation compare to the physiological concentration of receptor density? __
We agree that placing the tested receptor densities in a physiological context is valuable. As discussed in response to Reviewer #2, we have expanded the Discussion to compare our receptor densities with estimates for the erythrocyte glycocalyx (see lines 362-378). The maximum receptor density used in this study is approximately 15-fold lower than the total sialic acid density on the erythrocyte surface, although this comparison likely overestimates the number of physiologically relevant influenza receptors because it includes all sialic acid species irrespective of glycosidic linkage, glycan structure, or accessibility. The density of accessible influenza receptors within the endosomal lumen, where fusion occurs in vivo, is not known.
Reviewer #3: “4) Webster ER et al, PMID: 35143209 should be referenced as it showed similar approaches using DNA as a tether to study influenza membrane fusion.”
We thank Reviewer #3 for pointing out that we did not reference this study. We have modified the text to reference this study on lines 71-73 and 152.
Reviewer #3: “____A limitation is that only lipid mixing is monitored and no full fusion. Hence, one cannot conclude that all lipid mixing here leads to a full fusion pore opening. This should be addressed in the manuscript or at least noted as a limitation noted in the manuscript as a limitation. “
We thank Reviewer #3 for highlighting this important distinction. We agree that lipid mixing does not necessarily imply subsequent fusion pore formation and that our assay specifically reports on the lipid-mixing (hemifusion) intermediate rather than full fusion. We have revised the manuscript to make this point more explicit on lines 110–114 and 329-330. Specifically, we now state that our approach probes the lipid-mixing fusion intermediate (i.e., hemifusion), which reports on the preceding HA conformational changes and is an obligate intermediate on the pathway to fusion pore formation.
__ __
Description of analyses that authors prefer not to carry out
Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.
Reviewer #1: “The authors raise the interesting possibility that neuraminidase (NA) may contribute to virion attachment in the presence of NAI. Would it be worthwhile to test fusion of virions in the absence of NA? Or in the presence of NA where the sialic acid binding site has been mutated?”
We thank Reviewer #1 for this thoughtful suggestion. We agree that comparing lipid mixing of virions lacking NA or carrying mutations in the NA sialic acid-binding site would provide a direct test of whether receptor-NA interactions contribute to virion attachment and/or lipid mixing. Although NA is a relatively low-abundance virion glycoprotein (there are 4-5 HAs for each NA on the virion surface), it is possible that receptor-NA binding contributes in some way to lipid mixing. We are interested in exploring this in future work. However, production of virions without NA or with defective NA is expected to be challenging because of reduced viral fitness and infectivity. Moreover, this could lead to unintended (e.g. compensatory) differences in virion morphology or glycoprotein composition that would complicate interpretations of any observed effects on lipid mixing. We therefore believe that this represents a substantial follow-up study that is beyond the scope of the current manuscript.
References to works cited in this revision plan
Calder, L. J., S. Wasilewski, J. A. Berriman and P. B. Rosenthal (2010). "Structural organization of a filamentous influenza A virus." Proc Natl Acad Sci U S A 107(23): 10685–10690.
Das, D. K., R. Govindan, I. Nikic-Spiegel, F. Krammer, E. A. Lemke and J. B. Munro (2018). "Direct Visualization of the Conformational Dynamics of Single Influenza Hemagglutinin Trimers." Cell 174(4): 926–937 e912.
Floyd, D. L., J. R. Ragains, J. J. Skehel, S. C. Harrison and A. M. van Oijen (2008). "Single-particle kinetics of influenza virus membrane fusion." Proc Natl Acad Sci U S A 105(40): 15382–15387.
Ivanovic, T., J. L. Choi, S. P. Whelan, A. M. van Oijen and S. C. Harrison (2013). "Influenza-virus membrane fusion by cooperative fold-back of stochastically induced hemagglutinin intermediates." Elife 2: e00333.
