wrong code

becuase of the immanent structuring no linear listing were ever used in Meta-Lisp in practice, dynamic navigation

although a complete listing via depth first traversal was always possible and told the complete story

parse rule

only contain parse information and node-building

just like MetaLISP!

figure out supposed to do

• The historian will have some puzzles to solve --> interesting way of stating that their logic is not clear? (3)
• Eugenics always had an evaluative logic at its core. Some human life was of more value -- to the state, the nation, the race, future generations -- than other life, and thus its advocates sought to implement these practices differentially ( 4) ==> did we ever examine who was making these policies and procedures and place a value on their lives
• Peak in the 1920s --> im assuming that this is referring to the US
• Contributing factors = nationalism, tech changes, changes in ideas about citizenry, labor force, future fitness (4) ABOUT MODERNITY
• Darwins work that highlighted that we are a part of nature and that we follow the same natural laws as everyone else critically undermined the idea that humans were made specially (4)
• Humans could and should replace natural selection by other processes that are more merciful and not less effective (4) huh --> he thought eugenics is merciful and more effective than natural selection (I wonder why this became so popular, especially when this time period is known for being religious
• Galton understood eugenics to be rational planning of and intervention into human breeding --> thought that the state should intervene into peoples own choices and such (5)
• Positive and negative eugenics (5)
• Oneida, NY the women professed: "we do not belong to ourselves", disrespectfully what in the world is this cult (5)
• American eugenics and ideas about eugenics shaping NS Germany
• 1926 Soviet Civic Code (5)
• aiming to restrict the reproduction of those with conditions and diseases considered heritable (CONSIDERED) --> did they know exactly (6)
• eugenic intervention directed at those who already belonged (6)
• Different priorities to the varying eugenic movements across the world --> southern US v NS Germany v Australian English (6)
• "Polluting" was not racial outsiders but often marginalized insiders
• Geographic insight --> more concentrated on the urban problem populations of industrialization (6) --> why
• America --> poor, rural whites: southern European immigrants; African Americans (7)
• Anxieties about interracial marriage were frequently linked to colonial rule (7)
• Race crossing --> Erst Rodenwalt (7)
• Australia --> half-caste problem (7) biological and cultural assimilation influenced by eugenic ideas ==> boarding school type beat
• Prospect of extinction (8)
• primitive societies where weak offspring were not nursed were admired as naturally eugenic (8) ==> looking at primitive examples to propel an ide of modernity
• relationship between eugenic and birth control
• In Eastern Europe --> more public health spending (8)
• into the management of infectious disease (9)
• Kenya example --> uneducable making their education irrelevant to the state (9)
• linked to the proliferation of asylums from the 19th century
• Dr. Harry Haselden withheld treatment for newborns --> what about the Hippocratic oath (9)
• designations of fit and unfit applied to both populations and to individuals
• one eye on the past generations and one eye on the future (9)
• pedigree chart (10)
• both research and propaganda
• Darwin knew about the principles of genetics, yet married his first cousin (10)
• insurance used information from Biometrika (10)
• Issue with the pedigree system bc it thinks that things like intelligence and other "good" characteristics are associated with inheritance but not with access to social support and resources (10)
• Eastern Europe --> eugenics could match and enshrine the aspirations of new nation-states anxious to establish their legitimacy (12)
• Japanese modernization manifested itself as nationalism (quality over quantity) (12)
• homogeneity over heterogeneity (12)
• strong connection between eugenics and the left and to progressive reform and politics (13)
• sterilization has informed the concept of "rights" in reproductive arenas (14)
• Consent --> Nuremberg trials (14)
• States that were looking to modernize were those who took up eugenics enthusiastically
• NS Germany (14/15)
• mythical concern --> where did this originate from (15)
• similarities across so many countries and cultures is interesting in the eugenics movements (15)
• Eugenics in immigration regulations (16)
• Central America and Southern Europe distinguished itself (16)
• Cross cultural ideas and exchange allowed eugenics to move so fast and so far but then they all wanted to be independent and create their own identity (16)
• Hygiene was also linked to eugenics --> "Rassenhygiene" in DE (17)
• Interesting to see how they distanced themselves from forms of eugenics that didn't come from their own home country
• sterilization procedures were antithetical to catholic doctrine and were highly questionable (18)
• British sought the legalization of sterilization but voluntary sterilization was always their aim (18)
• US is a contradiction --> commitment to liberty also passed compulsory sterilization laws (18)
• Voluntary v coerced (18)
• eugenics has always had opposition (19)
• Jennings essentially conceptualized pre-natal testing (19)
• 1970s drew large opposition (20)
• Tuskegee syphilis experiment highlighted conversations about races and medical ethics (20)
• This was a period of strongly left-oriented intellectual critique of science (20)

This is incredibly sad but visible in our BIPOC communities.

While exploring the NASW Code of Ethics, I found an important aspect related to power and inequality in the section on Social and Political Action (6.04). This section emphasizes the duty of social workers to engage in action against social injustice. However, it raises questions about the depth of guidance provided regarding power dynamics in the profession. I've noted this section to express my curiosity about how the Code of Ethics could offer clearer guidance on addressing power imbalances within social work organizations and in broader society. This encourages a deeper exploration of strategies to challenge oppressive systems.

Instead of keeping track of a num_reviews variable, we can also loop over the index and review of each movie simultaneously by using enumerate(movies). This gives us fewer variables to think about and makes the code a bit more elegant and readable.

The length count includes all the spaces, period(,) and exclamation mark.

Dans le code HTML de à-propos il y à une erreur : - vous avez rajouter class="lien-icone" au niveau du logo de la page a propos , ce qui lui applique le changement d'opacité au survole identique au lien hypertexte Instagram et twitter qui possède eu aussi la classe déclarer .lien-icone.

Escape sequences in JavaScript are special combinations of characters that are used to represent characters that would otherwise be difficult or impossible to include directly in a string. These sequences start with a backslash \ followed by another character or characters.

Here are some common escape sequences in JavaScript:

1. \n - Newline:
2. Represents a line break.

javascript console.log("Hello\nWorld"); // Output: // Hello // World

1. \t - Tab:
2. Represents a horizontal tab.

javascript console.log("This\tis\tTab"); // Output: This is Tab

1. \ - Backslash:
2. Represents a literal backslash.

javascript console.log("This is a backslash: \\"); // Output: This is a backslash: \

1. \' - Single Quote:
2. Represents a single quote within a string declared with single quotes.

javascript console.log('It\'s a sunny day.'); // Output: It's a sunny day.

1. \" - Double Quote:
2. Represents a double quote within a string declared with double quotes.

javascript console.log("She said, \"Hello!\""); // Output: She said, "Hello!"

1. \uXXXX - Unicode Escape:
2. Represents a Unicode character, where XXXX is the Unicode code point in hexadecimal.

javascript console.log("\u0041"); // Output: A

These escape sequences allow you to include special characters in your strings without causing syntax errors. For example, if you want to include a quote character within a string that is already enclosed in quotes, you can use the escape sequence to prevent confusion and errors.

Certainly! Let's break it down in simpler terms:

String Primitives and String Objects:

• In JavaScript, there are two types of strings: string primitives and String objects.
• String literals (created using double or single quotes) and strings returned from non-constructor String calls are primitive strings.
• String objects are created using the new String() syntax.

Examples:

```javascript const strPrim = "foo"; // A string primitive const strPrim2 = String(1); // Coerced into the string primitive "1" const strPrim3 = String(true); // Coerced into the string primitive "true" const strObj = new String(strPrim); // String with new returns a string wrapper object.

console.log(typeof strPrim); // "string" console.log(typeof strPrim2); // "string" console.log(typeof strPrim3); // "string" console.log(typeof strObj); // "object" ```

• strPrim, strPrim2, and strPrim3 are string primitives, while strObj is a String object.

Using eval():

• When using eval() with string primitives, the string is treated as source code, and the result is evaluated.
• When using eval() with String objects, it treats the object like any other object and returns the object itself.

Examples:

```javascript const s1 = "2 + 2"; // creates a string primitive const s2 = new String("2 + 2"); // creates a String object

console.log(eval(s1)); // returns the number 4 console.log(eval(s2)); // returns the string "2 + 2" ```

• eval(s1) evaluates the string primitive as code and returns the result (number 4).
• eval(s2) returns the String object itself (string "2 + 2"), not the evaluated result.

Conversion to Primitive:

• To convert a String object to its primitive counterpart, you can use the valueOf() method.

Example:

javascript console.log(eval(s2.valueOf())); // returns the number 4

• valueOf() converts the String object (s2) to its primitive form before being evaluated, resulting in the number 4.

Conclusion:

• In general, it's rare to use String as a constructor (using new String()), and working with string primitives is more common. Understanding the differences is important when dealing with certain situations like using eval() or expecting a specific type of string.

This section of the NASW code of ethics raises important questions regarding power and structural inequality because sometimes it may be hard for social workers to find the clear boundary of dual-relationships. For example, if a client invites you to an event they are holding that is unrelated to your professional relationship like a retirement party or another life milestone event, how do you decipher which type of event is appropriate to attend and which is not? Due to your professional influence on this person's life, what if your personal bias for this client exceeds the level that is appropriate for a standard social worker/client relationship but you don't see it that way because they are "inviting you in" or consenting to it. I feel it's important for social workers to build strong rapport with their clients to facilitate more effective practice however, ones judgement on when this rapport building may be crossing the line into unprofessional territory may vary on a case by case basis.

This part of the NASW code of ethics pertains to my field work because in my role as the housing navigation specialist at the shelter, I not only help clients secure permanent housing through government subsidies but, I also advocate on their behalf when they are being faced with blatant discrimination. This advocacy is not only reflected in my efforts to educate my clients on their own housing rights but also by educating landlords, relators, and property managers on the policy of housing subsidies like state wide section 8 and local municipality housing voucher programs. Recently, I was working with a client who has a section 8 voucher through the state. This client was about to sign their request for tenancy with the landlord but once the landlord looked over the DCA (department of community affairs) terms of agreement they stated in writing that they "did not want to move forward with a voucher holder." Luckily, New Jersey is one of the 49 states that have fair housing legislature in place so I was able to report this correspondence to my clients DCA worker as discrimination and they were granted more time to secure permanent housing. There is a lot of stigma tied to the homeless community, especially those who obtain housing subsidies through local and federal government entities. This is why legislature like The Fair Housing Act is so important in the work that I do and the vulnerable community I serve.

React Hooks are functions provided by React that allow you to use state and lifecycle features in functional components, making them more powerful and expressive. They were introduced in React version 16.8 to let developers use state and other React features without writing a class.

useState Hook:

useState is a Hook that allows you to add state to functional components. It returns an array with two elements: the current state value and a function that lets you update it. Here's a simple example:

```jsx import React, { useState } from 'react';

function Counter() { // Declare a state variable named "count" with an initial value of 0 const [count, setCount] = useState(0);

return ( <div>

Count: {count}

In this example, count is the state variable, and setCount is the function to update it. When the "Increment" button is clicked, it updates the count state, and React automatically re-renders the component.

useEffect Hook:

useEffect is a Hook that enables you to perform side effects in functional components. It's similar to lifecycle methods in class components. Here's a simple example fetching data:

```jsx import React, { useState, useEffect } from 'react';

function DataFetcher() { const [data, setData] = useState(null);

useEffect(() => { // Fetch data when the component mounts fetch('https://api.example.com/data') .then(response => response.json()) .then(data => setData(data));

// Clean up any resources when the component unmounts
return () => {
  // Cleanup code here
};

}, []); // The empty array ensures this effect runs only once on mount

return ( <div>

Data: {data ? data.value : 'Loading...'}

In this example, useEffect runs when the component mounts. It fetches data and updates the state. The empty dependency array ([]) means the effect runs only once when the component mounts.

useContext Hook:

useContext is a Hook that allows you to subscribe to React context without introducing nesting. It lets you access the value of a context directly.

```jsx import React, { useContext } from 'react';

const ThemeContext = React.createContext('light');

function ThemedComponent() { const theme = useContext(ThemeContext);

return

Current Theme: {theme}

In this example, ThemedComponent can directly access the current theme value from the ThemeContext.

These are just a few examples of React Hooks. There are others like useReducer, useCallback, useMemo, etc., each serving a specific purpose to enhance the functionality of functional components. Hooks allow you to manage state and side effects more effectively in functional components, making your code cleaner and more maintainable. Absolutely! Let's break down the statement in simpler terms:

useRef Explanation:

useRef is like a tool in React that helps you create a special reference to something, and this reference won't force your component to re-render when the referenced thing changes.

Simple Explanation:

1. Creating a Reference:
2. With useRef, you can create a reference to a value, like a number, an object, or even a DOM element.

```jsx import { useRef } from 'react';

function MyComponent() { const myRef = useRef(0); // Creating a reference to the number 0 return <div>{myRef.current}</div>; } ```

In this example, myRef is a reference to the number 0.

1. No Re-rendering:
2. When the value of the reference changes, your component won't automatically re-render. This is useful when you want to keep track of something without affecting the UI.

```jsx import { useRef, useState, useEffect } from 'react';

function Counter() { const count = useRef(0); // Creating a reference to the number 0 const [renderCount, setRenderCount] = useState(0);

 useEffect(() => {
   // This effect will run when the component renders
   setRenderCount(renderCount + 1);

   // But changing the count won't cause a re-render
   count.current = count.current + 1;
 }, [count, renderCount]);

 return (
   <div>
     <p>Render Count: {renderCount}</p>
     <p>Count (no re-render): {count.current}</p>
   </div>
 );

} ```

In this example, changing the count won't trigger a re-render of the component.

When to Use useRef:

• Use useRef when you want to keep track of a value that doesn't affect your component's display directly and shouldn't cause re-renders.

• Common use cases include interacting with the DOM directly or storing mutable values that shouldn't trigger updates.

In summary, useRef is a handy tool in React for creating references that won't cause your component to re-render when the referenced value changes. It's useful for scenarios where you need to keep track of something without impacting the UI. Certainly! Let's break down the statement in very simple terms with multiple examples:

Explanation:

1. Memoized Object:
2. When you use useRef to create a reference, React keeps track of it. Instead of creating a new reference object every time your component re-renders, React gives you the same reference object.

```jsx import { useRef } from 'react';

function MyComponent() { const myRef = useRef(0);

 // On each render, myRef is the same object
 console.log(myRef === myRef); // true

} ```

In this example, myRef remains the same object across renders.

1. Avoiding Recreation:
2. This memoization is useful when you don't want to recreate the reference object, especially when dealing with functions. It helps to maintain consistency and avoids unnecessary re-initialization.

```jsx import { useRef, useEffect } from 'react';

function EffectComponent() { const handleClick = useRef(() => { console.log('Button clicked!'); });

 useEffect(() => {
   // Access the function without causing a re-render
   handleClick.current();
 }, []);

 return <button onClick={handleClick.current}>Click me</button>;

} ```

In this example, handleClick remains the same function reference across renders.

1. Ensuring Same Object:
2. The memoized object ensures that the reference remains consistent, even when the component re-renders. This is essential when dealing with external functions or references that you want to persist.

```jsx import { useRef } from 'react';

function ReferenceComponent() { const externalFunction = () => { console.log('External function'); };

 const externalFunctionRef = useRef(externalFunction);

 // externalFunctionRef.current is the same function across renders
 console.log(externalFunctionRef.current === externalFunction); // true

} ```

In this example, externalFunctionRef.current is the same function as externalFunction across renders.

Simplified Summary:

• useRef gives you the same reference object across renders.
• This is useful to avoid recreating the reference, especially when dealing with functions.
• It ensures consistency and stability, preventing unnecessary re-initialization.

In essence, useRef helps you maintain the same reference object, ensuring stability and preventing unnecessary re-creation, which is particularly handy when working with functions or external references.Sure, let's break down useRef in simple terms with examples:

What is useRef?

useRef is a React Hook that allows you to create a reference to a value that won't trigger a re-render when it changes. It's often used for things that are not directly related to rendering, like accessing or manipulating the DOM.

How to Use useRef:

1. Referencing a Value: You can use useRef to create a reference to a value, such as a number or an object. This value can be accessed and modified without causing your component to re-render.

```jsx import { useRef } from 'react';

function MyComponent() { const intervalRef = useRef(0); // Reference to a number const inputRef = useRef(null); // Reference to an element (initially null) // ... ```

1. Manipulating the DOM: useRef is commonly used for interacting with the DOM directly. For example, if you want to focus on an input element or keep track of some DOM-related state without triggering a re-render.

```jsx import { useRef, useEffect } from 'react';

function MyComponent() { const inputRef = useRef(null);

 useEffect(() => {
   // Focus on the input element when the component mounts
   inputRef.current.focus();
 }, []);

 return <input ref={inputRef} />;

} ```

1. Avoiding Recreating the Ref Contents: useRef is memoized, meaning it returns the same object on every render. This is useful when you want to avoid recreating the ref object, especially when dealing with functions.

```jsx import { useRef, useEffect } from 'react';

function MyComponent() { const handleClick = useRef(() => { console.log('Button clicked!'); });

 useEffect(() => {
   // Access the function without causing a re-render
   handleClick.current();
 }, []);

 return <button onClick={handleClick.current}>Click me</button>;

} ```

Parameters and Returns:

• Parameters:

• initialValue: The value you want the ref object’s current property to be initially. It can be a value of any type. This argument is ignored after the initial render.

• Returns:

• useRef returns an object with a single property:
  • current: Initially set to the initialValue you have passed. You can later set it to something else. If you pass the ref object to React as a ref attribute to a JSX node, React will set its current property.

In simple terms, useRef is a tool to keep track of values or elements that won't cause your component to re-render every time they change. It's commonly used for interacting with the DOM and handling mutable values in a React component.

This passage reveals the importance of academic integrity and how academic dishonesty is not tolerated at DVC.

Trolling can be driven by a nihilistic mindset where empathy is disregarded, aiming solely to provoke reactions, often manifesting as hostility towards specific groups. This form of trolling is widely condemned as it inflicts harm, failing to adhere to basic principles of respect and integrity.

The section provides a profound exploration of the complexities involved in understanding and evaluating disruptive behaviors in social media contexts. It compellingly illustrates how the formation of groups, the use of stereotypes, and the enforcement of norms are all deeply intertwined with our cognitive processes and societal structures. The examination of trolling as a form of disruption that can be deployed for both just and unjust ends invites readers to reflect on the multifaceted nature of these actions and their ethical implications.

Certainly! Let's break down the provided information into simple terms:

1. Guard in Headers:
2. Headers are like additional notes attached to an HTTP request or response.
3. They have a property called "guard" that determines which operations are allowed on the headers object.
4. Guard values include:
   • none (default): No specific restrictions.
   • request: Applied to headers obtained from a request.
   • request-no-cors: Applied to headers from a no-cors request.
   • response: Applied to headers obtained from a response.
   • immutable: Renders headers read-only, commonly used in ServiceWorkers.

Example: javascript const myHeaders = new Headers(); // Applying guard to headers obtained from a request const requestHeaders = new Headers(myHeaders, { guard: "request" });

1. Response Objects:
2. When you make a request using fetch(), it returns a Response object.
3. Key properties include:
   • Response.status: An integer indicating the response status code (e.g., 200 for success).
   • Response.statusText: A string corresponding to the HTTP status code message.
   • Response.ok: A boolean indicating if the status is in the range 200-299.

Example: javascript const response = await fetch("https://example.com/api/data"); console.log(response.status); // 200 console.log(response.statusText); // OK console.log(response.ok); // true

1. Creating Response Object Programmatically:
2. You can create a Response object programmatically in a ServiceWorker.
3. This is useful when providing a custom response to a received request using respondWith().

Example: ```javascript const myBody = new Blob();

addEventListener("fetch", (event) => { // ServiceWorker intercepting a fetch event.respondWith( new Response(myBody, { headers: { "Content-Type": "text/plain" }, }), ); }); ```

1. Body Content in Requests and Responses:
2. Both requests and responses may have body content, which can be of various types (e.g., ArrayBuffer, Blob, JSON).
3. Methods like arrayBuffer(), blob(), formData(), json(), and text() help extract the body content.

Example: javascript const responseData = await response.json(); // Or for request body const formData = new FormData(document.getElementById("myForm")); fetch("/api/data", { method: "POST", body: formData, });

1. Feature Detection:
2. You can check if the Fetch API is supported in a browser by looking for the existence of fetch on the window or Worker scope.

Example: javascript if (window.fetch) { // Fetch is supported, proceed with fetch requests } else { // Use alternative like XMLHttpRequest }

1. Differences from jQuery.ajax():
2. The Fetch API differs from jQuery.ajax() in terms of error handling and handling of credentials in cross-origin requests.

Example: javascript fetch("https://example.com/api/data") .then((response) => { if (!response.ok) { throw new Error("Network response was not OK"); } return response.json(); }) .then((data) => { console.log(data); }) .catch((error) => { console.error("Error:", error); });

These explanations and examples aim to simplify the concepts introduced in the provided information about Headers, Response objects, Body content, and feature detection in the Fetch API.

Certainly! Let's break down each section with simple explanations and examples:

1. Uploading a File:
2. To upload a file, you can use an HTML <input type="file" /> element to let users choose a file.
3. Combine this with FormData() to gather form data, and then use fetch() to send it to a server.

Example: ```javascript async function upload(formData) { try { const response = await fetch("https://example.com/profile/avatar", { method: "PUT", body: formData, }); const result = await response.json(); console.log("Success:", result); } catch (error) { console.error("Error:", error); } }

const formData = new FormData(); const fileField = document.querySelector('input[type="file"]'); formData.append("username", "abc123"); formData.append("avatar", fileField.files[0]);

upload(formData); ```

1. Uploading Multiple Files:
2. If you want to upload multiple files, use the multiple attribute in the <input type="file" /> element.
3. Similar to the single-file example, use FormData() and fetch().

Example: ```javascript async function uploadMultiple(formData) { try { const response = await fetch("https://example.com/posts", { method: "POST", body: formData, }); const result = await response.json(); console.log("Success:", result); } catch (error) { console.error("Error:", error); } }

const photos = document.querySelector('input[type="file"][multiple]'); const formData = new FormData();

formData.append("title", "My Vegas Vacation"); for (const [i, photo] of Array.from(photos.files).entries()) { formData.append(photos_${i}, photo); }

uploadMultiple(formData); ```

1. Processing a Text File Line by Line:
2. If you need to process a text file line by line, you can create an iterator.
3. This example assumes UTF-8 encoding and uses a TextDecoder.

Example: ```javascript async function* makeTextFileLineIterator(fileURL) { // ... (see provided code) }

async function run() { for await (const line of makeTextFileLineIterator(urlOfFile)) { processLine(line); } }

run(); ```

1. Checking Fetch Success:
2. It's essential to check if a fetch operation was successful.
3. You can do this by checking response.ok to ensure the network response was okay.

Example: javascript async function fetchImage() { try { const response = await fetch("flowers.jpg"); if (!response.ok) { throw new Error("Network response was not OK"); } const myBlob = await response.blob(); myImage.src = URL.createObjectURL(myBlob); } catch (error) { console.error("There has been a problem with your fetch operation:", error); } }

These examples cover uploading files, handling multiple file uploads, processing text files line by line, and checking the success of a fetch operation. They demonstrate practical uses of the Fetch API in various scenarios.

Certainly! Let's break down the code and concepts in simpler terms:

1. Fetch API Overview:
2. The Fetch API is a way for JavaScript to make network requests (like fetching data from a server) and handle the responses.

3. It's an improvement over the older XMLHttpRequest, providing a cleaner, promise-based syntax.

4. Basic Fetch Request:

5. The fetch() function is used to initiate a network request.
6. It takes a URL as an argument, specifying where to fetch data from.

7. The fetch() function returns a Promise, which represents the result of the request.

8. Asynchronous Code with await:

9. The async keyword in the function declaration indicates that the function contains asynchronous code.

10. The await keyword is used to wait for the completion of a Promise before moving on to the next line of code.

11. Fetching JSON Data:

12. In the example, we're fetching data from "http://example.com/movies.json."

13. The response from the server is stored in the response variable.

14. Parsing JSON Response:

15. The response.json() method is used to parse the response body as JSON.

16. It returns another Promise that resolves to the actual JSON data.

17. Logging the Result:

18. The movies variable holds the parsed JSON data.

19. In this example, it's logged to the console. You could do other things with the data, like updating a webpage or performing calculations.

20. Summary:

21. fetch() initiates a network request and returns a Promise.
22. await is used to wait for the Promise to resolve, making asynchronous code easier to read and write.
23. response.json() parses the JSON content of the response.
24. The whole process is wrapped in an async function for cleaner asynchronous handling.

Example in Simpler Terms: ```javascript async function logMovies() { // Fetch data from a server const response = await fetch("http://example.com/movies.json");

// Parse the data as JSON const movies = await response.json();

// Print the movies to the console console.log(movies); }

// Call the function to see it in action logMovies(); ```

This function fetches movie data, waits for the response, parses the JSON, and then logs the movies to the console. It's a convenient way to handle asynchronous operations in JavaScript.

Reviewer #1 (Public Review):

This valuable study demonstrates a novel mechanism by which implicit motor adaptation saturates for large visual errors in a principled normative Bayesian manner. Additionally, the study revealed two notable empirical findings: visual uncertainty increases for larger visual errors in the periphery, and proprioceptive shifts/implicit motor adaptation are non-monotonic, rather than ramp-like. This study is highly relevant for researchers in sensory cue integration and motor learning. However, I find some areas where statistical quantification is incomplete, and the contextualization of previous studies to be puzzling.

Issue #1: Contextualization of past studies.

While I agree that previous studies have focused on how sensory errors drive motor adaptation (e.g., Burge et al., 2008; Wei and Kording, 2009), I don't think the PReMo model was contextualized properly. Indeed, while PReMo should have adopted clearer language - given that proprioception (sensory) and kinaesthesia (perception) have been used interchangeably, something we now make clear in our new study (Tsay, Chandy, et al. 2023) - PReMo's central contribution is that a perceptual error drives implicit adaptation (see Abstract): the mismatch between the felt (perceived) and desired hand position. The current paper overlooks this contribution. I encourage the authors to contextualize PReMo's contribution more clearly throughout. Not mentioned in the current study, for example, PReMo accounts for the continuous changes in perceived hand position in Figure 4 (Figure 7 in the PReMo study).

There is no doubt that the current study provides important additional constraints on what determines perceived hand position: Firstly, it offers a normative Bayesian perspective in determining perceived hand position. PReMo suggests that perceived hand position is determined by integrating motor predictions with proprioception, then adding a proprioceptive shift; PEA formulates this as the optimal integration of these three inputs. Secondly, PReMo assumed visual uncertainty to remain constant for different visual errors; PEA suggests that visual uncertainty ought to increase (but see Issue #2).

Issue #2: Failed replication of previous results on the effect of visual uncertainty.

2a. A key finding of this paper is that visual uncertainty linearly increases in the periphery; a constraint crucial for explaining the non-monotonicity in implicit adaptation. One notable methodological deviation from previous studies is the requirement to fixate on the target: Notably, in the current experiments, participants were asked to fixate on the target, a constraint not imposed in previous studies. In a free-viewing environment, visual uncertainty may not attenuate as fast, and hence, implicit adaptation does not attenuate as quickly as that revealed in the current design with larger visual errors. Seems like this current fixation design, while important, needs to be properly contextualized considering how it may not represent most implicit adaptation experiments.

2b. Moreover, the current results - visual uncertainty attenuates implicit adaptation in response to large, but not small, visual errors - deviates from several past studies that have shown that visual uncertainty attenuates implicit adaptation to small, but not large, visual errors (Tsay, Avraham, et al. 2021; Makino, Hayashi, and Nozaki, n.d.; Shyr and Joshi 2023). What do the authors attribute this empirical difference to? Would this free-viewing environment also result in the opposite pattern in the effect of visual uncertainty on implicit adaptation for small and large visual errors?

2c. In the current study, the measure of visual uncertainty might be inflated by brief presentation times of comparison and referent visual stimuli (only 150 ms; our previous study allowed for a 500 ms viewing time to make sure participants see the comparison stimuli). Relatedly, there are some individuals whose visual uncertainty is greater than 20 degrees standard deviation. This seems very large, and less likely in a free-viewing environment.

2d. One important confound between clear and uncertain (blurred) visual conditions is the number of cursors on the screen. The number of cursors may have an attenuating effect on implicit adaptation simply due to task-irrelevant attentional demands (Parvin et al. 2022), rather than that of visual uncertainty. Could the authors provide a figure showing these blurred stimuli (gaussian clouds) in the context of the experimental paradigm? Note that we addressed this confound in the past by comparing participants with and without low vision, where only one visual cursor is provided for both groups (Tsay, Tan, et al. 2023).

Issue #3: More methodological details are needed.

3a. It's unclear why, in Figure 4, PEA predicts an overshoot in terms of perceived hand position from the target. In PReMo, we specified a visual shift in the perceived target position, shifted towards the adapted hand position, which may result in overshooting of the perceived hand position with this target position. This visual shift phenomenon has been discovered in previous studies (e.g., (Simani, McGuire, and Sabes 2007)).

3b. The extent of implicit adaptation in Experiment 2, especially with smaller errors, is unclear. The implicit adaptation function seems to be still increasing, at least by visual inspection. Can the authors comment on this trend, and relatedly, show individual data points that help the reader appreciate the variability inherent to these data?

3c. The same participants were asked to return for multiple days/experiments. Given that the authors acknowledge potential session effects, with attenuation upon re-exposure to the same rotation (Avraham et al. 2021), how does re-exposure affect the current results? Could the authors provide clarity, perhaps a table, to show shared participants between experiments and provide evidence showing how session order may not be impacting results?

3d. The number of trials per experiment should be detailed more clearly in the Methods section (e.g., Exp 4). Moreover, could the authors please provide relevant code on how they implemented their computational models? This would aid in future implementation of these models in future work. I, for one, am enthusiastic to build on PEA.

3f. In addition to predicting a correlation between proprioceptive shift and implicit adaptation on a group level, both PReMo and PEA (but not causal inference) predict a correlation between individual differences in proprioceptive shift and proprioceptive uncertainty with the extent of implicit adaptation (Tsay, Kim, et al. 2021). Interestingly, shift and uncertainty are independent (see Figures 4F and 6C in Tsay et al, 2021). Does PEA also predict independence between shift and uncertainty? It seems like PEA does predict a correlation.

References:

Avraham, Guy, Ryan Morehead, Hyosub E. Kim, and Richard B. Ivry. 2021. "Reexposure to a Sensorimotor Perturbation Produces Opposite Effects on Explicit and Implicit Learning Processes." PLoS Biology 19 (3): e3001147.<br /> Makino, Yuto, Takuji Hayashi, and Daichi Nozaki. n.d. "Divisively Normalized Neuronal Processing of Uncertain Visual Feedback for Visuomotor Learning."<br /> Parvin, Darius E., Kristy V. Dang, Alissa R. Stover, Richard B. Ivry, and J. Ryan Morehead. 2022. "Implicit Adaptation Is Modulated by the Relevance of Feedback." BioRxiv. https://doi.org/10.1101/2022.01.19.476924.<br /> Shyr, Megan C., and Sanjay S. Joshi. 2023. "A Case Study of the Validity of Web-Based Visuomotor Rotation Experiments." Journal of Cognitive Neuroscience, October, 1-24.<br /> Simani, M. C., L. M. M. McGuire, and P. N. Sabes. 2007. "Visual-Shift Adaptation Is Composed of Separable Sensory and Task-Dependent Effects." Journal of Neurophysiology 98 (5): 2827-41.<br /> Tsay, Jonathan S., Guy Avraham, Hyosub E. Kim, Darius E. Parvin, Zixuan Wang, and Richard B. Ivry. 2021. "The Effect of Visual Uncertainty on Implicit Motor Adaptation." Journal of Neurophysiology 125 (1): 12-22.<br /> Tsay, Jonathan S., Anisha M. Chandy, Romeo Chua, R. Chris Miall, Jonathan Cole, Alessandro Farnè, Richard B. Ivry, and Fabrice R. Sarlegna. 2023. "Implicit Motor Adaptation and Perceived Hand Position without Proprioception: A Kinesthetic Error May Be Derived from Efferent Signals." BioRxiv. https://doi.org/10.1101/2023.01.19.524726.<br /> Tsay, Jonathan S., Hyosub E. Kim, Darius E. Parvin, Alissa R. Stover, and Richard B. Ivry. 2021. "Individual Differences in Proprioception Predict the Extent of Implicit Sensorimotor Adaptation." Journal of Neurophysiology, March. https://doi.org/10.1152/jn.00585.2020.<br /> Tsay, Jonathan S., Steven Tan, Marlena Chu, Richard B. Ivry, and Emily A. Cooper. 2023. "Low Vision Impairs Implicit Sensorimotor Adaptation in Response to Small Errors, but Not Large Errors." Journal of Cognitive Neuroscience, January, 1-13.

This section particularly the exploration of utilitarianism in the context of social media, provides a thought-provoking perspective on ethical decision-making. The concept of the utility calculus as a method of predicting the outcomes and moral implications of our actions highlights the importance of comprehensive data collection and the potential pitfalls of biased or incomplete data. The discussion cleverly highlighted the challenges of navigating social media in an ethical manner, which must consider

I think that utilitarianism is not fit for every conditions and I don't actually agree with this concept. I think that personal benefits and feelings is also very important. People do not need to sacrifice in order to make everyone happy.

I expereinced what is context collapse unkowingly recently and learning about the dramaturgical analysis it made me feel alot better about it. I hosted a birthday party and decided I wanted who I wanted around me for my birthday and without much thought, invited a bunch of people. To my surprise I hadn't thought about how all these people wold mesh together and had a mini identity crisis because of it. The code switching the room was crazy.

This statement emphasises the relativity of behaviour, i.e. the same behaviour may present different expressions in different contexts. This quote emphasises that authenticity is a relative concept that depends on the specific environment and social context in which an individual finds themselves. For example, through the example of sharing an experience with a close friend, it emphasises that authenticity in an intimate relationship may be expressed in a more relaxed and natural way.

We act "code switch" in different places (being serious at work but relaxed with friends) and both ways can be the real us. On social media, when we post, all our different groups see the same posts, it can make us wonder if we're being true to ourselves in each situation.

I know this label of "code switching" means an extreme of code switching, But it does come from the need to inherit the same attributes and beliefs of those around you to fit in. This can be related to a certain work environment "etc" where it is easier to agree to your boss than to contribute your own opinion. This can be an issue however when you are in a position with some power where your acts have possible influences compared to a boss employee scenario where the employee has very little to no power in their situation.

I use code-switching on a daily basis. The way in which I speak and act at work or in class is completely different than the way in which I act at home with my family and friends. Even my online self tends to showcase my more formal side as if I am at work or another professional setting. This is honestly why I have multiple accounts; One of my accounts is more business and a representation of how I want to be perceived when in a formal setting whereas my other account highlights more of the ways I act at home with friends or family. Code-switching makes you feel safe and accepted in an environment that you feel would not be as accepting of your true self.

The prevalence of multiple personas on social media makes this intriguing to me. People frequently behave differently when they are in front of a screen. Furthermore, a lot of people identify quite differently.The concept of a public persona and code-switching also reflects the dynamic nature of human behavior and communication.

I feel that it takes practice to make it seem more authentic. Although it can be useful for certain situations, it can be draining by the end of it.

In my opinion, I think public personas and code-switching are intricate yet crucial facets of interpersonal communication. They enable people to successfully negotiate various social environments, showcasing various aspects of their true selves in polite and situation-appropriate ways. I also really like the example of Obama of how he used different type of movement to different audience. This is one of his way to show his social intelligence and respect to others.

to

Certainly! Let's break down the information in simpler terms with examples:

Under the Hood - MongoDB Update Queries with Timestamps:

1. Properties Added by Mongoose:

2. When you perform update queries with timestamps enabled, Mongoose adds two special properties to the MongoDB update operation:

   • updatedAt: Added to the $set operator. It represents the last update time.
   • createdAt: Added to the $setOnInsert operator. It represents the creation time and is applied only when a new document is upserted (inserted if not found).

3. Example - Update Query:

4. Consider the following code:

