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Critical Analysis – Types, Examples and Writing Guide

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Critical Analysis

Definition:

Critical analysis is a process of examining a piece of work or an idea in a systematic, objective, and analytical way. It involves breaking down complex ideas, concepts, or arguments into smaller, more manageable parts to understand them better.

Types of Critical Analysis

Types of Critical Analysis are as follows:

Literary Analysis

This type of analysis focuses on analyzing and interpreting works of literature , such as novels, poetry, plays, etc. The analysis involves examining the literary devices used in the work, such as symbolism, imagery, and metaphor, and how they contribute to the overall meaning of the work.

Film Analysis

This type of analysis involves examining and interpreting films, including their themes, cinematography, editing, and sound. Film analysis can also include evaluating the director’s style and how it contributes to the overall message of the film.

Art Analysis

This type of analysis involves examining and interpreting works of art , such as paintings, sculptures, and installations. The analysis involves examining the elements of the artwork, such as color, composition, and technique, and how they contribute to the overall meaning of the work.

Cultural Analysis

This type of analysis involves examining and interpreting cultural artifacts , such as advertisements, popular music, and social media posts. The analysis involves examining the cultural context of the artifact and how it reflects and shapes cultural values, beliefs, and norms.

Historical Analysis

This type of analysis involves examining and interpreting historical documents , such as diaries, letters, and government records. The analysis involves examining the historical context of the document and how it reflects the social, political, and cultural attitudes of the time.

Philosophical Analysis

This type of analysis involves examining and interpreting philosophical texts and ideas, such as the works of philosophers and their arguments. The analysis involves evaluating the logical consistency of the arguments and assessing the validity and soundness of the conclusions.

Scientific Analysis

This type of analysis involves examining and interpreting scientific research studies and their findings. The analysis involves evaluating the methods used in the study, the data collected, and the conclusions drawn, and assessing their reliability and validity.

Critical Discourse Analysis

This type of analysis involves examining and interpreting language use in social and political contexts. The analysis involves evaluating the power dynamics and social relationships conveyed through language use and how they shape discourse and social reality.

Comparative Analysis

This type of analysis involves examining and interpreting multiple texts or works of art and comparing them to each other. The analysis involves evaluating the similarities and differences between the texts and how they contribute to understanding the themes and meanings conveyed.

Critical Analysis Format

Critical Analysis Format is as follows:

I. Introduction

• Provide a brief overview of the text, object, or event being analyzed
• Explain the purpose of the analysis and its significance
• Provide background information on the context and relevant historical or cultural factors

II. Description

• Provide a detailed description of the text, object, or event being analyzed
• Identify key themes, ideas, and arguments presented
• Describe the author or creator’s style, tone, and use of language or visual elements

III. Analysis

• Analyze the text, object, or event using critical thinking skills
• Identify the main strengths and weaknesses of the argument or presentation
• Evaluate the reliability and validity of the evidence presented
• Assess any assumptions or biases that may be present in the text, object, or event
• Consider the implications of the argument or presentation for different audiences and contexts

IV. Evaluation

• Provide an overall evaluation of the text, object, or event based on the analysis
• Assess the effectiveness of the argument or presentation in achieving its intended purpose
• Identify any limitations or gaps in the argument or presentation
• Consider any alternative viewpoints or interpretations that could be presented
• Summarize the main points of the analysis and evaluation
• Reiterate the significance of the text, object, or event and its relevance to broader issues or debates
• Provide any recommendations for further research or future developments in the field.

VI. Example

• Provide an example or two to support your analysis and evaluation
• Use quotes or specific details from the text, object, or event to support your claims
• Analyze the example(s) using critical thinking skills and explain how they relate to your overall argument

VII. Conclusion

• Reiterate your thesis statement and summarize your main points
• Provide a final evaluation of the text, object, or event based on your analysis
• Offer recommendations for future research or further developments in the field
• End with a thought-provoking statement or question that encourages the reader to think more deeply about the topic

How to Write Critical Analysis

Writing a critical analysis involves evaluating and interpreting a text, such as a book, article, or film, and expressing your opinion about its quality and significance. Here are some steps you can follow to write a critical analysis:

• Read and re-read the text: Before you begin writing, make sure you have a good understanding of the text. Read it several times and take notes on the key points, themes, and arguments.
• Identify the author’s purpose and audience: Consider why the author wrote the text and who the intended audience is. This can help you evaluate whether the author achieved their goals and whether the text is effective in reaching its audience.
• Analyze the structure and style: Look at the organization of the text and the author’s writing style. Consider how these elements contribute to the overall meaning of the text.
• Evaluate the content : Analyze the author’s arguments, evidence, and conclusions. Consider whether they are logical, convincing, and supported by the evidence presented in the text.
• Consider the context: Think about the historical, cultural, and social context in which the text was written. This can help you understand the author’s perspective and the significance of the text.
• Develop your thesis statement : Based on your analysis, develop a clear and concise thesis statement that summarizes your overall evaluation of the text.
• Support your thesis: Use evidence from the text to support your thesis statement. This can include direct quotes, paraphrases, and examples from the text.
• Write the introduction, body, and conclusion : Organize your analysis into an introduction that provides context and presents your thesis, a body that presents your evidence and analysis, and a conclusion that summarizes your main points and restates your thesis.
• Revise and edit: After you have written your analysis, revise and edit it to ensure that your writing is clear, concise, and well-organized. Check for spelling and grammar errors, and make sure that your analysis is logically sound and supported by evidence.

When to Write Critical Analysis

You may want to write a critical analysis in the following situations:

• Academic Assignments: If you are a student, you may be assigned to write a critical analysis as a part of your coursework. This could include analyzing a piece of literature, a historical event, or a scientific paper.
• Journalism and Media: As a journalist or media person, you may need to write a critical analysis of current events, political speeches, or media coverage.
• Personal Interest: If you are interested in a particular topic, you may want to write a critical analysis to gain a deeper understanding of it. For example, you may want to analyze the themes and motifs in a novel or film that you enjoyed.
• Professional Development : Professionals such as writers, scholars, and researchers often write critical analyses to gain insights into their field of study or work.

Critical Analysis Example

An Example of Critical Analysis Could be as follow:

Research Topic:

The Impact of Online Learning on Student Performance

Introduction:

The introduction of the research topic is clear and provides an overview of the issue. However, it could benefit from providing more background information on the prevalence of online learning and its potential impact on student performance.

Literature Review:

The literature review is comprehensive and well-structured. It covers a broad range of studies that have examined the relationship between online learning and student performance. However, it could benefit from including more recent studies and providing a more critical analysis of the existing literature.

Research Methods:

The research methods are clearly described and appropriate for the research question. The study uses a quasi-experimental design to compare the performance of students who took an online course with those who took the same course in a traditional classroom setting. However, the study may benefit from using a randomized controlled trial design to reduce potential confounding factors.

The results are presented in a clear and concise manner. The study finds that students who took the online course performed similarly to those who took the traditional course. However, the study only measures performance on one course and may not be generalizable to other courses or contexts.

Discussion :

The discussion section provides a thorough analysis of the study’s findings. The authors acknowledge the limitations of the study and provide suggestions for future research. However, they could benefit from discussing potential mechanisms underlying the relationship between online learning and student performance.

Conclusion :

The conclusion summarizes the main findings of the study and provides some implications for future research and practice. However, it could benefit from providing more specific recommendations for implementing online learning programs in educational settings.

Purpose of Critical Analysis

There are several purposes of critical analysis, including:

• To identify and evaluate arguments : Critical analysis helps to identify the main arguments in a piece of writing or speech and evaluate their strengths and weaknesses. This enables the reader to form their own opinion and make informed decisions.
• To assess evidence : Critical analysis involves examining the evidence presented in a text or speech and evaluating its quality and relevance to the argument. This helps to determine the credibility of the claims being made.
• To recognize biases and assumptions : Critical analysis helps to identify any biases or assumptions that may be present in the argument, and evaluate how these affect the credibility of the argument.
• To develop critical thinking skills: Critical analysis helps to develop the ability to think critically, evaluate information objectively, and make reasoned judgments based on evidence.
• To improve communication skills: Critical analysis involves carefully reading and listening to information, evaluating it, and expressing one’s own opinion in a clear and concise manner. This helps to improve communication skills and the ability to express ideas effectively.

Importance of Critical Analysis

Here are some specific reasons why critical analysis is important:

• Helps to identify biases: Critical analysis helps individuals to recognize their own biases and assumptions, as well as the biases of others. By being aware of biases, individuals can better evaluate the credibility and reliability of information.
• Enhances problem-solving skills : Critical analysis encourages individuals to question assumptions and consider multiple perspectives, which can lead to creative problem-solving and innovation.
• Promotes better decision-making: By carefully evaluating evidence and arguments, critical analysis can help individuals make more informed and effective decisions.
• Facilitates understanding: Critical analysis helps individuals to understand complex issues and ideas by breaking them down into smaller parts and evaluating them separately.
• Fosters intellectual growth : Engaging in critical analysis challenges individuals to think deeply and critically, which can lead to intellectual growth and development.

Advantages of Critical Analysis

Some advantages of critical analysis include:

• Improved decision-making: Critical analysis helps individuals make informed decisions by evaluating all available information and considering various perspectives.
• Enhanced problem-solving skills : Critical analysis requires individuals to identify and analyze the root cause of a problem, which can help develop effective solutions.
• Increased creativity : Critical analysis encourages individuals to think outside the box and consider alternative solutions to problems, which can lead to more creative and innovative ideas.
• Improved communication : Critical analysis helps individuals communicate their ideas and opinions more effectively by providing logical and coherent arguments.
• Reduced bias: Critical analysis requires individuals to evaluate information objectively, which can help reduce personal biases and subjective opinions.
• Better understanding of complex issues : Critical analysis helps individuals to understand complex issues by breaking them down into smaller parts, examining each part and understanding how they fit together.
• Greater self-awareness: Critical analysis helps individuals to recognize their own biases, assumptions, and limitations, which can lead to personal growth and development.