Ivanovic, T. and S. C. Harrison (2015). "Distinct functional determinants of influenza hemagglutinin-mediated membrane fusion." Elife 4: e11009.
Li, T., Z. Li, E. E. Deans, E. Mittler, M. Liu, K. Chandran and T. Ivanovic (2021). "The shape of pleomorphic virions determines resistance to cell-entry pressure." Nat Microbiol 6(5): 617–629.
Li, Z., T. Li, M. Liu and T. Ivanovic (2022). "Hemagglutinin Stability Determines Influenza A Virus Susceptibility to a Broad-Spectrum Fusion Inhibitor Arbidol." ACS Infect Dis 8(8): 1543–1552.
Otterstrom, J. J., B. Brandenburg, M. H. Koldijk, J. Juraszek, C. Tang, S. Mashaghi, T. Kwaks, J. Goudsmit, R. Vogels, R. H. Friesen and A. M. van Oijen (2014). "Relating influenza virus membrane fusion kinetics to stoichiometry of neutralizing antibodies at the single-particle level." Proc Natl Acad Sci U S A 111(48): E5143–5148.
Partlow, E. A., A. Jaeggi-Wong, S. D. Planitzer, N. Berg, Z. Li and T. Ivanovic (2025). "Influenza A virus rapidly adapts particle shape to environmental pressures." Nat Microbiol 10(3): 784–794.
Vahey, M. D. and D. A. Fletcher (2019). "Influenza A virus surface proteins are organized to help penetrate host mucus." Elife 8.
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
Summary: The manuscript by Plamitzer et al. investigates influenza A virus hemagglutinin (HA)-mediated membrane fusion using bulk flow cytometry analysis. In the assay, they use red blood cells and liposomes carrying defined, variable densities of sialic acid residues. They used H3N2 and H1N1 model viruses with spherical morphology. The liposomes were produced using double-stranded DNA to attach sialic acid. Similar studies have been done using DNA-tethers to study membrane fusion between liposomes and influenza virions (PMID: 35143209, PMID: 27410740). Some previous studies have shown that reduced HA binding does not influence membrane fusion, while others have shown that binding increases membrane fusion. This work aims to reconcile prior conflicting observations. Based on their results, they conclude that HA-receptor binding avidity and the extent of neuraminidase (NA)-mediated receptor cleavage directly influence lipid mixing, which is used here as a proxy for membrane fusion and allows monitoring of the first step of fusion, namely the hemifusion intermediate. The results of the test whether receptor binding modulates the HA extended intermediate should be interpreted with more caution. The manuscript would benefit from more data on the characterisation of liposome carrying dsDNA sialic acid receptors and from using the TIRF SBP method to validate the flow cytometry. Major comments: 1) The conclusions are drawn from a flow cytometry, which allows to analyse samples in bulk. On one hand, it allows analysis of a large number of fusion events; on the other hand, it cannot resolve individual events or the relationship between binding and membrane fusion in individual binding states. The flow cytometry method used in the manuscript should be validated against single-particle fusion events in microscopy-based assays, which would also allow investigation of both the binding area and fusion (as done in the Fig S6). Here, authors could evaluate the effect of neutralizing antibody, NA inhibitor or the presence of soluble sialic acid pretreated virions at receptor concentrations higher than 16%. 2) In particular, authors do not provide data showing that flow cytometry can quantify time-resolved distributions of lipid mixing states among single virions bound to individual cells or vesicles, as claimed in the introduction (Page 3, line 85). Flow cytometry can not distinguish a single virion bound to a 1µm vesicle from multiple virions bound to a 1µm vesicle. Liposomes often can bind to multiple virions, as shown by previous cryo-EM studies. Since authors use liposomes extruded via a 1µm filter, it is expected that these liposomes will be heterogeneous in size and shape, and thus single liposomes can bind a variable amount of viruses, leading to a variable amount of dequnching. In addition, this will lead to aggregation - analogous to a hemagglutination assay, which will complicate the interpretation of flow cytometry results. 3) Liposomes with variying concentration of receptors should be characterized by cryo-EM as a quality check. It is not clear what the size distribution of liposomes and multilamellarity is. A fraction of small liposomes can skew the analysis as they would not be gated in the flow cytometry. In addition, cryo-ET could be used to analyze the contact zone area at different concentrations of DNA-linked sialic acid receptors. 