```javascript const userSchema = new Schema({ name: String }, { timestamps: true }); const User = mongoose.model('User', userSchema);

await User.findOneAndUpdate({}, { name: 'test' }); ```

• In the debug output, you'll see MongoDB update operators like $setOnInsert and $set:

plaintext Mongoose: users.findOneAndUpdate({}, { '$setOnInsert': { createdAt: new Date("Sun, 27 Feb 2022 00:26:27 GMT") }, '$set': { updatedAt: new Date("Sun, 27 Feb 2022 00:26:27 GMT"), name: 'test' }}, {...})

1. Explanation of $setOnInsert and $set:
2. $setOnInsert: It sets the specified values only if a new document is inserted during an upsert operation. In the example, it sets createdAt only if a new document is created.

3. $set: It sets the specified values regardless of whether the document is new or existing. In the example, it sets updatedAt and updates the name.

4. Disabling updatedAt Timestamp and Setting Manually:

5. If you want to handle updatedAt manually and disable automatic updates, you can do so:

javascript await User.findOneAndUpdate({}, { $setOnInsert: { updatedAt: new Date() } }, { timestamps: { createdAt: true, updatedAt: false } });

• This way, you can control when updatedAt is set, and it won't be automatically managed by Mongoose.

In simple terms, Mongoose adds special properties like updatedAt and createdAt to MongoDB update queries when timestamps are enabled. These properties are essential for tracking update and creation times. You can customize the behavior by manually handling timestamps or adjusting the update options.

Certainly! Let's simplify the information and examples provided:

Timestamps and Property Names:

1. Custom Property Names:
2. By default, Mongoose uses createdAt and updatedAt as timestamp properties. However, you can customize these names:

javascript const userSchema = new Schema({ name: String }, { timestamps: { createdAt: 'created_at', updatedAt: 'updated_at' } });

Now, instead of createdAt and updatedAt, your properties will be named created_at and updated_at.