About the author

Muhammad Hassan

Researcher, Academic Writer, Web developer

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How to write a critical analysis

Unlike the name implies a critical analysis does not necessarily mean that you are only exploring what is wrong with a piece of work. Instead, the purpose of this type of essay is to interact with and understand a text. Here’s what you need to know to create a well-written critical analysis essay.

What is a critical analysis?

A critical analysis examines and evaluates someone else’s work, such as a book, an essay, or an article. It requires two steps: a careful reading of the work and thoughtful analysis of the information presented in the work.

Although this may sound complicated, all you are doing in a critical essay is closely reading an author’s work and providing your opinion on how well the author accomplished their purpose.

Critical analyses are most frequently done in academic settings (such as a class assignment). Writing a critical analysis demonstrates that you are able to read a text and think deeply about it. However, critical thinking skills are vital outside of an educational context as well. You just don’t always have to demonstrate them in essay form.

How to outline and write a critical analysis essay

Writing a critical analysis essay involves two main chunks of work: reading the text you are going to write about and writing an analysis of that text. Both are equally important when writing a critical analysis essay.

Step one: Reading critically

The first step in writing a critical analysis is to carefully study the source you plan to analyze.

If you are writing for a class assignment, your professor may have already given you the topic to analyze in an article, short story, book, or other work. If so, you can focus your note-taking on that topic while reading.

Other times, you may have to develop your own topic to analyze within a piece of work. In this case, you should focus on a few key areas as you read:

• What is the author’s intended purpose for the work?
• What techniques and language does the author use to achieve this purpose?
• How does the author support the thesis?
• Who is the author writing for?
• Is the author effective at achieving the intended purpose?

Once you have carefully examined the source material, then you are ready to begin planning your critical analysis essay.

Step two: Writing the critical analysis essay

Taking time to organize your ideas before you begin writing can shorten the amount of time that you spend working on your critical analysis essay. As an added bonus, the quality of your essay will likely be higher if you have a plan before writing.

Here’s a rough outline of what should be in your essay. Of course, if your instructor gives you a sample essay or outline, refer to the sample first.

• Background Information

Critical Analysis

Here is some additional information on what needs to go into each section:

Background information

In the first paragraph of your essay, include background information on the material that you are critiquing. Include context that helps the reader understand the piece you are analyzing. Be sure to include the title of the piece, the author’s name, and information about when and where it was published.

“Success is counted sweetest” is a poem by Emily Dickinson published in 1864. Dickinson was not widely known as a poet during her lifetime, and this poem is one of the first published while she was alive.

After you have provided background information, state your thesis. The thesis should be your reaction to the work. It also lets your reader know what to expect from the rest of your essay. The points you make in the critical analysis should support the thesis.

Dickinson’s use of metaphor in the poem is unexpected but works well to convey the paradoxical theme that success is most valued by those who never experience success.

The next section should include a summary of the work that you are analyzing. Do not assume that the reader is familiar with the source material. Your summary should show that you understood the text, but it should not include the arguments that you will discuss later in the essay.

Dickinson introduces the theme of success in the first line of the poem. She begins by comparing success to nectar. Then, she uses the extended metaphor of a battle in order to demonstrate that the winner has less understanding of success than the loser.

The next paragraphs will contain your critical analysis. Use as many paragraphs as necessary to support your thesis.

Discuss the areas that you took notes on as you were reading. While a critical analysis should include your opinion, it needs to have evidence from the source material in order to be credible to readers. Be sure to use textual evidence to support your claims, and remember to explain your reasoning.

Dickinson’s comparison of success to nectar seems strange at first. However the first line “success is counted sweetest” brings to mind that this nectar could be bees searching for nectar to make honey. In this first stanza, Dickinson seems to imply that success requires work because bees are usually considered to be hard-working and industrious.

In the next two stanzas, Dickinson expands on the meaning of success. This time she uses the image of a victorious army and a dying man on the vanquished side. Now the idea of success is more than something you value because you have worked hard for it. Dickinson states that the dying man values success even more than the victors because he has given everything and still has not achieved success.

This last section is where you remind the readers of your thesis and make closing remarks to wrap up your essay. Avoid summarizing the main points of your critical analysis unless your essay is so long that readers might have forgotten parts of it.

In “Success is counted sweetest” Dickinson cleverly upends the reader’s usual thoughts about success through her unexpected use of metaphors. The poem may be short, but Dickinson conveys a serious theme in just a few carefully chosen words.

What type of language should be used in a critical analysis essay?

Because critical analysis papers are written in an academic setting, you should use formal language, which means:

• No contractions
• Avoid first-person pronouns (I, we, me)

Do not include phrases such as “in my opinion” or “I think”. In a critical analysis, the reader already assumes that the claims are your opinions.

Your instructor may have specific guidelines for the writing style to use. If the instructor assigns a style guide for the class, be sure to use the guidelines in the style manual in your writing.

Additional t ips for writing a critical analysis essay

To conclude this article, here are some additional tips for writing a critical analysis essay:

• Give yourself plenty of time to read the source material. If you have time, read through the text once to get the gist and a second time to take notes.
• Outlining your essay can help you save time. You don’t have to stick exactly to the outline though. You can change it as needed once you start writing.
• Spend the bulk of your writing time working on your thesis and critical analysis. The introduction and conclusion are important, but these sections cannot make up for a weak thesis or critical analysis.
• Give yourself time between your first draft and your second draft. A day or two away from your essay can make it easier to see what you need to improve.

Frequently Asked Questions about critical analyses

In the introduction of a critical analysis essay, you should give background information on the source that you are analyzing. Be sure to include the author’s name and the title of the work. Your thesis normally goes in the introduction as well.

A critical analysis has four main parts.

• Introduction

The focus of a critical analysis should be on the work being analyzed rather than on you. This means that you should avoid using first person unless your instructor tells you to do otherwise. Most formal academic writing is written in third person.

How many paragraphs your critical analysis should have depends on the assignment and will most likely be determined by your instructor. However, in general, your critical analysis paper should have three to six paragraphs, unless otherwise stated.

Your critical analysis ends with your conclusion. You should restate the thesis and make closing remarks, but avoid summarizing the main points of your critical analysis unless your essay is so long that readers might have forgotten parts of it.

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Writing a Critical Analysis

What is in this guide, definitions, putting it together, tips and examples of critques.

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This guide is meant to help you understand the basics of writing a critical analysis. A critical analysis is an argument about a particular piece of media. There are typically two parts: (1) identify and explain the argument the author is making, and (2), provide your own argument about that argument. Your instructor may have very specific requirements on how you are to write your critical analysis, so make sure you read your assignment carefully.

Critical Analysis

A deep approach to your understanding of a piece of media by relating new knowledge to what you already know.

Part 1: Introduction

• Identify the work being criticized.
• Present thesis - argument about the work.
• Preview your argument - what are the steps you will take to prove your argument.

Part 2: Summarize

• Provide a short summary of the work.
• Present only what is needed to know to understand your argument.

Part 3: Your Argument

• This is the bulk of your paper.
• Provide "sub-arguments" to prove your main argument.
• Use scholarly articles to back up your argument(s).

Part 4: Conclusion

• Reflect on  how  you have proven your argument.
• Point out the  importance  of your argument.
• Comment on the potential for further research or analysis.
• Cornell University Library Tips for writing a critical appraisal and analysis of a scholarly article.
• Queen's University Library How to Critique an Article (Psychology)
• University of Illinois, Springfield An example of a summary and an evaluation of a research article. This extended example shows the different ways a student can critique and write about an article
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• Last Updated: Feb 14, 2024 4:33 PM
• URL: https://libguides.pittcc.edu/critical_analysis

What Is a Critical Analysis Essay: Definition

Have you ever had to read a book or watch a movie for school and then write an essay about it? Well, a critical analysis essay is a type of essay where you do just that! So, when wondering what is a critical analysis essay, know that it's a fancy way of saying that you're going to take a closer look at something and analyze it.

So, let's say you're assigned to read a novel for your literature class. A critical analysis essay would require you to examine the characters, plot, themes, and writing style of the book. You would need to evaluate its strengths and weaknesses and provide your own thoughts and opinions on the text.

Similarly, if you're tasked with writing a critical analysis essay on a scientific article, you would need to analyze the methodology, results, and conclusions presented in the article and evaluate its significance and potential impact on the field.

The key to a successful critical analysis essay is to approach the subject matter with an open mind and a willingness to engage with it on a deeper level. By doing so, you can gain a greater appreciation and understanding of the subject matter and develop your own informed opinions and perspectives. Considering this, we bet you want to learn how to write critical analysis essay easily and efficiently, so keep on reading to find out more!

Meanwhile, if you'd rather have your own sample critical analysis essay crafted by professionals from our custom writings , contact us to buy essays online .

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Critical Analysis Essay Topics by Category

If you're looking for an interesting and thought-provoking topic for your critical analysis essay, you've come to the right place! Critical analysis essays can cover many subjects and topics, with endless possibilities. To help you get started, we've compiled a list of critical analysis essay topics by category. We've got you covered whether you're interested in literature, science, social issues, or something else. So, grab a notebook and pen, and get ready to dive deep into your chosen topic. In the following sections, we will provide you with various good critical analysis paper topics to choose from, each with its unique angle and approach.

Critical Analysis Essay Topics on Mass Media

From television and radio to social media and advertising, mass media is everywhere, shaping our perceptions of the world around us. As a result, it's no surprise that critical analysis essays on mass media are a popular choice for students and scholars alike. To help you get started, here are ten critical essay example topics on mass media:

• The Influence of Viral Memes on Pop Culture: An In-Depth Analysis.
• The Portrayal of Mental Health in Television: Examining Stigmatization and Advocacy.
• The Power of Satirical News Shows: Analyzing the Impact of Political Commentary.
• Mass Media and Consumer Behavior: Investigating Advertising and Persuasion Techniques.
• The Ethics of Deepfake Technology: Implications for Trust and Authenticity in Media.
• Media Framing and Public Perception: A Critical Analysis of News Coverage.
• The Role of Social Media in Shaping Political Discourse and Activism.
• Fake News in the Digital Age: Identifying Disinformation and Its Effects.
• The Representation of Gender and Diversity in Hollywood Films: A Critical Examination.
• Media Ownership and Its Impact on Journalism and News Reporting: A Comprehensive Study.