4) Data on receptor binding promote HA insertion is not cleanly explained and not experimentally justified. Increased binding of virion and liposomes, will potentially lead to an extended binding zone, which could give rise to multiple fusion pores, which could accelerate dequenching and the R18 fluorescence burst. Previous cryo-ET work has shown that interaction with liposomes can engage one to seven HA glycoproteins when 5 mol% gangliosides are present in 200 nm liposomes (PMID: 27572837). This should be considered in the model and the manuscript would largely benefit from having a resolution analysis of the liposomes at varying concentrations of the DNA-sialic acid and the binding area with a virus. 5) How does 60% receptor saturation compare to the physiological concentration of receptor density? Can strong binding induce liposome membrane rupture? 6) As in 3D space, viruses can also bind several vesicles; membrane fusion would lead to an increase in the size of the vesicles. Can this be detected by flow cytometry? If not, what is the benefit of the flow cytometer over the classical fluorimeter, which has been routinely used to monitor membrane fusion in bulk? Also, please comment on the sensitivity of the instrument and required volumes, as well as how acid is added. How was the final pH of the mixture determined so precisely? 7) How efficient is the membrane fusion compared to total disassembly included by a detergent? Normalisation to total dequnching should be used. 8) HA/NA functional balance is not only constrained by the distribution of sialic species but also by the ratio of the HA/NA and by the NA spatial distribution, which is clustered only at one end and hence it cannot interfere in the binding zone. This is not discussed in the manuscript or introduced. 9) Conclusions in the manuscript could be strengthened by providing data on binding and fusion using viruses that were pre-treated with different amounts of soluble sialic acid prior to the fusion kinetics measurement.<br /> Minor comments: 1) Figure 2 lacks information on time fo the three plots. A time stemp could be also included to the video. 2) It is not clear why the model is presented as figure 1 and the density of spikes does not reflect the density on the viruses - it is not drawn to the scale, which in this study might be important. 3) The manuscript structure could be improved, although it is understandable that the final format depends on the journal. Some information on methods is present in the main text, and some in the extended methods in the supplementary data. This makes the manuscript harder to review. Authors could consider redistributing some supplementary information to the main data, and consider publishing the first method and its validation, and another manuscript on biology analysis. 4) Webster ER et al, PMID: 35143209 should be referenced as it showed similar approaches using DNA as a tether to study influenza membrane fusion.
The study attempts to answer an important question and is of interest to the membrane fusion field and the virology field. It provides flow cytometry to study membrane fusion and a method for DNA-based sialic acid receptors that can be displayed at control density.
Limitations and Advances:
Expertise: virology, membrane fusion, cryo-EM
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
This manuscript by Planitzer et al. investigates how NA activity, HA receptor binding avidity, and the identity and density of sialic acid receptors within the target membrane influence membrane fusion. This is an important question, as functional balance between HA and NA drives the antigenic evolution of seasonal influenza viruses and contributes to the pandemic potential of zoonotic viruses. As discussed in this manuscript, prior studies of how sialic acid receptors contribute to fusion following initial viral attachment are relatively few, and these have frequently reached different conclusions. Many of these discrepancies are likely attributable to details of the experimental system used to address the question, highlighting the importance of approaches in which the receptor identity and density are precisely controlled. Within this context, the sensitive and quantitative method the authors have developed to measure the efficiency and kinetics of virion fusion using flow cytometry ('spCALM') stands out as an important contribution of this manuscript. Using this approach, the authors convincingly demonstrate that engagement of HA with sialic acid receptors during the fusion process promotes efficient fusion, and that conditions that disrupt this engagement (low receptor densities combined with NA activity or receptor-HA mismatch) lead to reduced fusion efficiency by reducing the probability of fusion peptide insertion. Overall, the methods and findings of this paper are a strong contribution to the field and will be of interest to researchers studying viral membrane fusion as well as HA-NA balance. The manuscript is well-written, and the figures are very clear. The supporting figures provide additional context that is valuable. Addressing the comments below would strengthen the manuscript further.