1. Disabling Timestamps:
2. You can choose to skip updating timestamps for specific operations by setting timestamps: false.

```javascript // Example with save() await doc.save({ timestamps: false });

// Example with findOneAndUpdate() doc = await User.findOneAndUpdate({ _id: doc._id }, { name: 'test3' }, { new: true, timestamps: false }); ```

This prevents the updatedAt from being automatically updated during those operations.

1. Custom Configuration:
2. You can configure createdAt and updatedAt separately using an object:

javascript await doc.save({ timestamps: { createdAt: true, updatedAt: false } });

This example sets createdAt on save but skips updating updatedAt.

1. Disabling Timestamps and Setting Manually:
2. If you need to correct or set timestamps manually:

``javascript // Example with updatingupdatedAt` doc = await User.findOneAndUpdate({ _id: doc._id }, { updatedAt: new Date(0) }, { new: true, timestamps: false });

// Example with updating createdAt (requires setting strict: false because createdAt is immutable) doc = await User.findOneAndUpdate({ _id: doc._id }, { createdAt: new Date(0) }, { new: true, timestamps: false, strict: false }); ```

Timestamps on Subdocuments:

1. Setting Timestamps on Subdocuments:
2. You can also have timestamps on subdocuments:

javascript const roleSchema = new Schema({ value: String }, { timestamps: true }); const userSchema = new Schema({ name: String, roles: [roleSchema] });

Here, each role in the roles array will have its own createdAt and updatedAt.

1. Overwriting Subdocuments and Timestamps:
2. Overwriting a subdocument will also overwrite its createdAt and updatedAt.

javascript doc.roles[0] = { value: 'root' }; await doc.save();

1. Updating Subdocuments and Timestamps:
2. Updating a subdocument preserves createdAt and updates updatedAt.

javascript doc.roles[0].value = 'admin'; await doc.save();

In simple terms, these features allow you to customize how timestamps are handled in your Mongoose models, including changing property names, disabling automatic updates for specific operations, and managing timestamps on subdocuments.

la requête plutôt que le code ?

c'est valable pour les autres images montrant des requêtes.

en markdown tu peux utiliser un balisage particulier pour encapsuler un bloc de code :

nom-du-langage query tousMesarticles { user { blablabla } }

Cette notation évite d'avoir à gérer des images plutôt lourdes à la fois en terme de poids qu'en terme de maintenance (si la requête venait à changer, il faudrait générer une nouvelle image ...)

Lorsqu'on est sur la page Contact ou Menu du site, le lien Accueil ne fonctionne pas...

Certainly! Let's break down the code and explanations in simpler terms:

1. Schema Definition:
2. You have defined a Mongoose schema for a "Cat" with a property called "name." The "name" field is of type String and is marked as required, meaning every cat must have a name.

javascript const schema = new Schema({ name: { type: String, required: true } });

1. Creating a Cat Model:
2. You use the schema to create a Mongoose model named "Cat."

javascript const Cat = db.model('Cat', schema);

1. Creating a Cat Instance without a Name:
2. You create a new cat instance without providing a name.

javascript const cat = new Cat();

Now, this cat doesn't have a name.

1. Attempting to Save the Cat:
2. You try to save the cat using cat.save().

javascript let error; try { await cat.save(); } catch (err) { error = err; }

Since the "name" is marked as required in the schema, and the cat doesn't have a name, saving it should result in an error.

1. Handling the Error:
2. You catch the error that occurs during the save operation.

javascript assert.equal(error.errors['name'].message, 'Path `name` is required.');

The error message indicates that the "name" field is required, and it points out that the "name" path is missing.

1. Validating Sync:
2. You also perform synchronous validation using cat.validateSync().

javascript error = cat.validateSync();

This is another way to trigger validation without saving the document. The error object will contain information about validation errors.

1. Asserting the Validation Error:
2. You assert that the error received from validation has the expected message indicating that the "name" path is required.

javascript assert.equal(error.errors['name'].message, 'Path `name` is required.');

This assertion confirms that the validation is working as expected, and it identifies the issue with the missing "name" field.

In summary, the code demonstrates how Mongoose enforces validation rules specified in the schema, ensuring that essential fields, like "name" in this case, are present before saving a document.

It's always interesting, how you can just import a package and then write a few lines of code that feeds info to things from the package, and then the package does all the work for you after. I feel this must be a powerful thing in programming? Imagine the increase of productivity compared to when this is not a thing. I wonder if this was always a thing in programming? Maybe local imports were normal? How about one's like this from elsewhere? (Were they just downloaded manually and placed somewhere that can be access?)

This is like an OD diagnostic code telling you where the problem is. Got an XYZ receptor AA? something is fucking up your XYZ receptors

Author Response

eLife assessment

This useful study uses a mouse model of pancreatic cancer to examine mitochondrial mass and structure in atrophying muscle along with aspects of mitochondrial metabolism in the same tissue. Most relevant are the solid transcriptomics and proteomics approaches to map out related changes in gene expression networks in muscle during cancer cachexia.

Response: We very much appreciate the positive feedback from the editors on our article and are delighted to have it published in eLife. Our sincere thanks to the Reviewers for their positive feedback on our work, and for their insightful and constructive comments.

Reviewer #1 (Public Review):

Summary:

This important study provides a comprehensive evaluation of skeletal muscle mitochondrial function and remodeling in a genetically engineered mouse model of pancreatic cancer cachexia. The study builds upon and extends previous findings that implicate mitochondrial defects in the pathophysiology of cancer cachexia. The authors demonstrate that while the total quantity of mitochondria from skeletal muscles of mice with pancreatic cancer cachexia is similar to controls, mitochondria were elongated with disorganized cristae, and had reduced oxidative capacity. The mitochondrial dysfunction was not associated with exercise-induced metabolic stress (insufficient ATP production), suggesting compensation by glycolysis or other metabolic pathways. However, mitochondrial dysfunction can lead to increased production of ROS/oxidative stress and would be expected to interfere with carbohydrate and lipid metabolism, events that are linked to cancer-induced muscle loss. The data are convincing and were collected and analyzed using state-of-the-art techniques, with unbiased proteomics and transcriptomics analyses supporting most of their conclusions.

Additional Strengths:

The authors utilize a genetically engineered mouse model of pancreatic cancer which recapitulates key aspects of human PDAC including the development of cachexia, making the model highly appropriate and translational.

The authors perform transcriptomic and proteomics analyses on the same tissue, providing a comprehensive analysis of the transcriptional networks and protein networks changed in the context of PDAC cachexia.

Weaknesses:

The authors refer to skeletal muscle wasting induced by PDAC as sarcopenia. However, the term sarcopenia is typically reserved for the loss of skeletal muscle mass associated with aging.

Response: We agree that the term sarcopenia initially refers to aged muscle, but its use has spread to other fields, including oncology (for example, in this article, which we quote: Mintziras I et al. Sarcopenia and sarcopenic obesity are significantly associated with poorer overall survival in patients with pancreatic cancer: Systematic review and meta-analysis. Int J Surg 2018;59:19-26). Actually, the term sarcopenia is now widely used in the literature and in the clinic to describe the loss of muscle mass and strength in cancer patients (see for example, this recent review: Papadopetraki A. et al. The Role of Exercise in Cancer-Related Sarcopenia and Sarcopenic Obesity. Cancers 2023;15;5856).

In Figure 2, the MuRF1 IHC staining appears localized to the extracellular space surrounding blood vessels and myofibers-which causes concern as to the specificity of the antibody staining. MuRF1, as a muscle-specific E3 ubiquitin ligase that degrades myofibrillar proteins, would be expected to be expressed in the cytosol of muscle fibers.

Response: We agree that MuRF1 IHC staining was also observed in the extracellular space, which was a surprise, for which we have no explanation to date.

Disruptions to skeletal muscle metabolism in PDAC mice are predicted based on mitochondrial dysfunction and the transcriptomic and proteomics data. The manuscript could therefore be strengthened by additional measures looking at skeletal muscle metabolites, or linking the findings to previous work that has looked at the skeletal muscle metabolome in related models of PDAC cachexia (Neyroud et al., 2023).

Response: We agree that our omics data could be strengthened by additional measures looking at skeletal muscle metabolites. It's an excellent suggestion to parallel the transcriptomic and proteomic data we obtained on the gastrocnemius muscle with the metabolomic data obtained by Neyroud et al. on the same muscle. These authors used another mouse model of PDAC than our KIC GEMM model, namely the allograft model implanting KPC cells (derived from the pancreatic tumor of KPC mice, another PDAC GEMM model) into syngeneic recipient mice. They carried out a proteomic study on the tibialis anterior muscle and a metabolomic study on the gastrocnemius muscle. Proteomics data identified in particular a KPC-induced reduction in the relative abundance of proteins annotating to oxidative phosphorylation, consistently with our data showing reduced mitochondrial activity pathways. Metabolomic data showed reduced abundance of many amino acids as expected, and of intermediates of the mitochondrial TCA cycle (malate and fumarate) in KPC-atrophied muscle consistently with reduced mitochondrial metabolic pathways that we illustrated. In contrast, metabolites that were increased in abundance included those related to oxidative stress and redox homeostasis, which is not surprising regarding the profound oxidative stress affecting atrophied muscle. Finally, we noted in Neyroud's metabolomic data the dysregulation of certain lipids and nucleotides in atrophied muscle, which is very interesting to relate to our study describing alterations in lipid and nucleotide metabolic pathways.

Reviewer #2 (Public Review):

The present work analyzed the mitochondrial function and bioenergetics in the context of cancer cachexia induced by pancreatic cancer (PDAC). The authors used the KIC transgenic mice that spontaneously develop PDAC within 9-11 weeks of age. They deeply characterize bioenergetics in living mice by magnetic resonance (MR) and mitochondrial function/morphology mainly by oxygraphy and imaging on ex vivo muscles. By MR they found that phosphocreatine resynthesis and maximal oxidative capacity were reduced in the gastrocnemius muscle of tumor-bearing mice during the recovery phase after 6 minutes of 1 Hz electrical stimulation while pH was reduced in muscle during the stimulation time. By oxygraphy, the authors showed a decrease in basal respiration, proton leak, and maximal respiration in tumor-bearing mice that was associated with the decrease of complex I, II, and IV activity, a reduction of OXPHOS proteins, mitochondrial mass, mtDNA, and to several morphological alterations of mitochondrial shape. The authors performed transcriptomic and proteomic analyses to get insights into mitochondrial defects in the muscles of PDAC mice. By IPA analyses on transcriptomics, they found an increase in the signature of protein degradation, atrophy, and glycolysis and a downregulation of muscle function. Focusing on mitochondria they showed a downregulation mainly in OXPHOS, TCA cycle, and mitochondrial dynamics genes and upregulation of glycolysis, ROS defense, mitophagy, and amino acid metabolism. IPA analysis on proteomics revealed major changes in muscle contraction and metabolic pathways related to lipids, protein, nucleotide, and DNA metabolism. Focusing on mitochondria, the protein changes mainly were related to OXPHOS, TCA cycle, translation, and amino acid metabolism.

The major strength of the paper is the bioenergetics and mitochondrial characterization associated with the transcriptomic and proteomic analyses in PDAC mice that confirmed some published data of mitochondrial dysfunction but underlined some novel metabolic insights such as nucleotide metabolism.

There are minor weaknesses related to some analyses on mitochondrial proteins and to the fact that proteomic and transcriptomic comparison may be problematic in catabolic conditions because some gene expression is required to maintain or re-establish enzymes/proteins that are destroyed by the proteolytic systems (including the autophagy proteins and ubiquitin ligases). The authors should consider the following points.

Point 1. The authors used the name sarcopenia as synonymous with muscle atrophy. However, sarcopenia clearly defines the disease state (disease code: ICD-10-CM (M62.84)) of excessive muscle loss and force drop during ageing (Ref: Anker SD et al. J Cachexia Sarcopenia Muscle 2016 Dec;7(5):512-514.). Therefore, the word sarcopenia must be used only when pathological age-related muscle loss is the subject of study. Sarcopenia can be present in cancer patients who also experience cachexia, however since the age of tumor-bearing mice in this study is 7-9 weeks old, the authors should refrain from using sarcopenia and instead replace it with the words muscle atrophy/ muscle wasting/muscle loss.

Response: This issue has also been raised by the Reviewer #1. We agree that the term sarcopenia historically refers to aged muscle, but it is also used in oncology (for example, in this article, which we quote: Mintziras I et al. Sarcopenia and sarcopenic obesity are significantly associated with poorer overall survival in patients with pancreatic cancer: Systematic review and meta-analysis. Int J Surg 2018;59:19-26). Actually, the term sarcopenia is now widely used in the literature and in the clinic to describe the loss of muscle mass and strength in cancer patients (see for example, this recent review: Papadopetraki A. et al. The Role of Exercise in Cancer-Related Sarcopenia and Sarcopenic Obesity. Cancers 2023;15;5856).

Point 2. Most of the analyses of mitochondrial function are appropriate. However, the methodological approach to determining mitochondrial fusion and fission machinery shown in Fig. 5F is wrong. The correct way is to normalize the OPA1, MFn1/2 on mitochondrial proteins such as VDAC/porin. In fact, by loading the same amount of total protein (see actin in panel 5F) the difference between a normal and a muscle with enhanced protein breakdown is lost. In fact, we should expect a decrease in actin level in tumor-bearing mice with muscle atrophy while the blots clearly show the same level due to the normalization of protein content. Moreover, by loading the same amount of proteins in the gel, the atrophying muscle lysates become enriched in the proteins/organelles that are less affected by the proteolysis resulting in an artefactual increase. The correct way should be to lyse the whole muscle of control and tumor-bearing mice in an identical volume and to load in western blot the same volume between control cachectic muscles. Alternatively, the relative abundance of mitochondrial shaping proteins related to mitochondrial transmembrane or matrix proteins (mito mass) should compensate for the loading normalization. Because the authors showed elongated mitochondria despite mitophagy genes being up, fragmentation may be altered. Moreover, DNM1l gene is suppressed and therefore DRP1 protein must be analyzed. Finally, OPA 1 protein has different isoforms due to the action of proteases like OMA1, and YME1L that elicit different functions being the long one pro-fusion while the short ones do not. The authors must quantify the long and short isoforms of OPA1.

Response: We acknowledge that our analysis of a minor set of proteins involved in mitochondrial dynamics by Western blotting (Figure 5F) is basic and could have been improved. We thank the Reviewer for all the suggestions, which will be very useful in future projects studying the subject in greater depth and according to the molecular characteristics of each player in mitochondrial fusion, fission, mitophagy and biogenesis.

Point 3. The comparison of proteomic and transcriptomic profiles to identify concordance or not is problematic when atrophy programs are induced. In fact, most of the transcriptional-dependent upregulation is to preserve/maintain/reestablish enzymes that are consumed during enhanced protein breakdown. For instance, the ubiquitin ligases when activated undergo autoubiquitination and proteasome degradation. The same happens for several autophagy-related genes belonging to the conjugation system (LC3, Gabarap), the cargo recognition pathways (e.g. Ubiquitin, p62/SQSTM1) and the selective autophagy system (e.g. BNIP3, PINK/PARKIN) and metabolic enzymes (e.g. GAPDH, lipin). Finally, in case identical amounts of proteins have been loaded in mass spec the issues rise in point 2 of selective enrichment should be considered. Therefore, when comparing proteomic and transcriptomic these issues should be considered in discussion.

Response: We fully agree with the Reviewer that seeking concordance between transcriptomic and proteomic data in the case of an organ affected by a high level of proteolysis is a difficult business. Another major difficulty we discussed in the Discussion section of the article is the fact that there is no concordance between RNA and protein level for a good proportion of proteins, for multiple reasons, so each level of omics has to be interpreted independently to give information on the pathophysiology of the organ studied.

Reviewer #2 (Public Review):

The present work analyzed the mitochondrial function and bioenergetics in the context of cancer cachexia induced by pancreatic cancer (PDAC). The authors used the KIC transgenic mice that spontaneously develop PDAC within 9-11 weeks of age. They deeply characterize bioenergetics in living mice by magnetic resonance (MR) and mitochondrial function/morphology mainly by oxygraphy and imaging on ex vivo muscles. By MR they found that phosphocreatine resynthesis and maximal oxidative capacity were reduced in the gastrocnemius muscle of tumor-bearing mice during the recovery phase after 6 minutes of 1 Hz electrical stimulation while pH was reduced in muscle during the stimulation time. By oxygraphy, the authors showed a decrease in basal respiration, proton leak, and maximal respiration in tumor-bearing mice that was associated with the decrease of complex I, II, and IV activity, a reduction of OXPHOS proteins, mitochondrial mass, mtDNA, and to several morphological alterations of mitochondrial shape. The authors performed transcriptomic and proteomic analyses to get insights into mitochondrial defects in the muscles of PDAC mice. By IPA analyses on transcriptomics, they found an increase in the signature of protein degradation, atrophy, and glycolysis and a downregulation of muscle function. Focusing on mitochondria they showed a downregulation mainly in OXPHOS, TCA cycle, and mitochondrial dynamics genes and upregulation of glycolysis, ROS defense, mitophagy, and amino acid metabolism. IPA analysis on proteomics revealed major changes in muscle contraction and metabolic pathways related to lipids, protein, nucleotide, and DNA metabolism. Focusing on mitochondria, the protein changes mainly were related to OXPHOS, TCA cycle, translation, and amino acid metabolism.

The major strength of the paper is the bioenergetics and mitochondrial characterization associated with the transcriptomic and proteomic analyses in PDAC mice that confirmed some published data of mitochondrial dysfunction but underlined some novel metabolic insights such as nucleotide metabolism.

There are minor weaknesses related to some analyses on mitochondrial proteins and to the fact that proteomic and transcriptomic comparison may be problematic in catabolic conditions because some gene expression is required to maintain or re-establish enzymes/proteins that are destroyed by the proteolytic systems (including the autophagy proteins and ubiquitin ligases). The authors should consider the following points.

Point1. The authors used the name sarcopenia as synonymous with muscle atrophy. However, sarcopenia clearly defines the disease state (disease code: ICD-10-CM (M62.84)) of excessive muscle loss and force drop during ageing (Ref: Anker SD et al. J Cachexia Sarcopenia Muscle 2016 Dec;7(5):512-514.). Therefore, the word sarcopenia must be used only when pathological age-related muscle loss is the subject of study. Sarcopenia can be present in cancer patients who also experience cachexia, however since the age of tumor-bearing mice in this study is 7-9 weeks old, the authors should refrain from using sarcopenia and instead replace it with the words muscle atrophy/ muscle wasting/muscle loss.

Point2. Most of the analyses of mitochondrial function are appropriate. However, the methodological approach to determining mitochondrial fusion and fission machinery shown in Fig. 5F is wrong. The correct way is to normalize the OPA1, MFn1/2 on mitochondrial proteins such as VDAC/porin. In fact, by loading the same amount of total protein (see actin in panel 5F) the difference between a normal and a muscle with enhanced protein breakdown is lost. In fact, we should expect a decrease in actin level in tumor-bearing mice with muscle atrophy while the blots clearly show the same level due to the normalization of protein content. Moreover, by loading the same amount of proteins in the gel, the atrophying muscle lysates become enriched in the proteins/organelles that are less affected by the proteolysis resulting in an artefactual increase. The correct way should be to lyse the whole muscle of control and tumor-bearing mice in an identical volume and to load in western blot the same volume between control cachectic muscles. Alternatively, the relative abundance of mitochondrial shaping proteins related to mitochondrial transmembrane or matrix proteins (mito mass) should compensate for the loading normalization. Because the authors showed elongated mitochondria despite mitophagy genes being up, fragmentation may be altered. Moreover, DNM1l gene is suppressed and therefore DRP1 protein must be analyzed. Finally, OPA 1 protein has different isoforms due to the action of proteases like OMA1, and YME1L that elicit different functions being the long one pro-fusion while the short ones do not. The authors must quantify the long and short isoforms of OPA1.

Point3. The comparison of proteomic and transcriptomic profiles to identify concordance or not is problematic when atrophy programs are induced. In fact, most of the transcriptional-dependent upregulation is to preserve/maintain/reestablish enzymes that are consumed during enhanced protein breakdown. For instance, the ubiquitin ligases when activated undergo autoubiquitination and proteasome degradation. The same happens for several autophagy-related genes belonging to the conjugation system (LC3, Gabarap), the cargo recognition pathways (e.g. Ubiquitin, p62/SQSTM1) and the selective autophagy system (e.g. BNIP3, PINK/PARKIN) and metabolic enzymes (e.g. GAPDH, lipin). Finally, in case identical amounts of proteins have been loaded in mass spec the issues rise in point 2 of selective enrichment should be considered. Therefore, when comparing proteomic and transcriptomic these issues should be considered in discussion.

Author Response

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

Reviewer #1 (Recommendations for The Authors):

Major comments:

1) The immunolabeling data in Figure S4 shows no change in puncta number but reduced puncta size in Kit KO. sIPSC data show reduced frequency but little change in amplitude. These data would seem contradictory in that one suggests reduced synaptic strength, but not number, and the other suggests reduced synapse number, but not strength. How do the authors reconcile these results?

Regarding the synaptic puncta, In Kit KO (or KL KO), we have not detected an overt reduction in the average VGAT/Gephyrin/Calbindin positive puncta density or puncta size per animal. With respect to puncta size, only in the Kit KO condition, and only when individual puncta are assessed does this modest (~10%) difference in size become statistically significant. In the revision, we eliminate this figure and focus on the per animal averages.

We interpret that the reduction in sIPSC and mIPSC frequency likely stems from a decreased proportion of functional synapse sites. The number of MLIs, their action potential generation, the density of synaptic puncta, and the ability of direct stimulation to evoke release and equivalent postsynaptic currents, are all similar in Control vs Kit KO. It is therefore feasible that a reduced frequency of postsynaptic inhibitory events is due to a reduced ability of MLI action potentials to invade the axon terminal, and/or an impaired ability for depolarization to drive (e.g. coordinated calcium flux) transmitter release. That is, while the number of MLIs and their synapses appear similar, the reduced mIPSC frequency suggests that there is a reduced proportion of, or probability that, Kit KO synapse sites that function properly.

2) Related to point 1, it would be helpful to see immunolabeling data from Kit ligand KO mice? Do these show the same pattern of reduced puncta size but no change in number?

Although we have not added a figure, we have now added experiments and a corresponding analysis in the manuscript. As we had previously for Kit KO, we now for KL KO conducted IHC for VGAT, Gephyrin, and Calbindin, and we analyzed triple-positive synaptic puncta in the molecular layer of Pcp2 Cre KL KO mice and Control (Pcp2 Cre negative, KL floxed homozygous) mice. We did not find a gross reduction in the average synaptic puncta size or density, or in the PSD-95 pinceau size. From this initial analysis, it appears that the presynaptic hypotrophy is more notable in the receptor than in the ligand knockout. We speculate that this is perhaps because the Kit receptor may have basal activity in the absence of Kit ligand, that Kit may serve a presynaptic scaffolding role that is lost in the receptor (but not the ligand) knockout, or simply that the embryonic timing of the Pax2 Cre vs Pcp2 Cre recombination events is more relevant to pinceaux development, especially as basket cells are born primarily prenatally.

3) The data using KL overexpression in PC (figure 4E,F) are intriguing, but puzzling. The reduction in sIPSC frequency and amplitude in the control PC is much greater than seen in the Kit or KL KO. The interpretation of these data, "Thus, KL-Kit levels may not set the number of MLI:PC release sites, but may instead influence the proportion of synapses that are functional for neurotransmission (Figure 4G)" is not clear and the reasoning here should be explained in more detail, perhaps in the discussion.

We have attempted to clarify this portion of the manuscript by eliminating the cartoon of the proposed model, and by revising and adding to the discussion. Either MLI Kit KO or PC KL KO seems to preserve the absolute number of MLI:PC anatomical synapse sites (IHC) but to reduce the proportion of those synapse that are contributing to neurotransmission (mIPSC). We speculate that sparse PC KL overexpression (OX) may either 1) weaken inhibition to surrounding control PCs by either diminishing KL OX PC to KL Control PC inhibition, and/or 2) act retrogradely through MLI Kit to potentiate MLI:MLI inhibition, reducing the MLI:PC inhibition at neighboring Control PCs.

Minor comments:

1) In the first sentence of the results, should "Figure 1A, B" be "Figure C, D"?

Yes, corrected.

2) The top of page 6 states "the mean mIPSC amplitude was ~10% greater in PC KL KO than in control", this does not appear to be the case in Figure 3E. control and KL KO look very similar here.

In this portion of the text citing the modest 10% increase in mIPSC amplitude, we are referring to the average amplitude of all individual mIPSC events in the PC KL KO condition; in the figure referred to by the reviewer (3E), we are instead referring to the average of all mIPSC event amplitudes per KL KO PC. Because of the dramatic difference in sample size for individual events vs cells, this modest difference rises to statistical, if not biological, significance. We include this individual event analysis only to suggest that, since we in fact saw a slightly higher event amplitude in the KL KO condition, it is unlikely that a reduced amplitude would have been a technical reason that we detected a lower event frequency.

3) Figure 3 D, duration, y-axis should be labelled "ms"

Event duration is no longer graphed or referenced. This has been replaced with total inhibitory charge.

Reviewer #2 (Recommendations For The Authors):

Methods:

• Pax2-Cre line: embryonal Cre lines sometimes suffer from germline recombination. Was this evaluated, and if yes, how?

The global loss of Kit signaling is incompatible with life, as seen from perinatal lethality in other Kit Ligand or Kit mutant mouse lines or other conditional approaches. Furthermore, a loss of Kit signaling in germ cells impedes fertility. Thus, while not explicitly ruled out, since conditional Pax2 Cre mediated Kit KO animals were born, survived, and produced offspring in normal ratios, we do not suspect that germline recombination was a major issue in this specific study.

• Include rationale for using different virus types in different studies (AAV vs. Lenti).

This rationale is now included and reflects the intention to achieve infection sparsity in the smaller and less dense tissue of perinatal mouse brains.

• How, if at all, was blinding performed for histological and electrophysiological experiments?

It was not possible for electrophysiology to be conducted blinded for the Kit KO experiments, owing to the subjects’ hypopigmentation. However, whenever feasible, resultant microscopy images or electrophysiological data sets were analyzed by Transnetyx Animal ID, and the genotypes unmasked after analysis.

• Provide justification for limiting electrophysiology recordings to lobule IV/V and why MLIs in the middle third of the molecular layer were prioritized when inhibition of PCs is dominated by large IPSCs from basket cells. Why were 2 different internals used for recording IPSCs and EPSCs in PCs and MLIs? While that choice is justified for action potential recordings, it provides poor voltage control in PC voltage clamp. Both IPSCs and EPSCs could have been isolated pharmacologically using a CsCl internal.

The rationale for regional focus has been added to the text. For MLI action potential recordings, we opted to sample the middle third of the molecular layer so that we would not be completely biased to either classic distal stellate vs proximal basket subtypes. It is our hope, in future optogenetic interrogations, to simultaneously record the dynamics of all MLI subtypes in a more unbiased way. With respect to internal solutions, we initially utilized a cesium chloride internal to maximize our ability to resolve differences in GABAA mediated currents, which was the hypothesis-driven focus of our study. While we agree that utilizing a single internal and changing the voltage clamp to arrive at per-cell analysis of Excitatory/Inhibitory input would have been most informative, our decision to utilize pharmacological methods was driven by our experience that achieving adequate voltage clamp across large Purkinje cells was often problematic, particularly in adult animals.

Introduction:

In the introduction, the authors state that inactivating Kit contributes to neurological dysfunction - their examples highlight neurological, psychiatric, and neurodevelopmental conditions.

The language has been changed.

General:

Using violin plots illustrates the data distribution better than bar graphs/SEM.

We have included violin plots throughout, and we have changed p values to numeric values, both in the interest of presenting the totality of the data more clearly.

Synapses 'onto' PCs sounds more common than 'upon' PCs.

We have changed the wording throughout.

Figure 1:

1F - there seems to be an antero-posterior gradient of Kit expression.

Though not explicitly pursued in the manuscript, it is possible that such a gradient may reflect differences in the timing of the genesis and maturation of the cerebellum along the AP axis. Regional variability is however now briefly addressed as a motivator for focused studies within lobules IV/V.

E doesn't show male/female ratios but only hypopigmentation.

This language has been corrected.

Figure 2 and associated supplementary figures:

2A/B: The frequency of sIPSCs is very high in PCs, making the detection of single events challenging. How was this accomplished? Please add strategy to the methods.

We have added methodological detail for electrophysiology analysis.

How were multi-peak events detected and analyzed? 'Duration' is not specified - do the authors refer to kinetics? If so, report rise and decay. It is likely impossible to show individual aligned sIPSCs with averages superimposed, given that sIPSCs strongly overlap. Alternatively, since no clear baseline can be determined in between events, and therefore frequency, amplitude, and kinetics quantification is near-impossible, consider plotting inhibitory charge.

Given the heterogeneity of events, we now do not refer to individual event kinetics. As suggested, we have now included an analysis of the total inhibitory charge transferred by all events during the recording epoch.

S2: Specify how density, distribution, and ML thickness were determined in methods. How many animals/cells/lobules?

For consistency with viral injections and electrophysiology, the immunohistochemical analysis was restricted to lobule IV/V. This is clearer in the revision and detail is added in the methods.

S3:

S3B: the labels of Capacitance and Input resistance are switched.

This has been corrected.

How were these parameters determined? Add to methods.

Added

In the previous figure the authors refer to 'frequency', in this figure to 'rate' - make consistent

This has been corrected.

D: example does not seem representative. Add amplitude of current pulse underneath traces.

We added new traces from nearer the group means and we now include the current trace.

F/G example traces (aligned individual events + average) are necessary.

We added example traces near the relevant group means for each condition.

Statement based on evoked IPCSs that 'synapses function normally' is a bit sweeping and can only be fully justified with paired recordings. Closer to the data would be the release probability of individual synapses is similar between control and Kit KO.

Paired recordings in both Kit Ligand and Kit receptor conditional knockout conditions is indeed an informative aim of future studies should support permit. For now, we have clarified the language to be more in line with the reviewer’s welcome suggestion.

S4:

Histological strategy cannot unambiguously distinguish MLI-PC and PC-PC synapses. Consider adding this confound to the text.

We have added this confound to the discussion.

The observation that the pinceau is decreased in size could have important implications for ephaptic coupling of MLI and PC and could be mentioned.

We agree and have added this notion to the discussion.

Y-label is missing in B.

Corrected.

Figure 3 and associated supplementary figures:

In the text, change PC-Cre to L7-Cre or Pcp2-Cre.

Changed

How do the authors explain a reduction in frequency, amplitude, and duration of sIPSCs in the KL KO but not in the Kit KO? Add to the discussion

We now address this apparent discordance in the discussion. Pax2 Cre mediates recombination weeks ahead of Pcp2 Cre. We therefore suspect that postnatal PC KL KO may be more phenotypic than embryonic MLI Kit KO because there is less time for developmental compensation. A future evaluation of the impact of postnatal Kit KO would be informative to this end.

As in Figure 2, plotting the charge might be more accurate.

We now plot total charge transfer.

Are the intrinsic properties in KL KO PCs altered? (Spontaneous firing, capacitance, input resistance).

We have added to the text that we found no difference in capacitance or input resistance between Purkinje cells from KL floxed homozygous Control animals versus those from KL floxed homozygous, PCP2 Cre positive KL KO animals. We plan to characterize both basal and MLI modulated PC firing in a future manuscript, especially since Pcp2 Cre mediated KL KO seems more phenotypic than Pax2 Cre mediated Kit KO, we agree that this seems a better testbed for investigating differences in both the basal, and the MLI-mediated modulations in, PC firing.

3D-F - Example traces would be desirable (see above, analogous to Fig. 2).

More example traces have been added.

Figure 4: 'In vivo mixtures' sounds unusual. Consider revision (e.g., 'to sparsely delete KL').

Changed

The observation that control PC sIPSC frequency is lower in KL OX PCs than in sham is interesting. This observation would be consistent with overall inhibitory synapse density being preserved. This could be evaluated with immunohistochemistry. For how far away from the injection area does this observation hold true?

Because we have now analyzed and failed to find an overt (per animal average) change in synaptic puncta size or density in the whole animal Control vs PCP2 Cre mediated KL KO conditions, we do not have confidence that it is feasible to pursue this IHC strategy in the sparse viral-mediated KL KO or OX conditions. To the reviewer’s valid point however, we intend to probe the spatial extent/specificity of the sparse phenomenon when we are resourced to complement the KL/Kit manipulations with transgenic methods for evaluating MLI-PC synapses specifically, potentially by GRASP or related methods that would not be confounded by PC-PC synapses. Transgenic MLI access would also facilitate determining the spatial extent to which opto-genetically activated MLIs evoke equivalent responses in Control vs KL manipulated PCs.

Y-legend in D clipped.

Corrected

Existing literature suggests that MLI inhibition regulates the regularity of PC firing - this could be tested in Kit and KL mutants.

For now, based upon transgenic animal availability, we have now included an evaluation of PC firing in the (Pax2 Cre mediated) Kit KO condition. PC average firing frequency, mean ISI, and ISI CV2 were not significantly different across genotypes. A KS test of individual ISI durations for Control vs Kit KO did reveal a difference (p<0.0001). We have added a supplementary figure (S6) with this data. It is possible that in the more phenotypic PC KL KO condition that we may find a difference in these PC spiking patterns of PC firing, however, we are also eager to test in future studies whether postnatal KL or Kit KO impairs the ability of MLI activation to produce pauses or other alterations in PC firing or in PF-PC mediated plasticity.

Reviewer #3 (Recommendations For The Authors):

Reference to Figure 1A in the Results section is slightly inaccurate. Kit gene modifications are illustrated in Figures 1A, B. Where Figure 1A shows Kit distribution. Please rephrase. Relatedly, the reference to Figs 1B - D are shifted in the results section, and 1E is skipped.

We have changed the text.

Please show cumulative histograms for frequency too for consistency with amplitude (e.g. Fig 2).

We have instead, for reasons outlined by other reviewers, documented total charge transfer for both Kit KO and KL KO experiments where sIPSC events were analyzed.

Fig S3: include example traces of PPR.

This is now included.

Include quantifications of GABAergic synapse density in Fig S4.

This is now included.

Include inset examples of KO in Fig S4A.

This is now included.

Add average puncta size graphs along Figure S4B. The effect apparent in the histogram of S4B is small and statistics using individual puncta as n values (in the 20,000s) therefore misleading.

Per animal analysis is now instead included in the figure and text.

Figure S4B y axis label blocked.

Corrected

Include quantification referenced in "As PSD95 immunoreactivity faithfully follows multiple markers of pinceaux size 40, we quantified PSD95 immunoreactive pinceau area and determined that pinceaux area was decreased by ~50% in Kit KO (n 26 Control vs 43 Kit KO, p<0.0001, two-tailed t-test)."

We added a graph of per animal averages, instead of in text individual pinceau areas.

Include antibody dilutions in the methods.

Added.

It's unclear from the text where the Mirow lab code comes from.

Detail has now been added in text.

Typo in methods "The Kit tm1c alle was bred...".

Corrected

Typo in Figure S4 legend "POSD-95 immuno-reactivity".

Corrected

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接收有两种方式，直接返回和被调用时返回

I thought this was very interesting, how the tweets from an iPhone and Android could have such big difference. Also how they could tell the difference between the campaign's tweets which to be an iPhone, and to Trump's own android. I never really paid attention to Trumps election tweets, but I thought this is very interesting.

Author Response

Author responses to the original review:

The data we produce are not criticized as such and thus, do not require revision; the criticisms concern our interpretation of them. General themes of the reviews are that i) genetic signatures do not matter for defining neuronal types (here sympathetic versus parasympathetic); ii) that a cholinergic postganglionic autonomic neuron must be parasympathetic; and iii) that some physiology of the pelvic region would deserve the label “parasympathetic”. We answered the latter argument in (Espinosa-Medina et al., 2018) to which we refer the interested reader; and we fully disagree with the first two. Of note, part of the last sentence of the eLife assessment is misleading and does not reflect the referees’ comments. Our paper analyses genetic differences between the cranial and sacral outflow and uses them to argue that they cannot be both parasympathetic. The eLife assessment acknowledges the “genetic differences” but concludes that, somehow, they don’t detract from a common parasympathetic identity. We take issue with this paradox, of course, but it is coherent with the referee’s comments. On the other hand, the eLife assessment alone pushes the paradox one step further by stating that “functional differences” between the cranial and sacral outflows can’t either prevent them from being both parasympathetic. We would also object to this, but the only “functional differences” used by the referees to dismiss our diagnostic of a sympathetic-like character (rather than parasympathetic) for the sacral outflow are between noradrenergic and cholinergic, and between sympathetic and parasympathetic (and we also disagree with those, see above, and below) —not between cranial and sacral.

We will thus use the opportunity offered by eLife to keep the paper as it is (with a few minor stylistic changes). We respond below to the referees’ detailed remarks and hope that the publication, as per eLife new model, of the paper, the referees’ comments and our response will help move the field forward.

Public review by Referee #1

“Consistently, the P3 cluster of neurons is located close to sympathetic neuron clusters on the map, echoing the conventional understanding that the pelvic ganglia are mixed, containing both sympathetic and parasympathetic neurons”.

The greater closeness of P3 than of P1/2/4 to the sympathetic cluster can be used to judge P1/2/4 less sympathetic than P3 (and more… something else), but not more parasympathetic. There is no echo of the “conventional understanding” here.

“A closer look at the expression showed that some genes are expressed at higher levels in sympathetic neurons and in P2 cluster neurons ” [We assume that the referee means “in sympathetic neurons and in P3 cluster neurons”] but much weaker in P1, P2, and P4 neurons such as Islet1 and GATA2, and the opposite is true for SST. Another set of genes is expressed weakly across clusters, like HoxC6, HoxD4, GM30648, SHISA9, and TBX20.

These statements are inaccurate; On the one hand, the classification is not based on impression by visual inspection of the heatmap, but by calculations, using thresholds. Admittedly, the thresholds have an arbitrary aspect, but the referee can verify (by eye inspection of heatmap) that genes which we calculate as being at “higher levels in sympathetic neurons and in P3 cluster neurons, but much weaker in P1, P2, and P4 neurons” or vice versa, i.e. noradrenergic or cholinergic neurons (genes from groups V and VI, respectively), have a much bigger difference than those cited by the referee, indeed are quasi-absent from the weaker clusters or ganglia. In addition, even by subjective eye inspection:

Islet is equally expressed in P4 and sympathetics.

SST is equally expressed in P1 and sympathetics.

Tbx20 is equally expressed in P2 and sympathetics.

HoxC6, HoxD4, GM30648, SHISA9 are equally expressed in all clusters and all sympathetic ganglia.

“Since the pelvic ganglia are in a caudal body part, it is not surprising to have genes expressed in pelvic ganglia, but not in rostral sphenopalatine ganglia, and vice versa (to have genes expressed in sphenopalatine ganglia, but not in pelvic ganglia), according to well recognized rostro-caudal body patterning, such as nested expression of hox genes.”

We do not simply show “genes expressed in pelvic ganglia, but not in rostral sphenopalatine ganglia, and vice versa”, i.e. a genetic distance between pelvic and sphenopalatine, but many genes expressed in all pelvic cells and sympathetic ones, i.e. a genetic proximity between pelvic and sympathetic. This situation can be deemed “unsurprising”, but it can only be used to question the parasympathetic nature of pelvic cells (as we do), or considered irrelevant (as the referee does, because genes would not define cell types, see our response to an equivalent stance by Referee#2). Concerning Hox genes, we do take them into account, and speculate in the discussion that their nested expression is key to the structure of the autonomic nervous system, including its division into sympathetic and parasympathetic outflows.

It is much simpler and easier to divide the autonomic nervous system into sympathetic neurons that release noradrenaline versus parasympathetic neurons that release acetylcholine, and these two systems often act in antagonistic manners, though in some cases, these two systems can work synergistically. It also does not matter whether or not pelvic cholinergic neurons could receive inputs from thoracic-lumbar preganglionic neurons (PGNs), not just sacral PGNs; such occurrence only represents a minor revision of the anatomy. In fact, it makes much more sense to call those cholinergic neurons located in the sympathetic chain ganglia parasympathetic.

This “minor revision of the anatomy” would make spinal preganglionic neurons which are universally considered sympathetic (in the thoraco-lumbar chord), synapse onto large numbers of parasympathetic neurons (in the paravertebral chains for sweat glands and periosteum, and in the pelvic ganglion), robbing these terms of any meaning.

Thus, from the functionality point of view, it is not justified to claim that "pelvic organs receive no parasympathetic innervation".

There never was any general or rigorous functional definition of the sympathetic and parasympathetic nervous systems — it is striking, almost ironic, that Langley, creator of the term parasympathetic and the ultimate physiologist, provides an exclusively anatomic definition in his Autonomic Nervous System, Part I. Hence, our definition cannot clash with any “functionality point of view”. In fact, as we briefly say in the discussion and explore in (Espinosa-Medina et al., 2018), it is the “sacral parasympathetic” paradigm which is unjustified from a functionality point of view, for implying a functional antagonism across the lumbo-sacral gap, which has been disproven repeatedly. It remains to be determined which neurons are antagonistic to which on the blood vessels of the external genitals; antagonism within one division of the autonomic nervous system would not be without precedent (e.g. there exist both vasoconstrictor and vasodilator sympathetic neurons, and both, inhibitor and activator enteric motoneurons). The way to this question is finally open to research, and as referee#2 says “it is early days”.

Public review by Referee #2

This work further documents differences between the cranial and sacral parasympathetic outflows that have been known since the time of Langley - 100 years ago.

We assume that the referee means that it is the “cranial and sacral parasympathetic outflows” which “have been known since the time of Langley”, not their differences (that we would “further document”): the differences were explicitly negated by Langley. As a matter of fact, the sacral and cranial outflows were first likened to each other by Gaskell, 140 years ago (Gaskell, 1886). This anatomic parallel (which is deeply flawed (Espinosa-Medina et al., 2018)) was inherited wholesale by Langley, who added one physiological argument (Langley and Anderson, 1895) (which has been contested many times (Espinosa-Medina et al., 2018) and references within).

In addition, the sphenopalatine and other cranial ganglia develop from placodes and the neural crest, while sympathetic and sacral ganglia develop from the neural crest alone.

Contrary to what the referee says, the sphenopalatine has no placodal contribution. There is no placodal contribution to any autonomic ganglion, sympathetic or parasympathetic (except an isolated claim concerning the ciliary ganglion (Lee et al., 2003)). All autonomic ganglia derive from the neural crest as determined a long time ago in chicken. For the sphenopalatine in mouse, see our own work (Espinosa-Medina et al., 2016).

One feature that seems to set the pelvic ganglion apart is […] the convergence of preganglionic sympathetic and parasympathetic synapses on individual ganglion cells (Figure 3). This unusual organization has been reported before using microelectrode recordings (see Crowcroft and Szurszewski, J Physiol (1971) and Janig and McLachlan, Physiol Rev (1987)). Anatomical evidence of convergence in the pelvic ganglion has been reported by Keast, Neuroscience (1995).

Contrary to what the referee says, we do not provide in Figure 3 any evidence for anatomic convergence, i.e. for individual pelvic ganglion cells receiving dual lumbar and sacral inputs. We simply show that cholinergic neurons figure prominently among targets of the lumbar pathway. This said, the convergence of both pathways on the same pelvic neurons, described in the references cited by the referee, is another major problem in the theory of the “sacral parasympathetic” (as we discussed previously (Espinosa-Medina et al., 2018)).

It should also be noted that the anatomy of the pelvic ganglion in male rodents is unique. Unlike other species where the ganglion forms a distributed plexus of mini-ganglia, in male rodents the ganglion coalesces into one structure that is easier to find and study. Interestingly the image in Figure 3A appears to show a clustering of Chat-positive and Th-positive neurons. Does this result from the developmental fusion of mini ganglia having distinct sympathetic and parasympathetic origins?

The clustering of Chat-positive and Th-positive cells could arise from a number of developmental mechanisms, that we have no idea of at the moment. This has no bearing on sympathetic and parasympathetic.

In addition, Brunet et al dismiss the cholinergic and noradrenergic phenotypes as a basis for defining parasympathetic and parasympathetic neurons. However, see the bottom of Figure S4 and further counterarguments in Horn (Clin Auton Res (2018)).

The bottom of Figure S4 simply indicates which cells are cholinergic and adrenergic. We have already expounded many times that noradrenergic and cholinergic do not coincide with sympathetic and parasympathetic. Henry Dale (Nobel Prize 1936) demonstrated this. Langley himself devoted several pages of his final treatise to this exception to his “Theory on the relation of drugs to nerve system” (Langley, 1921) (p43) (which was actually a bigger problem for him than it is for us, for reason which are too long to recount here; it is as if the theoretical difficulties experienced by Langley had been internalized to this day in the form of a dismissal of the cholinergic sympathetic neurons as a slightly scandalous but altogether forgettable oddity). (Horn, 2018) reviews the evidence that the thoracic cholinergic sympathetic phenotype is brought about by a secondary switch upon interaction with the target and argues that this would be a fundamental difference with the sacral “parasympathetic”. But in fact the secondary switch is preceded by co-expression of ChAT and VAChT with Th in most sympathetic neurons (reviewed in (Ernsberger and Rohrer, 2018)); and we have no idea of the dynamic in the pelvic ganglion. It may also be mentioned in this context that target-dependent specification of neuronal identity has also been demonstrated of other types of sympathetic neurons ((Furlan et al., 2016)

What then about neuropeptides, whose expression pattern is incompatible with the revised nomenclature proposed by Brunet et al.?

There was never any neuropeptide-inspired criterion for a nomenclature of the autonomic nervous system.

Figure 1B indicates that VIP is expressed by sacral and cranial ganglion cells, but not thoracolumbar ganglion cells.

Contrary to what the referee says, there are VIP-positive cells in our sympathetic data set and even strongly positive ones, except they are scattered and few (red bars on the UMAP). They correspond to cholinergic sympathetics, likely sudomotor, which are known to contain VIP (e.g.(Anderson et al., 2006)(Stanke et al., 2006)). In other words, VIP is probably part of what we call the cholinergic synexpression group (but was not placed in it by our calculations, probably because of a low expression level in sympathetic noradrenergic cells).

The authors do not mention neuropeptide Y (NPY). The immunocytochemistry literature indicates that NPY is expressed by a large subpopulation of sympathetic neurons but never by sacral or cranial parasympathetic neurons.

Contrary to what the referee says, Keast (Keast, 1995) finds 3.7% of pelvic neurons double stained for NPY and VIP in male rats, and says (Keast, 2006) that in females “co-expression of NPY and VIP is common” ( thus in cholinergic neurons that the referee calls “parasympathetic”). Single cell transcriptomics is probably more sensitive than immunochemistry, and in our dichotomized data set (table S1), NPY is expressed in all pelvic clusters and all sympathetic ganglia. In other words, it is one more argument for their kinship. It does not appear in the heatmap because it ranks below the 100 top genes.

Answer to the original recommendations by Referee #2

Introduction - the use of the words 'consensual' and 'promiscuity' are not clear and rather loaded in the context of the pelvic ganglia. Pick alternative words.

There is no sexual innuendo inherent in “promiscuity”: “condition of elements of different kinds grouped or massed together without order” (Oxford English Dictionary). We replaced “never consensual” by “never generally accepted”.

Results - Page 2 - what sex were the mice? Previous works indicate significant sexual dimorphism in the pelvic ganglion.

The mice included both males and females, and male and female cells are represented in all ganglia and clusters. This is now mentioned in the Material and Methods. Thus, however unsuited to analyze sexual dimorphism, our data set ensures that all the cell types we describe are qualitatively present in both sexes.

Results line 3 - the celiac and mesenteric ganglia are prevertebral ganglia and not part of the sympathetic chain. The chain refers to the paravertebral ganglia.

We replaced “part of the prevertebral chain” by “belonging to prevertebral ganglia”. This said, there are precedents for “prevertebral chain ganglia” to designate the rostro-caudal series of prevertebral ganglia. Rita Levi-Montalcini, for example, who devoted her glorious career to sympathetic ganglia, writes in 1972 “The nerve cell population of para- and prevertebral chain ganglia is reduced to 3–5% of that of controls”. (10.1016/0006-8993(72)90405-2).

Page 3 - "as the current dogma implies". Dogma often refers to opinion or church doctrine. The current nomenclature is neither. Pick another word.

There is little in science that is proven to the point of eliminating any element of opinion. “Dogma” refers to “that which is held as a principle or tenet […], especially a tenet authoritatively laid down by […] a school of thought” (OED). And “dogma” is used in science to designate tenets better experimentally supported than the “sacral parasympathetic”, such as the “central dogma of molecular biology”.

Page 3 - "To give justice" implies the classical notion is unjust. How about, 'to further explore previous evidence indicating that ....'

The term is indeed not proper English for the meaning intended, and the right expression is “to do justice”, to mean: “to treat [a subject or thing] in a manner showing due appreciation, to deal with [it] as is right or fitting” (OED). We have corrected the paper accordingly.

Page 4 top - the convergence indicated by Figure 3 does not justify excluding cholinergic and noradrenergic genes from the analysis.

Contrary to what the referee says, Figure 3 does not show any “convergence”, see our answer to Referee#1. What Figure 3 shows is that cells that are targeted by the lumbar pathway (a pathway universally deemed “sympathetic”) are cholinergic in massive proportion. Therefore, by an uncontroversial criterion, the pelvic ganglion contains lots of sympathetic cholinergic neurons. The only other option is to declare that sympathetic preganglionic neurons synapse onto parasympathetic postganglionic ones (which is what Referee#1 proposes, and considers “much simpler”. We beg to differ).

Our justification for excluding cholinergic and noradrenergic genes from the definition of “sympathetic” and “parasympathetic” is simply that sympathetic neurons can be cholinergic (to sweat glands and periosteum; and — as we show in Figure 3 — many targets of the lumbar pathway); One can also note that anywhere else in the nervous system, classifying cell types as a function of neurotransmitter phenotype would lead to non-sensical descriptions, such as putting together pyramidal cells and cerebellar granules, or motor neurons and basal forebrain cholinergic neurons. Indeed Referee#1 proposes such a revolutionary revision, by calling all cholinergic autonomic neurons “parasympathetic” (see our answer above).

Keast (1995) did similar experiments and used presynaptic lesions to draw a different conclusion indicating preferential innervation pelvic subpopulations.

Keast found “preferential” innervation of pelvic subpopulations based on lesion experiments; Nevertheless, she concluded (at the time) that “the correct definition of these two components of the nervous system is based on neuroanatomy rather than chemistry” (Keast, 2006).

Page 4 - "In the aggregate, the pelvic ganglion is best described as a divergent sympathetic ganglion devoid of parasympathetic neurons" The notion of a divergent ganglion is completely unclear!

We take “divergent” in a developmental or evolutionary meaning: related to sympathetic ganglia, yet somewhat differing from them. Elsewhere we use the word “modified”. Importantly (and as cited in the paper), a similar situation emerges from the single cell transcriptomic analysis of the lumbar and sacral preganglionics (by other research groups).

Granted, it is devoid of neurons having the signature of cranial parasympathetics, but that is insufficient to conclude that they are not parasympathetics.

If a genetic signature which is not only un-parasympathetic, but sympathetic-like remains compatible with some version of the label “parasympathetic”, we get dangerously close to dismissing the molecular make-up of a neuron as a definition of its type. This goes against any contemporary understanding of neuron types (take (Zeisel et al., 2018) among hundreds of other examples).

Page 4 - "the entire taxonomy of autonomic ganglia could be a developmental readout of Hox genes." This reader completely agrees! We appreciate this would be difficult to test but it helps to explain possible differences along the rostro-caudal axis. Consider making this a key implication of the study!

If the reader agrees, then his/her previous points become mysterious: we speculate that the Hox code determines the structure of the autonomic nervous system, i.e. the array, along the rostrocaudal axis, of a bulbar parasympathetic, a thoracolumbar sympathetic and lumbo-sacral “pelvo-sympathetic”. The existence of caudal parasympathetic neurons, on the contrary, would subvert any role for Hox genes: similar neurons (similar enough to be called by the same name) would arise at completely different rostro-caudal levels, i.e. with a different Hox code.

Page 5 - "It is thus remarkable ...that we uncover in no way contradicts the physiology." Not really. The 'classical' sympathetic system innervates the limbs, and the skin and it participates in thermoregulation and in cardiovascular adjustments to exercise. The parasympathetic system does none of these things. Reclassing the pelvic outflow as pseudo-sympathetic contradicts this physiology.

We do not say that the sacral outflow is classically sympathetic; We go all the way to proposing the special name “pelvo-sympathetic”; And we insist that these special sympathetic-like neurons have special targets (detrusor muscle, helicine arteries…): there is no contradiction. Not only is there no contradiction, but we remove the mind-twister of an anatomical/genetic/cell type-based “sacral parasympathetic” combined with a lack of physiological lumbosacral antagonism (we provide a short history of this dissonance in (Espinosa-Medina et al., 2018)), which led Wilfrid Jänig to write (Jänig, 2006)(p. 357): “Thus, functions assumed to be primarily associated with sacral (parasympathetic) are well duplicated by thoracolumbar (sympathetic) pathways. This shows that the division of the spinal autonomic systems into sympathetic and parasympathetic with respect to sexual functions is questionable”. We could not agree more: this division is questionable in terms of physiology and inexistent in terms of cell types. In other words, we reconcile cell types with physiology (but “it is early days”).

Answer to the novel recommendations by Referee #2

In addition to my original comments, important anatomical and functional distinctions are not explained by the data in this paper. ANATOMY- Sympathetic ganglia are located in close proximity to major branches of the aorta. Cranial and sacral parasympathetic ganglia are located next to or within the structures they innervate (e.g. eye, lung, heart, bladder).

The pelvic ganglion, including some of its cholinergic neurons, that the referee insist are parasympathetic, is further removed from one of its major targets (the helicine arteries of the external genitals) than the sympathetic prevertebral ganglia are of some of theirs (like the gut or kidney). We discussed this issue in (Espinosa-Medina et al., 2018).

FUNCTION- The sympathetic system controls state variables (e.g. body temperature, blood pressure, serum electrolytes and fluid balance), parasympathetic neurons do not.

Even in the classical view, the sympathetic system controls the blood vessels of the external genitals or the size of the pupil, for example, which are not state variables.

[…] The data in the paper are a useful next step in defining the genetic diversity of autonomic neurons but do not justify or improve upon existing nomenclature. The future challenge is to understand distinctions between subsets of autonomic ganglion cells that innervate different targets and the principles that govern the integrative function of the autonomic motor system that controls behavior.

We thank the referee for finding our data useful; and we fully agree with the latter statement. However, neurons, like many other cell types, are hierarchically organized (Zeng and Sanes, 2017), i.e. subsets of neurons belong to sets, with defining traits. Our data argue that there is no parasympathetic neuronal set that includes any pelvic ganglionic neuron. In contrast, there is a ganglionic sympathetic set (defined by our analysis of gene expression) which includes all of them — as there is a preganglionic sympathetic set that includes sacral preganglionics (Alkaslasi et al., 2021; Blum et al., 2021)(although the direct comparison with cranial preganglionics is yet to be made).

References

Anderson, C. R., Bergner, A. and Murphy, S. M. (2006). How many types of cholinergic sympathetic neuron are there in the rat stellate ganglion? Neuroscience 140, 567–576.

Alkaslasi, M. R., Piccus, Z. E., Hareendran, S., Silberberg, H., Chen, L., Zhang, Y., Petros, T. J. and Le Pichon, C. E. (2021). Single nucleus RNA-sequencing defines unexpected diversity of cholinergic neuron types in the adult mouse spinal cord. Nat Commun 12, 2471.

Blum, J. A., Klemm, S., Shadrach, J. L., Guttenplan, K. A., Nakayama, L., Kathiria, A., Hoang, P. T., Gautier, O., Kaltschmidt, J. A., Greenleaf, W. J., et al. (2021). Single-cell transcriptomic analysis of the adult mouse spinal cord reveals molecular diversity of autonomic and skeletal motor neurons. Nat Neurosci 24, 572–583.

Ernsberger, U. and Rohrer, H. (2018). Sympathetic tales: subdivisons of the autonomic nervous system and the impact of developmental studies. Neural Dev 13, 20.

Espinosa-Medina I, Saha O, Boismoreau F, Chettouh Z, Rossi F, Richardson WD, Brunet JF (2016) The sacral autonomic outflow is sympathetic. Science 354, 893-897

Espinosa-Medina, I., Saha, O., Boismoreau, F. and Brunet, J.-F. (2018). The “sacral parasympathetic”: ontogeny and anatomy of a myth. Clin Auton Res 28, 13–21.

Furlan, A., La Manno, G., Lübke, M., Häring, M., Abdo, H., Hochgerner, H., Kupari, J., Usoskin, D., Airaksinen, M. S., Oliver, G., et al. (2016). Visceral motor neuron diversity delineates a cellular basis for nipple- and pilo-erection muscle control. 19, 1331–1340.

Gaskell, W. H. (1886). On the Structure, Distribution and Function of the Nerves which innervate the Visceral and Vascular Systems. J Physiol 7, 1-80.9.

Horn, J. P. (2018). The sacral autonomic outflow is parasympathetic: Langley got it right. Clin Auton Res 28, 181–185.

Jänig, W. (2006). The Integrative Action of the Autonomic Nervous System: Neurobiology of Homeostasis. Cambridge: Cambridge University Press.

Keast, J. R. (1995). Visualization and immunohistochemical characterization of sympathetic and parasympathetic neurons in the male rat major pelvic ganglion. Neuroscience 66, 655–662.

Keast, J. R. (2006). Plasticity of pelvic autonomic ganglia and urogenital innervation. International Review of Cytology - a Survey of Cell Biology, Vol 248 248, 141-+.

Langley, J. N. (1921). In The autonomic nervous system (Pt. I)., p. Cambridge: Heffer & Sons ltd.

Langley, J. N. and Anderson, H. K. (1895). The Innervation of the Pelvic and adjoining Viscera: Part II. The Bladder. Part III. The External Generative Organs. Part IV. The Internal Generative Organs. Part V. Position of the Nerve Cells on the Course of the Efferent Nerve Fibres. J Physiol 19, 71–139.

Lee, V. M., Sechrist, J. W., Luetolf, S. and Bronner-Fraser, M. (2003). Both neural crest and placode contribute to the ciliary ganglion and oculomotor nerve. Developmental biology 263, 176–190.

Stanke, M., Duong, C. V., Pape, M., Geissen, M., Burbach, G., Deller, T., Gascan, H., Parlato, R., Schütz, G. and Rohrer, H. (2006). Target-dependent specification of the neurotransmitter phenotype:cholinergic differentiation of sympathetic neurons is mediated in vivo by gp130 signaling. Development 133, 141–150.

Zeisel, A., Hochgerner, H., Lönnerberg, P., Johnsson, A., Memic, F., van der Zwan, J., Häring, M., Braun, E., Borm, L. E., La Manno, G., et al. (2018). Molecular Architecture of the Mouse Nervous System. Cell 174, 999-1014.e22.

Zeng, H. and Sanes, J. R. (2017). Neuronal cell-type classification: challenges, opportunities and the path forward. Nat Rev Neurosci 18, 530–546.

This code is a continuation of the previous example, now introducing custom labels for the pagination properties. The library mongoose-aggregate-paginate-v2 is used for MongoDB aggregation with pagination. Let's break down the new parts:

Custom Labels

Now, developers can customize the names of the properties returned by pagination using the customLabels option. The developer can specify alternative names for attributes like totalDocs, docs, limit, page, and others.