Critical Analysis Essay Topics on Sports

Sports are a ubiquitous aspect of our culture, and they have the power to unite and inspire people from all walks of life. Whether you're an athlete, a fan, or just someone who appreciates the beauty of competition, there's no denying the significance of sports in our society. If you're looking for an engaging and thought-provoking topic for your critical analysis essay, sports offer a wealth of possibilities:

• The Role of Sports in Diplomacy: Examining International Relations Through Athletic Events.
• Sports and Identity: How Athletic Success Shapes National and Cultural Pride.
• The Business of Sports: Analyzing the Economics and Commercialization of Athletics.
• Athlete Activism: Exploring the Impact of Athletes' Social and Political Engagement.
• Sports Fandom and Online Communities: The Impact of Social Media on Fan Engagement.
• The Representation of Athletes in the Media: Gender, Race, and Stereotypes.
• The Psychology of Sports: Exploring Mental Toughness, Motivation, and Peak Performance.
• The Evolution of Sports Equipment and Technology: From Innovation to Regulation.
• The Legacy of Sports Legends: Analyzing Their Impact Beyond Athletic Achievement.
• Sports and Social Change: How Athletic Movements Shape Societal Attitudes and Policies.

Critical Analysis Essay Topics on Literature and Arts

Literature and arts can inspire, challenge, and transform our perceptions of the world around us. From classic novels to contemporary art, the realm of literature and arts offers many possibilities for critical analysis essays. Here are ten original critic essay example topics on literature and arts:

• The Use of Symbolism in Contemporary Poetry: Analyzing Hidden Meanings and Significance.
• The Intersection of Art and Identity: How Self-Expression Shapes Artists' Works.
• The Role of Nonlinear Narrative in Postmodern Novels: Techniques and Interpretation.
• The Influence of Jazz on African American Literature: A Comparative Study.
• The Complexity of Visual Storytelling: Graphic Novels and Their Narrative Power.
• The Art of Literary Translation: Challenges, Impact, and Interpretation.
• The Evolution of Music Videos: From Promotional Tools to a Unique Art Form.
• The Literary Techniques of Magical Realism: Exploring Reality and Fantasy.
• The Impact of Visual Arts in Advertising: Analyzing the Connection Between Art and Commerce.
• Art in Times of Crisis: How Artists Respond to Societal and Political Challenges.

Critical Analysis Essay Topics on Culture

Culture is a dynamic and multifaceted aspect of our society, encompassing everything from language and religion to art and music. As a result, there are countless possibilities for critical analysis essays on culture. Whether you're interested in exploring the complexities of globalization or delving into the nuances of cultural identity, there's a wealth of topics to choose from:

• The Influence of K-Pop on Global Youth Culture: A Comparative Study.
• Cultural Significance of Street Art in Urban Spaces: Beyond Vandalism.
• The Role of Mythology in Shaping Indigenous Cultures and Belief Systems.
• Nollywood: Analyzing the Cultural Impact of Nigerian Cinema on the African Diaspora.
• The Language of Hip-Hop Lyrics: A Semiotic Analysis of Cultural Expression.
• Digital Nomads and Cultural Adaptation: Examining the Subculture of Remote Work.
• The Cultural Significance of Tattooing Among Indigenous Tribes in Oceania.
• The Art of Culinary Fusion: Analyzing Cross-Cultural Food Trends and Innovation.
• The Impact of Cultural Festivals on Local Identity and Economy.
• The Influence of Internet Memes on Language and Cultural Evolution.

How to Write a Critical Analysis: Easy Steps

When wondering how to write a critical analysis essay, remember that it can be a challenging but rewarding process. Crafting a critical analysis example requires a careful and thoughtful examination of a text or artwork to assess its strengths and weaknesses and broader implications. The key to success is to approach the task in a systematic and organized manner, breaking it down into two distinct steps: critical reading and critical writing. Here are some tips for each step of the process to help you write a critical essay.

Step 1: Critical Reading

Here are some tips for critical reading that can help you with your critical analysis paper:

• Read actively : Don't just read the text passively, but actively engage with it by highlighting or underlining important points, taking notes, and asking questions.
• Identify the author's main argument: Figure out what the author is trying to say and what evidence they use to support their argument.
• Evaluate the evidence: Determine whether the evidence is reliable, relevant, and sufficient to support the author's argument.
• Analyze the author's tone and style: Consider the author's tone and style and how it affects the reader's interpretation of the text.
• Identify assumptions: Identify any underlying assumptions the author makes and consider whether they are valid or questionable.
• Consider alternative perspectives: Consider alternative perspectives or interpretations of the text and consider how they might affect the author's argument.
• Assess the author's credibility : Evaluate the author's credibility by considering their expertise, biases, and motivations.
• Consider the context: Consider the historical, social, cultural, and political context in which the text was written and how it affects its meaning.
• Pay attention to language: Pay attention to the author's language, including metaphors, symbolism, and other literary devices.
• Synthesize your analysis: Use your analysis of the text to develop a well-supported argument in your critical analysis essay.

Step 2: Critical Analysis Writing

Here are some tips for critical analysis writing, with examples:

• Start with a strong thesis statement: A strong critical analysis thesis is the foundation of any critical analysis essay. It should clearly state your argument or interpretation of the text. You can also consult us on how to write a thesis statement . Meanwhile, here is a clear example:
• Weak thesis statement: 'The author of this article is wrong.'
• Strong thesis statement: 'In this article, the author's argument fails to consider the socio-economic factors that contributed to the issue, rendering their analysis incomplete.'
• Use evidence to support your argument: Use evidence from the text to support your thesis statement, and make sure to explain how the evidence supports your argument. For example:
• Weak argument: 'The author of this article is biased.'
• Strong argument: 'The author's use of emotional language and selective evidence suggests a bias towards one particular viewpoint, as they fail to consider counterarguments and present a balanced analysis.'
• Analyze the evidence : Analyze the evidence you use by considering its relevance, reliability, and sufficiency. For example:
• Weak analysis: 'The author mentions statistics in their argument.'
• Strong analysis: 'The author uses statistics to support their argument, but it is important to note that these statistics are outdated and do not take into account recent developments in the field.'
• Use quotes and paraphrases effectively: Use quotes and paraphrases to support your argument and properly cite your sources. For example:
• Weak use of quotes: 'The author said, 'This is important.'
• Strong use of quotes: 'As the author points out, 'This issue is of utmost importance in shaping our understanding of the problem' (p. 25).'
• Use clear and concise language: Use clear and concise language to make your argument easy to understand, and avoid jargon or overly complicated language. For example:
• Weak language: 'The author's rhetorical devices obfuscate the issue.'
• Strong language: 'The author's use of rhetorical devices such as metaphor and hyperbole obscures the key issues at play.'
• Address counterarguments: Address potential counterarguments to your argument and explain why your interpretation is more convincing. For example:
• Weak argument: 'The author is wrong because they did not consider X.'
• Strong argument: 'While the author's analysis is thorough, it overlooks the role of X in shaping the issue. However, by considering this factor, a more nuanced understanding of the problem emerges.'
• Consider the audience: Consider your audience during your writing process. Your language and tone should be appropriate for your audience and should reflect the level of knowledge they have about the topic. For example:
• Weak language: 'As any knowledgeable reader can see, the author's argument is flawed.'
• Strong language: 'Through a critical analysis of the author's argument, it becomes clear that there are gaps in their analysis that require further consideration.'

Master the art of critical analysis with EssayPro . Our team is ready to guide you in dissecting texts, theories, or artworks with depth and sophistication. Let us help you deliver a critical analysis essay that showcases your analytical prowess.

Creating a Detailed Critical Analysis Essay Outline

Creating a detailed outline is essential when writing a critical analysis essay. It helps you organize your thoughts and arguments, ensuring your essay flows logically and coherently. Here is a detailed critical analysis outline from our dissertation writers :

I. Introduction

A. Background information about the text and its author

B. Brief summary of the text

C. Thesis statement that clearly states your argument

II. Analysis of the Text

A. Overview of the text's main themes and ideas

B. Examination of the author's writing style and techniques

C. Analysis of the text's structure and organization

III. Evaluation of the Text

A. Evaluation of the author's argument and evidence

B. Analysis of the author's use of language and rhetorical strategies

C. Assessment of the text's effectiveness and relevance to the topic

IV. Discussion of the Context

A. Exploration of the historical, cultural, and social context of the text

B. Examination of the text's influence on its audience and society

C. Analysis of the text's significance and relevance to the present day

V. Counter Arguments and Responses

A. Identification of potential counterarguments to your argument

B. Refutation of counterarguments and defense of your position

C. Acknowledgement of the limitations and weaknesses of your argument

VI. Conclusion

A. Recap of your argument and main points

B. Evaluation of the text's significance and relevance

C. Final thoughts and recommendations for further research or analysis.

This outline can be adjusted to fit the specific requirements of your essay. Still, it should give you a solid foundation for creating a detailed and well-organized critical analysis essay.

Useful Techniques Used in Literary Criticism

There are several techniques used in literary criticism to analyze and evaluate a work of literature. Here are some of the most common techniques:

• Close reading: This technique involves carefully analyzing a text to identify its literary devices, themes, and meanings.
• Historical and cultural context: This technique involves examining the historical and cultural context of a work of literature to understand the social, political, and cultural influences that shaped it.
• Structural analysis: This technique involves analyzing the structure of a text, including its plot, characters, and narrative techniques, to identify patterns and themes.
• Formalism: This technique focuses on the literary elements of a text, such as its language, imagery, and symbolism, to analyze its meaning and significance.
• Psychological analysis: This technique examines the psychological and emotional aspects of a text, including the motivations and desires of its characters, to understand the deeper meanings and themes.
• Feminist and gender analysis: This technique focuses on the representation of gender and sexuality in a text, including how gender roles and stereotypes are reinforced or challenged.
• Marxist and social analysis: This technique examines the social and economic structures portrayed in a text, including issues of class, power, and inequality.

By using these and other techniques, literary critics can offer insightful and nuanced analyses of works of literature, helping readers to understand and appreciate the complexity and richness of the texts.