Major comments:
Minor comments:
Line 49: "3-4 neighboring HA2 molecules" should be clarified to indicate that this means 3-4 trimers (as stated a few lines later).
Line 278: "presence of absence" should read "presence or absence".
Line 296: I am surprised that 450nM MEDI8852 is a sub-neutralizing dose; this seems higher than what has been reported elsewhere, although it may be a specific feature of the virus used here. Can the authors clarify how this was determined, i.e., is this based on an infection assay or a lipid mixing assay?
This study establishes a powerful experimental approach to quantitatively dissect fusion between influenza viruses and target membranes across a wide range of experimental conditions. This approach represents a significant methodological advance and its high throughput allows the authors to quantify rare events. Additionally, the manuscript presents convincing data showing that, within the regimes tested here, sustained receptor engagement promotes membrane fusion by increasing the likelihood of fusion peptide insertion. The observation that this is reduced by NA activity at low receptor densities carries implications for HA-NA functional balance.
While the specific findings may be restricted to the range of experimental conditions tested here, the results will be of interest to researchers studying virus-membrane fusion and functional balance between the influenza virus glycoproteins.
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
Planitzer et al. have developed a clever flow cytometry assay to monitor hemagglutinin (HA)-mediated membrane fusion. This novel approach provides several advantages over bulk and single-particle fusion assays, including dramatically higher throughput and detection of lipid mixing under low efficiency conditions. As such, on purely technical grounds, this manuscript provides a significant advance to the field. The authors then apply their new approach to probe the role of HA-sialic acid binding in the membrane fusion reaction. This reflects an important open question in the field, which, as the authors point out, has been elusive. They conclude that sialic acid binding promotes efficient membrane fusion, probably by increasing the likelihood of the fusion peptide engaging the target membrane after acid-induced conformational changes. Overall, the manuscript is very well written; the data are rigorously analyzed and nicely presented; and the results are important and informative. My comments are, for the most part, minor.
A couple points in the text, the authors use the term "distributional dynamics". This term is not defined and could use clarification.
In the spCALM assay, the authors indicate that labeled virions were incubated with cells or liposomes at a ratio of "0.15 virions per target". How were the numbers of virions determined for these experiments? The Methods refer to MOIs in units of PFU/cell but do not indicate how the number of physical particles were determined.
Why have the authors used this technique using lipid-ssDNA and receptor-DNA to display sialic acid on the liposomes? In principle, this allows control over the distance between the sialic acid and the membrane. But in the spCALM assay the length of the spacer DNA (12 or 24 nt) had not effect. So, why then not use the simpler approach of incorporating sialylated lipids (e.g. GD1a, GD1b) in the membrane?
The authors raise the interesting possibility that neuraminidase (NA) may contribute to virion attachment in the presence of NAI. Would it be worthwhile to test fusion of virions in the absence of NA? Or in the presence of NA where the sialic acid binding site has been mutated?
The authors indicate that the MEDI antibody "increased binding across all tested conditions." But this was not mentioned again. By what mechanism do they expect the antibody enhances virus binding?
Planitzer et al. have developed a clever flow cytometry assay to monitor hemagglutinin (HA)-mediated membrane fusion. This novel approach provides several advantages over bulk and single-particle fusion assays, including dramatically higher throughput and detection of lipid mixing under low efficiency conditions. As such, on purely technical grounds, this manuscript provides a significant advance to the field. The authors then apply their new approach to probe the role of HA-sialic acid binding in the membrane fusion reaction. This reflects an important open question in the field, which, as the authors point out, has been elusive. They conclude that sialic acid binding promotes efficient membrane fusion, probably by increasing the likelihood of the fusion peptide engaging the target membrane after acid-induced conformational changes. Overall, the manuscript is very well written; the data are rigorously analyzed and nicely presented; and the results are important and informative.
a criminogenic environment
I recommend including a definition for students.