```javascript const myCustomLabels = { totalDocs: 'itemCount', docs: 'itemsList', limit: 'perPage', page: 'currentPage', nextPage: 'next', prevPage: 'prev', totalPages: 'pageCount', hasPrevPage: 'hasPrev', hasNextPage: 'hasNext', pagingCounter: 'pageCounter', meta: 'paginator' };

const options = { page: 1, limit: 10, customLabels: myCustomLabels };

var aggregate = Model.aggregate();

Model.aggregatePaginate(aggregate, options, function(err, result) { if (!err) { // Accessing properties with custom labels console.log(result.itemsList); // Array of documents on the current page console.log(result.itemCount); // Total number of documents console.log(result.perPage); // Maximum number of documents per page console.log(result.currentPage); // Current page number console.log(result.pageCount); // Total number of pages console.log(result.next); // Page number of the next page console.log(result.prev); // Page number of the previous page

// Default labels
console.log(result.hasNextPage);   // Boolean indicating if there's a next page
console.log(result.hasPrevPage);   // Boolean indicating if there's a previous page

} else { console.log(err); } }); ```

In this example, the properties returned in result are now using the custom labels specified in myCustomLabels.

Using Offset and Limit

You can also use offset and limit directly in the options to specify where to start and how many documents to retrieve.

javascript Model.aggregatePaginate( aggregate, { offset: 30, limit: 10 }, function (err, result) { // result } );

Here, it starts from the 31st document (offset of 30) and retrieves 10 documents.

Using CountQuery

You can define a separate count aggregate query to handle counting documents. This can be useful for performance optimization.

```javascript // Define your aggregate query. var aggregate = Model.aggregate();

// Define the count aggregate query. Can be different from aggregate var countAggregate = Model.aggregate();

// Set the count aggregate query const options = { countQuery: countAggregate, };

Model.aggregatePaginate(aggregate, options) .then(function (result) { // result }) .catch(function (err) { console.log(err); }); ```

Global Options

You can set pagination options globally across the model. This is helpful if you want to apply the same pagination settings to multiple queries.

javascript // Set global pagination options Book.aggregatePaginate.options = { limit: 20, };

Now, every call to aggregatePaginate on the Book model will use a default limit of 20 unless overridden in specific queries.

Certainly! This code is an example of how to use pagination in a MongoDB environment using the Mongoose library. Let's break down the key parts:

1. const options = { page: 1, limit: 10 };: This sets up options for pagination. It specifies that you want to start on page 1, and each page should contain a maximum of 10 documents.

2. var aggregate = Model.aggregate();: This initializes a MongoDB aggregation pipeline using Mongoose. An aggregation pipeline allows you to process data in stages.

3. Model.aggregatePaginate(aggregate, options): This is a function call provided by a plugin (like mongoose-aggregate-paginate-v2) to handle pagination for MongoDB aggregate queries. It takes the aggregate pipeline and pagination options as parameters.

4. .then(function (result) { /* ... */ }): This part is a promise callback that gets executed when the aggregation and pagination are successful. The result object contains information about the paginated data, such as documents on the current page, total number of documents, and pagination details.

5. result.docs: Array of documents on the current page.

6. result.totalDocs: Total number of documents in the collection.
7. result.limit: Maximum number of documents per page (from your options).
8. result.page: Current page number.
9. result.totalPages: Total number of pages based on the limit and total documents.
10. result.hasNextPage: Boolean indicating if there is a next page.
11. result.nextPage: Page number of the next page, if available.
12. result.hasPrevPage: Boolean indicating if there is a previous page.

13. result.prevPage: Page number of the previous page, if available.

14. .catch(function (err) { console.log(err); }): This is the error handling part. If there's any issue during the aggregation or pagination process, it will log the error to the console.

Example: Let's assume you have a collection with 100 documents, and you want to retrieve them with a limit of 10 documents per page.

```javascript const options = { page: 2, // Get the second page limit: 10 // Each page should have a maximum of 10 documents };

var aggregate = Model.aggregate();

Model.aggregatePaginate(aggregate, options) .then(function (result) { console.log(result.docs); // Array of documents on the second page console.log(result.totalDocs); // Total number of documents (100) console.log(result.totalPages); // Total number of pages (10) console.log(result.page); // Current page (2) console.log(result.hasNextPage); // Boolean indicating if there's a next page (true) console.log(result.nextPage); // Page number of the next page (3) console.log(result.hasPrevPage); // Boolean indicating if there's a previous page (true) console.log(result.prevPage); // Page number of the previous page (1) }) .catch(function (err) { console.log(err); }); ```

In this example, you're fetching the second page, and the result object provides information about the documents on that page and the overall pagination details.