Sample Critical Analysis Essay

Now that you know how to write a critical analysis, take a look at the critical analysis essay sample provided by our research paper writers and better understand this kind of paper!

Final Words

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How to write a critical analysis essay, what is a critical analysis essay, related articles.

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Critical analysis is a formal evaluation of someone else’s work. In academia, this work is most often a book, article, poem, play or piece of visual art. However, in business, you might need to carry out a critical analysis of a proposal for a project or grant, a policy or white paper, an industry handbook or even a research study. Broadly speaking, critical analysis involves examining the work to see how well the author has carried out their purpose, or how well the project or policy will or does carry out its purpose.

Critical analysis is therefore an extension of both critical thinking and critical reading . Critical thinking is the ability to engage in reflective and independent thinking about the information that you encounter. Critical reading is engaging with what you read by asking yourself questions about the author’s intention and your reaction to that. Critical analysis is the formalisation of both these processes, coupled with a written analysis for others.

There are therefore two elements to critical analysis. The first is carrying out the analysis itself: deciding what you think. The second is writing up your findings and judgement for others.

Steps in a Critical Analysis

There are several steps that you need to take to carry out a critical analysis. These include:

1. Critical reading of your text or examination of your piece.

This is a matter of carefully reading your text, possibly several times.

As you do so, consider (and note down) what you think is important and relevant. It will also be helpful to note any controversial points, or areas where you disagree.

There is more about this process in our page on Critical Reading .

During this process, you should aim to identify the main thesis, point or purpose, and then sub-themes or issues.

In a piece of business writing or an essay, the purpose or thesis is usually set out early on, often in the introduction. In a piece of fiction writing, it may be necessary to read the text fully to identify themes and then highlight the most important theme.>

It is also worth taking note of any evidence that supports the themes and purpose.

Finally, it is worth writing yourself a one-paragraph summary of the text. This is likely to be a good starting point for your analysis, because your readers may not have read the text themselves.

2. Analysing the text or piece

The purpose of your analysis is to make an overall judgement about how well the text has met its objectives, based on the evidence available to you.

There are five useful aspects to consider in analysing the text or piece:

Your reaction to the text . This has two purposes. First, it affects how you approach the analysis. For example, if the ideas in the text make you angry, you will find it harder to see their benefits. Second, writers often want to evoke certain emotions in their audience. This is part of the purpose of the piece—and therefore assessing this issue is an important part of judging whether it has met its objectives.

The background to the text . It is worth considering the backdrop against which the text was written. For a policy paper, for example, what has gone before? How urgent is the need to address the situation? For a piece of creative writing, when was it written and what was happening in the world at the time? How might this have affected the way that the author was writing, or what they wanted to achieve?

The author’s background and the possible implications of this . The author’s background is likely to have informed their opinions and views—and therefore what they have written. It is worth considering the text in this light. This is part of the background, but specific enough to consider as a separate category.

The definitions and concepts in the text . Consider how well the author has defined concepts and ideas. It is much easier to assess ideas if they are clearly defined and described in simple language. Similarly, poor definitions may mean that the author is not clear about their own meaning, or that your understanding is different from theirs.

The use of evidence . You should consider the evidence presented in the text in two senses. First, examine its general validity and reliability. For example, in a proposal or paper, are the ideas supported by peer reviewed studies published in reputable journals? Second, you should consider how well the evidence supports the author’s points. It is also worth considering what evidence is NOT cited, but which might support or undermine the author’s points. It follows that you should also have evidence to support your own arguments in your analysis.

3. Writing up your analysis

The final stage of a critical analysis is to write up your analysis to present it to others.

The precise form that you use is likely to depend both on your preferences, and on any guidelines provided by your organisation or institution (see box).

TOP TIP! Check your guidelines

Your organisation or institution may have guidelines for carrying out a critical analysis. Check them carefully for the structure that you are expected to use, or any essential sections that must be included. For example, some organisations require a summary paragraph upfront (like an executive summary).

You are likely to need to include:

A brief summary of the text or proposal.

A brief summary of your assessment of the text . This should usually be structured around a main point or thesis against which you will consider various aspects of the text.

• For example, if you are analysing a business proposal, you might be concerned that the concepts are not defined very clearly, and that this may demonstrate that the author has not clearly understood the issues. Your main thesis is therefore this lack of clarity.

A more expanded version of  your analysis, with the evidence for each of your points . Again, this should be structured around your main thesis. It should also set your analysis in the wider context, including what else is known about the subject.

• The example from the previous bullet described concerns about the lack of clarity of definitions and therefore ambiguity. In this example, your expanded analysis would focus on areas that are not clear, and the problems that might arise from the ambiguity.

A conclusion that sums up your argument and reiterates your judgement on the text.

TOP TIP! You don’t have to write it in order—just sort it afterwards

It is often easier to prepare your introduction and conclusion once you have finished your analysis, and you are absolutely clear on the points you want to highlight.

It is also a good idea to use headings to show divisions between sections.

Summing Up Critical Analysis

Ultimately, critical analysis is about asking questions—and then setting the answers into context.

The most important questions are What, How, Why and So what? The answers will provide a clear and succinct critique of a text, project or idea, and allow you to form a judgement about the text.

Continue to: Critical Thinking and Fake News Analytical Skills

See also: Assessing Internet Information Styles of Writing Understanding and Addressing Conspiracy Theories

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Critical analysis: home.

• Reading Critically

What is Critical Analysis?

Analysis is a word that is also often used when taking a critical approach to something. It could be that you look at some evidence and if you think it is good quality, you may choose to include that in your essay or writing to help support your argument. When you have analysed different sets of evidence you may  synthesize all the ideas gathered from multiple sources bringing together the relevant information into a different argument or idea. 

To evaluate something or someone, you think and consider it or them in order to make a judgment about it/them; this could be as simple as how good or bad they are. When you critically evaluate something or someone you consider how judgments vary from different perspectives and how some judgments are stronger than others. This often means creating an objective, reasoned argument for your overall case, based on the evaluation from different perspectives.

Taking a critical approach when you are studying involves constantly asking questions and keeping an open mind.

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How to Write a Critical Analysis

Last Updated: March 27, 2024 Fact Checked

This article was co-authored by Jake Adams . Jake Adams is an academic tutor and the owner of Simplifi EDU, a Santa Monica, California based online tutoring business offering learning resources and online tutors for academic subjects K-College, SAT & ACT prep, and college admissions applications. With over 14 years of professional tutoring experience, Jake is dedicated to providing his clients the very best online tutoring experience and access to a network of excellent undergraduate and graduate-level tutors from top colleges all over the nation. Jake holds a BS in International Business and Marketing from Pepperdine University. This article has been fact-checked, ensuring the accuracy of any cited facts and confirming the authority of its sources. This article has been viewed 1,462,412 times.

Reading Critically

• You may need to read the text more than once, especially if it is dense or complicated.

• It might be easier to find the thesis in an academic article than in a creative work, movie, or painting. If you’re critiquing a work of fiction or creative nonfiction, in either written form or film form, identify one of the main themes of the story instead. For a painting, identify what the painter may be trying to get across with their work of art.

• In an academic article, identify the topic sentences of each paragraph or section.
• For works of fiction or paintings, look for scenes and imagery that seem to support the thesis.

• If the text was a film or work of art, write a brief 1 to 2 paragraph synopsis of the film or description of the painting.

Analyzing the Text

• For example, if the text made you angry, what was it about the text that made you angry?
• If you found yourself laughing at the text, what about it was laughable?

• For example, if the author is an outspoken proponent of healthcare reform, then this would likely explain any bias in an argumentative essay on universal healthcare.
• The author’s background may also include credentials, such as a doctorate or medical degree. This is part of the ethos of the text since having credentials may help to bolster an author’s credibility.

• For example, if the author’s explanation of greenhouse gasses is long, full of jargon, and confusing, then you might focus on this as part of your critique.

Tip : Keep in mind that you can also have a positive critique of the text if you think it was effective. For example, if the author’s description of greenhouse gasses was written in simple, easy to understand language, you might note this as part of your analysis.

• For example, if the author has used a website that is known for being biased in favor of their argument, then this would weaken their position. However, if the author used sources that were fair and unbiased, then this would strengthen their position.
• Not all texts will incorporate evidence. For example, if you’re doing a critical analysis of a film or work of art, it probably won’t include secondary sources.

Drafting the Analysis

• For example, in the first sentence of your essay, provide the basic information on the text. Then, describe text’s argument in about 1 to 2 sentences.

• For example, you might write, “Darcy Gibbons’ essay on the environmental impact of consumerism provides a thorough and valuable overview of the problem.”
• Or, you might write, “Shannon Duperty’s mixed media painting, “Dove on Heroin,” falls short of its attempt at edgy political commentary.”

• Keep in mind that the summary paragraph is the only place in your essay where you may include summary. The rest of the essay should provide analysis of the essay.

• Organization. How did the author organize their argument? Was this a good strategy or not? Why?
• Style. What style did the author use to get their point across? How did the style hurt or help their argument?
• Effectiveness. In general, was the text effective at getting its point across? Why or why not?
• Fairness or bias. Did the author demonstrate a fair or biased perspective on their topic? How could you tell?
• Appeal to a specific audience. Did the author seem to have a specific audience in mind? If so, who were they and how well did the author meet their needs?

Tip : Check with your teacher for details on how to cite sources. They may want you to use a specific citation style, such as MLA, Chicago, or APA.

• For example, you might conclude by talking about how the author made a good effort in some regards, but ultimately their argument was ineffective, and then explain why in 2 to 3 sentences.