Sawyer and Wagner 202
This citation is missing a digit in the date.
In the United States more than 2 out of 3 people in jail are legally innocent and simply awaiting trial because they cannot pay bail (Sawyer and Wagner 202, Sawyer et al 2026).
I'm happy to see this information included.
For each of the following statements, circle T for true or F for false:
T, F, F, T, T
) but
spacing issue.
Illinois Law Enforcement Training and Standards Board (ILETSB)
I recommend adding a link to the ILETSB website.
Men of African, indigenous, or other non-white ancestry would have endured restrictions on their ability to participate in policing efforts, and women’s involvement in policing would have been inconceivable until the middle of the 20th century
I recommend creating two sentences. The first sentence should include a date.
Example = Men of African, indigenous, or other non-white ancestry endured restrictions on their ability to participate in policing efforts until...
Also, women’s involvement in policing was prohibited until the middle of the 20th century.
when Illinois was part of the Northwest Territory
I think including a range of years here would be helpful.
Building and Location Responsible Police Department Rockford City Hall (425 E State Street) Rockford Police Department Alpine Pool (4310 Newburg Rd) Rockford Park District Police Winnebago County Jail at Winnebago County Criminal Justice Center (650 W State Street) Winnebago County Sheriff’s Office Brookview Elementary School (1750 Madron Road) Rockford Police Department Estelle M. Black Library (Rock Valley College, 3301 N. Mulford Road) Rock Valley College Police Department
This table is very helpful. I wonder if it would be beneficial to include a comparison to Baltimore to help students understand how unique the structure of government is in Illinois.
FF=(20lb/ft2)(200ft2)=18,000lb=18k
Correction FF=(20lb/ft2)(900ft2)=18,000lb=18k
Increase Text Decrease TextDecrease Text GrayscaleGrayscale High ContrastHigh Contrast High ContrastHigh Contrast Light BackgroundLight Background Links UnderlineLinks Underline Readable FontReadable Font Reset Reset
Robust: Maximize compatibility with current and future user tools.
A2. Co-deploy Ory Hydra + its DB; configure opaque tokens, DCR, PKCE-required, refresh rotation, revocation, and TTLs (~1h / ~30d).
Can you dig into it and explain what exact implemented by this lib vs what need to add around it?
Ory Hydra
this is GO library, need to use oidc-provider library
Implementation
What missing for me is when and how to get the apikey of the account, and how allow choosing which apikey to use? Maybe we need to add step to the consent page to show all account's apikey and allow user to choose which one to use.
redirect back with code
What the purpose of this step, why not sending the secret in this step?
Andrej Karpathy's popular critique
contrasting standard RAG with persistent, evolving knowledge bases.
💻/thinkpad/🧊/me/📓/2026/7/2/~/documents.looks.like.rags/~/search/@/google/p
search@google?"documents looks like rag"
,
Delete. No new subject.
How can other types of foods have their recipes modified to create fewer crumbs?
How can the recipes for other types of foods be modified to create fewer crumbs?
double baking soda
double the baking soda
here
delete
here
you tested
recipe(s) generate
This does create an awkward subject verb agreement issue. You could most easily get around it by shifting to "generated" (which works for both single and plural recipe/recipes). That still fits the context.
Or, you could just leave it as "recipes" ... you are asking which ones (of seven) generated the most crumbs.... that's also accurate for the project.
Or, just leave it, and assume most won't care that recipe/generate is an issue and treat "recipe(s)" as inherently plural.
as
because
,
No new subject
percent remaining
For reading clarity, it might make sense to reference it the way you did "Before" and "After"...