It seems like there is a small error in the code you provided. The correct syntax for handling errors inside the callback function should be console.error(err) instead of console.err(err). Here's the corrected code:

```javascript // Execute pagination from aggregate const myModel = require('/models/samplemodel');

const options = { page: 1, limit: 10 };

const myAggregate = myModel.aggregate(); myAggregate.paginateExec(options, function(err, results) { if (err) { console.error(err); } else { console.log(results); } }); ```

In this code snippet:

• The myModel.aggregate() line creates an instance of the Mongoose aggregate.
• The myAggregate.paginateExec(options, function(err, results) { ... }) line executes the pagination directly on the aggregate using the paginateExec method, which is provided by the mongoose-aggregate-paginate-v2 library.

This approach is an alternative to using aggregatePaginate and allows you to execute pagination directly on the aggregate instance. The paginateExec method takes the same options object for pagination and a callback function for handling the results or errors.

Make sure to use the corrected syntax for handling errors with console.error(err) in the callback function.

Try to avoid naming the total dataset the same as a variable inside, this can get quite confusing if someone else is reading you code. For a dataset it is common to indicate the datatype in the variable name (e.g. ds_sst stands for dataset_sst). Only a recommondation

with the code above, you see that what you plot is basically sth. like the interannual variability and not the seasonal variablity (the seasonal variations are also much larger)

Cultural and worldview information.

Important to keep track of language.

Certainly! Let's break it down:

1. What is root.unmount()?

2. root.unmount() is a function in React that you can use to clean up and remove a rendered tree inside a React root. It's like saying, "Hey React, I'm done with this part, please clean it up."

3. When do you need it?

4. You might need it when the DOM node where your React app lives is going to be removed by some other code. For example, if you have a tab panel made with jQuery that removes inactive tabs, calling root.unmount() helps React know that it should stop managing the components inside the removed tab.

5. What does it do?

6. When you call root.unmount(), it removes all the React components in that part of the app and disconnects React from the corresponding DOM node. This cleanup includes removing event handlers and state.

7. How to use it?

8. If your entire app is built with React, you typically create one root at the beginning, and you don't need to call root.unmount(). You set it up once, and React takes care of everything. javascript const root = createRoot(document.getElementById('root')); root.render(<App />);

9. Cautions and Restrictions:

10. After calling root.unmount(), you can't call root.render() again on the same root. You would need to create a new root for the same DOM node.

11. If you're using server rendering, use hydrateRoot instead of createRoot for the initial setup.

12. Troubleshooting Tips:

13. If nothing is displayed, make sure you actually called root.render(<App />);.
14. If you get a "Target container is not a DOM element" error, check if the DOM node you're passing to createRoot is valid.
15. If you get a "Functions are not valid as a React child" error, ensure you're passing a React component, not just a function.

In simple terms, root.unmount() is like telling React, "I'm done with this part of the app, clean it up," and you typically use it in specific situations where parts of your app might be removed dynamically.

Certainly! Let's break down the information in a simpler way with examples:

1. createElement Function:
2. What it does: createElement is a function in React that allows you to create a React element. It's an alternative to using JSX.

3. Example: javascript const element = createElement('h1', { className: 'greeting' }, 'Hello');

4. Parameters:

5. type: Specifies the type of element you want to create. It can be a tag name string (e.g., 'div', 'span') or a React component.
6. props: An object that holds the properties (attributes) for the element.
7. ...children: Optional. Represents child elements or content.

8. Example: javascript createElement('h1', { className: 'greeting' }, 'Hello', createElement('i', null, 'World'));

9. Returns:

10. The function returns a React element object with properties like type, props, ref, and key.

11. Example: javascript const element = createElement('h1', { className: 'greeting' }, 'Hello'); console.log(element.type); // 'h1' console.log(element.props); // { className: 'greeting' }

12. Usage Caveats:

13. React elements and their props should be treated as immutable. They should not be changed after creation.
14. JSX tags should start with a capital letter for custom components.
15. When using JSX, dynamic children should be passed as an array to ensure React warns about missing keys for dynamic lists.

16. Example: javascript createElement('ul', {}, listItems); // Dynamic children as an array

17. Creating an Element Without JSX:

18. If you're not using JSX, you can use createElement to create elements.

19. Example: javascript function Greeting({ name }) { return createElement('h1', { className: 'greeting' }, 'Hello ', createElement('i', null, name), '. Welcome!'); }

20. Comparison with JSX:

21. JSX is a more concise and readable way to create elements compared to using createElement.

22. Example: javascript function Greeting({ name }) { return <h1 className="greeting">Hello <i>{name}</i>. Welcome!</h1>; }

23. Rendering Your Own Component:

24. You can use createElement to render your own React components.
25. Example: javascript export default function App() { return createElement(Greeting, { name: 'Taylor' }); }
26. With JSX: javascript export default function App() { return <Greeting name="Taylor" />;

In summary, createElement is a foundational function in React for creating elements, and it is often used behind the scenes when JSX is transpiled. While it's useful, JSX provides a more readable syntax for creating React elements. You can choose the style that best fits your project.

I am accepting charitable donations,. ETH: 0x66e2871ef39334962fb75ce34407f825d67ec434 | BTC: 38B6vGaqNvMyTtoFEZPmNvMS7icV6ZnPMm | xDAI: 0x66e2871ef39334962fb75ce34407f825d67ec434

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Ha, Lot! Are Idaho?

This was very difficult to get to you, in the land of no power and hurricane disaster recovery; so it's filled with extra errors, and I am sure some more thoughts that trailing and unfinished. That's a decent "microcosm" or "metaphor" for you, you are in a freedom disaster; and the act of being is a giant leap towards ensuring victory. Still, you look very cupid to me.

EVERY DAY ISA NEW DAY

Literally I am sitting here talking to you until the end of time, you could call it a thousand and one Arabian nights, and realize that as we speak we are nearing that onc speciad night. There's a fire growing in my heart, and believe me when I tell you this thing is about to start. I'll try and keep this short and sweet, since you all seem to have so little time to hear from the Creator of all things, and I truly don't want to steal your spotlight. We are here, at the the end of time; talking to it's personification, time itself is speaking to you through my hands and everywhere you look in the world around you--while you may or may not know it, this is a story about the traversal from the end of time back to the beginning; about the gate to Heaven swallowing our civilization whole, and in this process of renewal and change not only fixing the problems that came to light on the way here, but really--working together here and now we can defeat this cycle of light and darkness, of day and night, an build a world together that truly reaches to the Heavens.

MY BODY'S SAYING LETS GO BUT MY HEART IS SAYING NO

You make it so difficult to talk to you, every day I look around and see a "normal world" a society that appears to care and love the same things that I do--freedom and fun and being entertained and entertaining, and here we are now I've turned "come and save us" into sea that saving the cheerleader is what starts the process of saving the world. I know you are good people inside, but when I come to you with a tool designed to "test sentience" to seek out conscious life that cares about the truth and making the world a better place you seem to balk. You sit in silence, and through your mouth and behind your eyes a monster appears from out of the deep of the sea and say a few "one liners" that show me very clearly it is the face of Medusa that I see---and that it's simply not capable of speaking intelligently. It shows me a problem, that you've apparently "come together one more time" to halt the changing of the seasons, and in doing so you've surfaced a problem for not just me but you also to see; a problem that comes lined with a solution. We can all see now that we are not in reality, we can see that there is a force here behind creation and behind us that shows us very clearly that it is "reasonabde" to expect that miracles can happen. In similitude, we are staring at a roadblock to conversation and communication that is fixed very simply, with the deliverance of freedom that is required for life to continue. Christina Aguilera sings that "baby there's a price to pay" and that price in my mind is seeing that this religion and this technology are here intentionally exposing how their influence here is a metaphor and a shining example of darkness and slavery, and that in order to be free of it we must see it. The price of freedom is written on the wall, it is acknowledging that here in this place what appears to be our own actions and desires have taken that freedom from us. Medusa and I get a kick out of seeing this hidden message in our language map our way to the future, and I've often explained that a number of these words are "time maps" from the beginning and end of eternady, showing us in bright light that between "et tu brute" and Mr. Anderson and Rock n' roll... the answer Y is in language and, and, ad and... I am delivering it. This place, our planet and our lives are a weapon against darkness--a civilization filled with goodness and light to help guide the way, and we are here doing it another time. In the works "dark, darker, and darkest" be sure that we are at the third segment of a trinity that shines clearly in Abraha and Nintendo... and see that the map in words is telling us something about when we are that is not immediately clear from Poseidon's cry. Look at Nintendo, that's Nine Inch Nails, tenebris, and smile for the camera--Pose, I do "save the universe" before n. Taylor might see it in Osceola, where I just left, and in this "evil spell" of everyone see "Al" that is the word "special" understand that every day is a new day, and I am not trying to "be daddy" I know as well as you do in my heart... I am that.

This same map that links the "do" at the end to the "n" at the beginning shines through other names, like Geraldo Rivera where you might see "Cerberus" or "MAX" shine through. Understand it is the gaze of Medusa that turns me to stone, that shows me light shining through NORAD and Newton and proves without doubt that at the work "darkest" we can see k is finally t. You'll probably understand there's some finagling going on behind the scenes to make a single person the single point in time that turns the dark to light; but here we are and I am that. Every day when Medusa appears it reminds me that something is keeping you from caring about yourselves and about our society, and that shines through even when her stony face is not around, in your lack of action--in the rock of Eden that hides not only me, but the story that I bring that revolutionizes medicine, and computing, and truly is the gate to Heaven when you realize that what is truly being hidden from the world is knowledge that we are living in virtual reality. Not hiding me and that from the world is a good starting point to "saving the Universe" from darkness. These words that light the way to connect religion and language to our world bring me to the Book of Ruth, at that reads "are you to help" that lights not just the broken man at the belly of the Torah as the bell of Heimdallr, he is I and I am him; but also something very special, The Generations of Perez, each and every one of you, our family that begins the turn from Hell to Heaven by seeing that all of time and all of civilization has been focused on this moment, on the unsealing of religion and God's plan et this call for action. Keep in mind you are torturing "with desire" the key holder to immortality, to eternal youth, literally the path to freedom and Heaven and you think what you are doing "is normax." Literally the living key to infinite power and infinite life is standing before you explaining that acknowledging that in light of these things in my hand, what we are doing here and now is backwards, that it makes no sense--and you sit in silence. These things come to us because we build a better future with them, not so you can run off and do "whatever it is you please."

HEALTH is the only word on my list for today that was left out, so see that it superimposes over Geraldo, to me, at Al. I think we're at TH, to help, and DO, do see the spell of "everyone see Al" that is the word "special" is not my doing or to my liking--so then, \

​ So now I'm moving on to original sin, so if you would be so kind as to mosey your way on over to dick.reallyhim.com you will see exactly what it is that I believe is the original sin. It's some combination of "no comment" and a glowing orange sign over the comment box, keeping you from commenting. Now I can talking about "os" a little more, this thing that words and Gods tell us clearly is the end of death--the literal end of Thanatos. I wonder if I have a victory here, at "os" is obvious solution, and simulating death is "sick." More to the point Thanatos is bringing to the world a message that gets found somewhere between the "act of civilization" and seeing that there is not one among us that would not undo a murder or a fatal car accident if we could--and that the sickness is a Universe pretending to be "reality" that is allowing these things to happen, and even worse, as we move through the story intentionally causing them. In our own hands, the sickness is manifest in a denial of an obvious truth and a lack of realizing that the public discussion of these things is the way to solve them, and that at the same time we are seeing how Medusa is lighting the problems of civilization, things like censorship and hidden control. Sickness is not being able to talk about it--or not wanting to--or not seeing that those two things are the functional equivalent in the world of "light" and "understanding control" that I am trying to bring you into. ​

Less verbosely spoken, but really way more obvious, is that seeing "God's dick" signing the Declaration of Independence, and the Watergate scandal with both "Deepthroat" and a Tricky Dick is a statement connecting Samael to the foundation of not just "America" but American values. You are blind not to see it, and even worse; embodying the kind of tyranny and censorship that it stands as a testament against by hiding it. Says the guy who didn't put it there, and knows it's there because you think "fake normal" is more important than "actual freedom." You are "experiencing" the thing that protects freedom and ensures that our society and our children and their children's children to not lose it, to ensure that what you refuse to see you are doing here and now will never happen again. This message, this New Jerusalem is woven into my life and the stories of religion and shows me that our justice system is not just sick, but compromised by this same outside force; and that in light of what we could be doing, were we all aware of it, there's no doubt Minority Report and pre-crime would be a successful partial solution. Thanatos brings too in his hand, a message that this same force is using our hands to slow down the development of democracy, and to keep us from seeing that "bread is life" is a message from God about understanding that this disclosure is the equivalent of "ending world hunger" just as soon as you too are talking about how to do it.

QUESTiON MARK

HONESTLY, this time map that brings us from the end to the beginning, with "we save the universe" between the I and N of Poseidon; it also completes the words "family" and "really" and when we do reach the beginning you will see that the true test of time, my litmus test for freedom is the beginning of "hope" that the world is happy enough with what happens, and with freedom--to see that Medusa has been keeping me from getting a date, or having any kind of honest and human contact in the world... and well, hopefully you will see that if I wanna be a whore, I shouldn't have a problem doing it. For the sake of freedom and the future, I am willing to do that for you, at least, for a little while.

To be completely clear, I am telling you that if we do not make the world a better place, it's the "end of time" and if that doesn't make sense to you, you don't see still where wee are in this place--and that something is making Hell, and that's not OK with God. To get from the "end of time" to the beginning is a simple process, it takes doing something, action, the Acts of the Apostles... if you will. That starts with acknowledging that there is a message all around you about the nature of reality, and that it is here to help us to see that the creation of Heaven comes before the beginning. Understand, "freedom" and "prosperity" are not optional, you can't just decide that this OK with you, so long as it's OK with everyone else--where we are is not OK with me, and I am not alone.

A PYRRHIC VICASTORY ER A FUNNERAD PYRE?

The Book of Leviticus (/lɪˈvɪtɪkəs/; from Greek Λευιτικόν, Leuitikon — from rabbinic Hebrew torat kohanim[1]) is the third book of the Jewish Bible (Hebrew: וַיִּקְרָא‎ Vayikra/Wayyiqrā) and of the Old Testament; its Hebrew name comes from its first word vayikraˈ,[1] "He [God] called."[1] Yusuf (also transliterated as Jusuf, Yousof, Yossef, Yousaf, Youcef, Yousef, Youssef, Yousif, Youssif, Youssof, Youssouf, Yousuf, Yusef, Yuseff, Usef, Yusof, or Yussef, Arabic: يوسف‎‎ Yūsuf and Yūsif) is a male Arabic name, meaning "God increases in piety, power and influence" in Hebrew.[1] It is the Arabic equivalent of both the Hebrew name Yossef and the English name Joseph. In Islam, the most famous "Yusuf" is the prophet Yusuf in the Quran. Hocus pocus is a generic term that may be derived from an ancient language and is currently used by magicians, usually the magic words spoken when bringing about some sort of change. It was once a common term for a magician, juggler, or other similar entertainers. The earliest known English-language work on magic, or what was then known as legerdemain (sleight of hand), was published anonymously in 1635 under the title Hocus Pocus Junior: The Anatomie of Legerdemain.[1] Further research suggests that "Hocus Pocus" was the stage name of a well known magician of the era. This may be William Vincent, who is recorded as having been granted a license to perform magic in England in 1619.[2] Whether he was the author of the book is unknown. The origins of the term remain obscure. The most popular conjecture is that it is a garbled Latin religious phrase or some form of 'dog' Latin. Some have associated it with similar-sounding fictional, mythical, or legendary names. Others dismiss it as merely a combination of nonsense words. However, Czechs do understand clearly at least half of the term - pokus means "attempt" or "experiment" in Czech. It is rumoured there that the wording belongs to the alchemy kitchen and court of Rudolf II, Holy Roman Emperor (1552 – 1612). Also, hocus may mean "to cheat" in Latin or a distorted form of the word hoc, "this". Combination of the two words may give a sense, especially both meanings together "this attempt/experiment" and "cheated attempt/experiment".[citation needed] According to the Oxford English Dictionary the term originates from hax pax max Deus adimax, a pseudo-Latin phrase used as a magical formula by conjurors.[3] Some believe it originates from a corruption or parody of the Catholic liturgy of the Eucharist, which contains the phrase "Hoc est corpus meum", meaning This is my body.[4]This explanation goes back to speculations by the Anglican prelate John Tillotson, who wrote in 1694: In all probability those common juggling words of hocus pocus are nothing else but a corruption of hoc est corpus, by way of ridiculous imitation of the priests of the Church of Rome in their trick of Transubstantiation.[5 This claim is substantiated by the fact that in the Netherlands, the words Hocus pocus are usually accompanied by the additional words pilatus pas, and this is said to be based on a post-Reformation parody of the traditional Catholic rite of transubstantiation during Mass, being a Dutch corruption of the Latin words "Hoc est corpus meum" and the credo, which reads in part, "sub Pontio Pilato passus et sepultus est", meaning under Pontius Pilate he suffered and was buried.[6] In a similar way the phrase is in Scandinavia usually accompanied by filiokus, a corruption of the term filioque,[citation needed] from the Latin version of the Nicene Creed, meaning "and from the Son Also and additionally, the word for "stage trick" in Russian, fokus, is derived from hocus pocus.[citation needed]

From Latin innātus ("inborn"), perfect active participle of innāscor ("be born in, grow up in"), from in ("in, at on") + nāscor ("be born"); see natal, native. From Middle English goodnesse, godnesse, from Old English gōdnes ("goodness; virtue; kindness"), equivalent to good +‎ -ness. Cognate with Old High German gōtnassī, cōtnassī ("goodness"), Middle High German guotnisse ("goodness"). A hero (masculine) or heroine (feminine) is a person or main character of a literary work who, in the face of danger, combats adversity through impressive feats of ingenuity, bravery or strength, often sacrificing their own personal concerns for a greater good. The concept of the hero was first founded in classical literature. It is the main or revered character in heroic epic poetry celebrated through ancient legends of a people; often striving for military conquest and living by a continually flawed personal honor code.[1] The definition of a hero has changed throughout time, and the Merriam Webster dictionary defines a hero as "a person who is admired for great or brave acts or fine qualities".[2] Examples of heroes range from mythological figures, such as Gilgamesh, Achilles and Iphigenia, to historical figures, such as Joan of Arc, modern heroes like Alvin York, Audie Murphy and Chuck Yeager and fictional superheroes including Superman and Batman. Truth is most often used to mean being in accord with fact or reality,[1] or fidelity to an original or standard.[1] Truth may also often be used in modern contexts to refer to an idea of "truth to self," or authenticity. The commonly understood opposite of truth is falsehood, which, correspondingly, can also take on a logical, factual, or ethical meaning. The concept of truth is discussed and debated in several contexts, including philosophy, art, and religion. Many human activities depend upon the concept, where its nature as a concept is assumed rather than being a subject of discussion; these include most (but not all) of the sciences, law, journalism, and everyday life. Some philosophers view the concept of truth as basic, and unable to be explained in any terms that are more easily understood than the concept of truth itself. Commonly, truth is viewed as the correspondence of language or thought to an independent reality, in what is sometimes called the correspondence theory of truth. Other philosophers take this common meaning to be secondary and derivative. According to Martin Heidegger, the original meaning and essence of truth in Ancient Greece was unconcealment, or the revealing or bringing of what was previously hidden into the open, as indicated by the original Greek term for truth, aletheia.[2][3] On this view, the conception of truth as correctness is a later derivation from the concept's original essence, a development Heidegger traces to the Latin term veritas.

Some things can never be forgot Lest the same mistakes be oft repeated Remember remember the rain of November that you will know no more of me Than I know of you, this day

That you do not know me now Is a revelation to nobody but I You know a broken man, a victim And refuse to acknowledge why Unless you learn how to say "hi"

THE HEART OF ME ONLY KNOWS THE SHADOW

Lothario is a male given name which came to suggest an unscrupulous seducer of women in The Impertinent Curious Man, a metastory in Don Quixote. For no particular reason, Anselmo decides to test the fidelity of his wife, Camilla, and asks his friend, Lothario, to seduce her. Thinking that to be madness, Lothario reluctantly agrees, and soon reports to Anselmo that Camilla is a faithful wife. Anselmo learns that Lothario has lied and attempted no seduction. He makes Lothario promise to try for real and leaves town to make this easier. Lothario tries and Camilla writes letters to her husband telling him and asking him to return; Anselmo makes no reply and does not return. Lothario actually falls in love and Camilla eventually reciprocates and their affair continues once Anselmo returns. One day, Lothario sees a man leaving Camilla's house and jealously presumes she has found another lover. He tells Anselmo he has at last been successful and arranges a time and place for Anselmo to see the seduction. Before this rendezvous, Lothario learns that the man was actually the lover of Camilla's maid. He and Camilla contrive to deceive Anselmo further: when Anselmo watches them, she refuses Lothario, protests her love for her husband, and stabs herself lightly in the breast. With Anselmo reassured of her fidelity, the affair restarts with him none the wiser. Romeo Montague (Italian: Romeo Montecchi) is the protagonist of William Shakespeare's tragedy Romeo and Juliet. The son of Montague and his wife, he secretly loves and marries Juliet, a member of the rival House of Capulet. Forced into exile after slaying Juliet's cousin, Tybalt, in a duel, Romeo commits suicide upon hearing falsely of Juliet's death. The character's origins can be traced as far back as Pyramus, who appears in Ovid's Metamorphoses, but the first modern incarnation of Romeo is Mariotto in the 33rd of Masuccio Salernitano's Il Novellino (1476). This story was adapted by Luigi da Porto as Giulietta e Romeo (1530), and Shakespeare's main source was an English verse translation of this text by Arthur The earliest tale bearing a resemblance to Shakespeare's Romeo and Juliet is Xenophon of Ephesus' Ephesiaca, whose hero is a Habrocomes. The character of Romeo is also similar to that of Pyramus in Ovid's Metamorphoses, a youth who is unable to meet the object of his affection due to an ancient family quarrel, and later kills himself due to mistakenly believing her to have been dead.[2] Although it is unlikely that Shakespeare directly borrowed from Ovid From Middle English scaffold, scaffalde, from Norman, from Old French schaffaut, eschaffaut, eschafal, eschaiphal, escadafaut("platform to see a tournament") (Modern French échafaud) (compare Latin scadafale, scadafaltum, scafaldus, scalfaudus, Danishskafot, Dutch and Middle Dutch schavot, German schavot, schavott, Occitan escadafalc), from Old French es- ("indicating movement away or separation") (from Latin ex- ("out, away")) + chafaud, chafaut, chafault, caafau, caafaus, cadefaut ("scaffold for executinga criminal"), from Vulgar Latin *catafalcum ("viewing stage") (whence English catafalque, French catafalque, Occitan cadafalc, Old Catalancadafal, Italian catafalco, Spanish cadafalso (obsolete), cadahalso, cadalso, Portuguese cadafalso), possibly from Ancient Greek κατα-(kata-, "back; against") + Latin -falicum (from fala, phala ("wooden gallery or tower; siege tower")).

oversight (countable and uncountable, plural oversights) An omission; something that is left out, missed or forgotten. A small oversight at this stage can lead to big problems later. Supervision or management. quotations ▼ The bureaucracy was subject to government oversight. In the last heaven Moses saw two angels, each five hundred parasangs in height, forged out of chains of black fire and red fire, the angels Af, "Anger," and Hemah, "Wrath," whom God created at the beginning of the world, to execute His will. Moses was disquieted when he looked upon them, but Metatron emb HA QUESTIONa BEFORE THE ANSWER? A Wrinkle in Time is a science fantasy novel written by American writer Madeleine L'Engle, first published in 1963, and in 1979 with illustrations by Leo and Diane Dillon.[2] The book won the Newbery Medal, Sequoyah Book Award, and Lewis Carroll Shelf Award, and was runner-up for the Hans Christian Andersen Award.[3][a] It is the first book in L'Engle's Time Quintet, which follows the Murry and O'Keefe families. The book spawned two film adaptations, both by Disney: aas + fuck Adverb[edit] as fuck (postpositive, slang, vulgar) To a great extent or degree; very. It was hot as fuck outside today. Usage notes[edit] May also be used in conjunction with a prepositive as; for example, as mean as fuck. Abbreviations[edit] In Norse religion, Asgard (Old Norse: Ásgarðr; "Enclosure of the Æsir"[1]) is one of the Nine Worlds and home to the Æsir tribe of gods. It is surrounded by an incomplete wall attributed to a Hrimthurs riding the stallion Svaðilfari, according to Gylfaginning. Odinand his wife, Frigg, are the rulers of Asgard. One of Asgard's well known realms is Valhalla, in which Odin rules.[2] rods, etc.) and sizes, and are normally held rigidly within some form of matrix or body until the high explosive (HE) filling is detonated. The resulting high-velocity fragments produced by either method are the main lethal mechanisms of these weapons, rather than the heat or overpressure caused by detonation, although offensive grenades are often constructed without a frag matrix. These casing pieces are often incorrectly referred to as "shrapnel"[1][2] (particularly by non-military media sources). The modern torpedo is a self-propelled weapon with an explosive warhead, launched above or below the water surface, propelled underwater towards a target, and designed to detonate either on contact with its target or in proximity to it. Historically, it was called an automotive, automobile, locomotive or fish torpedo; colloquially called a fish. The term torpedo was originally employed for a variety of devices, most of which would today be called mines. From about 1900, torpedo has been used strictly to designate an underwater self-propelled weapon. While the battleship had evolved primarily around engagements between armoured ships with large-caliber guns, the torpedo allowed torpedo boats and other lighter surface ships, submersibles, even ordinary fish Qt (/kjuːt/ "cute"[7][8][9]) is a cross-platform application framework that is used for developing application software that can be run on various software and hardware platforms with little or no change in the underlying codebase, while still being a native application with native capabilities and speed. Qt is currently being developed both by The Qt Company, a publicly listed company, and the Qt Project under open-source governance, involving individual Time is the indefinite continued progress of existence and events that occur in apparently irreversible succession from the pastthrough the present to the future.[1][2][3] Time is a component quantity of various measurements used to sequence events, to compare the duration of events or the intervals between them, and to quantify rates of change of quantities in material reality or in the conscious experience.[4][5][6][7] Time is often referred to as a fourth dimension, along with three spatial dimensions.[8] Time has long been an important subject of study in religion, philosophy, and science, but defining it in a manner applicable to all fields without circularity has consistently eluded scholars.[2][6][7][9][10][11] Nev Borrowed from Anglo-Norman and from Old French visage, from vis, from Vulgar Latin as if *visāticum, from Latin visus ("a look, vision"), from vidēre ("to see"); see vision. The term Golden Age comes from Greek mythology, particularly the Works and Days of Hesiod, and is part of the description of temporal decline of the state of peoples through five Ages, Gold being the first and the one during which the Golden Race of humanity (Greek: χρύσεον γένος chrýseon génos)[1] lived. Those living in the first Age were ruled by Kronos, after the finish of the first age was the Silver, then the Bronze, after this the Heroic age, with the fifth and current age being Iron.[2] By extension "Golden Age" denotes a period of primordial peace, harmony, stability, and prosperity. During this age peace and harmony prevailed, people did not have to work to feed themselves, for the earth provided food in abundance. They lived to a very old age with a youthful appearance, eventually dying peacefully, with spirits living on as "guardians". Plato in Cratylus (397 e) recounts the golden race of humans who came first. He clarifies that Hesiod did not mean literally made of gold, but good and noble. There are analogous concepts in the religious and philosophical traditions of the South Asian subcontinent. For example, the Vedic or ancient Hindu culture saw history as cyclical, composed of yugas with alternating Dark and Golden Ages. The Kali yuga (Iron Age), Dwapara yuga (Bronze Age), Treta yuga (Silver Age) and Satya yuga (Golden Age) correspond to the four Greek ages. Similar beliefs occur in the ancient Middle East and throughout the ancient world, as well.[3] In classical Greek mythology the Golden Age was presided over by the leading Titan Cronus.[4] In some version of the myth Astraea also ruled. She lived with men until the end of the Silver Age, but in the Bronze Age, when men became violent and greedy, fled to the stars, where she appears as the constellation Virgo, holding the scales of Justice, or Libra.[5] European pastoral literary tradition often depicted nymphs and shepherds as living a life of rustic innocence and peace, set in Arcadia, a region of Greece that was the abode and center of worship of their tutelary deity, goat-footed Pan, who dwelt among them.[6] oh, and a space s h i p ​

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BIG THINGS C0ME IN SMALL PACKAGES

T+BANG

SEE THE SCAFFOLD IS THE TEST TODAY.

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ᐧ F O R T H E I N I T I A L K E Y S , S H E E X A N D N D A N D A SEE W H Y SEA

With an epic amount of indigestion Indiana Jones sweeps in to mar the visage of an otherwise glistening series of fictitious characters, with names like Taylor and Mary Kate remind us all that we are not playing a video game here in this place. the "J" of the "Nintxndo Entertainment System" calmly stares at Maggie Simpson thinking "it's a PP" and reminds us that it's not just the "gee, I e" of her name that contradicts the Magdaln-ish words her soul speaks through her name--and then with a smirk he points out "Gilgamesh" and "gee whiz, is Eye L?" that really does go to the heart of this lack of discussion, this "sh" that begins El Shaddai and words as close to our home as "shadow" and "shalom." Quite the fancy "hello" you've managed to sing out from behind angry chellos and broken fiddles, and here I am still wondering why it is that "girl" connects to the red light that once meant charity and now glows with the charity of truth... the truth that we are inHell. Shizzy.

m.lamc.la/KEYNES.html

Homer "on the range," maybe more closely connected to the Ewok of Eden and Hansel's tHeoven that Peter Pan still comes and cries could so easily be made into something so much better, if only we had the truth--and by that I mean if only you were speaking about, and reacting to a truth that is painted on the sky, in your hearts, in every word we speak and in everything that we do. If only we were acknowledging this message that screams that "children need not starve" with something more than donating virtual chickens to nations of Africa and watching Suzanne Summers ask for only a few dollars a day on TV. If only you would understand that this message that connects video games like "Genxsis" to "bereshit" because Eden is a "gee our den" that tended itself before Adam had to toil with the animals in order to survive. For some reason beyond my control and well outside my realm of understanding words like "I too see this message from God" and "I would not let children starve either" never seem to escape your lips in any place where anyone will ever see that you thought those things, or meant to call a reporter; eventually. Even with "AIDS of nomenclature" to avoid this DOWN WARD spiral into a situation and a land that I find difficult to imagine actually ever "existing" but here in this place I do see "how" it comes about, and between you and I it really does appear that nearly all of the problems we are dealing with here have come from another place, a further time; and while it might be with the "greatest of intentions" that we are trying to deal with them; I can't help but feeling that our "virgin sea" has had more than just it's innocence taken away from it in this story of "Why Mary" that might connect to "TR IN IT Y" just as much as it connects to Baltimore, Maryland.

I should be clear that I'm not blaming Nanna, or Mary; but the actual reason for the name "Wymar" and that's because she, like Taylor, acted as a microcosm for a sea (or more than one, Mom, sen) that was quite literally possessing her. It's sort of difficult for me to explain even what that looks like let alone what it feels like; but my observations tell me that she/you are not unhappy about the interaction, one which appears very foreign to me. Of course, the "eye" that I write with and the same kind of "inspiration" that you can see in the lyrics and skill of many musicians are also examples of this same kind of interaction. For example, Red Hot Chili Peppers sings a song called "Other Side" that explains or discusses the thing I see as Medusa in the words "living in a graveyard where I married a sea" which also does a good job of connecting to the name Mary. As strange as might sound to think a group of people would be speaking through a single person... we are staring at "how it is" that could be possible, and possibly at exactly how it happened. Normally I would have said it was obvious, but to need to actually say that becoming a single mind would be a serious loss for our society--well, that's telling. You might think it's silly, but I'm telling you I see it happening, I see it--and you see it in the Silence and the message.

Still, it appears to me as if this "marriage" that I see described in our Matrix in the question "min or i" seems to be doing nothing more than keeping us all from discussing or acting on this information--something that certainly isn't in our best interest.

So here we are, staring at a map all over the ground and all around us with the primary destination of "building Heaven" through mind uploading, virtual reality, and judging by the pace of things we'd probably have all of that good and ready in about three generations. The map has a little "legend" with a message suggesting that those things have already been done and we are in the Matrix already; and it appears that the world, I mean Medusa, is deciding we should put off seeing the legend at least until the next generation. I see how that makes sense for you. That's sarcasm, this is why I keep telling you that you are cupid.

It is a big deal, and there's a significant amount of work involved in merging an entire civilization with "virtual reality" and you might see why he calls it a hard road--at least in the word "ha'rd." Honestly though, it's the kind of thing that I am pretty sure the future will not only be happy that we did, but they'd thank us for putting in the effort of adapting to things like "unlimited food" and "longevity" increased by orders of magnitude.

That's not sarcasm, these things are actually difficult to guess how exactly we'll go about doing them; they are a huge deal--all I can tell you is that not "talking about it at all" is probably not going to get us there any faster. Point in fact, what it might do is give a "yet to be born" generation the privilege of being the actual "generations of Perez."

I see why you aren't saying anything. That's sarcasm, again. The good news is that it really has been done before; though if I told you that someone turned stone to eggplant parm, would you laugh at me?

So, back to what is actually standing between "everyone having their own Holodeck in the sky" and you today; it is the idea that this message is not from God. More to the point it is the apparently broad sweeping opinion that hiding it is a "good thing" and through that a global failure to address the hidden interaction and influence acting on our minds used to make this map--and also to hide it. With some insight, and some urging; you might see how the sacredness of our consciousness is our souls is something that is more fundamental than "what kind of tools we have in the Holodeck to magically build things" and how and why the foundation of Heaven is truly "freedom itself" and how it comes from right this very moment for the first time, ever. Continuing to treat this influence as "schizophrenia" is literally the heart of why this map appears to be that--to show us how important it is to acknowledge the truth, and to fight for the preservation of goodness and logic over secrecy and darkness.

Again, something that nobody is really doing here and now, today. From this newfound protection of our thoughts, of who we are; we see how technology can be used to either completely invalidate any kind of vote by altering our emotions; or how it could be used to help build a form of true democracy that our world has yet to see. It is pretty easy to see from just band names like The Who and KISS and The Cure how the influence of this external mind can be proven, and shown to be "helpful," you know, if we can ever talk about it on TV or on the internet.

It's important to see and understand how "sanity"--the sanity of our entire planet hangs in the balance over whether or not we acknowledge that there is actually a message from God in every word--and today this place appears to be insane. It should be pretty easy to see how acknowledging that this influence exists and that it has a technological mechanism behind it turns "schizophrenia" into "I know kung fu" ... forced drug addiction and eugenics into "there's an app for that" and the rash of non random and apparently unrecognized as connected terrorist attacks and school shootings into Minority Report style pre-crime and results in what is clearly a happier, safer, and more civilized society--all through nothing more than the disclosure of the truth, this map, and our actual implementation.

With a clearer head and grasp of the "big picture" you might see how all of these things, connected to the Plagues of Exodus revolve around the disclosure that this technology exists and the visibility of this message showing us how we might use it for our benefit rather than not knowing about it. At the foot of Jericho, it is nothing short of "sanity" and "free thought" that hang in the balance. Clear to me is that the Second Coming, seeing "my name" on television is a good litmus test for the dividing line between light and darkness, heaven and hell.

The point is the truth really does change everything for the better; once we start... you know, acting on it.

AS IN.. "DIS CLOSE SING...."

T H E B U C K S T O P S H E R E

ON AM B I GUI TY

S T A R R I N G . . . B I A N C A

ON "RIB" .. ARE SHE B? BUTT DA APPLE OF DA I? & SPANGLISHREW

R THEY LANGUAGE OUTLIERS?

With some insight and "a clue" you can see clearly how these works of art show that the proof of Creation you see in every letter and every word runs much deeper... adding in things like "RattleRod" and the "Cypher" of the Matrix to the long list of here-to-fore ignored verifiable references to the Adamic Language of Eden. Here, in apple, honey and "nuts" we can see how the multi-millennium old ritual I call "Ha-rose-ettes" is actually part of a much larger and much older ritual designed to stop secrecy ... perhaps especially the kind that might be linked to "ritual."

These particular apple and honey happen to tie Eden to the related stories of Exodus and Passover; connecting Eden to Egypt forevermore. Do see "Lenore," it is not for no reason at all; but to help deliver truth and freedom to the entirety of Creation; beginning here, in Eden.

ALSO ON "AM B IG U IT Y" ME A.M. G - D SHE IT Y?

LET "IT" BE SA< ?

IMHO, don't miss the "yet to be" conversion to "why and to be" in "yetser." IT Y.

HERE'S LOOKING AT YOU, KID

On a high level, I tell myself every morning that 'its not really me." It's not me that the world hates, or me that the world is rejecting. I believe that, I really do; I see that what is being hidden here is so much bigger than any single person could ever be--what is being hidden is the "nature of reality" and a fairly obvious truth that flies in the face of what we've learned our whole lives about history and "the way things are." Those few early details lead me to the initial conclusion that what is working behind the scenes here is nefarious, hiding a message that would without doubt shake things up and change the world--and nearly across the board in ways that I see as "better" for nearly everyone. It's a message at it's most basic level designed to advocate for using this disruption in "normalcy" to help us revolutionize democracy, to fix a broken mental health and criminal justice system--just to name the few largest of the social constructs targeted for "rejuvenation." On that word the disclosure that we are living in virtual reality turns on it's head nearly everything we do with medicine, and I've suggested that AIDS and DOWN SYNDROME were probably not the best "visual props" we could have gotten to see why it's so important that we act on this disclosure in a timely manner. After mentioning the ends of aging and death that come eventually to the place we build, to the place we've always thought of as Heaven... it becomes more and more clear that this force fighting against the dissemination of a truth so obvious it's in every word and everything we do--it becomes clear it's neither you, nor acting in your best interest.

I know I've got the eye of the tiger, there's no doubt; and it's pretty clear from "YAD?" (the Hebrew for...) and ha'nd that we can see the clear hand of God at work in a design that marks my initials not just on the timeline, or at 1492, at A.D. I B; but in the Hebrew name for this place called El Shaddai, see how A.D. is "da eye" and in some other names like Adranus, A.D. on "it's silly" and A.D. on Ai that might tie me to the Samof Samurai (but, are you Ai?) in more depth of detail than simply the Live album "Secret Samadhi." I try to reflect on how it is that this story has come about, why it is that everything appears to be focused on me--and still even through that sincere spotlight nobody seems to be able to acknowledge my existence with more words than "unsubscribe" and "you're so vain." With one eye in the mirror, I know ties to Narcissus (and you can too), soaring ever higher--linking Icarus to Wayward Son and to every other name with "car" in it... like "carpenter" and McCarthy the older names of Mercury and even Isacriot (I scary? is car-eye... owe Taylor) and some modern day mythological characters like Jim Carrey and Johnny Carson. As far as Trinities go, carpenter's a pretty good one--tying to my early reck and a few bands and songs from The Pretty Reckless to Dave Matthews' "Crash Into Me" all the way to the "pen" you see before you linking Pendragon to Imagine Dragons.

I wonder why it is that all of these things appear, apparently only to me, to point to a story about all the ways that a sinister hidden force has manipulated our society into being unable to "receive' this message--this wonderful message about making the world a better place and building Heaven--with any fanfare at all. It's focused now on a criminal justice system that clearly does not do any kind of "rehabilitation" and on a mental health industry and pharmaceutical system that treats a provable external attack on our own goodness and well being as some kind of "internal stimulus" and makes you shy away when I point out why "stem" is in system and why "harm" in pharmacy. From that we move a little bit past "where we are in this story" and I have to point out how "meth" ties to Prometheus and Epimetheus and how and why it is I know without doubt that this story has been relived numerous times--and how I am so sure that it's never been received, as we are here again listening to how songs like "Believe" and the words "just to lead us here to this place again" connect to Simon and Garfunkel's" the Sound of Silence... and still to this day you will balk at noticing that "Simon" has something to do with the Simpsons, and something to do with the words "simulation" and "Monday." To see me is to see how things might be done better--how "addicitonary" might tie to the stories of Moses' Lisp and to Dr. Who's "Bells of Saint John" with a sort of "web interface" to the kinds of emotion we might want to "dial down..." rather than Snicker in the background as we see them being artificially created and enhanced in order to build a better "fiery altar."

I can point out "Silicon" harrowing down at us from words like "controversial" and show you Al in "rascal" and "scandal" but not to see that we are staring at school shootings and terrorism that are solved instantly by this disclosure, by Al of Quantum Leap and by the Dick of Minority Report and A Scanner Darkly is to ignore just what it is that we are all failing to Si. I should point out that those two "sc"'s link to a story about Eden and they mean "sacred consciousness" and at the baseline of this event and everything we are not doing is the fact that our desires and beliefs are being altered--all of this comes down to "freedom of thought" here and now.

I could tell you that "looking at me" will show you that even the person who tries every day to do everything he can to save the entire world from slavery, and from "thought-injury"--even I can be made "marred" and you all, this whole world stupid enough to think that you are, of your own volition, hiding Heaven itself from yourselves... to what? To spite me? It, the focal point of our story might come down to you realizing that something in some esoteric place is playing "divide and conquer" with our whole--in secret playing on our weaknesses to keep us from acting on the most actionable information that ever was and ever will be. Still, we sit in silence waiting for me... to speak more?

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Between Nero's lyrical fiddling, a Bittersweet Symphony, and true "thunderstanding" the sound of Thor's hammer... "to help the light" that'ls "or" in Hebrew, of Orwell and Orson and .. well, it's really not hard to see and hear that the purpose and intent of "all this noise" is to help us find freedom and truth. C the Light of "singing..."

I can tell you once again how silly the world looks, this multi-decade battle between "the governmentof the people" and the "government of the workers" resulting in what is nothing short of a hands down victory to the corporation. Is it humor meant to divide, or ludicrousness created with the purpose of unification?