Sample Analyses

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• ↑ Jake Adams. Academic Tutor & Test Prep Specialist. Expert Interview. 20 May 2020.
• ↑ https://www.tru.ca/__shared/assets/Critical_Analysis_Template30565.pdf
• ↑ https://depts.washington.edu/pswrite/Handouts/CriticalAnalysisPapers.pdf
• ↑ https://content.nroc.org/DevelopmentalEnglish/unit09/Foundations/creating-a-thesis-and-an-outline-for-a-critical-analysis-essay.html
• ↑ https://writingcenter.fas.harvard.edu/pages/ending-essay-conclusions

About This Article

To write a critical analysis, first introduce the work you’re analyzing, including information about the work’s author and their purpose in writing it. As part of the introduction, briefly state your overall evaluation of the work. Then, summarize the author’s key points before you use the bulk of your paper to provide your full critique of the work. Try to put each point you want to make in a separate paragraph for clarity. Finally, write a concluding paragraph that restates your opinion of the work and offers any suggestions for improvement. To learn how to balance positive and negative comments in your critical analysis, keep reading! Did this summary help you? Yes No

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How to Write a Critical Analysis Essay?

So, you want to write a critical analysis essay, but it feels a bit overwhelming, right? No worries! We're here to guide you through the process step by step. Whether you're a pro or a newbie, we've got practical tips and insights to help you nail your critical analysis essay. Let's make this academic adventure a bit less scary and a lot more exciting.

Have you ever wondered, "How to write a critical analysis essay?" We get it. It's not as easy as just summarizing stuff. But fear not! In this article, we're going to break down the whole process for you. Whether you're a seasoned thinker or just dipping your toes into critical analysis, we've got your back. Get ready for some straightforward advice to help you tackle that critical analysis essay like a champ. Let's do this together! For additional reading, please consult our guide on how to write an essay in general.

What Is Critical Analysis Essay?

A critical analysis essay is a type of academic writing in which the writer evaluates and interprets a piece of literature, artwork, film, music, or any other work of art. The primary purpose of a critical analysis essay is to assess the effectiveness or merit of the work in question, providing insights into its strengths, weaknesses, and overall impact.

Writing critical analysis essay demands a thoughtful, informed, and evaluative approach to a work, with a focus on details, evidence, and interpretation. It's an exercise in understanding the layers and complexities within creation and expressing your informed perspective on its merits and shortcomings.

• The primary purpose of a critical analysis essay is to assess and evaluate a specific work, such as a piece of literature, art, film, or any other cultural creation. Unlike a summary, where you merely recap the content, a critical analysis delves deeper. It aims to explore the work's meaning, effectiveness, and significance.

Depth of Research

• To conduct a thorough critical analysis, you often need to go beyond surface-level understanding. This might involve researching the background of the author or artist, understanding the historical context, and exploring any relevant cultural influences. The depth of research ensures that your analysis is well-informed and provides a nuanced perspective.
• Learning how to write critical analysis paper involves paying attention to subtle details and nuances within the work. This could include examining the use of literary devices, artistic techniques, symbolism, or the choices made by the creator. Identifying these nuances allows you to offer a more insightful and detailed analysis.

Evidence-Based Insights

• A strong critical analysis is not merely an expression of personal opinion but is supported by evidence from the work itself. This might involve quoting specific lines from a text, describing particular scenes in a film, or pointing to elements within a piece of art. By using evidence, you strengthen your argument and make your analysis more persuasive.

Judgment and Evaluation

• The analysis goes beyond observation; it includes forming a judgment about the work. This involves assessing its strengths and weaknesses. You might discuss what the creator did well and where improvements could be made. This evaluative aspect adds depth to your analysis and demonstrates your engagement with the work.

Insights and Interpretation

• A critical analysis invites interpretation. It's an opportunity to express your unique perspective on the work and offer insights that might not be immediately apparent. This could involve exploring symbolism, thematic connections, or the impact of specific artistic choices.

Critical Analysis Essay Outline

Here's a basic outline for critical analysis essay. Remember, this is a general outline, and you may need to adjust it based on the specific requirements of your assignment or the nature of the work you are analyzing.

Introduction

Introduction to the Work

• Briefly introduce the work being analyzed.
• Provide necessary background information about the author, artist, or creator.

Thesis Statement

• State the main argument or perspective you will present in your analysis.
• Provide a concise summary of the work.
• Ensure that readers have a basic understanding of the content and context.

Introduction to Analysis

• Briefly explain the specific elements you will analyze (e.g., themes, characters, techniques).

Element 1 Analysis

• Discuss the first element in detail.
• Provide evidence from the work to support your analysis.

Element 2 Analysis

• Repeat the process for the second element.
• Again, support your analysis with relevant evidence.

Additional Elements (if needed)

• Include additional elements you plan to analyze.
• Provide analysis and evidence for each.

Introduction to Evaluation

• Briefly explain the criteria you will use to evaluate the work.

Positive Aspects

• Discuss the strengths or positive aspects of the work.
• Support your evaluation with evidence.

Areas for Improvement

• Discuss weaknesses or areas where the work could be improved.
• Again, support your evaluation with evidence.

Summary of Analysis and Evaluation

• Summarize the main points of your analysis.
• Recap your evaluation.

Final Thoughts

• Offer any final insights or reflections.
• Restate the significance of your analysis.

Writing critical analysis essay involves a systematic and thoughtful approach. Remember, the critical analysis essay is an opportunity to showcase your ability to analyze and evaluate a work thoughtfully. It's essential to provide clear reasoning and support your arguments with evidence from the work itself. 

To learn how to start a critical analysis essay, it is crucial to begin with a compelling introduction that captivates the reader's attention and sets the stage for the subsequent analysis. Start by providing contextual information about the work under scrutiny, including the author, artist, or creator, and briefly outlining the overall content. Engage your audience by posing a thought-provoking question, presenting a relevant quote, or offering a striking observation of the subject matter. 

Clearly articulate the main purpose of your analysis and introduce your thesis statement, which succinctly encapsulates the central argument you will be exploring in the essay. By establishing a solid foundation in the introduction, you invite readers to delve into your critical examination with a clear understanding of your perspective and the significance of the work at hand. Here's a step-by-step guide to help you through the entire writing process:

Understand the Assignment

• Read the assignment instructions carefully.
• Identify the specific work (e.g., a book, film, artwork) you need to analyze.
• Note any guidelines or criteria provided by your instructor.

Choose a Literary Work

• If the work is not assigned, select one that interests you and aligns with the assignment requirements.

Read/View/Experience the Work

• Engage with the work attentively.
• Take notes on significant details, themes, characters, and any elements that stand out.

Research Background Information

• Gather information about the author, artist, or creator.
• Understand the historical and cultural context if relevant.

Develop a Thesis Statement

• Formulate a clear thesis statement that presents the main argument or perspective you will explore in your analysis.

Create an Outline

• Structure your essay with an introduction, body paragraphs for analysis, an evaluation section, and a conclusion.
• Use the outline provided earlier as a template.

Write the Introduction

• Introduce the work and provide essential background information.
• Present your thesis statement, outlining the focus of your analysis.

Write the Summary

• Provide a concise summary of the work, ensuring readers have a basic understanding of its content and context.

Analyze Specific Elements

• Follow your outline to analyze specific elements of the work.
• Discuss themes, characters, techniques, or any other relevant aspects.

Evaluate the Work

• Introduce your criteria for evaluation.
• Discuss the positive aspects of the work, supporting your points with evidence.
• Address areas for improvement, again using evidence to support your evaluation.

Write the Conclusion

• Summarize the main points of your analysis and evaluation.
• Restate your thesis and its significance.
• Offer final thoughts or reflections.

Revise and Edit

• Review your essay for clarity, coherence, and consistency.
• Check for grammatical errors, typos, and proper citation if applicable.

Seek Feedback (Optional)

• If possible, get feedback from peers, instructors, or writing centers to refine your essay.

Finalize Your Essay

• Make any necessary revisions based on feedback.
• Ensure your essay meets the assignment requirements.

Cite Sources (If Required)

• If you used external sources for background information, quotes, or references, ensure proper citation according to the specified style guide (e.g., APA, MLA).

If this information seems too challenging at the moment or your deadline is short, simply say, ‘ write my essay for me ,’ and our expert writers will take it from here.

Critical Analysis Essay Topics

Students can explore a myriad of thought-provoking topics for critical analysis essay for their across various disciplines. For example, you might dissect the portrayal of power dynamics in dystopian novels or scrutinize the evolution of a character's identity throughout a classic work. Venturing into the cinematic landscape, students could critically analyze the symbolism and visual storytelling techniques in a renowned film or examine the cultural commentary embedded in a contemporary piece. For those inclined toward the visual arts, delving into the complexities of a particular artwork or artistic movement offers an opportunity to unravel hidden meanings. Moreover, students may consider such critical analysis essay ideas as the representation of gender roles, the impact of technology on human connections, or the exploration of mental health in literature. Consider the following topic examples:

• Identity and self-discovery in 'The Catcher in the Rye.'
• The impact of social media on interpersonal relationships.
• Symbolism in Edgar Allan Poe's 'The Raven.'
• Gender roles in the film 'Wonder Woman.'
• Environmental conservation messages in Nike advertisements.
• Historical accuracy in 'The Crown' TV series.
• Technology's role in shaping modern higher education.
• Cultural significance of traditional Japanese Noh theatre.
• Portrayal of mental health in 'BoJack Horseman.'
• Ethical implications of CRISPR gene editing.
• Metaphor in Martin Luther King Jr.'s 'I Have a Dream' speech.
• Impact of Affordable Care Act on healthcare accessibility.
• Symbolism in Leonardo da Vinci's 'Mona Lisa.'
• Influence of jazz on American society.
• Representation of diversity in 'The Sneetches' by Dr. Seuss.
• Role of humor in addressing social issues in Dave Chappelle's comedy special.
• Effectiveness of anti-smoking public service announcements.
• Portrayal of cultural clashes in 'The Joy Luck Club.'
• Impact of artificial intelligence on daily life.
• Role of education in addressing economic inequality.

Tips for Writing a Critical Analysis Essay

Thoroughly understand the work.

Before diving into a critical analysis essay, ensure a comprehensive understanding of the work. Read a literary piece multiple times, watch a film attentively, or study an artwork closely. Take notes on key elements, themes, and characters to form a solid foundation for your analysis.

Focus on Specific Elements

Instead of attempting to analyze the entire work, narrow down your focus to specific elements. This could include examining the use of symbolism, characterization, narrative structure, or visual techniques. By concentrating on specific aspects, you can provide a more in-depth and focused analysis.