"Percent remaining"
(That matches the way it appears on the chart" -- or just use "percent remaining" lowercase)
, and is the same as 92%
"which equals 0.92 or 92%"
3 trials
Noticing it here.... in multiple surrounding spots in this section, you've spelled out three... "three different crumb tests"... "three cookies"
Might look back to see how much of this you've done. I see two instances of "3 trials" right here... 3.g and 4.e... but if you've spelled numbers out elsewhere... be consistent.
"cookies," and/or
Seems like you're second clause justifies just leaving this as "or"
of
about?
in the oven
Delete
You should bake the cookies for a total of 9 to 11 minutes, or until golden brown.
... unless they are burning as the previous sentence indicates.... obvious. But the sentence is contradictory.
And this suggests that if they are not burning at 5 minutes... you leave them to 9 or 11..... again... there's a bit of a logic gap here. This seems to "suggest" they might be burning at 5 minutes.... (which may be true for one recipe).
whisk,
No comma; no new subject
In a small bowl, combine the flour, baking soda, and salt and whisk, as shown in Figure 1, below.
The whisk isn't being thrown into the bowl with the ingredients.
"In a small bowl, combine the flour, baking soda, and salt and then whisk, as shown in Figure 1."
(Need the "and then" -- otherwise, break it to two sentences: In a small bowl, combine the flour, baking soda, and salt. Whisk the ingredients together, as shown in Figure 1. )
measure
weigh?
or you
Run-on: (comma required)
in one day, or you
Nestle
Nestlé
having
using?
having
using?
You will test the following three independent variables:
Again... issue of presentation.
i.e.
e.g.,
which of these ingredients are
If the end result is that "one" is most important, I would suggest correcting the grammar -- which --> is
If the implication is that more than one (of the three) is "most" important... just leave it.
test three independent variables (i.e., amount of flour, butter, and egg)
We don't typically "tell" them what their variables are. Particularly at this level, they should be able to determine that. I would suggest revising how this is phrased.
Lastly
Rethink "Lastly" twice in the same paragraph.
agitating (i.e., stirring up)
using "i.e.," here is cumbersome... at the high school level... this may not be needed
or just rephrase it: by agitating (by shaking or stirring)
the resultant
suggested change:
"that a cookie produces"
,
While this comma can technically be used here, in this case, the sentence will read better without it. (This sentence appears in the abstract, too. Same change suggested.) -- And, yes, grammarly will suggest ", such as" - which isn't the voice here.
crumby risk
cute... but may or may not work
"crumbly" might work
a "crumb risk" could work
or "the risk of crumbs"
Because crumbs can float in zero gravity, they can easily move around once they are loose, and then squeeze into places they should not be, such as electrical panels, where they may cause fires, and air filtration systems, clogging and breaking them.
First -- the comma before "and then" needs to be removed. There is no new verb. Second -- there is a parallelism issue with the ending. Two suggested alternatives:
Because crumbs can float in zero gravity, they can easily move around once they are loose and then squeeze into places they should not be, such as electrical panels, where they may cause fires, and air filtration systems, where they may clog the system and cause damage.
Or... "where they may clog and break the system" (if you want to stay with "break")
Crumbs float around in zero gravity, can squeeze into electrical panels, where they may cause fires, and can get sucked into air filtration systems, clogging them.
Suggested edit to improve flow and parallelism: "Crumbs can float around in zero gravity, squeeze into electrical panels, where they may cause fires, and get sucked into and clog air filtration systems."