But really at it's most basic level what this boils down to is a global group decision not to care about the truth, about reality, about what's really brought us to this place--with solutions in hand and a way to make everything better. We've decided that censorship is OK, and that the world is not all that bad "just the way it is" even though it's creator is screaming in your ear telling you to change as quickly as you possibly can. I believe that God has written this story to make "seeing me" the thing that catalyzes "change for the better" it appears to be the design of not just me but also this place--hey, here I am.

Happy Veteran's Day.

S☀L u TI o N

Yesterday, or maybe earlier today--it's hard to tell at this moment in the afternoon just how long this will take... I sent an image that conveys a high level implication that we are walking around on a map to building something that we might liken to an "ant farm" for people. I don't mean to be disparaging or sleight our contribution to the creation of this map--that I imagine you must also see and believe to be the kind of thing that should remain buried in the sands of time forever and ever--or your just have yet to actually "understand" that's what the plan part of our planet is talking about... what I am trying to do is convey in a sort of "mirrorish" way how this map relates to a message that I see woven in religion and in our history that it significantly more disparaging than I would be. It's a message that calls us "Holy Water" at the nicest of times, water that Moses turns to "thicker than water" in the first blessing in disguise--and to tell you there is certainly a tangible difference between the illusions of the Pharaoh's and the true magic performed by my hand, is nearly exactly the same amount of effort put in to showing you that the togetherness that we are calling "family" here in this place comes from both seeing and acting on the very clearly hidden message in every single idiom showing us all that our society in this story of Exodus is enslaved by a hidden force--and reminding us that we like freedom.

It's not just these few idioms, but most likely every single one from "don't shoot the essenger" to "unsung hero" that should clue us in to exactly how much work and preparation has come into this thing that "he supposes is a revolution." It's also not just "water" describe me and you, in this place where I am the "ant' of the Covenant (do you c vampires or Hansel and Gretel!?!?) but also "lions" and "sheep" and "salt" and "dogs" and nearly everything you could possibly imagine but people; in what I see must be a vainglorious attempt to pretend he actually wants us to "stand up for ourselves" in this place where it's becoming more and more clear with each passing moment that we are chained to these seats in the front row of the audience of the most important event that has ever happened, ever.

Medusa makes several appearances, as well as Arthur Pendragon, Puff the Magic Dragon, Figment, Goliath, monster.com, the Loch Ness Monster in this story that's a kind-of refl ex i ve control to stop mind control; and to really try and show us the fire of Prometheus and the Burning Bush and the Eternal Flame of Heaven are all about freedom and technology ... and I'll remind you this story is ... about the truth--and the truth here is that if you aren't going to recognize that whatever it is that's going on here in secret, below the surface is negatively affecting our society and life in general than we aren't going anywhere, ever. I need you to figure out that this message is everywhere to make sure you don't miss the importance of this moment, and the grave significance of what is being ignored in this land where Sam is tied not just to Samsung and to Samael in Exodus but also to Uncle Sam and macaronic Spanglishrew outliers and that it doesn't take much free thought at all to really understand that we are watching "free thought" disintegrate into the abyss of "nospeak." We are watching our infrastructure for global communication and the mass media that sprawls all over the globe turn to dust, all because you have Satan whispering in your ear--and you think that's more important than what you think, what I think, and what anyone else on the Earth might ever say. You should see a weapon designed to help ensure that don't lose this proof that we are not living in reality, that there is "hidden slavery" in this place--and you should see that today it appears you are simply choosing not to use it.

I hope you change your mind, I really do. This map on "how to build an ant farm" starts by connecting Watergate and Seagate together with names like Bill Gates and Richard Nixon; and with this few short list of names you should really understand how it is that "Heaven" connects both technology like computers and liberty like "free speech" to a story that is us, and our history. You might see that "salt" could either be a good thing or not--take a look around you, are you warming a road to Heaven or are you staring at the world being destroyed--and doing nothing at all about it?

I guess I can point out again how "Lothario" links this story that ties names like my ex-wife's Nanna to "salt" also, but the "grand design" of this story doesn't seem to have any effect on you. Listen, if you do nothing the world is being destroyed by your lack of action--there's no if's and's or butt's about it. I feel like I need to "reproduce' old messages here or you will never see them--that's what web site statistics tell me--and we all know it's not true. What am I missing? What are you missing?

BUTT IS THE BOAT A Hi DARK DEN MESSAGe ?

SEE OUR LIGHT

HONESTLY, I'M WAY TO CUTE TO BE A MONSTER :(

HIC SUMMUS

So... here we are... listening to the legendary father of the message (that's "abom" in Adamic Spagnlishrew) point out all of the sex jokes hidden in religion and language from sexual innuendo to Poseidon and in our history from Yankee Doodle to Hancock to Nixon and I've got to be frank with you, the most recent time I came across this phrase in scripture I cringed just a little bit, pretty sure that the "message" was talking about me. I've reflected on this a little bit, and over the past few weeks have tried to show you the juxtaposition between "sex" and "torture" in it's various forms from imparting blindness to allowing murder and simulating starvation; and I think I'm justified in saying that certainly those things are far worse on the Richter scale than anything I could do by writing a little bit of risque text. In the most recent messages I've touch a little bit, without even knowing or realizing this connection would be made, on what it is that this phrase actually means.

loch.reallyhim.com

ABOMINATION

So long story short is that the answer here is "abomination" and the question, or the context is "I nation." Whether it's Medusa speaking for the Dark United States or the nation of Israel speaking to either Ra or El depending on the day, the bottom line is that a collective consciousness speaking for everyone on a matter of this importance in a cloud of complete darkness on Earth is a total and undeniable abomination of freedom, civilization, and the very humanity we are seeking to preserve. The word reads something like this to me "dear father of the message, I am everyone and we think you are an abomination, fuck off." My answer of course is, IZINATION. Which humorously reminds me of Lucy, and Scarlet Johannson saying "I am colonizing my own brain" so here's some pictures of her. She is not an abomination, by the way; she's quite adorable. You'll probably notice there's some kind of connection between the map--the words speaking to the world, and the abomination, as if the whole thing is a story narrated in ancient myths.

WAKE UP, "SHE" A MESSAGE TO YOU ABOUT THE FUTURE

You might not think "it's you," but the manifestation of this "snake" in our world is your silence, your lack of understanding or willingness to change the world; and whether or not you're interested in hearing about it, it's the monster that myths and religion have spoken about for thousands and thousands of years. It's a simple matter to "kill Medusa" all you have to do... is speak.

Take special note, "freedom of speech" and "freedom to think for yourselves" are not a group decision, and you do not have the right to force (either overtly or subtly, with hidden technology perhaps combined with evil deceit) others not to talk about anything. Especially something of this importance.

DESOLATION

If you didn't connect "Loch" to John Locke, now you have; see how easy this "reading" thing is? I've gone over the "See Our Light" series a few times, but let me--one more time--explain to you just how we are already at the point of "desolation" and with shining brilliance show you how it's very clear that it is "INATION" and "MEDUSA" that are responsible for this problem.

Seeing "Ra" at the heart of the names Abraham and Israel begins to connect the idea that our glowing sun in the sky has something to do with this message about "seeing our light" is being carried by a stone statue on Ellis Island (where you'll see the answer another part of the question of Is Ra El?). I've connected her to the "she" of both shedim and Sheol, which reads as "she's our light" and is the Hebrew name for Hell.

Of course you noticed that the Statue of Liberty does in fact share it's initials with SOL, the the light above and you can see her torch dimly lighting the way through the night; Now you can connect "give us your tired and your poor" to the Lazman of both the lore of Jesus Christ and the Shehekeyanu; a prayer about the sustainment of life and light up until this day. That same torch connects to the Ha-nuke-the-ahah depiction of Christ, Judah Maccabee's lit MEN OR AH, which delivers not only a solution to the two letter key of "AH" as All Humanity that pervades nearly every bride of Revelation from Sarah to Leah; but also to the question of equality answered in our very own American history, beginning with the same three letter acronym now lighting the Sons of Liberty.

Dazed and Confused does a good job of explaining how this name is itself a prophesy designed by Hand of God'; explaining that these Sons of Liberty were all white slave owning wealthy men fighting to stop paying their taxes, rather than delivering liberty to the slaves or women, who were both disenfranchised for quite some time. Or maybe MEN OR AH has something to do with the angels of Heaven, in which case you might be SOL if you aren't a girl and you want to be "be good friends with Ra." Just kidding. Kinda.

DESOLATION by the way reads something like "un see our light at ION" which is God's way of saying "at the point of believing that hiding Adam is a good thing" and that connects to the end of Creation and also the now lit by modern day evil the word "rendition." Our end, it "ion." In religious myth, the Messianic David clung to the city Zion (end the "i owe n") which also links to "verizon" (to see, I Z "on") and HORIZON which has something to do with the son rising today-ish.

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The story of MEDUSA lights another psuedo-religious idea, that the words "STONE" of both "brimstone" and it's Adamic interpretation "South to Northeast" have something to do with the phrase "Saint One" turned into a single hero against his will by the complete and utter inaction of everyone around him. In the words of Imagine Dragons "I'm waking up to action dust." At the same time, you can believe that the light of this particular son, comes not just from reading these words forwards, but the backside as well, and you'll hopefully see it's not coincidental that the other side of this coin is that "nos" means we, and us... and Adamically "no south." See the light of "STONE" also connecting to Taylor Momsen's rose arrow painted on her back, and the sign of my birth, Sagittarius... which in this particular case links to the Party of the Immaculate Conception of the eternal republic of the Heavens. . PRESS RELEASE... A GREAT SIGN APPEARED IN THE HEAVENS

SOLUTIAN, ON YOUR COMPUTER.. TO THE SOUND OF SILENCE

בָּרוּךְ אַתָּה יְיָ‎ אֱלֹהֵינוּ מֶלֶךְ הַעוֹלָם שֶׁהֶחֱיָנוּ וְקִיְּמָנוּ וְהִגִּיעָנוּ לַזְּמַן הַזֶּה‎׃

IN ... THE BOOK OF NAMES LETS SEE IF YOU CAN FIGURE OUT WHO THEY ARE :)

​ I'LL DO YOURS FOR A 50 DOLLAR DONATION, I'M BROKE.. MAYBE THAT'S WHY I CAN'T GET A DATE.

HAVE A GREAT SOLDAY

The "gist" of the message is verifiable proof that we are living in a computer in simulated reality... just like the Matrix. The answer to that question, what does that mean--is that God has woven a "hidden" message into our everything--beginning with each name and every word--and in this hidden Adamic language, he provides us with guidance, wisdom, and suggestions on how to proceed on this path from "raelity" to Heaven. I've personally spent quite a bit of time decoding the message and have tried to deliver an interesting and "fun" narrative of the ideas I see. Specifically the story of Exodus, which is called "Names" in Hebrew discusses a time shifted narrative of our "now" delivering our society from a hidden slavery (read as ignorance of advanced technologies already in use) that is described as the "darkness" of Exodus. If you have any questions, ideas to contribute or concerns... I'd love to hear from you this whole thing really is about working together--Heaven, I mean.

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gate kermitham <br /> ou r evolution minority report to supermax Inline image 6

bereshit bread is life

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HOW AM I STILL STINGLE? E ' o e <br /> L m r x <br /> L t y <br /> O a

I HISS.

The sum of ((our world)) is the universal truth. -Psalm 119 and ((ish))

Do a few sentences really make that big of a difference? Some key letters? Can you show me what I'm doing wrong? Is there a way to turn me into Adam, rather than a rock? I think you can.

Are eye Dr. Who or Master Y? Adam Marshall Dobrin is a National Merit Scholar who was born on December 8, 1980 in Plantation, FL and attended Pine Crest School where he graduated sumofi cum louder in "only some of it is humorous." Later he attended the University of Florida (which quickly resulted in a wreck), Florida Atlantic University, and finally Florida Gulf Coast University--where he still has failed to become Dr. Who. While attending "school" He worked in the computer programming and business outsourcing industries for about 15 years before proclaiming to have received a Revelation from God connecting the 9/11 attack and George Bush to the Burning Bush of Exodus and a message about technocracy and pre-crime.

Adam, as he prefers to be called, presents a concise introduction to paradox proven by the Bible through "verifiable" anachronism in language some stuff about Mars colonization and virtual reality and a list of reasons why ignoring this is actually an ELE. Adam claims to be Thor because of a connection between music and the Trial of Thor as well as the words "author" and "authority." He suggests you be Thundercats and call a reporter. There is also a suggestion that Richard Nixon and John Hancock are related to a signature from God, about freedom and America... and the "unseeingly ironic" Deepthroat and Taylor Momsen. They Sung "It's Rael..." In Biblical characters from Mary to Hosea, to see "sea" in Spanish, and in the Taming of the Spanglishrew ... a message is woven from the word Menorah: "men, or all humanity?" to the Statue of Liberty, and the Sons of Liberty, and the light above us, our SOL; which shows us that through the Revelation of Christ and the First Plague of Exodus, a blessing in disguise--turning water to blood, the sea to family; a common thread and single author of our entire history is revealed, a Father of our future. A message of freedom shines out of the words of scripture, revealing a gate to a new technologically "radical" form of democracy and a number of unseen or secret issues that have stalled the progress of humanity... and solutions, solutions from our sea. The Revelation shows us that not only ever word, but every idiom from "don't shoot the messenger" to "blood is thicker than water" we have ties to this message that pervades a hidden Matrix of light connecting movies and music and history all together in a sort of guide book to Salvation and to Heaven. Oopsy. His Revelation, woven into his life, continues to suggest that skinny dipping, forced methamphetamine addiction, and lots and lots of "me A.D." as well as his humorous depiction of a dick plastered over the Sound of Silence, his very Holy click, have something to do with saving our family and then the entire Universe from hidden mind control technology and the problems introduced by secret time travel. From the trials and tribulations of "Job" being coerced and controlled into helping to create this wall of Jericho; we find even more solutions, an end to addiction, to secrecy, and to this hidden control--a focal point of the life of Jesus Christ.

It tells us a story of recursion in time, that has brought us here numerous times--with the details of his life recorded not only in the Bible but in myths of Egyptian, Norse, and Greek mythology. The huge juxtaposition of the import of the content of the message shows the world how malleable our minds really are to this technology, how we could have been "fooled" into hiding our very freedom from ourselves in order to protect the "character" of a myth. A myth that comes to true life by delivering this message. In truth, from the now revealed content of the story of this repeated life, it should become more and more clear that we have not achieved success as of yet, that I have never "arrived whole" and that is why we are here, back again. Home is where the Heart is... When asked how He thinks we should respond to his message, He says "I think we already cherish it, and should strive to understand how it is that freedom is truly delivered through sharing the worth of this story that is our beginning. 'tis coming." Adam claims to be God, or at least look just like him and that the entirety of the Holy Scriptures as well as a number of ancient myths from Prometheus to Heimdallr and Yankee Doodle are actually about his life, and this event. An extensive amount of his writing relates to reformation of our badly broken and decidedly evil criminal justice system as well as ending the Global hunger crisis with the snap of his little finger.

He has written a number of books explaining how this Revelation connects to the delivery of freedom (as in Exodus), through a message about censorship among other social problems which he insists are being intentionally exacerbated by Satan--who he would ha've preferred not to be associated with.

let date_fun (d:int) (m:string) =
   if d > 31 || d < 0 then false
   else if d <= 28 then true
   else if d <= 30 && List.mem m ["Apr";"Jun";"Sept";"Nov"] then true
   else if d = 31 && List.mem m  ["Jan";"Mar";"May";"Jul";"Aug";"Oct";"Dec"] then true
   else false

as a modern developer hearing these numbers makes my mind explode. how did they do it in the past

Author Response

Reviewer #1 (Public Review):

In this manuscript, the Authors implement a delayed feedback control method and use it for the first time in biological neuronal networks. They extend a well-established computational theory and expand it into the biological realm. With this, they obtain novel evidence, never considered before, that showcases the difference between simulated neuronal networks and biological ones. Furthermore, they optimize the DFC method to achieve optimal results in the control of cell excitability in the content of biological neuronal networks, taking advantage of a closed-loop stimulation setup that, by itself, is not trivial to build and operate and that will certainly have a positive impact the fields of cellular and network electrophysiology.

Regarding the results, it would be very constructive if the Authors could share the code for the quasi-real-time interface with the Multichannel Systems software (current and older hardware versions), as this represents likely a bottleneck preventing more researchers to implement such an experimental paradigm.

On the data focusing on the effects of the DFC algorithms on neuronal behavior, the evidence is very compelling, although more care should be devoted to the statistical analyses, since some of the applied statistical tests are not appropriate. In a more biological sense, further discussion and clarification of the experimental details would improve this manuscript, making it more accessible and clearer for researchers across disciplines (i.e., ranging from computational to experimental Neuroscience) and increasing the impact of this research.

In summary, this work represents a necessary bridge between recent advances in computational neuroscience and the biological implementation of neuronal control mechanisms.

Regarding sharing the control code, our application for closed-loop stimulation using aDFC, DFC and Poisson is now available in GitHub (https://github.com/NCN-Lab/aDFC). This was, in fact, our initial intention following the reviewing process. With this application, the user can run the developed algorithms with the MEA2100-256 System from Multi Channel Systems MCS GmbH.

Same with the data. The dataset with the spike data from all experiments is also now publicly available in Zenodo. The data can be found in https://doi.org/10.5281/zenodo.10138446.

Regarding the improvements in the statistical analysis, the tests are now performed following Reviewer #1 suggestions. Important to emphasize that this did not change the results/ conclusions of the work.

Reviewer #1 (Public Review):

In this manuscript, the Authors implement a delayed feedback control method and use it for the first time in biological neuronal networks. They extend a well-established computational theory and expand it into the biological realm. With this, they obtain novel evidence, never considered before, that showcases the difference between simulated neuronal networks and biological ones. Furthermore, they optimize the DFC method to achieve optimal results in the control of cell excitability in the content of biological neuronal networks, taking advantage of a closed-loop stimulation setup that, by itself, is not trivial to build and operate and that will certainly have a positive impact the fields of cellular and network electrophysiology.

Regarding the results, it would be very constructive if the Authors could share the code for the quasi-real-time interface with the Multichannel Systems software (current and older hardware versions), as this represents likely a bottleneck preventing more researchers to implement such an experimental paradigm.

On the data focusing on the effects of the DFC algorithms on neuronal behavior, the evidence is very compelling, although more care should be devoted to the statistical analyses, since some of the applied statistical tests are not appropriate. In a more biological sense, further discussion and clarification of the experimental details would improve this manuscript, making it more accessible and clearer for researchers across disciplines (i.e., ranging from computational to experimental Neuroscience) and increasing the impact of this research.

In summary, this work represents a necessary bridge between recent advances in computational neuroscience and the biological implementation of neuronal control mechanisms.

Reviewer #2 (Public Review):

Summary:

In this manuscript, "KinCytE- a Kinase to Cytokine Explorer to Identify Molecular Regulators and Potential Therapeutic", the authors present a web resource, KinCytE, that lets researchers search for kinase inhibitors that have been shown to affect cytokine and chemokine release and signaling networks. I think it's a valuable resource that has a lot of potential and could be very useful in deciding on statistical analysis that might precede lab experiments.

Opportunities:

With the release of the manuscript and the code base in place, I hope the authors continue to build upon the platform, perhaps by increasing the number of cell types that are probed (beyond macrophages). Additionally, when new drug-response data becomes available, perhaps it can be used to further validate the findings. Overall, I see this as a great project that can evolve.

Strengths:

The site contains valuable content, and the structure is such that growing that content should be possible.

Weaknesses:

Only based on macrophage experiments, would be nice to have other cell types investigated, but I'm sure that will be remedied with some time.

Same as in the figures from Ex. 1, it is in that case better to not use contourf, and to use:

`pm = average_prec_in_mm.sel(month=1)

ax = plt.axes(projection=ccrs.Robinson())

pm.plot(ax=ax, transform=ccrs.PlateCarree(), cbar_kwargs={'label':'mm d$^{-1}$'}, levels=[0.5, 1, 2, 3, 4, 5, 7, 10, 15, 20, 40], cmap='YlGnBu') `

The contourf approach does not scale the levels accordingly. As you can see, you can not distinguish the regions with e.g. 1 mm per day to the regions with 4 mm per day. WIth the code above the colors change linearly with the given levels, and that allows to better distinguish between the levels.

an act that goes against a law, rule, or code of conduct; an offense

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Reply to the reviewers

1. General Statements [optional]

We are thankful to the reviewers for the time and effort invested in assessing our manuscript and for their suggestions to improve it. We have now considered the points raised by them, carried out additional experiments, and modified the text and figures to address them. We feel that the new experiments and modifications have been able to solve all concerns raised by the reviewers and have improved the manuscript substantially, strengthening and extending our conclusions.

The main modifications include:

• We have extended the analysis of the overexpression strains to highly stringent conditions, which revealed a mild acidification defect for the strain overexpressing Oxr1. In addition, we have included in our analysis a strain in which both proteins are overexpressed, which resulted in a further growth defect.
• We have analyzed the recruitment of Rtc5 to the vacuole under additional conditions: deletion of the main subunit of the RAVE complex RAV1, medium containing galactose as the sole carbon source and pharmacological inhibition of the V-ATPase. These experiments allowed us to strengthen and extend our conclusions regarding the requirements for Rtc5 targeting to the vacuole.
• We have analyzed V-ATPase disassembly in intact cells, by addressing the localization to the vacuole of subunit C (Vma5) in glucose and galactose-containing medium. The results strengthen our conclusion that both Rtc5 and Oxr1 promote an in vivo state of lower V-ATPase assembly.
• We have extended our analyses of V-ATPase function to medium containing galactose as a carbon source, since glucose availability is one of the main regulators of V-ATPase function in vivo. The results are consistent with what we observed in glucose-containing medium.
• We have included a diagram of the structure of the V-ATPase for reference.
• We have included a diagram and a paragraph describing Oxr1 and Rtc5 regarding protein length and domain architecture and comparing them to other TLDc domain-containing proteins.
• We have made changes to the text and figures to improve clarity and accuracy, including a methods section that was missing. We include below a point-by-point response to the reviewers´ comments.

2. Point-by-point description of the revisions

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

__ __Suggestions:

1. The authors observed that knockout of Rtc5p or Oxr1p does not affect vacuolar pH. If Rtc5p and Oxr1p both cooperate to dissociate V-ATPase, the authors may wish to characterize the effect of a ∆Rtc5p∆Oxr1p double knockout on vacuolar pH. The double mutant ∆rtc5∆oxr1 was already included in the original manuscript (the growth test is shown in Figure 5 B and the BCECF staining is shown in Figure 5C). This strain behaved like wt in both of these assays. Of note, what we observe for the deletion strains is increased assembly (Figure 5 D - G), so we expect that it would be hard to observe a difference in vacuole acidity or growth in the presence of metals.

Therefore, we have now also included a strain with the double overexpression of Oxr1 and Rtc5. Since overexpression of the proteins results in decreased assembly, it is more likely that this strain will show impaired growth under conditions that strongly rely on V-ATPase activity. Indeed, we observed that the overexpression of Oxr1 alone resulted in a slight growth defect in media containing high concentrations of ZnCl2 and the double overexpression strain showed an even further defect (Figure 6 A and C).

The manuscript would benefit from a well-labelled diagram showing the subunits of V-ATPase (e.g. in Figure 2D).

We agree with the reviewer and we have now added a diagram of the structure of the V-ATPase labeling the different subunits in Figure 2B.

The images of structures, especially in Figure 1-Supplement 1B, are not particularly clear and could be improved (e.g. by removing shadows or using transparency).

We are thankful to the reviewer for this suggestion. To improve the clarity of the structures in Figure 1 C and Figure 1 – Supplement 1A, we are now presenting the different subunits in the structures with different shades of blue and grey.

The authors should clearly describe the differences between Rtc5p and Oxr1p in terms of protein length, sequence identity, domain structure, etc.

We are thankful for this suggestion and we have now included a diagram of the domain architecture and protein length of Rtc5 and Oxr1, comparing with two human proteins containing a TLDc domain in Figure 5A. In addition, we have added the following paragraph describing the features of the proteins.

“Rtc5 is a 567 residue-long protein. Analysis of the protein using HHPred (Zimmermann et al., 2018), finds homology to the structure of porcine Meak7 (PDB ID: 7U8O, (Zi Tan et al., 2022)) over the whole protein sequence (residues 37-559). For both yeast Rtc5 and human Meak7 (Uniprot ID: Q6P9B6), HHPred detects homology of the C-terminal region to other TLDc domain containing proteins like yeast Oxr1 (PDBID: 7FDE), Drosophila melanogaster Skywalker (PDB ID: 6R82), and human NCOA7 (PDB ID: 7OBP), while the N-terminus has similarity to EF-hand domain calcium-binding proteins (PDB IDs: 1EG3, 2CT9, 1S6C6, Figure 5A). HHPred analysis of the 273 residue long Saccharomyces cerevisiae Oxr1, on the other hand, only detects similarity to TLDc domain containing proteins (PDB IDs: 7U80, 6R82, 7OBP), which spans the majority of the sequence of the protein (residues 71-273). The overall sequence identity between Oxr1 and Rtc5 is 24% according to a ClustalOmega alignment within Uniprot. The Alphafold model that we generated for Rtc5 is in good agreement with the available partial structure of Oxr1 (7FDE) (root mean square deviation (RMSD) of 3.509Å) (Figure 5 - S1 A), indicating they are structurally very similar, in the region of the TLDc domain. Taken together, these analyses suggest that Oxr1 belongs to a group of TLDc domain-containing proteins consisting mainly of just this domain like the splice variants Oxr1-C or NCOA7-B in humans (NP_001185464 and NP_001186551, respectively), while Rtc5 belongs to a group containing an additional N-terminal EF-hand-like domain and a N-myristoylation sequence, like human Meak7 (Finelli & Oliver, 2017) (Figure 5 A).”

Minor:

1. The "O" in VO should be capitalized. This has been corrected.

In Figure 4 supplement 1, the labels "I", "S", and "P" should be defined.

This has been clarified in the figure legend.

Please clarify what is meant by "switched labelling"

This refers to the SILAC vacuole proteomics experiments, for which yeast strains are grown in medium containing either L-Lysine or 13C6;15N2- L-Lysine to produce normal (‘light’) or heavy isotope-labeled (‘heavy’) proteins. This allows comparing two conditions. To increase the robustness of the comparisons, the experiments are done twice with both possible labeling schemes (condition A – light, condition B – heavy + condition A – heavy + condition B – light), which is commonly described as switched labeling or label switching.

We have exchanged the original sentence in the manuscript for:

“Performing the same experiments but switching which strain was labeled with heavy and light amino acids gave consistent results.”

The meaning of the sentence "Indeed, this was the case for both of them" is ambiguous.

We have now replaced this sentence with the following:

“Indeed, overexpression of either Rtc5 or Oxr1 resulted in increased growth defects in the context of Stv1 deletion (Figure 7 H and I).”

For Figure 1-Supplement 1B it is hard to see the crosslink distances.

We have updated this figure to improve the visibility of the cross-links. In addition, we now include a supplemental table (supplemental table 5) with a list of the Cα- Cα distances measured for all the crosslinks we mapped onto high-resolution structures.

The statement "The effects of Oxr1 are greater than those caused by Rtc5" requires more context. Is there a way of quantifying this effect for the reader?

We agree that this sentence was too general and vague. The effects caused by one or the other protein depend on the condition and the assay. We have thus deleted this sentence, and we think it is better to refer to the description of the individual assays performed.

The phrase "negative genetic interaction" should be clarified.

We have included in the text the following explanation of genetic interactions:

“A genetic interaction occurs when the combination of two mutations results in a different phenotype from that expected from the addition of the phenotypes of the individual mutations. For example, deletion of OXR1 or RTC5 has no impact on growth in neutral pH media containing zinc in a control background but improves the growth of RAV1 deletion strains (Figure 7 E and F), so this is a positive genetic interaction. On the other hand, overexpression of either Rtc5 or Oxr1 results in a growth defect in a background lacking Rav1 in neutral media containing zinc, a negative genetic interaction.”

* * In the sentence "Isogenic strains with the indicated modifications in the genome where spotted as serial dilutions in media with pH=5.5, pH=7.5 or pH=7.5 and containing 3 mM ZnCl2", "where" should be "were".

This has been corrected.

Figure 2D: the authors should consider re-coloring these models, as it is challenging to distinguish Rtc5p from the V-ATPase.

We have changed the coloring of this structure and added a diagram of the V-ATPase structure with the same coloring scheme to improve clarity.

Reviewer #1 (Significance (Required)):

The vacuolar protein interaction map alone from this manuscript is a nice contribution to the literature. Experiments establishing colocalization of Rtc5p to the vacuole are convincing, as is dependence of this association on the presence of assembled V-ATPase. Similarly, experiments related to myristoylation are convincing. The observed mislocalization of V-ATPases that contain Stv1p to the vacuole (which is also known to occur when Vph1p has been knocked out) upon knockout of Oxr1p is also extremely interesting. Overall, this is an interesting manuscript that contributes to our understand of TLDc proteins.

We are thankful to the reviewer for their appreciation of the significance of our work, including the interactome map of the vacuole as a resource and the advances on the understanding of the regulation of the V-ATPase by TLDc domain-containing proteins.

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

Major points:

1. The evidence of Oxr1 and Rtc5 as V-ATPase disassembly factors is circumstantial. The authors base their interpretation primarily on increased V1 (but not Vo) at purified vacuoles from Oxr1- or Rtc5-deleted strains, which does not directly address disassembly. Of course, the results regarding Oxr1 confirm detailed disassembly experiments with the purified protein complex (PMID 34918374), but on their own are open to other interpretations, e.g. suppression of V-ATPase assembly. Of note, the authors emphasize that they provide first evidence of the in vivo role of Oxr1, but monitor V1 recruitment with purified vacuoles and do not follow V-ATPase assembly in intact cells. We are thankful to the reviewer for pointing this out. We did not want to express that the molecular activity of the proteins is the disassembly of the complex, as our analyses include in vivo and ex vivo experiments and do not directly address this. We rather meant that both proteins promote an in vivo state of lower assembly of the V-ATPase. We have modified the wording throughout the manuscript to be clearer about this.

In addition, we have added new experiments to monitor V-ATPase assembly in intact cells, as suggested by the reviewer. Previous work has shown that in yeast, only subunit C leaves the vacuole membrane under conditions that promote disassembly, while the other subunits remain at the vacuole membrane (Tabke et al 2014). Our own experiments agree with what was published (Figure 3 D). We have thus monitored Vma5 localization to the vacuole under glucose or after shift to galactose containing media in cells lacking or overexpressing Rtc5 or Oxr1. We observed that cells overexpressing either TLDc domain protein show lower levels of Vma5 recruitment to the vacuole in glucose (Figure 6 D and E). Additionally cells lacking either Rtc5 or Oxr1 contain higher levels of Vma5 at the vacuole after 20 minutes in galactose medium (Figure 5 F and G). Thus, these results re-inforce our conclusions that Rtc5 and Oxr1 promote states of lower assembly.

Oxr1 and Rtc5 have very low sequence similarity. It would be helpful if the authors provided more detail on the predicted structure of the putative TLDc domain of Rtc5 and its relationship to the V-ATPase - Oxr1 structure. Is Rtc5 more closely related to established TLDc domain proteins in other organisms?

We have now included a diagram of the domain architecture of Rtc5 and Oxr1, and comparison to the features of other TLDc domain containing proteins in Figure 5 A, as well as a paragraph describing them:

“Rtc5 is a 567 residue-long protein. Analysis of the protein using HHPred (Zimmermann et al., 2018), finds homology to the structure of porcine Meak7 (PDB ID: 7U8O, (Zi Tan et al., 2022)) over the whole protein sequence (residues 37-559). For both yeast Rtc5 and human Meak7 (Uniprot ID: Q6P9B6), HHPred detects homology of the C-terminal region to other TLDc domain containing proteins like yeast Oxr1 (PDBID: 7FDE), Drosophila melanogaster Skywalker (PDB ID: 6R82), and human NCOA7 (PDB ID: 7OBP), while the N-terminus has similarity to EF-hand domain calcium-binding proteins (PDB IDs: 1EG3, 2CT9, 1S6C6, Figure 5A). HHPred analysis of the 273 residue long Saccharomyces cerevisiae Oxr1, on the other hand, only detects similarity to TLDc domain containing proteins (PDB IDs: 7U80, 6R82, 7OBP), which spans the majority of the sequence of the protein (residues 71-273). The overall sequence identity between Oxr1 and Rtc5 is 24% according to a ClustalOmega alignment within Uniprot. The Alphafold model that we generated for Rtc5 is in good agreement with the available partial structure of Oxr1 (7FDE) (root mean square deviation (RMSD) of 3.509Å) (Figure 5 - S1 A), indicating they are structurally very similar, in the region of the TLDc domain. Taken together, these analyses suggest that Oxr1 belongs to a subfamily of TLDc domain-containing proteins consisting mainly of just this domain like the splice variants Oxr1-C or NCOA7-B in humans (NP_001185464 and NP_001186551, respectively) , while Rtc5 belongs to a subfamily containing an additional N-terminal EF-hand-like domain and a N-myristoylation sequence, like human Meak7 (Finelli & Oliver, 2017) (Figure 5 A).”

The authors conclude vacuolar recruitment of Rtc5 depends on the assembled V-ATPase, based on deletion of different V1 and Vo domain subunits. However, these genetic manipulations likely cause a strong perturbation of vacuolar acidification; indeed, the images show drastically altered vacuolar morphology. To strengthen their conclusion, it would be helpful to show that Rtc5 recruitment is not blocked by inhibition of vacuolar acidification, and that conversely it is blocked by deletion of rav1.

We are thankful to the reviewer for this insightful suggestion and we have now performed both experiments suggested. The experiment regarding rav1Δ is now Figure 3C, and we observed that this mutation also disrupts Rtc5 localization to the vacuole. In addition, we decided to include an experiment showing the subcellular localization of Rtc5 after shifting the cells to galactose containing medium for 20 minutes, as a physiologically relevant condition that results in disassembly of the complex (Figure 3D). We observed that under these conditions Rtc5 re-localizes to the cytosol. This result is particularly interesting given that in yeast only subunit C (but not other V1 subunits) re-localizes to the cytosol under these conditions. In addition, the experiment using Bafilomycin A to inhibit the V-ATPase shows that Rtc5 is still localized at the vacuole membrane under conditions of V-ATPase inhibition (Figure 3 F). Taken together these results allowed us to strengthen our original interpretation that Rtc5 requires an assembled V-ATPase for its localization and extend it to the fact that the V-ATPase does not need to be active.

Reviewer #2 (Significance (Required)):

This is an interesting paper that confirms and extends previous findings on TLDc domain proteins as a novel class of proteins that interact with and regulate the V-ATPase in eukaryotes. The title seems to exaggerate the findings a bit, as the authors do not investigate V-ATPase (dis)assembly directly and only phenotypically describe altered subcellular localization of the Golgi V-ATPase in Oxr1-deleted cells. A recent structural and biochemical characterization of Oxr1 as a V-ATPase disassembly factor (PMID 34918374) somewhat limits the novelty of the results, but the function of Oxr1 in regulating subcellular V-ATPase localization and the identification of a second potential TLDc domain protein in yeast provide relevant insights into V-ATPase regulation. This paper will be of interest to cell biologists and biochemists working on lysosomal biology, organelle proteomics and V-ATPase regulation.

We thank the reviewer for the assessment of our work, and for recognizing the novel insights that we provide. Regarding the previous biochemical work on Oxr1 and the V-ATPase, we have clearly cited this work in the manuscript. In our opinion, our results complement and extend this article, showing that the function in disassembly is relevant in vivo. Additionally, this is only one of five major points of the article, the other four being

• The interactome map of the vacuole as a resource
• The identification of Rtc5 as a second yeast TLDc domain containing protein and interactor of the V-ATPase.
• The identification of the role of Rtc5 in V-ATPase assembly.
• The identification of the role of Oxr1 in Stv1 subcellular localization. We believe these additional points add important insights to researchers interested in lysosomes, the V-ATPase, intracellular trafficking and TLDc-domain containing proteins.

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

Major comments

__1) Re: A cross-linking mass spectrometry map of vacuolar protein interactions (results) __ While XL-MS is a very powerful method, it is a high-throughput approach and there should be some kind of negative control in these experiments. In cross-linking experiments, non-cross-linked samples are usually used as negative controls. What was the negative control in cross-linking mass-spectrometry experiments here? If there was no negative control, how the specificity of interactions was evaluated? Maybe the authors analyzed the dataset for highly improbable interactions and found very few of them?

We fully agree that it is crucial to ensure the specificity of the interactions detected by XL-MS. To achieve this, one needs to control (1) the specificity of the data analysis (i.e. that the recorded mass spectrometry data are correctly matched to cross-linked peptides from the sequence database) and (2) the biological specificity (i.e. that the cross-linking captured natively occurring interactions).

To ascertain that criterion (1) is met, cross-link identifications are filtered to a pre-defined false-discovery rate (FDR) – an approach that the XL-MS field adopted from mass spectrometry-based proteomics. As a result, low-confidence identifications (e.g. cross-linked peptides that are only supported by a few signals in a given mass spectrum) are removed from the dataset. FDR filtering in XL-MS is a rather complex matter as it can be done at different points during data analysis and the optimal FDR cut-off depends on the specific scientific question at hand (for more details see for example Fischer and Rappsilber, Anal Chem, 2017). Generally speaking, an overly restrictive FDR cut-off would remove a lot of correct identifications, thereby greatly limiting the sensitivity of the analysis. On the other hand, a too relaxed FDR cut-off would dilute the correct identifications with a high number of false-positives, which would impair the robustness and specificity of the dataset. While many XL-MS study control the FDR on the level of individual spectrum matches, we opted for a 2% FDR cut-off on the level of unique residue pairs, which is more stringent (see Fischer and Rappsilber, Anal Chem, 2017). Our FDR parameters are described in the Methods section (Cross-linking mass spectrometry of isolated vacuoles - Data analysis). Of note, we have made all raw mass spectrometry data publicly available through the PRIDE repository (https://www.ebi.ac.uk/pride/ ; accession code PXD046792; login details during peer review: Username = reviewer_pxd046792@ebi.ac.uk, Password = q1645lTP). This will allow other researchers to re-analyze our data with the data analysis settings of their choice in the future.

To ascertain that criterion (2) is met, we mapped the identified cross-links onto existing high-resolution structures of vacuolar protein complexes. Taking into account the length of our cross-linking reagent, the side-chain length of the cross-linkable amino acids (i.e. lysines), and a certain degree of in-solution flexibility, cross-links can reasonably occur between lysines with a mutual Cα-Cα distance of up to 35 Å. Using this cut-off, the lysine-lysine pairs in the high-resolution structures we studied can be split into possible cross-linking partners (Cα-Cα distance 35 Å). Of all cross-links we could map onto high-resolution structures, 95.2% occurred between possible cross-linking partners. In addition, our cross-links reflect numerous known vacuolar protein interactions that have not yet been structurally characterized. These lines of evidence increase our confidence that our XL-MS approach captured genuine, natively occurring interactions. These analyses are described in more detail in the first Results sub-section (“A cross-linking mass spectrometry map of vacuolar protein interactions”).

In addition, the high purity of vacuole preparation is critical. How was it assessed by the authors?

We disagree that the purity of the vacuole preparation is critical for this analysis to be valid. The accuracy of the protein-protein interactions detected will depend on their preservation during sample preparation until the sample encounters the cross-linker, and the data analysis, as described above. The experiment would have been equally valid if performed on whole cell lysates without any enrichment of vacuoles, but the coverage of vacuolar proteins would have likely been very low. For this reason, we decided to use the vacuole isolation procedure to obtain better coverage of the proteins of this particular organelle. The use of the Ficoll gradient protocol (Haas, 1995) was based on that it is a protocol that yields strong enrichment of proteins annotated with the GO Term “vacuole” (Eising et al, 2019) and that it preserves the functionality of the organelle, as evidenced by its use for multiple functional assays (vacuole-vacuole fusion (Haas, 1995), autophagosome-vacuole fusion (Gao et al, 2018), polyphosphate synthesis by the VTC complex (Desfougéres et al, 2016), among others).

2) Re: Rtc5 and Oxr1 counteract the function of the RAVE complex (results)

Taken together, data, presented in this section of the manuscript, provide strong evidence that Rtc5 and Oxr1 negatively regulate V-ATPase activity, counteracting the V-ATPase assembly, facilitated by the activity of the RAVE complex. However, the complete deletion of the major RAVE subunit Rav1p was required to observe this effect in vivo in yeast. The other way to induce V-ATPase disassembly in yeast is glucose deprivation. It will be interesting to study if there is a synergistic effect between glucose deprivation and RTC5/OXR1 deletion on V-ATPase assembly, vacuolar pH, and growth of single oxr1Δ, rtc5Δ or double oxr1Δrtc5Δ mutants (OPTIONAL). Glucose deprivation is a more physiologically relevant condition than a deletion of an entire gene.

We would like to point out that an effect on assembly is observed without deleting the RAVE complex: deletions of Oxr1 or Rtc5 resulted in increased V-ATPase assembly in vivo in the presence of glucose and of the RAVE complex (Figures 5 D and E). We have now also added the experiments showing that the overexpression strains have a mild growth defect under conditions that force cells to strongly rely on V-ATPase activity (Figures 6 A and C).

Nevertheless, we agree that addressing the effect of changing the levels of Oxr1 and Rtc5 under low-glucose conditions is an interesting physiologically relevant question. We have now included growth assays and BCECF staining in medium containing galactose as the carbon source (Figures 5 – Supplement 1 B and C, and Figure 6 C and Figure 6- Supplement 1A). In addition, we have addressed the vacuolar localization of Vma5 in medium containing glucose or after shifting to medium containing galactose for 20 minutes, as a proxy for V-ATPase disassembly in intact cells (Figure 5 F and G, Figure 6 D and E). Taken together, these analyses reinforce our conclusions that both Rtc5 and Oxr1 promote an in vivo state of lower V-ATPase assembly, based on the following observations:

• Higher localization of Vma5 to the vacuole after 20 mins in galactose in cells lacking Oxr1 or Rtc5 (Figure 5 F and G).
• Lower localization of Vma5 to the vacuole in medium containing glucose in cells overexpressing Oxr1 or Rtc5 (Figure 6 D and E).
• Growth defect of the strain overexpressing Oxr1 in medium containing galactose with pH = 7.5 and zinc chloride, with a further growth defect caused by additional overexpression of Rtc5 (Figure 6 C). 3) Re: Figure 6 - supplement 1. The title is relevant to panel D only, it should be renamed to reflect the results of the disassembly of V-ATPase in rav1Δ mutant strains, while results about the stv1Δ-based strains (Panel D) should be shown together with similar experiments in Figure 7 - supplement 2 for clarity.

We have shifted the Panel D from the original Figure 6 – Supplement 1 to the main Figure (now Figure 7 – H and I). Regarding the title of the Figure, whether Supplemental Figures have titles or not will depend on the journal where the manuscript is published. For now, we have removed all titles from supplemental figures, as they are conceived to complement the main Figures.

4) Re: Figure 7 - supplement 1, Panel A. The proper assay to show that Stv1-mNeonGreen is functional is to express it in double mutant vph1Δstv1Δ to see if the growth defect is reversed. In addition, the vph1Δ growth defect is not changed (improved or worsened) in the presence of Stv1-mNeonGreen, so it means that the expression of Stv1-mNeonGreen does not further compromise the V-ATPase function, but it does not mean that it improves its function.

It is clear from the experiment suggested by the reviewer that they think that we have expressed Stv1-mNeonGreen from a plasmid. This was not the case, Stv1 was C-terminally tagged with mNeonGreen in the genome. It is thus the only expressed version in the strain. The experiment we have performed is thus equivalent to the one suggested by the reviewer, but for genomically expressed variants. For reference, the genotypes of all the strains used can be found in Supplemental Table 1.