Support Your Analysis with Evidence

A critical analysis is strengthened by providing evidence from the work itself. Quote specific lines from a text, reference particular scenes in a film, or describe elements in an artwork. This evidence supports your interpretation and demonstrates a deep engagement with the work.

Consider the Context

Acknowledge the context in which the work was created. Consider the historical, cultural, and social background that might influence its meaning. Understanding the context allows you to offer a more nuanced analysis, recognizing the creator's intent and the work's relevance within a broader framework.

How to Format a Critical Analysis Essay?

Critical analysis essay format follows general guidelines for academic writing. Always refer to your assignment guidelines and any specific instructions from your instructor, as formatting requirements can vary. Here's a typical structure in terms of formatting:

• Include the title of your essay, your name, the course title, the instructor's name, and the date.
• Follow any specific formatting instructions provided by your instructor.
• Begin with a strong introduction that introduces the work and provides essential background information.
• State your thesis clearly and concisely.

Body Paragraphs

• Organize your analysis into well-structured paragraphs.
• Each paragraph should focus on a specific element or aspect of the work.
• Begin each paragraph with a clear topic sentence that relates to your thesis.

Analysis and Evidence

• Analyze the chosen elements of the work thoroughly.
• Support your analysis with evidence, such as quotes, examples, or specific scenes.
• Ensure that your analysis is logical and well-connected.
• Include a section where you evaluate the strengths and weaknesses of the work.
• Discuss what works well and areas that could be improved.
• Provide thoughtful judgments and support them with evidence.
• Restate your thesis and highlight the significance of your analysis.
• Offer any final thoughts or reflections.

References or Works Cited

• Include a list of all the sources you cited in your essay.
• Follow the citation style (APA, MLA, Chicago, etc.) specified by your instructor.

Formatting Style

• Use a standard font (e.g., Times New Roman, Arial) in 12-point size.
• Double-space the entire essay.
• Set 1-inch margins on all sides.
• Align text to the left; do not justify.

Page Numbers

• Number pages consecutively, starting from the title page (if applicable).
• Place page numbers in the header or footer.

Additional Instructions

• Follow any additional formatting guidelines provided by your instructor.
• Check for any specific requirements regarding headers, footers, or additional elements.

Following a clear and consistent format ensures that your critical analysis essay is well-organized and easy to read.

Critical Analysis Essay Example

We believe that a critical analysis essay sample can help students master the intricacies of a particular assignment. In this article, we’ve decided to give you two different examples that will definitely inspire you to move the needle when tackling this task.

The Symbolism of Light and Dark in Shakespeare's "Macbeth"

William Shakespeare's tragedy "Macbeth" explores the intricate interplay of light and dark imagery as symbols throughout the play. In this critical analysis, we will delve into how Shakespeare employs these symbols to convey complex themes and character motivations. The juxtaposition of light and dark in "Macbeth" serves as a powerful metaphor for the moral and psychological transformations undergone by the characters.

Shakespeare strategically employs light and dark imagery to signify moral clarity and corruption. In the opening scenes, the protagonist – Macbeth – associates darkness with deception and evil intentions. For instance, when contemplating regicide, Macbeth implores the stars to "hide [their] fires; Let not light see my black and deep desires" (1.4.50-51). The contrast between light and dark intensifies as the narrative progresses, underscoring the characters' moral dilemmas.

As Macbeth succumbs to his ambition, the imagery of darkness deepens. The murder of King Duncan occurs under the cover of night, symbolizing the moral obscurity of Macbeth's actions. The once honorable protagonist descends into darkness both metaphorically and literally, as evidenced by Lady Macbeth's plea to "come, thick night, / And pall thee in the dunnest smoke of hell" (1.5.49-50). The darkness that initially masked his ambitions becomes a pervasive element in Macbeth's psyche.

Interestingly, the play also presents a paradoxical relationship between light and darkness. While darkness represents evil, light is not always synonymous with goodness. The false sense of security in the daylight becomes apparent as Macbeth's heinous deeds unfold. Banquo's murder, planned in broad daylight, challenges the traditional association of light with virtue, revealing the deceptive nature of appearances.

In conclusion, Shakespeare's adept use of light and dark imagery in "Macbeth" contributes to the thematic richness of the play. The evolving symbolism underscores the moral complexities the characters face, particularly Macbeth, as they navigate the consequences of their choices. By examining the nuanced interplay of light and dark, we gain profound insights into the psychological and moral dimensions of Shakespeare's tragic narrative.

The Dystopian Mirror: A Critical Analysis of Societal Portrayal in George Orwell's "1984"

George Orwell's "1984" stands as a dystopian masterpiece, a haunting vision of a totalitarian society that raises profound questions about power, control, and individual agency. In this critical analysis, we will delve into how Orwell's portrayal of society in "1984" serves as a reflection of his concerns about government overreach and the erosion of personal freedoms.

Orwell's vision of an omnipresent surveillance state in "1984" serves as a chilling forecast of the erosion of privacy in contemporary society. The ubiquitous presence of telescreens and the Thought Police in the novel underscores the dangers of unchecked governmental power. Through the character of Winston Smith, Orwell illustrates the psychological impact of living under constant surveillance and the pervasive fear of dissent.

Orwell introduces the concept of Newspeak, a language designed to eliminate rebellious thoughts, further illustrating the manipulative power of the state. The controlled language in "1984" becomes a tool for thought control, limiting the ability of individuals to articulate dissenting ideas. This linguistic manipulation echoes Orwell's concerns about the potential distortion of truth and the stifling of independent thought in a society governed by authoritarian rule.

The portrayal of Big Brother in "1984" serves as a powerful commentary on the creation of a cult of personality to consolidate power. The Party's ability to shape and control public perception through the iconic figure of Big Brother reflects Orwell's apprehensions about manipulating truth and the dangers of blind loyalty to charismatic leaders. The relentless propaganda in the novel underscores the potential consequences of unchecked authority on societal values.

To sum up, George Orwell's "1984" remains a poignant critique of societal structures that compromise individual freedoms in the pursuit of unchecked power. Through the vivid portrayal of surveillance, linguistic manipulation, and the cult of personality, Orwell prompts readers to reflect on the fragility of democratic values. As we navigate the complexities of our own society, the warnings embedded in "1984" continue to resonate, urging us to remain vigilant against threats to individual autonomy and the integrity of truth.

Learning how to write critical analysis essays is crucial for students as it cultivates essential skills vital for academic and professional success. Firstly, it hones their analytical thinking abilities, enabling them to dissect complex ideas, texts, or artworks and discern underlying themes, motifs, and messages. This skill is invaluable in academia, where critical analysis forms the cornerstone of scholarly discourse and research. 

Secondly, critical analysis writing fosters effective communication skills as students learn to articulate their thoughts coherently, provide evidence-based arguments, and engage with diverse perspectives. Such proficiency not only enhances their academic writing but also prepares them for future endeavors in fields such as research, journalism, or policymaking. Does that sound interesting, or maybe you would like to do something different today rather than critique literary works? In that case, pay for an essay and have a wonderful evening! 

Frequently asked questions

What is a critical analysis essay structure, what type of language should be used in a critical analysis essay, what are the benefits of writing a critical analysis essay.

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How To Write a Critical Analysis Essay?

13 April, 2020

8 minutes read

Author:  Tomas White

All works of art are subject to criticism. That criticism comes in the form of this essay type. Every time you read or watch a review or a discussion on a piece, you are exposing yourself to a critical analysis. Handmadewriting team has prepared this guide for You to learn how to write a critical analysis essay. Let's rock!

What is a Critical Analysis Essay?

A critical analysis essay puts you, the writer, in the position of judging creative work. Generally, these essays cover what you believe the work did well and what it did wrong while adding some personal insight. You will use examples from the piece to support your claims.

Having problems with your critical analysis? Our  essay writer help provides services 24\7!

How to Start A Critical Analysis Essay

Before you start the writing portion of your essay, you’ll need to spend some time with the creative work and get a good idea of the messages it attempts to portray.

Identify the Author’s Thesis

What is the main message behind the creative piece? Identify what the author is trying to argue. Which side of the argument do they fall on? Do they offer solutions to the argument or simply present to show awareness? This isn’t always clear in the work, and some outside research may be needed to fully understand the message.

Outline the Main Ideas

Go through the piece and note down any overarching messages. How does the creator appeal to your emotions? Is it done effectively, or could it have been done better? In most creative works, all of the main ideas come together to form the thesis statement.

A good way of searching for these is to work backwards from the previously established thesis. View the piece with the thesis in mind, and jot down each and every point that contributes to it.

Evaluate the Author’s Points

Summarize these points and state how well they support the thesis. Are they the strongest possible arguments for the thesis? What examples does the creator present against the thesis? Are they fair and objective, or do they lean heavily to one side? Like a good critical analysis, a good creative piece evaluates all sides of the presented argument and supports each side with evidence.

Check this guide in case you need to learn more about a rhetorical analysis .

Organizing your research into an outline is the best way to prepare for the writing. A proper outline will allow your writing to flow and give you a structure to adhere to.

How to write a Critical Analysis Essay Outline:

Introduction.

When starting a critical analysis essay, you need to state the name of the creative work, the creator, and any relevant publication/distribution details. Then, outline what parts of the work you’re going to discuss. This is when you should introduce the creator’s thesis and how they supported that thesis.

Once that’s done, end the introduction with your thesis statement . Your thesis should consist of your original thoughts and reactions to the creation and its message. Come up with a main idea about the work and use different examples from it to support your statement.

This is your opinion and it should not be the same as the creator’s thesis. A good thesis example would state what the creator’s argument is, and then what it does well/badly and how it accomplishes that.

Here, you’re going to go more in-depth on the main ideas you outlined in your introduction. Break down what the piece is about. Talk about the characters, the themes, how the creator utilizes these to show their vision and argue their points.

Pretend the reader hasn’t been exposed to the piece of creative work. Don’t hold back, show them what the piece is so they can understand where you are coming from.

The part you’ve waited for. The analysis is broken into multiple body paragraphs, much like other types of essays. Break down your ideas that you stated in your thesis, giving each their own paragraph. Do your best to state both positive and negative parts of the creative work. If you really feel strongly one way or the other, highlight the opposite feeling early on before getting into your opinions. If you hate the film, discuss what it did well at the beginning of your analysis, then get into what it did wrong.