Crumbs created by food
"Crumbs from food" -- more natural/typical phrasing (still true)
up
Delete
the
delete
roles
role (and before you argue it... you have "roles is"... -- while you are listing two things here, this will read better as role (otherwise, you need "roles are" -- this is a grammar error as is))
You and the LLM co-evolve this over time as you figure out what works for your domain.
co-evolve this
PLEX
Knowledge as a living wiki
It is more than knowledge
It is People Centered discovey/serendipity at the edge of Human Mutual Learning =
in the real InterPersonal commons on the IndyWeb
powered by IndyWiki
completing Retrieval Augmented Generation (RAG) powered by LLM
with HyperPost powered by IndyWiki PLEX
Doing the Job of exapnding not just organizing
Human Mutual Learning
for the People, by the People, augmenting Human InterIntellect for the Benefit of the People not the just the Providers of Centralized Services
not cometing but for mutual completion
WE'RE RAISING $25,000
**Bad practice:
**Text cuts of at "$25,0" and the page won't let me scroll horizontally to see what is being cut off. It's only through this annotation that Iam able to see that the number is "$25, 000".
Principle 1 – Perceivable: Guideline 1.4 – Distinguishable - 1.4.4 Resize Text - * Level AA: Except for captions and images of text, text can be resized without assistive technology up to 200 percent without loss of content or functionality.*
PartnershipsArt CartICA PAS ScholarshipUp:Rising Literary AnthologyRendezvous with Madness Artist in ResidencePartner With UsABOUT
*Bad Practice:
*Text "Partnerships" and "About" overlap when using the websites built-in Accessibility Menu's "Bigger Text" function to the maximum level with Chrome set to 100% zoom. For reference, I am using a standard TMU desktop screen. The end of and beginning of the 2 labels become illegible, not sure how the overlap would affect access to the subsections/menus that drop down from the labels. Could be a coding error.
Principle 1 – Perceivable: Guideline 1.4 – Distinguishable 1.4.4 Resize Text - Level AA: Except for captions and images of text, text can be resized without assistive technology up to 200 percent without loss of content or functionality.
IN CRISIS? Distress Centre: 416-408-4357
Bad practice:
Hot pink/red colour is used to emphasize text, or when the mouse hovers over a hyperlink. This emphasize is noticeable when using Colorblindly extension set to monochromacy / achromatopsia. This is the brand colour, but as mental health NGO, these particular text must always be emphasized and perceivable by all website users.
Principle 1 – Perceivable - Guideline 1.4 – Distinguishable - 1.4.1 - Use of Color - Level A: Color is not used as the only visual means of conveying information, indicating an action, prompting a response, or distinguishing a visual element.
Good practice:
Accessibility Menu available through "CTRL + U" immediately upon loading page. No need for a mouse or to go searching for the menu. All website users can adapt the page to fit their individual accessibility needs.
Operable: Make all functionality available from a keyboard.
Good Practice:
Logo and background have clear contrast. There's no greater colour contrast combination that black and white.
Understandable: Make text readable and understandable.
PLEX - wiki - llm - rag - document
It is more than knowledge
It is People Centered Personal first
in the real InterPersonal commons on the IndyWeb
Symathetic Egine
conversations
powered by IndyWiki
completing Retrieval Augmented Generation (RAG) powered by LLM
with HyperPost powered by IndyWiki PLEX
Doing the Job of exapnding not just organizing
Human Mutual Learning
for the People, by the People, augmenting Human InterIntellect for the Benefit of the People not the just the Providers of Centralized Services
not cometing but for mutual completion
250. Tackling ‘Big History’ feat. Ian Morris<br /> unSILOed Podcast with Greg LaBlanc
250. Tackling ‘Big History’ feat. Ian Morris<br /> unSILOed Podcast with Greg LaBlanc
Watched 2026-07-29
a Staff Engineer at Datadog, has written about using skills built around pup, a CLI for querying Datadog metrics, tailing logs, and managing dashboards
https://medium.com/jonathans-musings/agent-skills-the-cheat-codes-for-claude-code-b8679f0c3c4d
The idea is that a skill is essentially a useful context that can be re-used.
This preprint was published as Over half of threatened species require targeted recovery actions to avert human-induced extinction and can be found here: https://esajournals.onlinelibrary.wiley.com/doi/full/10.1002/fee.2537