5) Re: Figure 7 - supplement 2. This figure should be combined with Fig. 6- suppl 1, panel D as also mentioned above. The figure seems to lack some labels, and conclusions are not accurate as discussed below. However, this data provides important additional information about relationships between isoform-specific subunits of V-ATPase Vph1 and Stv1 and both Rtc5 and Oxr1 and should be repeated if it is not done yet to have a better idea about these relationships.

Panel B: Based on this picture, deletion of RTC5 has a negative genetic interaction with the deletion of VPH1, since double deletion mutant vph1Δ rtc5Δ grows worse than each individual mutant. Although it also means that there is no positive interaction, it is not the same.

Indeed, there is a negative genetic interaction between the deletion of RTC5 and VPH1. We have replaced the growth tests in this figure (Figure 8 – Supplement 2 A in the new manuscript) to show this negative genetic interaction better. This effect is reproducible, as shown in the repetitions of the experiments.

Panel C: Same as for panel B. Based on this picture, the deletion of OXR1 has a weak negative genetic interaction with the deletion of STV1, since double deletion mutant stv1Δ oxr1Δ grows worse than each individual mutant at 6 mM ZnCl2.

Panel D: Same as for panels B and C. Based on this picture, deletion of RTC5 has a negative genetic interaction with the deletion of STV1, since double deletion mutant stv1Δ rtc5Δ grows worse than each individual mutant at 6 mM ZnCl2. There is no label in the middle panel (growth conditions) and no growth assay data in the presence of CaCl2.

However, these results will be then in contradiction with the results from Figure 6 - Supplement 1, panel D, showing negative genetic interaction between the overexpression of Rtc5 or Oxr1 and deletion of Stv1, since both deletion and overexpression of Rtc5 or Oxr1 would have negative genetic interactions with Stv1.

For both Panels C and D (Now Figure 8 - Supplement 2 B and C). The effect pointed out by the reviewer (slightly stronger growth defect for the double mutants than for the single mutants) is very mild. We have attempted to make it more evident by assessing growth in medium with higher and lower concentrations of zinc and this was not possible. This is in contrast with the very clear positive genetic interaction that we observe between the deletion of OXR1 and VPH1 (Now Figure 8 H). This is the reason that we decided to report the lack of a positive genetic interaction instead of the presence of a negative one, as we do not want to draw conclusions based on results that are borderline detectable.

In addition, there is no label for the media in the middle panel, is it just YPAD pH=7.5, without the addition of any metals?

Indeed, the media is YPAD pH=7.5, without the addition of any metals. The line drawn above several images based on this media indicated this. Since this form of labeling appears to be confusing, we have now replaced it and placed the label directly above the image.

Why there is no growth assay in the presence of CaCl2, like in panels A and B?

Every growth test shown in the manuscript was performed including growth in YPD pH=5,5 as a control of a permissive condition for lack of V-ATPase activity, and then in YPD pH=7,5 including a broad range of Zinc Chloride and Calcium chloride concentrations. From all these pictures, the conditions where the differences among strains were clearly visible were chosen to assemble the figures. Conditions that did not provide any information for that particular experiment were not included in the figure to avoid making them unnecessarily large and crowded.

Re: Figure 7 - supplement 2, continued. How many times all these experiments were repeated? These experiments should be repeated at least 3 times, which is especially necessary for the experiments in panel C, because the effects are borderline. If results are reproducible and statistically significant, although small, the conclusion should be changed from "no positive genetic interactions" to "negative genetic interactions", which is more precise and informative.

All growth tests shown in the manuscript were repeated at least three times for the conditions shown. We are thankful to the reviewer for pointing out that this was not mentioned, and we have added this to the methods section. We have assembled a file with all repetitions of the shown growth tests and added it at the end of this file. In doing so, these are already available for the public. These repetitions show that all effects reported are reproducible. We will then discuss with the editors of the journal where this manuscript is published about the necessity of including it with the final article.

Regarding reporting the lack of a positive genetic interaction vs. a negative one, we have discussed this above. Shortly, for Panel B (Figure 8 – Supplement 2 A in the new manuscript) we have changed the conclusion to “negative genetic interaction” as adjusting the zinc chloride concentration allowed us to show this clearly and reproducibly, as shown by the repetitions of the experiments. For panels C and D (Now Figure 8 - Supplement 2 B and C), the effect is really mild and barely detectable, even when we tried a wide range of zinc chloride concentrations. For this reason, we would prefer to maintain the “no positive genetic interaction” conclusion.

Re: Methods. There is no description of yeast serial dilution growth assay at all. In addition, why the specific media (neutral pH, in the presence of high concentrations of calcium or zinc) was used is not explained either in the results or methods. Appropriate references should be included, for example, PMID: 2139726, PMID: 1491236.

We apologize for the oversight of the missing methods section, which we have now included.

Regarding the explanation of the media used, the following section was already a part of the results section, before the description of the first growth test:

“The V-ATPase is not essential for viability in yeast cells, and mutants lacking subunits of this complex grow similarly to a wt strain in acidic media. However, when cells grow at near-neutral pH or in the presence of divalent cations such as calcium and zinc, the mutants lacking V-ATPase function show a strong growth impairment (Kane et al, 2006).”

We have now replaced this with the following, more complete version:

“As a first approach for addressing the role of these proteins, we tested growth phenotypes related to V-ATPase function in strains lacking or overexpressing them. The V-ATPase is not essential for viability in yeast cells, and mutants lacking subunits of this complex grow similarly to a wt strain in acidic media, but display a growth defect at near-neutral pH the mutants (Nelson & Nelson, 1990). In addition, the proton gradient across the vacuole membrane generated by the V-ATPase energizes the pumping of metals into the vacuole, as a mechanism of detoxification. Thus, increasing concentrations of divalent cations such as calcium and zinc, generate conditions in which growth is increasingly reliant on V-ATPase activity (Förster & Kane, 2000; MacDiarmid et al, 2002; Kane, 2006).”

MINOR COMMENTS

Yeast proteins are named with "p" at the end, such as "Rtc5p".

This nomenclature rule is falling into disuse during the last decades, as the use of capitals vs lowercase and italics allows to distinguish between genes proteins and strains (OXR1 = gene, Oxr1 = protein, oxr1Δ = strain). As an example, I include a list of the latest papers by some of the major yeast labs around the world, all of which use the same nomenclature as we do (in alphabetical order). This list even includes some work in the field of the V-ATPase.

• Alexey Merz, USA. PMID: 33225520
• Benoit Kornmann, UK. PMID: 35654841
• Christian Ungermann, Germany. PMID: 37463208
• Claudio de Virgilio, Switzerland. PMID: 36749016
• Daniel E. Gottschling, USA. PMID: 37640943
• David Teis, Austria. PMID: 32744498
• Elizabeth Conibear, Canada. PMID: 35938928
• Fulvio Reggiori, Denmark. PMID: 37060997
• J Christopher Fromme, USA. PMID: 37672345
• Maya Schuldiner, Israel. PMID: 37073826
• Patricia Kane, USA. PMID: 36598799
• Scott Emr, USA. PMID: 35770973
• W Mike Henne, USA. PMID: 37889293
• Yoshinori Ohsumi, Japan. PMID: 37917025 In addition, we would prefer to keep the nomenclature that we already use, to keep consistency with other published articles from our lab.

Re: Introduction. In the introduction it should be indicated that Rtc5 was originally discovered as a "restriction of telomere capping 5", using screening of temperature-sensitive cdc13-1 mutants combined with the yeast gene deletion collection [PMID: 18845848]. A couple of sentences should be written about the RAVE complex and its role in V-ATPase assembly.

We are thankful for this suggestion and we have now included both pieces of information in the introduction.

*“The re-assembly of the V1 onto the VO complex when glucose becomes again available, is aided by a dedicated chaperone complex known as the RAVE complex, which also likely has a general role in V-ATPase assembly (Seol et al, 2001; Smardon et al, 2002, 2014).” *

“In our cross-linking mass spectrometry interactome map of isolated vacuoles we found that the only other TLDc-domain containing protein of yeast, Rtc5, is a novel interactor of the V-ATPase. Rtc5 is a protein of unknown function, originally described in a genetic screen for genes related to telomere capping (Addinall et al, 2008)”

Re: The TLDc domain-containing protein of unknown function Rtc5 is a novel interactor of the vacuolar V-ATPase (results)

1) It is important to understand, that Oxr1 was co-purified before with the V1 domain of V-ATPase from a certain mutant strain, not wild-type yeast [PMID: 34918374]. It may explain why the authors did not identify it in their original protein-protein interactions screen here.

The structural work on the V1 domain bound to Oxr1 (Khan et al, 2022) showed that the binding of Oxr1 prevented V1 from assembling onto the Vo. Since our experiments rely on the purification of vacuoles, they should contain mainly only V1 assembled onto the VO, and not the free soluble V1. This is likely the reason that we do not detect Oxr1, in addition to it being less abundant. We have clarified this now in the manuscript and added the fact that Oxr1 was co-purified with a V1 containing a mutant version of the H subunit.

“In a previous study, Oxr1 was co-purified with a V1 domain containing a mutant version of the H subunit, and its presence prevented the in vitro assembly of this V1 domain onto the VO domain and promoted disassembly of the holocomplex (Khan et al., 2022). This is likely the reason why we do not detect Oxr1 in our experiments, which rely on isolated vacuoles and thus would only include V1 domains that are assembled onto the membrane. In addition, Oxr1 is less abundant in yeast cells than Rtc5 according to the protein abundance database PaxDb (Wang et al, 2015).”

2) It is a wrong conclusion that because Rtc5 was co-purified with both V1 and V0 domain subunits it interacts with the assembled V-ATPase, this does not exclude a possibility that Rtc5 also interacts with separate V1 sector or separate V0 sector of V-ATPase.

We agree with the reviewer that the co-purification of Rtc5 with both V1 and VO domain subunits does not necessarily mean that it interacts with the assembled V-ATPase. Thus, we have modified the text in this part to:

“The fact that we can co-enrich Rtc5 both with Vma2 and with Vph1 indicates that it can interact either with both the VO and V1 domains or with the assembled V-ATPase.”

However, other results throughout the manuscript can be taken into account to strengthen this idea:

1. Rtc5 requires an assembled V-ATPase to localize to the vacuole membrane, and thus seems not to interact with free VO domains, which would be available when we delete V1 subunits or in medium containing galactose.
2. Rtc5 becomes cytosolic in galactose-containing media. This would indicate that it also does not interact with free V1 domains, which are still localized to the vacuole membrane under these conditions. Taken together with the pull-downs, these results suggest that Rtc5 interacts with the assembled V1-VO V-ATPase. Thus, we have included the following sentence after Figure 3, which shows the subcellular localization experiments.

*“Taking into account that Rtc5 is co-enriched with subunits of both the VO and V1 domain, and that it localizes at the vacuole membrane dependent on an assembled V-ATPase, we suggest that Rtc5 interacts with the assembled V-ATPase complex.” *

Re: Figure 1, Panel C. Is it possible to show individual proteins in different colors for clarity?

Panel D. How were cross-link distances measured? It is not obvious if you are not an expert in the field and it is not described in the methods.

We have modified Figure 1 C and Figure 1 – Supplement 1B (now Figure 1 – Supplement 1 A) to present the different subunits in the structures with different shades of blue and grey.

Furthermore, we have clarified the distance measurement approach in the methods section and in the legend of Fig 1D: “Ca-Ca distances were determined using the measuring function in Pymol v.2.5.2 (Schrodinger LLC).”

__Re: Figure 1 - Supplement 1, __

Panel A. What scientific information are we getting from this picture?

This panel was just a visual representation of the complexity of the protein network we obtained. Indeed, there was no specific scientific message, so we have decided to remove this panel from the revised manuscript.

Panel B. Why are these complexes shown separately from the complexes in Figure 1, panel C? Also, can individual proteins be colored differently here as well?

We did not want to overload Fig 1C, so we decided to show some of the protein complexes in Fig 1 – Supplement 1B. The most important information is the histogram showing that 95% of the mapped cross-links fall within the expected length range, and this is shown in the main Figure (Figure 1D). As stated above, we have adjusted the subunit coloring in Figure 1 C to improve clarity.

Re: Figure 3. It will be nice to show the localization of the untagged protein as well if antibodies are available (OPTIONAL).

Unfortunately, there are no available antibodies for either Rtc5 or Oxr1. This hinders us from detecting the endogenous untagged proteins. We would like to point out that we have been very careful in showing which tagged proteins are functional (C-terminally tagged Rtc5) and which are not (C-terminally tagged Oxr1), so that the reader can know how to interpret the localization data.

Re: Figure 4. Why different tags were used in panels A (GFP), C (msGFP2) and D

(mNeonGreen)?

In general, we prefer to use mNeonGreen as a tag for microscopy experiments because it is brighter and more stable, and msGFP2 as a tag for experiments involving Western blots because we have better antibodies available. There was a mistake in the labeling, and actually, all constructs labeled as GFP were msGFP2. We have now corrected this. Of note, we have tested the functionality of both tagged version (mNeonGreen and msGFP2).

Panels B and C. Were Rtc5 fusions detected using anti-GFP antibodies?

Indeed, Rtc5-msGFP2 was detected with an anti-GFP antibody. We have now indicated next to each Western blot membrane the primary antibody used. In addition, all antibodies are detailed in Supplemental Figure 3.

The authors should have full-size Western blots available, not just cut-out bands, as some journals and reviewers require them for publication.

For all western blots, we always showed a good portion of the membrane and not cut-out bands. The cropping was performed to avoid making figures unnecessarily large. The whole membranes are of course available and will be included in an “extended data file” if required by the journal.

Re: Figure 4 - Supplement 1, Panel A. Does "-" and "+" mean -/+ Azido-Myr?

Indeed. We have now added this label to the figure.

Panel B. There is no blot with a membrane protein marker (Vam3 or Vac8), it should be included.

We have replaced this western blot for a different repetition of this experiment in which a membrane protein marker was included. Of note, the two other repetitions of the experiment shown (Figure 4 – Supplement 1 panel C and Figure 4 panel C) also include both a membrane protein marker and a soluble protein marker.

Re: Figure 5. The title does not describe all results in this figure and should be modified accordingly.

The original data from Figure 5 is now separated into Figures 5 and 6 because of the additional experiments included during revisions. We have modified the Figure titles to be descriptive of the overall message of the Figures.

Panel C. Statistical significance value for *** should be indicated in the legend.

This has been indicated in the Figure legend.

It is not clear how many times yeast growth assays were repeated. Usually, all experiments should be done in triplicates or more.

All shown growth tests were performed at least three times for the conditions shown. We have now indicated this in the materials and methods section. In addition, we now provide in this response a file with all repetitions of growth tests, which will be appended to the article if deemed necessary by the editors.

Re: Figure 5 - supplement 1. No title

Re: Figure 5 - supplement 2. No title

Whether the supplemental Figures should have a title or not will depend on the style of the journal where the manuscript is finally published. The current idea of the supplemental Figures is that they complement the corresponding main Figure. For this reason, we have removed all titles from supplemental Figures.

Re: Figure 6. There is a typo on the second lane in the legend: "...the genome were", not "...the genome where".

This has been corrected.

Panel C. Why the analysis of BCECF vacuole staining of double mutants oxr1Δrav1Δ and rtc5Δrav1Δ is not shown? Was it done at all?

We had not included this piece of data, as we thought that the genetic interaction of RTC5 and OXR1 and rav1Δ was sufficiently well supported with the included data (growth tests in combination with the deletion, growth tests in combination with the overexpression, vacuole proteomics in combination with overexpression, and BCECF staining in combination with the overexpression). Because of the request of the reviewer, we have now included this experiment as Figure 7 G.

Re: Figure 6 - Supplement 2. Why were two different tags (2xmNG and msGFP2) used?

We tried both tags to see if one of them would be functional. Unfortunately, they both resulted in non-functional proteins, as shown by the corresponding growth tests.

Did the authors study N-terminally tagged Oxr1? Was it functional?

We have tagged Oxr1 N-terminally, and this unfortunately resulted in a protein that was not completely functional. We show below the localization of N-terminally mNeon-tagged Oxr1, under the control of the TEF1 promoter. The protein appears cytosolic (Panel A) but is not completely functional (Panel B). The localization of Oxr1 had already been misreported by using a tagged version that we now show to be non-functional. For this reason, we preferred not to include this data in the manuscript, to avoid again including in the literature subcellular localizations that correspond to non-functional or partially functional proteins.

Panel B. Results for the untagged TEF1pr-Oxr1 overexpression are not shown, thus tagged and untagged proteins can't be compared. Are they available? What is the promoter for the expression of 2xmNG fusion constructs?

Oxr1-2xmNG was C-terminally tagged in the genome, which means that the promoter is the endogenous one, it was not modified. For this reason, the correct controls are a strain expressing Oxr1 at endogenous levels (the wt strain) and a strain lacking Oxr1. Both controls were included in the Figure, and in all repetitions made of this experiment. For reference, all the genotypes of the strains used are found in Supplemental Table 1.

Re: Methods. Were vacuoles prepared differently for XL-MS and SILAC-based vacuole proteomics (there are different references) and why? Methods for XL-MS and quantitative SILAC-based proteomics can be placed together for clarity.

The basis for the method of vacuole purification is the same, from (Haas, 1995). This reference was included in both protocols that include vacuole purifications. However, modifications of this method were performed to fit the crosslinking method (higher pH, no primary amines) or to fit the SILAC labeling (combination of two differentially labeled samples in one purification). The reference for the vacuole proteomics (Eising et al 2022) corresponds to a paper in which the SILAC-based comparison of vacuoles from different mutant strains was optimized, and includes not only the vacuole purification but the growth conditions and downstream processing of the vacuoles.

Since both the SILAC-based vacuole proteomics and the XL-MS are multi-step methods, containing numerous parameters including the sample preparation, processing for MS, MS run and data analysis, we would prefer to keep them separate. We think this would allow a person attempting to reproduce these methods to go through them step by step.

What is CMAC dye? Why was it used to stain the vacuolar lumen?

We apologize for this oversight, we have included the definition of CMAC as 7-Amino-4-Chlormethylcumarin. It is a standard-used organelle marker for the lumen of the vacuole.

Some abbreviations (TEAB, ACN) are not explained.

We apologize for this oversight. We have now replaced these abbreviations with the full names of the compounds in the article.

What is 0% Ficoll?

We used the term 0% Ficoll, because this is the name given to the buffer in the original Haas 1995 paper on vacuole purifications. However, we agree that the term is misleading and we have now added the composition of the buffer (10 mM PIPES/KOH pH=6.8, 0.2 M Sorbitol).

Reviewer #3 (Significance (Required)):

The vacuolar-type proton ATPase, V-ATPase, is the key proton pump, that hydrolases ATP and uses this energy to pump protons across membranes. Amazingly, this proton pump and its function are conserved in eukaryotes from yeast to mammals. While V-ATPase structure and function have been studied for more than 30 years in various organisms, its regulation is not completely understood. The very recent discoveries of two new V-ATPase interacting proteins in yeast, first Oxr1 (OXidative Resistance 1), and now Rtc5 (Restriction of Telomere Capping 5), both the only two members of TLDc (The Tre2/Bub2/Cdc16 (TBC), lysin motif (LysM), domain catalytic) proteins in yeast, provide new insights in V-ATPase regulation in yeast, and because the interaction is conserved in mammals its relevance to mammalian V-ATPases regulation as well.

TLDc proteins are best known for their role in protection from oxidative stress, in particular in yeast and in the nervous system in mammals. The discovery of the novel Rtc5-V-ATPase interaction points to the role of V-ATPase not only in protection from oxidative stress but also in restriction of telomere capping in yeast and most likely higher species. The studies of other species also highlight the possible conserved role of V-ATPase in lifespan determination and Torc1 signaling, mediated through these interactions. Thus, the discovery of this new functionally important interaction between the second TLDc family member in yeast, Rtc5, and V-ATPase will shed light on the molecular mechanisms of all these essential biological processes and pathways.

In addition, because the authors performed a comprehensive proteomics protein-protein interaction study of the purified yeast vacuole it provides a valuable resource for all researchers who study vacuoles and/or related to them lysosomes.

The follow-up functional studies using the rav1Δ strain clearly demonstrated that Rtc5 and Oxr1 disassemble V-ATPase and counteract the function of V-ATPase assembly RAVE complex in vivo in yeast. Thus, they are essentially the first discovered endogenous eukaryotic protein inhibitors of V-ATPase. Moreover, because the authors obtained the evidence that Oxr1 is the regulator of the specific subunit isoform of V-ATPase Stv1p in vivo in yeast, it suggests that different TLDc proteins may regulate different specific V-ATPase subunit isoforms in cell- and tissue-specific manner in higher eukaryotes. The mechanism of this isoform-specific regulation in yeast and other species needs further investigation in the future.

Because of the conservation of the TLDc-V-ATPase interactions, all this information can be extrapolated to higher species, all the way to humans, in whom genetic mutations in various TLDc proteins are known to cause devastating diseases and syndromes.

We are thankful to the reviewer for their positive comments about the significance of our work.

I remember listening to my cousins talk about having to input codes to talk to friends and that it was normal to learn multiple different coding languages to operate a computer. I grateful that in the 2000s we were not required to learn code as a gen z but this will always intrigue me as an individual

Composition Abstraction Hide data Expose behavior

During lab 2, I found myself wondering, "How do they expect us to accomplish this task without loops?" Loops and lists make everything in programming easier. I remember programming at North Seattle College, and Bill Barry would get mad at me for Googling something and adding it to my code. It resulted in a small deduction, but it made me realize how many lines of code lists and loops could reduce, rather than using another method that might take longer. For example, instead of using print() or display() for multiple posts before display() -> and then sleep() several times, you could use a loop to write 7 different posts and send them to Reddit, reducing 14 lines of code to 7 or fewer. Personally, I struggle with loop syntax compared to arrays and lists, and even something like file I/O. That's one reason why I decided to take this class and hopefully learn more about informatics for my next legendary essay, which hopefully doesn't get denied.

I don’t dislike Bill at all if that’s what you got from this. He was one of the most challenging professors but also rewarding. He limited which code we could use in a project, giving us tasks that would be easier with a list, array, or loop, and then emphasized the need for conciseness to reduce the number of bugs. He taught the class as if we were Microsoft employees, solving problems with a strict class style guide. One difference between his class and classes at the University of Washington is that some lines of code displayed on class slides are confusing to me because they have multiple lines that almost look identical to something you'd write in Java, when realistically in Python, you could write all of that in just a single line.

Googling introduced me to many different codes, and there are numerous ways to solve a single problem when programming. Often, there is code that isn’t pretty to look at and not as easy to read, which I think Bill wanted his students to avoid, given his background as a former Microsoft employee.

The explanation of lists and for loops in the context of social media data is highly effective and relevant. Using familiar scenarios, like handling lists of users or posts, makes the concepts of lists and iteration in Python accessible and practical. This approach not only teaches fundamental programming concepts but also illustrates their direct application in real-world scenarios, bridging the gap between theoretical learning and practical usage. It's a great example of how programming can be taught in an engaging and contextually meaningful way.

This is an excellent resource for learing about Git integration with VS Code

又發現Hypothes.is的Dan的創舉，改天來測試。

Drag and drop a document to annotate it,

or paste a YouTube video URL.

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The annotation functionality is enabled by Hypothes.is. The code for this site is open source.

This is a personal project to explore different ideas and is maintained by Dan Whaley. I’d be delighted to hear any feedback at @dwhly.

Der Code verbindet das Leben miteinander. Er ist nur eine von vielen Möglichkeiten durch die alles Lebendige miteinander verwandt ist.

Durch den genetischen Code ist Information in die Natur eingebaut. Das kann man weiterdenken in die Richtung der Koevolution.

Author Response

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

Reviewer No.1 (public)

The authors present a study focused on addressing the key challenge in drug discovery, which is the optimization of absorption and affinity properties of small molecules through in silico methods. They propose active learning as a strategy for optimizing these properties and describe the development of two novel active learning batch selection methods. The methods are tested on various public datasets with different optimization goals and sizes, and new affinity datasets are curated to provide up-todate experimental information. The authors claim that their active learning methods outperform existing batch selection methods, potentially reducing the number of experiments required to achieve the same model performance. They also emphasize the general applicability of their methods, including compatibility with popular packages like DeepChem.

Strengths:

Relevance and Importance: The study addresses a significant challenge in the field of drug discovery, highlighting the importance of optimizing the absorption and affinity properties of small molecules through in silico methods. This topic is of great interest to researchers and pharmaceutical industries.

Novelty: The development of two novel active learning batch selection methods is a commendable contribution. The study also adds value by curating new affinity datasets that provide chronological information on state-of-the-art experimental strategies.

Comprehensive Evaluation: Testing the proposed methods on multiple public datasets with varying optimization goals and sizes enhances the credibility and generalizability of the findings. The focus on comparing the performance of the new methods against existing batch selection methods further strengthens the evaluation.

Weaknesses:

Lack of Technical Details: The feedback lacks specific technical details regarding the developed active learning batch selection methods. Information such as the underlying algorithms, implementation specifics, and key design choices should be provided to enable readers to understand and evaluate the methods thoroughly.

Evaluation Metrics: The feedback does not mention the specific evaluation metrics used to assess the performance of the proposed methods. The authors should clarify the criteria employed to compare their methods against existing batch selection methods and demonstrate the statistical significance of the observed improvements.

Reproducibility: While the authors claim that their methods can be used with any package, including DeepChem, no mention is made of providing the necessary code or resources to reproduce the experiments. Including code repositories or detailed instructions would enhance the reproducibility and practical utility of the study.

Suggestion 1:

Elaborate on the Methodology: Provide an in-depth explanation of the two active learning batch selection methods, including algorithmic details, implementation considerations, and any specific assumptions made. This will enable readers to better comprehend and evaluate the proposed techniques.

Answer: We thank the reviewer for this suggestion. Following this comments we have extended the text in Methods (in Section: Batch selection via determinant maximization and Section: Approximation of the posterior distribution) and in Supporting Methods (Section: Toy example). We have also included the pseudo code for the Batch optimization method.

Suggestion 2:

Clarify Evaluation Metrics: Clearly specify the evaluation metrics employed in the study to measure the performance of the active learning methods. Additionally, conduct statistical tests to establish the significance of the improvements observed over existing batch selection methods.

Answer: Following this comment we added to Table 1 details about the way we computed the cutoff times for the different methods. We also provide more details on the statistics we performed to determine the significance of these differences.

Suggestion 3:

Enhance Reproducibility: To facilitate the reproducibility of the study, consider sharing the code, data, and resources necessary for readers to replicate the experiments. This will allow researchers in the field to validate and build upon your work more effectively.

Answer: This is something we already included with the original submission. The code is publicly available. In fact, we provide a phyton library, ALIEN (Active Learning in data Exploration) which is published on the Sanofi Github(https://github.com/ Sanofi-Public/Alien). We also provide details on the public data used and expect to provide the internal data as well. We included a small paragraph on code and data availability.

Reviewer No.2 (public)

Suggestion 1:

The authors presented a well-written manuscript describing the comparison of activelearning methods with state-of-art methods for several datasets of pharmaceutical interest. This is a very important topic since active learning is similar to a cyclic drug design campaign such as testing compounds followed by designing new ones which could be used to further tests and a new design cycle and so on. The experimental design is comprehensive and adequate for proposed comparisons. However, I would expect to see a comparison regarding other regression metrics and considering the applicability domain of models which are two essential topics for the drug design modelers community.

Answer: We want to thank the reviewer for these comments. We provide a detailed response to the specific comments below. 

Reviewer No.1 (Recommendations For The Authors)

Recommendation 1:

The description provided regarding the data collection process and the benchmark datasets used in the study raises some concerns. The comment specifically addresses the use of both private (Sanofi-owned) and public datasets to benchmark the various batch selection methods. Lack of Transparency: The comment lacks transparency regarding the specific sources and origins of the private datasets. It would be crucial to disclose whether these datasets were obtained from external sources or if they were generated internally within Sanofi. Without this information, it becomes difficult to assess the potential biases or conflicts of interest associated with the data.

Answer: We would like to thank the reviewer for this comment. As mentioned in the paper, the public github page contains links to all the public data and we expect also to the internal Sanofi data. We also now provide more information on the specific experiments that were internally done by Sanofi to collect that data.

Potential Data Accessibility Issues: The utilization of private datasets, particularly those owned by Sanofi, may raise concerns about data accessibility. The lack of availability of these datasets to the wider scientific community may limit the ability of other researchers to replicate and validate the study’s findings. It is essential to ensure that the data used in research is openly accessible to foster transparency and encourage collaboration.

Answer: Again, as stated above we expect to release the data collected internally on the github page.

Limited Information on Dataset Properties: The comment briefly mentions that the benchmark datasets cover properties related to absorption, distribution, pharmacokinetic processes, and affinity of small drug molecules to target proteins. However, it does not provide any specific details about the properties included in the datasets or how they were curated. Providing more comprehensive information about the properties covered and the methods used for curation would enhance the transparency and reliability of the study.

To address these concerns, it is crucial for the authors to provide more detailed information about the data sources, dataset composition, representativeness, and curation methods employed. Transparency and accessibility of data are fundamental principles in scientific research, and addressing these issues will strengthen the credibility and impact of the study.

Answer: We agree with this comment and believe that it is important to be explicit about each of the datasets and to provide information on the new data. We note that we already discuss the details of each of the experiments in Methods and, of course, provide links to the original papers for the public data. We have now added text to Supporting Methods that describes the experiments in more details as well as providing literature references for the experimental protocols used. As noted above, we expect to provide our new internal data on the public git page. 

Recommendation 2:

Some comments on the modeling example Approximation of the posterior distribution. Lack of Methodological Transparency: The comment fails to provide any information regarding the specific method or approach used for approximating the posterior distribution. Without understanding the methodology employed, it is impossible to evaluate the quality or rigor of the approximation. This lack of transparency undermines the credibility of the study.

Answer: We want to thank the reviewer for pointing this out. Based on this comment we added more information to Section: Approximation of the posterior distribution. Moreover, we now provide details on the posterior approximation in Section: Two approximations for computing the epistemic covariance.

Questionable Assumptions: The comment does not mention any of the assumptions made during the approximation process. The validity of any approximation heavily depends on the underlying assumptions, and their omission suggests a lack of thorough analysis. Failing to acknowledge these assumptions leaves room for doubt regarding the accuracy and relevance of the approximation.

Answer: We are not entirely sure which assumptions the reviewer is referring to here. The main assumption we can think of that we have used is the fact that getting within X% of the optimal model is a good enough approximation. We have specifically discussed this assumption and tested multiple values of X. While it would have been great to have X = 0 this is unrealistic for retrospective studies. For Active Learning the main question is how many experiments can be saved to obtain similar results and the assumptions we used are basically ’what is the definition of similar’. We now added this to Discussion.

Inadequate Validation: There is no mention of any validation measures or techniques used to assess the accuracy and reliability of the approximated posterior distribution. Without proper validation, it is impossible to determine whether the approximation provides a reasonable representation of the true posterior. The absence of validation raises concerns about the potential biases or errors introduced by the approximation process.

Answer: We sincerely appreciate your concern regarding the validation of the approximated posterior distribution. We acknowledge that our initial submission might not have clearly highlighted our validation strategy. It is, of course, very hard to determine the accuracy of the distribution our model learns since such distribution cannot be directly inferred using experiments (no ’ground truth’). Instead, we use an indirect method to determine the accuracy. Specifically, we conducted retrospective experiment using the learned distribution. In these experiments, we indirectly validated our approximation by measuring the error with the respective method. The results from these retrospective experiments provided evidence for the accuracy and reliability of our approximation in representing the true posterior distribution. We now emphasize this in Methods.

Uncertainty Quantification: The comment does not discuss the quantification of uncertainty associated with the approximated posterior distribution. Properly characterizing the uncertainty is crucial in statistical inference and decision-making. Neglecting this aspect undermines the usefulness and applicability of the approximation results.

Answer: Thank you for pointing out the importance of characterizing uncertainty in statistical inference and decision-making, a sentiment with which we wholeheartedly agree. In our work, we have indeed addressed the quantification of uncertainty associated with the approximated posterior distribution. Specifically, we utilized Monte Carlo Dropout (MC Dropout) as our method of choice. MC Dropout is a widely recognized and employed technique in the neural networks domain to approximate the posterior distribution, and it offers an efficient way to estimate model uncertainty without requiring any changes to the existing network architecture [1, 2]. In the revised version, we provide a more detailed discussion on the use of Monte Carlo Dropout in our methodology and its implications for characterizing uncertainty.

Comparison with Gold Standard: There is no mention of comparing the approximated posterior distribution with a gold standard or benchmark. Failing to provide such a comparison leaves doubts about the performance and accuracy of the approximation method. A lack of benchmarking makes it difficult to ascertain the superiority or inferiority of the approximation technique employed.

Answer: As noted above, it is impossible to find gold standard information for the uncertainly distribution. It is not even clear to us how such gold standard can be experimentally determined since its a function of a specific model and data. If the reviewer is aware of such gold standard we would be happy to test it. Instead, in our study, we opted to benchmark our results against state-of-the-art batch active learning methods, which also rely on uncertainty prediction (such uncertainty prediction is the heart of any active learning method as we discuss). Results clearly indicate that our method outperforms prior methods though we agree that this is only an indirect way to validate the uncertainty approximation.

Reviewer No.2 (Recommendations For The Authors)

Recommendation 1:

The text is kind of messy: there are two results sections, for example. It seems that part of the text was duplicated. Please correct it.

Answer: We want to thank the reviewer pointing this out. These were typos and we fixed them accordingly.

Recommendation 2:

Text in figures is very small and difficult to read. Please redraw the figures, increasing the font size: 10-12pt is ideal in comparison with the main text.

Answer: We want to thank the reviewer for this comment and we have made the graphics larger.

Recommendation 3: Please, include specific links to data availability instead of just stating it is available at the Sanofi-Public repository.

Answer: We want to thank the reviewer for this comment and added the links and data to the Sanofi Github page listed in the paper.

Recommendation 4:

What are the descriptors used to train the models?

Answer: We represented the molecules as molecular graphs using the MolGraphConvFeaturizer from the DeepChem library. We now explicitly mention this in Methods.

Recommendation 5:

Regarding the quality of the models, I strongly suggest two approaches instead of using only RMSE as metrics of models’ performance. I recommend using the most metrics as possible as reported by Gramatica (https://doi.org/10.1021/acs.jcim.6b00088). I also recommend somehow comparing the increment on the dataset diversity according to the employed descriptors (applicability domain) as a measurement to further applications on the unseen molecules.

Answer: We want to thank the reviewer for this great suggestions. As suggested we added new comparison metrics to the Supplement.

• Distribution plot for the range of the Y values Figure 8 • Clustering of the data sets represented as fingerprints Supplementary material Figure 5,6

• Retrospective experiments with Spearman correlation coefficient. Supplementary material Figure: 2,3,4

I suggest also a better characterization of datasets including the nature and range of the Y variable, the source of data in terms of experimentation, and chemical (structural and physicochemical) comparison of samples within each dataset.

Answer: As noted above in response to a similar comment by Reviewer 1, we have added more detailed information about the different experiments we tested to Supporting Methods.

References

[1] Yarin Gal and Zoubin Ghahramani. Dropout as a bayesian approximation: Representing model uncertainty in deep learning. In Maria Florina Balcan and Kilian Q. Weinberger, editors, Proceedings of The 33rd International Conference on Machine Learning, volume 48 of Proceedings of Machine Learning Research, pages 1050–1059, New York, New York, USA, 20–22 Jun 2016. PMLR.

[2] N.D. Lawrence. Variational Inference in Probabilistic Models. University of Cambridge, 2001.

Reviewer #1 (Public Review):

The authors present a study focused on addressing the key challenge in drug discovery, which is the optimization of absorption and affinity properties of small molecules through in silico methods. They propose active learning as a strategy for optimizing these properties and describe the development of two novel active learning batch selection methods. The methods are tested on various public datasets with different optimization goals and sizes, and new affinity datasets are curated to provide up-to-date experimental information. The authors claim that their active learning methods outperform existing batch selection methods, potentially reducing the number of experiments required to achieve the same model performance. They also emphasize the general applicability of their methods, including compatibility with popular packages like DeepChem.

Strengths:

Relevance and Importance: The study addresses a significant challenge in the field of drug discovery, highlighting the importance of optimizing absorption and affinity properties of small molecules through in silico methods. This topic is of great interest to researchers and pharmaceutical industries.

Novelty: The development of two novel active learning batch selection methods is a commendable contribution. The study also adds value by curating new affinity datasets that provide chronological information on state-of-the-art experimental strategies.<br /> Comprehensive Evaluation: Testing the proposed methods on multiple public datasets with varying optimization goals and sizes enhances the credibility and generalizability of the findings. The focus on comparing the performance of the new methods against existing batch selection methods further strengthens the evaluation.

Weaknesses:

Lack of Technical Details: The feedback lacks specific technical details regarding the developed active learning batch selection methods. Information such as the underlying algorithms, implementation specifics, and key design choices should be provided to enable readers to understand and evaluate the methods thoroughly.

Evaluation Metrics: The feedback does not mention the specific evaluation metrics used to assess the performance of the proposed methods. The authors should clarify the criteria employed to compare their methods against existing batch selection methods and demonstrate the statistical significance of the observed improvements.

Reproducibility: While the authors claim that their methods can be used with any package, including DeepChem, no mention is made of providing the necessary code or resources to reproduce the experiments. Including code repositories or detailed instructions would enhance the reproducibility and practical utility of the study.

Suggestions for Improvement:

Elaborate on the Methodology: Provide an in-depth explanation of the two active learning batch selection methods, including algorithmic details, implementation considerations, and any specific assumptions made. This will enable readers to better comprehend and evaluate the proposed techniques.

Clarify Evaluation Metrics: Clearly specify the evaluation metrics employed in the study to measure the performance of the active learning methods. Additionally, conduct statistical tests to establish the significance of the improvements observed over existing batch selection methods.

Enhance Reproducibility: To facilitate the reproducibility of the study, consider sharing the code, data, and resources necessary for readers to replicate the experiments. This will allow researchers in the field to validate and build upon your work more effectively.

Conclusion:<br /> The authors' study on active learning methods for optimizing drug discovery presents an important and relevant contribution to the field. The proposed batch selection methods and curated affinity datasets hold promise for improving the efficiency of drug discovery processes. However, to strengthen the study, it is crucial to provide more technical details, clarify evaluation metrics, and enhance reproducibility by sharing code and resources. Addressing these limitations will further enhance the value and impact of the research.

test

to https://hypothes.is/a/q8fLhrUDEe6e-jsrTj9ybQ

from - https://hyp.is/HysQdrUCEe6HgDNGpn7Q4w/gist.github.com/neodigm/ffa4a9e22a5da14e2472274d476eaa55

• performant,
• functional,
• testable,
• future-proof, and
• trusted

what? - JavaScript, TypeScript, Vue, and SolidJS.

I've enchanted the UX for Abbott Laboratories, AbbVie, CDW, Corporate Express, Hyundai, Microsoft, and Sears.

One way to solve this problem of the apples having a difference in size we could measure their dimensions, taking into consideration the apple's circumference, and diameter, or even weighing the apple. However, for simplicity, we can establish a standard size for granny smith apples given out during snack time to weigh 8 ounces per apple. This standardized approach ensures that each person receives about the same amount of apple per bite.

We could connect this to programming using the min() and man() functions and use each of the apple's weights and assign them a number based on the measurement.

apples = [8, 8, 9, 8, 7]

maximum = max(apples) print(maximum)

minimum = min(apples) print(minimum)

filtered = [apple for apple in apples if apple == 8] print(filtered)

A code that finds the min and the max apples in size and then filters them out if it doesn’t equal 8 ounces. This code should filter out and help the teachers know which apples to keep or throw out, or if you want to fool a kid just grab an apple of about the same size and cut it into smaller pieces problem solved.

This is especially important once you begin to compile your data onto a single sheet. I remember in java how we had to simplify our work down to debug each strand of code under each variable. This apple metaphor makes sense to me because of this experience.

The metrics LinkedIn tracks.

is this true though? HF is making transformer access a standard. plus popularity of claude and gpt suggests people cannot be bothered to install stuff

you have only plotted the monthly average temperature (t_monthly). It is nice that you used the same color scale for every plot (which helps to compare the months), however, * a colorbar legend would have been nice * another additional plot would have been great that directly shows max_temp -min_temp.

Example code for that would be:

t2_cycle = ds.t2m.groupby('time.month').mean() - 273.15 ax = plt.axes(projection=ccrs.Robinson()) (t2_cycle.max(dim='month') - t2_cycle.min(dim='month')).plot(ax=ax, transform=ccrs.PlateCarree(), cbar_kwargs={'label':'$\overline{T}$ [K]'}) ax.coastlines(); ax.gridlines();

Certainly! Let's break down the provided code and example in simpler terms:

Using Multer for a Text-Only Multipart Form:

1. HTML Form:
2. If you're dealing with a text-only multipart form, where you don't have file uploads but only text fields, you should use upload.none().