When breaking down your ideas, be sure to discuss the three different types of appeals:

• Ethos: How the creator shows their credibility in their art. Why should they be trusted, or how did they fail to establish credibility?
• Pathos: All art is based on emotion. How did the creators work to appeal to your emotions specifically? Did you agree with how the creator wanted you to feel, or did they miss the mark completely?
• Logos: How does the creator use logic to get you on their side?

At the end of your analysis paragraphs, your paper should clearly state your thoughts and opinions on the creation, have those thoughts supported with examples from the piece, and address whether or not the creator achieved their goal.

Restate your thesis. Say it in a different, but similar, way to reaffirm it. Summarize the main point of each body paragraph, strengthening your thesis even more. Finally, your conclusion should show what the reader shall take away from the creation:

• You shouldn’t watch this movie because it’s offensive to all genders.
• You should read this book because it is an incredibly informative retelling of the Watergate scandal

While working on the planning and pre-planning stages of your essay, be sure to keep these tips in mind for a stronger end product:

Critical Analysis Essay Writing Tips from HandmadeWriting 

• Provoke your reader to rethink their opinions on the piece. Strive to make your opinion so sound that they have no choice but to consider it.
• Be fair. If you’re just whining or mindlessly praising the piece, no one is going to take your analysis seriously. Provide a quality argument with strong examples. Create a strong foundation for your opinion.
• Have fun. You’re speaking your mind on a creation. Really get into why you feel the way you do. If you’re having fun, it will show, and the reader will too. Check out our  paper writing guide  to learn how to write other essay types.

And finally, check your essay upon completion and make sure it follows these guidelines. If it does, you’re set:

Do’s and Don’ts From Our Writing Team

• Take the time to find strong examples both for and against your argument.
• Ensure the reader has a clear vision on what the piece is about before you get into the analysis.
• Be honest. Share your emotions with the reader. It will build a stronger argument, and an apt reader will see right through you being fake.
• Research the piece. Reading up on it will give you some insight you may not have considered, leading to a stronger argument.
• Lean too far one way without discussing the other.
• Write “My thoughts” or “My opinion is”. Just state your opinions.
• Use first or second pronouns. Say “the audience” or “the reader”.
• Spend too much time on the summary. Your analysis should be the biggest part of your paper.

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Critical Analysis in Composition

Glossary of Grammatical and Rhetorical Terms

• An Introduction to Punctuation
• Ph.D., Rhetoric and English, University of Georgia
• M.A., Modern English and American Literature, University of Leicester
• B.A., English, State University of New York

In composition , critical analysis is a careful examination and evaluation of a text , image, or other work or performance.

Performing a critical analysis does not necessarily involve finding fault with a work. On the contrary, a thoughtful critical analysis may help us understand the interaction of the particular elements that contribute to a work's power and effectiveness. For this reason, critical analysis is a central component of academic training; the skill of critical analysis is most often thought of in the context of analyzing a work of art or literature, but the same techniques are useful to build an understanding of texts and resources in any discipline.

In this context, the word "critical" carries a different connotation than in vernacular, everyday speech. "Critical" here does not simply mean pointing out a work's flaws or arguing why it is objectionable by some standard. Instead, it points towards a close reading of that work to gather meaning, as well as to evaluate its merits. The evaluation is not the sole point of critical analysis, which is where it differs from the colloquial meaning of "criticize."

Examples of Critical Essays

• "Jack and Gill: A Mock Criticism" by Joseph Dennie
• "Miss Brill's Fragile Fantasy": A Critical Essay About Katherine Mansfield's Short Story "Miss Brill" and "Poor, Pitiful Miss Brill"
• "On the Knocking at the Gate in Macbeth " by Thomas De Quincey
• A Rhetorical Analysis of Claude McKay's "Africa"
• A Rhetorical Analysis of E B. White's Essay "The Ring of Time"
• A Rhetorical Analysis of U2's "Sunday Bloody Sunday"
• "Saloonio: A Study in Shakespearean Criticism" by Stephen Leacock
• Writing About Fiction: A Critical Essay on Hemingway's Novel The Sun Also Rises

Quotes About Critical Analysis

• " [C]ritical analysis involves breaking down an idea or a statement, such as a claim , and subjecting it to critical thinking in order to test its validity." (Eric Henderson, The Active Reader: Strategies for Academic Reading and Writing . Oxford University Press, 2007)​
• "To write an effective critical analysis, you need to understand the difference between analysis and summary . . . . [A] critical analysis looks beyond the surface of a text—it does far more than summarize a work. A critical analysis isn't simply dashing off a few words about the work in general." ( Why Write?: A Guide to BYU Honors Intensive Writing . Brigham Young University, 2006)
• "Although the main purpose of a critical analysis is not to persuade , you do have the responsibility of organizing a discussion that convinces readers that your analysis is astute." (Robert Frew et al., Survival: A Sequential Program for College Writing . Peek, 1985)

Critical Thinking and Research

"[I]n response to the challenge that a lack of time precludes good, critical analysis , we say that good, critical analysis saves time. How? By helping you be more efficient in terms of the information you gather. Starting from the premise that no practitioner can claim to collect all the available information, there must always be a degree of selection that takes place. By thinking analytically from the outset, you will be in a better position to 'know' which information to collect, which information is likely to be more or less significant and to be clearer about what questions you are seeking to answer." (David Wilkins and Godfred Boahen, Critical Analysis Skills For Social Workers . McGraw-Hill, 2013)

How to Read Text Critically

 "Being critical in academic enquiry means: - adopting an attitude of skepticism or reasoned doubt towards your own and others' knowledge in the field of enquiry . . . - habitually questioning the quality of your own and others' specific claims to knowledge about the field and the means by which these claims were generated; - scrutinizing claims to see how far they are convincing . . .; - respecting others as people at all times. Challenging others' work is acceptable, but challenging their worth as people is not; - being open-minded , willing to be convinced if scrutiny removes your doubts, or to remain unconvinced if it does not; - being constructive by putting your attitude of skepticism and your open-mindedness to work in attempting to achieve a worthwhile goal." (Mike Wallace and Louise Poulson, "Becoming a Critical Consumer of the Literature." Learning to Read Critically in Teaching and Learning , ed. by Louise Poulson and Mike Wallace. SAGE, 2004)

Critically Analyzing Persuasive Ads

"[I]n my first-year composition class, I teach a four-week advertisement analysis project as a way to not only heighten students' awareness of the advertisements they encounter and create on a daily basis but also to encourage students to actively engage in a discussion about critical analysis by examining rhetorical appeals in persuasive contexts. In other words, I ask students to pay closer attention to a part of the pop culture in which they live. " . . . Taken as a whole, my ad analysis project calls for several writing opportunities in which students write essays , responses, reflections, and peer assessments . In the four weeks, we spend a great deal of time discussing the images and texts that make up advertisements, and through writing about them, students are able to heighten their awareness of the cultural 'norms' and stereotypes which are represented and reproduced in this type of communication ." (Allison Smith, Trixie Smith, and Rebecca Bobbitt, Teaching in the Pop Culture Zone: Using Popular Culture in the Composition Classroom . Wadsworth Cengage, 2009)

Critically Analyzing Video Games

 "When dealing with a game's significance, one could analyze the themes of the game be they social, cultural, or even political messages. Most current reviews seem to focus on a game's success: why it is successful, how successful it will be, etc. Although this is an important aspect of what defines the game, it is not critical analysis . Furthermore, the reviewer should dedicate some to time to speaking about what the game has to contribute to its genre (Is it doing something new? Does it present the player with unusual choices? Can it set a new standard for what games of this type should include?)." (Mark Mullen, "On Second Thought . . ." Rhetoric/Composition/Play Through Video Games: Reshaping Theory and Practice , ed. by Richard Colby, Matthew S.S. Johnson, and Rebekah Shultz Colby. Palgrave Macmillan, 2013)

Critical Thinking and Visuals

 "The current critical turn in rhetoric and composition studies underscores the role of the visual, especially the image artifact, in agency. For instance, in Just Advocacy? a collection of essays focusing on the representation of women and children in international advocacy efforts, coeditors Wendy S. Hesford and Wendy Kozol open their introduction with a critical analysis of a documentary based on a picture: the photograph of an unknown Afghan girl taken by Steve McCurry and gracing the cover of National Geographic in 1985. Through an examination of the ideology of the photo's appeal as well as the 'politics of pity' circulating through the documentary, Hesford and Kozol emphasize the power of individual images to shape perceptions, beliefs, actions, and agency." (Kristie S. Fleckenstein, Vision, Rhetoric, and Social Action in the Composition Classroom . Southern Illinois University Press, 2010)

Related Concepts

• Analysis  and  Critical Essay
• Book Report
• Close Reading
• Critical Thinking
• Discourse Analysis
• Evaluation Essay
• Explication
• Problem-Solution
• Rhetorical Analysis
• Definition and Examples of Explication (Analysis)
• Rhetorical Analysis Definition and Examples
• Quotes About Close Reading
• How to Write a Critical Essay
• What Is a Critique in Composition?
• Definition and Examples of Analysis in Composition
• Definition and Examples of Evaluation Essays
• Understanding the Use of Language Through Discourse Analysis
• What Is a Written Summary?
• Critical Thinking in Reading and Composition
• Critical Thinking Definition, Skills, and Examples
• Miss Brill's Fragile Fantasy
• 6 Skills Students Need to Succeed in Social Studies Classes
• Stylistics and Elements of Style in Literature
• Informal Logic
• literary present (verbs)

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Your chance of acceptance, your chancing factors, extracurriculars, what's the general structure of the ap seminar course and exam.

I'm considering taking AP Seminar next year, but I'd like to know more about the course structure and exam format. Can anyone give a brief overview of what to expect in this course and how the exam works? Thanks!

AP Seminar is a unique course that focuses on developing students' research, analytical, and interdisciplinary thinking skills. It's part of the AP Capstone™ program, which also includes AP Research, and is designed to help prepare students for college-level research and academic success.