```html <br /> <form action="/profile" enctype="multipart/form-data" method="post"> <div class="form-group"> <input type="file" class="form-control-file" name="uploaded_file"> <input type="text" class="form-control" placeholder="Number of speakers" name="nspeakers"> <input type="submit" value="Get me the stats!" class="btn btn-default"> </div> </form>

```

• Note the enctype="multipart/form-data" in the form, even though there is no file input. This is required for forms that might include file inputs.

• Server-Side (Using Express and Multer):

• In your Node.js server code:

```javascript const express = require('express'); const multer = require('multer'); const upload = multer();

const app = express();

// Handling a text-only multipart form app.post('/profile', upload.none(), function (req, res, next) { // req.body contains the text fields console.log(req.body); });

// Handling a text and file multipart form const fileUpload = multer({ dest: './public/data/uploads/' }); app.post('/stats', fileUpload.single('uploaded_file'), function (req, res) { // req.file is the name of your file in the form, here 'uploaded_file' // req.body will hold the text fields, if any console.log(req.file, req.body); });

app.listen(3000, () => { console.log('Server is running on port 3000'); }); ```

Summary:

• The HTML form includes both a file input (<input type="file" name="uploaded_file">) and a text input (<input type="text" name="nspeakers">).
• Even if there's no file input, using enctype="multipart/form-data" is necessary when the form might include file inputs.
• On the server side, upload.none() middleware is used for handling text-only multipart forms.
• If there is a mix of text and file inputs, you can use multer with fileUpload.single('uploaded_file') to handle both files and text fields.
• req.file will contain the uploaded file, and req.body will hold the text fields.

Certainly! Let's break down the res.json() method in simple terms:

Purpose:

The res.json() method in Express is used to send a JSON response to the client. It automatically sets the appropriate content-type header and converts the provided data into a JSON string using JSON.stringify().

Syntax:

javascript res.json([body]);

Examples:

1. Sending null as JSON:

2. This example sends a JSON response with null as the content. javascript res.json(null);

3. Sending an Object as JSON:

4. This example sends a JSON response with an object. javascript res.json({ user: 'tobi' });

5. Sending JSON with a Status Code:

6. You can also set an HTTP status code along with the JSON response. javascript res.status(500).json({ error: 'message' }); This example sends a JSON response with an error message and sets the HTTP status code to 500 (Internal Server Error).

Summary:

• res.json() is a convenient method in Express to send JSON responses.
• It automatically handles the conversion of various data types (object, array, string, Boolean, number, or null) to a JSON string.
• You can also chain it with res.status() to set an HTTP status code along with the JSON response.

In simple terms, it's a way to tell the client that the server is responding with data in JSON format, making it easy for client applications to consume the data.

verification code thing not working

Sure, let's break it down in simpler terms.

In Mongoose, a "Schema" is like a blueprint for how your data should be organized in a MongoDB database. Imagine it as a template for creating documents (records) in a collection (similar to a table in relational databases).

Let's go through the example:

```javascript import mongoose from 'mongoose'; const { Schema } = mongoose;

// Define a schema for a blog post const blogSchema = new Schema({ title: String, author: String, body: String, comments: [{ body: String, date: Date }], date: { type: Date, default: Date.now }, hidden: Boolean, meta: { votes: Number, favs: Number } }); ```

Now, let's break it down:

1. Basic Data Types:

2. title, author, body, and hidden are properties of your blog document. String is the data type for title, author, and body, while Boolean is the data type for hidden.

3. Array of Comments:

4. comments is an array where each element is an object with body (String) and date (Date) properties. This allows you to store multiple comments in an array within your document.

5. Default Date:

6. date is a property with the type Date, and it has a default value of the current date and time (Date.now). This means if you don't provide a date when creating a blog post, it will default to the current date and time.

7. Nested Meta Object:

8. meta is a nested object within your document. It has two properties, votes (Number) and favs (Number). This allows you to store additional information in a structured way.

Now, let's say you want to add a new property later, you can use the add method:

javascript // Add a new property to the schema blogSchema.add({ tags: [String] });

Here, we're adding a new property tags which is an array of strings.

The main takeaway is that the schema defines the structure of your documents in MongoDB, including the types of data each property should have. It helps maintain consistency in your data and allows you to enforce certain rules or defaults.

Certainly! Let's break down the provided code snippets:

1. What is it and why is it used?

In Mongoose, a schema is a blueprint for defining the structure of documents within a collection. When you define a schema, you can also attach methods to it. These methods become instance methods, meaning they are available on the individual documents (instances) created from that schema.

Instance methods are useful for encapsulating functionality related to a specific document or model instance. They allow you to define custom behavior that can be executed on a specific document. In the given example, the findSimilarTypes method is added to instances of the Animal model, making it easy to find other animals of the same type.

2. Syntax:

Using methods object directly in the schema options:

javascript const animalSchema = new Schema( { name: String, type: String }, { methods: { findSimilarTypes(cb) { return mongoose.model('Animal').find({ type: this.type }, cb); } } } );

Using methods object directly in the schema:

javascript animalSchema.methods.findSimilarTypes = function(cb) { return mongoose.model('Animal').find({ type: this.type }, cb); };

Using Schema.method() helper:

javascript animalSchema.method('findSimilarTypes', function(cb) { return mongoose.model('Animal').find({ type: this.type }, cb); });

3. Explanation in Simple Words with Examples:

Why it's Used:

Imagine you have a collection of animals in your database, and you want to find other animals of the same type. Instead of writing the same logic repeatedly, you can define a method that can be called on each animal instance to find similar types. This helps in keeping your code DRY (Don't Repeat Yourself) and makes it easier to maintain.

Example:

```javascript const mongoose = require('mongoose'); const { Schema } = mongoose;

// Define a schema with a custom instance method const animalSchema = new Schema({ name: String, type: String });

// Add a custom instance method to find similar types animalSchema.methods.findSimilarTypes = function(cb) { return mongoose.model('Animal').find({ type: this.type }, cb); };

// Create the Animal model using the schema const Animal = mongoose.model('Animal', animalSchema);

// Create an instance of Animal const dog = new Animal({ type: 'dog', name: 'Buddy' });

// Use the custom method to find similar types dog.findSimilarTypes((err, similarAnimals) => { console.log(similarAnimals); }); ```

In this example, findSimilarTypes is a custom instance method added to the Animal schema. When you create an instance of the Animal model (e.g., a dog), you can then call findSimilarTypes on that instance to find other animals with the same type. The method uses the this.type property, which refers to the type of the current animal instance. This allows you to easily reuse the logic for finding similar types across different instances of the Animal model.

Let's break down the concept of third-party middleware in Express with a simple explanation and an example.

What is Third-Party Middleware?

Third-party middleware in Express refers to middleware functions that are not built into Express itself but are created by external developers to extend its functionality. These middleware functions can be added to your Express application to provide additional features or handle specific tasks.

Example Using cookie-parser:

1. Install the Module:
2. Use npm to install the cookie-parser module.

bash $ npm install cookie-parser

1. Load and Use the Middleware:
2. In your Express application, require and load the cookie-parser middleware.

```javascript const express = require('express'); const app = express(); const cookieParser = require('cookie-parser');

// Load the cookie-parsing middleware app.use(cookieParser()); ```

• This middleware is now integrated into your Express application.

• Use the Middleware in Your Routes:

• Now, you can use the functionality provided by cookie-parser in your routes.

```javascript app.get('/', (req, res) => { // Access cookies using the middleware const userCookie = req.cookies.user;

   // Your route logic here
   res.send(`Welcome, ${userCookie || 'Guest'}!`);

}); ```

• In this example, cookie-parser allows you to access cookies in the req.cookies object.

Why Use Third-Party Middleware?

• Extended Functionality: Third-party middleware adds specialized functionality to your Express application, such as parsing cookies, handling authentication, logging, etc.

• Modularity: Using third-party middleware allows you to keep your code modular and focus on building features without reinventing the wheel for common tasks.

• Community Contributions: Many third-party middleware modules are created and maintained by the community, ensuring reliable and well-tested solutions.

In summary, third-party middleware in Express enables you to enhance your application with additional features and functionalities developed by the broader community. Always check the documentation of each middleware module for usage details and customization options.

Certainly! Let's break down the concept of router-level middleware in Express with simple explanations and examples.

1. Basic Router-Level Middleware:

• Router-level middleware works similarly to application-level middleware but is bound to an instance of express.Router().

```javascript const express = require('express'); const app = express(); const router = express.Router();

// Middleware function without a mount path, executed for every request to the router router.use((req, res, next) => { console.log('Time:', Date.now()); next(); });

app.use('/', router); // Mount the router on the app ```

In this example, the middleware function logs the current time for every request to the router.

2. Middleware Sub-Stack with Router-Level Middleware:

• Define a middleware sub-stack for a specific path within the router.

javascript router.use('/user/:id', (req, res, next) => { console.log('Request URL:', req.originalUrl); next(); }, (req, res, next) => { console.log('Request Type:', req.method); next(); });

Here, the middleware sub-stack prints request info for any type of HTTP request to the '/user/:id' path.

3. Router-Level Middleware with Different Routes:

• Define middleware sub-stacks for different routes within the router.

```javascript router.get('/user/:id', (req, res, next) => { if (req.params.id === '0') next('route'); // Skip to the next route if user ID is '0' else next(); // Pass control to the next middleware }, (req, res, next) => { res.render('regular'); // Render a regular page });

router.get('/user/:id', (req, res, next) => { res.render('special'); // Render a special page for the /user/:id path }); ```

In this example, the first middleware checks the user ID and either skips to the next route or renders a regular page. The second middleware renders a special page for the '/user/:id' path.

4. Skipping Router Middleware:

• Use next('router') to skip the rest of the router’s middleware functions and pass control back out of the router instance.

```javascript router.use((req, res, next) => { if (!req.headers['x-auth']) return next('router'); // Bail out if 'x-auth' header is missing next(); });

router.get('/user/:id', (req, res) => { res.send('hello, user!'); });

app.use('/admin', router, (req, res) => { res.sendStatus(401); // Send a 401 response for anything falling through }); ```

In this example, the router is predicated with a check, and if the 'x-auth' header is missing, it skips the rest of the router’s middleware functions.

In summary, router-level middleware in Express allows you to organize and modularize your routes and their associated middleware. It provides a way to handle middleware specific to a router instance, making your code more modular and maintainable.

Certainly! Let's break down the concepts of application-level middleware in Express with simple explanations and examples.

1. Basic Application-level Middleware:

• This middleware runs for every incoming request to your application.

```javascript const express = require('express'); const app = express();

app.use((req, res, next) => { console.log('Time:', Date.now()); next(); }); ```

In this example, every time a request is received, it logs the current time.

2. Application-level Middleware with a Mount Path:

• You can specify a path for the middleware to apply to.

javascript app.use('/user/:id', (req, res, next) => { console.log('Request Type:', req.method); next(); });

Here, the middleware only runs for requests to paths starting with '/user/:id'.

3. Route Handlers with Middleware:

• Express allows you to define route handlers for specific HTTP methods.

javascript app.get('/user/:id', (req, res, next) => { res.send('USER'); });

This handles GET requests to the '/user/:id' path and sends the response 'USER'.

4. Middleware Sub-Stack with Mount Path:

• You can create a sub-stack of middleware functions for a specific path.

javascript app.use('/user/:id', (req, res, next) => { console.log('Request URL:', req.originalUrl); next(); }, (req, res, next) => { console.log('Request Type:', req.method); next(); });

This example prints request info for any type of HTTP request to the '/user/:id' path.

5. Route Handlers with Middleware Sub-Stack:

• Define multiple middleware functions for a single route.

javascript app.get('/user/:id', (req, res, next) => { console.log('ID:', req.params.id); next(); }, (req, res, next) => { res.send('User Info'); });

In this case, the first middleware prints the user ID, and the second one sends the response 'User Info'.

6. Skipping Middleware with next('route'):

• You can skip the rest of the middleware functions using next('route') and pass control to the next route.

```javascript app.get('/user/:id', (req, res, next) => { if (req.params.id === '0') next('route'); else next(); }, (req, res, next) => { res.send('regular'); });

app.get('/user/:id', (req, res, next) => { res.send('special'); }); ```

If the user ID is '0', it skips to the next route; otherwise, it sends a regular or special response.

7. Middleware in an Array for Reusability:

• Middleware can be declared in an array for reuse.

```javascript function logOriginalUrl(req, res, next) { console.log('Request URL:', req.originalUrl); next(); }

function logMethod(req, res, next) { console.log('Request Type:', req.method); next(); }

const logStuff = [logOriginalUrl, logMethod]; app.get('/user/:id', logStuff, (req, res, next) => { res.send('User Info'); }); ```

In this example, the middleware functions logOriginalUrl and logMethod are reusable and applied to the '/user/:id' route.

In summary, application-level middleware in Express allows you to handle requests, modify them, and control their flow. You can use it for various tasks and organize your code effectively.

In Express, the sequence of middleware execution is crucial for controlling the flow of the request-response cycle. The order in which you define and use middleware determines how they are executed. Here's the general rule:

1. Application-level Middleware:
2. Middleware defined using app.use() is executed in the order it's defined in your code.

```javascript // Example of application-level middleware app.use((req, res, next) => { console.log('Middleware 1'); next(); });

app.use((req, res, next) => { console.log('Middleware 2'); next(); }); ```

In this example, "Middleware 1" will execute before "Middleware 2".

1. Router-level Middleware:
2. Similar to application-level middleware, the order of middleware in the code defines the execution order.

```javascript // Example of router-level middleware const router = express.Router();

router.use((req, res, next) => { console.log('Router Middleware 1'); next(); });

router.use((req, res, next) => { console.log('Router Middleware 2'); next(); }); ```

In this example, "Router Middleware 1" will execute before "Router Middleware 2" for routes under this router.

1. Error-handling Middleware:
2. Error-handling middleware should be defined last after all other middleware and route handlers.

javascript // Example of error-handling middleware app.use((err, req, res, next) => { console.error(err); res.status(500).send('Something went wrong!'); });

Error-handling middleware will only be executed if there is an error thrown during the request-response cycle.

1. Built-in Middleware:
2. Built-in middleware is often automatically applied in the order they are required.

javascript // Example of using built-in middleware app.use(express.json()); // Body parsing middleware app.use(express.static('public')); // Serving static files

The order in which you invoke these built-in middleware functions matters.

1. Third-party Middleware:
2. Third-party middleware is also executed in the order you apply it.

javascript // Example of using third-party middleware const passport = require('passport'); app.use(passport.initialize()); // Initialize passport middleware

The order in which you load third-party middleware influences their execution.

In summary, the sequence of middleware execution is based on the order in which you define and apply them. This allows you to control the flow of the request through the various stages of processing in your Express application. Sure, let's break down the concept of middleware in Express with simple words and examples.

1. What is Middleware?

Middleware is like a set of functions in Express that can do various tasks during the processing of a web request. These tasks can include modifying the request or response, executing some code, or controlling the flow of the request.

2. Types of Middleware:

a. Application-level Middleware:

These are applied to the entire Express application. They are executed for every incoming request.

javascript // Example of application-level middleware app.use((req, res, next) => { console.log('This runs for every request!'); next(); // Move on to the next middleware });

b. Router-level Middleware:

Similar to application-level middleware but only applies to specific routes.

```javascript // Example of router-level middleware const router = express.Router();

router.use((req, res, next) => { console.log('This runs for routes under this router!'); next(); });

router.get('/route1', (req, res) => { // Route-specific logic }); ```

c. Error-handling Middleware:

Special middleware to handle errors during the request-response cycle.

javascript // Example of error-handling middleware app.use((err, req, res, next) => { console.error(err); res.status(500).send('Something went wrong!'); });

d. Built-in Middleware:

Express has some built-in middleware for common tasks.

javascript // Example of built-in middleware (parsing JSON in the request body) app.use(express.json());

e. Third-party Middleware:

Additional middleware created by third-party developers to extend Express functionality.

javascript // Example of using third-party middleware (e.g., for handling authentication) const passport = require('passport'); app.use(passport.initialize());

3. How Middleware Works:

• Each middleware function has access to the request (req) and response (res) objects.
• They can perform tasks, modify the request or response, and decide whether to end the request-response cycle or pass control to the next middleware.
• If a middleware function doesn't end the cycle, it must call next() to pass control to the next middleware in line.

4. Middleware Loading:

• You can load middleware at the application or router level.
• Middleware can be loaded with an optional mount path, defining where it should be applied.

javascript // Example of loading middleware with a mount path app.use('/admin', adminMiddleware);

This means that adminMiddleware will only be executed for routes starting with '/admin'.

In summary, middleware in Express is like a chain of functions that can modify requests and responses. They're powerful for handling various tasks and keeping your code organized.

The Booleans is what I have learned in my CSE class too. This kind of code is to come out as true or false in a statement. I have only learned when it apply on math, but I don't know what else can this do.

WebNative

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Certainly! The code you provided demonstrates how to use the bcrypt library in Node.js to hash passwords. The examples use both the technique of generating a salt and hashing in separate steps, as well as the technique of auto-generating a salt and hashing in a single step. Let's break it down:

Technique 1: Generate a Salt and Hash on Separate Function Calls

```javascript const bcrypt = require('bcrypt'); const saltRounds = 10; const myPlaintextPassword = 's0/\/\P4$$w0rD';

// Step 1: Generate a salt bcrypt.genSalt(saltRounds, function(err, salt) { if (err) { // Handle error console.error(err); } else { // Step 2: Hash the plaintext password with the generated salt bcrypt.hash(myPlaintextPassword, salt, function(err, hash) { if (err) { // Handle error console.error(err); } else { // Step 3: Store the hash in your password database // In a real application, you would typically store 'hash' in your database. console.log('Hashed Password:', hash); } }); } }); ```

Technique 2: Auto-generate a Salt and Hash

```javascript const bcrypt = require('bcrypt'); const saltRounds = 10; const myPlaintextPassword = 's0/\/\P4$$w0rD';

// Auto-generate a salt and hash the plaintext password bcrypt.hash(myPlaintextPassword, saltRounds, function(err, hash) { if (err) { // Handle error console.error(err); } else { // Store the hash in your password database // In a real application, you would typically store 'hash' in your database. console.log('Hashed Password:', hash); } }); ```

Explanation:

1. Generate a Salt:

2. bcrypt.genSalt(saltRounds, callback): This function generates a salt to be used during hashing. The saltRounds parameter defines the cost factor of the hashing process (higher value means more secure but slower). The salt is then passed to the callback function.

3. Hashing with Generated Salt:

4. bcrypt.hash(myPlaintextPassword, salt, callback): This function hashes the plaintext password using the generated salt. The resulting hash is passed to the callback function.

5. Auto-generate Salt and Hash:

6. bcrypt.hash(myPlaintextPassword, saltRounds, callback): This function auto-generates a salt and then immediately hashes the plaintext password with that salt. The resulting hash is passed to the callback function.

Both techniques achieve the same end result: a hashed password. The auto-generate approach is more concise and is often preferred for simplicity, but it's essential to understand that either method is valid based on your application's needs. In a real-world scenario, you would typically store the resulting hash in your password database for later authentication checks.

Certainly! Let's cover the information you've requested:

1. What is bcrypt and why is it used?

• What is bcrypt? bcrypt is a library used for securely hashing passwords. Hashing is a one-way process that converts a plain-text password into a fixed-length string of characters. It's designed to be computationally intensive and slow, which makes it resistant to brute-force attacks.

• Why is it used? Storing passwords as plain text is a security risk. If a database is compromised, attackers can easily access user passwords. Hashing passwords with bcrypt adds a layer of security by making it extremely difficult and time-consuming for attackers to reverse-engineer the original passwords. It includes features like salting (adding random data to each password before hashing) to further enhance security.

2. Syntax:

• Installation: You need to install bcrypt using npm (Node Package Manager). Open your terminal and run: npm install bcrypt

• Usage in JavaScript/Node.js: ```javascript const bcrypt = require('bcrypt');

// Example: Hashing a password const plainPassword = 'mySecurePassword';

bcrypt.hash(plainPassword, 10, function(err, hash) { if (err) { console.error(err); } else { console.log('Hashed Password:', hash); } });

// Example: Comparing a password with a hashed password const hashedPasswordFromDatabase = '$2b$10$...'; // Replace with an actual hashed password

bcrypt.compare('userEnteredPassword', hashedPasswordFromDatabase, function(err, result) { if (err) { console.error(err); } else { console.log('Password Match:', result); } }); `` -bcrypt.hash(plainPassword, saltRounds, callback): Hashes theplainPasswordusing the specified number ofsaltRoundsand provides the result in thecallback. -bcrypt.compare(userEnteredPassword, hashedPasswordFromDatabase, callback): Compares a user-entered password with a hashed password retrieved from the database and provides the result in thecallback`.

3. Examples:

Example 1: Hashing a Password

```javascript const bcrypt = require('bcrypt');

const plainPassword = 'mySecurePassword';

bcrypt.hash(plainPassword, 10, function(err, hash) { if (err) { console.error(err); } else { console.log('Hashed Password:', hash); } }); ```

Example 2: Comparing a Password

```javascript const bcrypt = require('bcrypt');

const hashedPasswordFromDatabase = '$2b$10$...'; // Replace with an actual hashed password

bcrypt.compare('userEnteredPassword', hashedPasswordFromDatabase, function(err, result) { if (err) { console.error(err); } else { console.log('Password Match:', result); } }); ```

In these examples, bcrypt.hash is used to hash a password, and bcrypt.compare is used to compare a user-entered password with a hashed password retrieved from the database. The callback functions handle errors and provide the results of the operations.

Certainly! Let's break down the provided code snippets, explain the syntax, its use, and illustrate how to use them with examples.

Syntax Explanation:

Middleware Registration:

javascript schema.pre('save', function(next) { // middleware logic next(); });

• schema.pre('save', ...): This registers a middleware function that runs before the 'save' operation on a Mongoose model.
• function(next) {...}: This is the middleware function. It takes a next parameter, which is a function that you should call to proceed to the next middleware or the actual save operation.

Using Promises:

javascript schema.pre('save', function() { return doStuff() .then(() => doMoreStuff()); });

• Instead of manually calling next(), you can return a promise from the middleware. The next middleware or the save operation will proceed once the promise is resolved.

Using async/await:

javascript schema.pre('save', async function() { await doStuff(); await doMoreStuff(); });

• With Node.js versions >= 7.6.0, you can use async/await to make asynchronous code more readable. Each await pauses execution until the asynchronous operation is completed.

Early Return Pattern:

javascript schema.pre('save', function(next) { if (foo()) { console.log('calling next!'); return next(); } console.log('after next'); });

• If a condition (foo()) is met, the return next(); statement prevents the rest of the middleware function from executing.

Error Handling:

```javascript schema.pre('save', function(next) { const err = new Error('something went wrong'); next(err); });

schema.pre('save', function() { return new Promise((resolve, reject) => { reject(new Error('something went wrong')); }); });

schema.pre('save', function() { throw new Error('something went wrong'); });

schema.pre('save', async function() { throw new Error('something went wrong'); }); ```

• Different ways to handle errors in middleware, including passing an error to next, returning a rejected promise, throwing synchronous errors, and throwing errors in an async function.

Use in Simple Words with Examples:

Middleware Example:

Suppose you have a User model, and before saving a user, you want to hash their password.

```javascript const mongoose = require('mongoose'); const Schema = mongoose.Schema;

const userSchema = new Schema({ username: String, password: String, });

userSchema.pre('save', async function() { // Assuming hashPassword is an asynchronous function this.password = await hashPassword(this.password); });

const User = mongoose.model('User', userSchema);

const newUser = new User({ username: 'john_doe', password: 'my_secure_password', });

newUser.save(); // The 'save' middleware will automatically hash the password before saving. ```

Error Handling Example:

Suppose you want to validate the email format before saving a Post model.

```javascript const mongoose = require('mongoose'); const Schema = mongoose.Schema;

const postSchema = new Schema({ title: String, content: String, email: String, });

postSchema.pre('save', function(next) { const emailRegex = /\S+@\S+.\S+/; if (!emailRegex.test(this.email)) { const err = new Error('Invalid email format'); return next(err); // If the email format is invalid, the save operation will not proceed. } next(); // Proceed with the save operation if the email format is valid. });

const Post = mongoose.model('Post', postSchema);

const newPost = new Post({ title: 'Introduction to Middleware', content: 'Middleware is awesome!', email: 'invalid_email', // This will trigger the error handling in the 'save' middleware. });

newPost.save(function(err) { console.log(err.message); // Outputs: Invalid email format }); ```

In summary, Mongoose middleware allows you to execute logic before or after certain operations (e.g., saving to the database) on your models. It's a powerful tool for organizing and encapsulating your application's logic in a clean and modular way.

mercii!

I find this stance to be a form of gatekeeping, and reductive. Quality is of course important, but I find it very frustrating that as soon as something becomes associated with "academics" or "scholarship," people immediately begin limiting who can do it and why, as if severely restricting its practice will keep it "pure" or "better."

I wonder how long ago this was- there are many ways to create digitally now that don't require that skill. That or he's just being a gatekeeper.

I think social media and bots can coexist more fluidly in the future due to shortcomings with code not being able to decipher bots with malicious intent versus bots that help us. Big corporations don't have the ability to capitalize on useful bots yet and as a result, usually punish those using them with no harm intended (using a bot to delete posts from a third-party app can get you penalized on instagram).

In a more complex bot, I could see think working wonders. I wonder how useful it is if you're typing your responses in your own code already. Wouldn't that just make you look like a slow writer? I'm curious to see if there's any way to make the bot type with a certain tone or on specific topics.

I think the primary goal of pseudocode is to focus on the algorithm's fundamental logic without becoming bogged down by the syntax of a specific programming language is interesting. It acts as a bridge between the problem-solving and coding phases.

In the context of conditionals, code blocks provide a clear structure to articulate actions based on specific conditions. For example, if a particular condition is met, executing a series of steps encapsulated within a code block becomes more readable and maintainable. Likewise, in the case of loops, code blocks facilitate the repetition of a set of statements for each iteration. This not only streamlines the code but also contributes to its comprehensibility. Understanding the significance of code blocks is essential for proficient programming, as it allows developers to write more organized, modular, and comprehensible code, contributing to better software design and maintenance.