Course Structure:

In AP Seminar, you will undertake several major projects and participate in regular class discussions throughout the year. The course includes exploring various topics, themes, and viewpoints by critically analyzing varied sources. You will work individually and in teams to research, discuss, and synthesize different perspectives, creating evidence-based arguments to demonstrate your understanding. Students will develop and present research projects, honing their oral communication skills too.

Exam Format:

The AP Seminar exam is divided into three main components:

1. Team Project and Presentation (Team PTP): This component constitutes 20% of the overall AP Seminar score. You and your teammates will collaborate on a research project, selecting a theme and crafting a team question or problem based on that theme. You'll then divide the work and carry out research individually before coming together to create a team presentation, addressing various perspectives and implications.

2. Individual Research Report (IRR): Making up 35% of the AP Seminar score, the IRR is a 2,000-word research paper, developed by each student individually. The paper should delve into a topic related to the team project, providing evidence-based arguments and analysis while demonstrating critical thinking and research skills.

3. End-of-Course Exam: The last component, which constitutes 45% of the overall score, is a 2-hour exam typically taken in May. The exam has two main sections, both involving reading, analyzing, and responding to stimulus material, such as articles, infographics, and data sets.

Section 1 (Short Answer Responses): In this section, you'll analyze various sources and write three short, focused responses to questions provided. You'll need to examine, evaluate and synthesize the given information to form evidence-based arguments.

Section 2 (Essay Question): Here, you'll be asked to develop a well-reasoned argument with a strong thesis statement based on provided stimulus material. You'll need to synthesize the information into a coherent essay, incorporating evidence to support your position.

In conclusion, AP Seminar offers a diverse learning experience that diverges from the typical content-specific AP courses. It focuses on honing research, analytical, and presentation skills, which are valuable for college and beyond. Assessments will challenge you to conduct research, work collaboratively, and develop strong arguments through various mediums such as reports, presentations, and timed exams. Good luck!

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• Open access
• Published: 14 May 2024

Identification of novel single nucleotide variants in the drug resistance mechanism of Mycobacterium tuberculosis isolates by whole-genome analysis

• Weiye Qian 1 ,
• Xi Zeng 2 ,
• Mai Shi 3 ,
• Mingqiang Wang 4 ,
• Zhiyuan Yang 1 , 3 &
• Stephen Kwok-Wing Tsui 3 , 5  

BMC Genomics volume  25 , Article number:  478 ( 2024 ) Cite this article

Metrics details

Tuberculosis (TB) represents a major global health challenge. Drug resistance in Mycobacterium tuberculosis (MTB) poses a substantial obstacle to effective TB treatment. Identifying genomic mutations in MTB isolates holds promise for unraveling the underlying mechanisms of drug resistance in this bacterium.

In this study, we investigated the roles of single nucleotide variants (SNVs) in MTB isolates resistant to four antibiotics (moxifloxacin, ofloxacin, amikacin, and capreomycin) through whole-genome analysis. We identified the drug-resistance-associated SNVs by comparing the genomes of MTB isolates with reference genomes using the MuMmer4 tool.

We observed a strikingly high proportion (94.2%) of MTB isolates resistant to ofloxacin, underscoring the current prevalence of drug resistance in MTB. An average of 3529 SNVs were detected in a single ofloxacin-resistant isolate, indicating a mutation rate of approximately 0.08% under the selective pressure of ofloxacin exposure. We identified a set of 60 SNVs associated with extensively drug-resistant tuberculosis (XDR-TB), among which 42 SNVs were non-synonymous mutations located in the coding regions of nine key genes (ctpI, desA3, mce1R, moeB1, ndhA, PE_PGRS4, PPE18, rpsA, secF). Protein structure modeling revealed that SNVs of three genes (PE_PGRS4, desA3, secF) are close to the critical catalytic active sites in the three-dimensional structure of the coding proteins.

This comprehensive study elucidates novel resistance mechanisms in MTB against antibiotics, paving the way for future design and development of anti-tuberculosis drugs.

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Introduction

Tuberculosis (TB), primarily caused by Mycobacterium tuberculosis (MTB), is one of the major epidemics worldwide, with a high mortality rate surpassing that of any other infectious disease [ 1 ]. In 2021, the World Health Organization estimated that around 10 million people were affected by tuberculosis, leading to 1.6 million deaths [ 2 ]. TB is a treatable and curable disease, often managed through a combination of antibiotics. However, a critical challenge in TB treatment lies in the widespread drug-resistance mechanisms manifested by MTB [ 3 ].

Drug-resistant tuberculosis can be classified into various categories, including multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) [ 4 ]. MDR-TB exhibits resistance to isoniazid and rifampicin, while XDR-TB exhibits resistance to all first-line drugs and at least one second-line drug [ 5 ]. Gupta et al. reported that ofloxacin resistance is significantly high among multidrug-resistant MTB strains across India, most of which were especially associated with the Beijing genotype and carried gyrA mutations [ 6 ]. Fluoroquinolone is the classical representative of first-line drugs in TB treatment. MTB acquires resistance to fluoroquinolones mainly through mutations in the quinolone resistance-determining region of the pknB gene [ 7 ]. Moxifloxacin is a common second-line drug in the treatment of pneumonia and tuberculosis infections [ 8 ]. Clinical trials have shown that moxifloxacin can improve standard treatment regimens with better bactericidal activity [ 9 ]. Understanding the mechanisms of drug resistance in MTB is crucial for developing new treatment strategies and improving the management of drug-resistant TB cases.

MTB is characterized by several genomic features that contribute to its adaptability, virulence, and ability to survive within host organisms. In 2013, the reference genome assembly ASM19595v2 of MTB H37Rv was published. Within the genomic landscape of MTB, high-frequency genomic mutations play pivotal roles in drug-resistance mechanisms [ 10 ]. In our previous studies, we have identified the function of hypothetical proteins in the MTB genome and explored the drug-resistance mechanism of insertions and deletions in 1110 MTB isolates [ 11 , 12 ]. In bacteria, gene mutations can contribute to genetic diversity and lead to the emergence of strains resistant to specific drugs. For instance, it was reported that mutations in gene rpoB, which codes for the RNA polymerase beta subunit, were associated with rifampin resistance in MTB [ 13 ]. Mutations in gene katG were linked to isoniazid resistance [ 14 ]. Thus, investigation of genomic mutations in drug-resistant isolates could help uncover novel drug-resistance mechanisms in MTB.

Single nucleotide variant (SNV) is a mutation at a single position of the genome sequence, often used as a marker to study the association between drug-resistance features [ 15 ]. SNVs can be divided into synonymous mutations and non-synonymous mutations, according to their functional consequences. Non-synonymous mutations are genetic variations that result in a change to the amino acid sequence of the encoded protein, potentially altering the structure and function of the protein.

In this study, we analyzed the whole genome of 716 clinical MTB isolates to identify the non-synonymous SNVs in XDR strains. We determined SNVs associated with drug resistance by comparing the whole genomes of XDR isolates against the MTB reference genome. We compared the frequencies of resistant and susceptible strains for different antibiotics using Fisher’s exact test. The non-synonymous mutations in some key genes were reported and discussed. Our findings suggested a novel view of drug-resistance mechanisms in MTB.

WGS data analysis on antibiotic resistance

Our focus centered on exploring the resistance mechanisms of MTB isolates resistant to two fluoroquinolones, namely moxifloxacin and ofloxacin, as well as two second-line drugs, amikacin and capreomycin. The whole-genome sequences of 716 MTB isolates which were tested with at least one drug mentioned above were collected from the BV-BRC database (Fig.  1 ). Resistance to different drugs varies significantly between geographical locations. For example, for amikacin, Belarus, Russia, and South Korea show relatively high resistance, accounting for 40.01%, 29.2%, and 21.9% respectively, while Iran, Romania, and Uzbekistan have lower resistance. Other drugs such as capreomycin, moxifloxacin, and ofloxacin showed similar trends (Fig.  1 C). In this study, a group of 83 MTB isolates were tested by all four antibiotics. The testing results revealed that a set of 251 MTB isolates exhibited resistance to moxifloxacin, while a larger set of 645 isolates demonstrated resistance to ofloxacin (Table  1 ). Additionally, 283 MTB isolates exhibited resistance to amikacin, and the same number of MTB isolates displayed resistance to capreomycin. The isolates resistant to ofloxacin accounted for the highest proportion, with 94.2% in testing samples, indicating that MTB is more prone to mutations induced by exposure to ofloxacin.

Whole-genome sequencing samples obtained in this study. ( A ) Number of MTB isolates tested by at least 1, 2, 3, or 4 antibiotics; ( B ) Venn diagram of samples tested by four antibiotics; ( C ) prevalence of drug resistance

SNV calling by MuMer4

The WGS data of MTB isolates resistant to antibiotics were compared against the H37Rv reference genome using MUMmer4 to identify possible SNVs. The frequency and proportion of SNVs in MTB isolates resistant to the four antibiotics are also calculated (Table  2 ). Frequency represents the count of a certain mutation that occurs, while proportion represents the proportion of this mutation in the total count of mutations. As a result, we found that the mutation patterns were similar in the MTB isolates resistant to the four antibiotics. Notably, the proportions of T-> A mutations were consistently low across all antibiotics, accounting for only 1.6%, 1.53%, 1.55%, and 1.61%, respectively (Fig.  2 A). In contrast, A-> G mutations were the most predominant, which accounted for around 14% of isolates resistant to three antibiotics (ofloxacin, moxifloxacin, and amikacin). Proportions of all the other mutations fell within the range of 2%-14%.

The average mutation number of the four antibiotics is presented in Fig.  2 B. The greatest number of mutations were found in the ofloxacin-resistant MTB (an average of 3529 mutations in each MTB isolate). The fewest mutation number was found in the moxifloxacin-resistant MTB (an average of 1370 mutations in each MTB isolate). Considering the size of MTB genome (4,411,532 base pairs [ 16 ]), we estimated the mutation rate of one base as 0.080% in ofloxacin-resistant isolates and 0.031% in moxifloxacin-resistant MTB.

Mutation summary in antibiotic-resistant isolates. ( A ) Average proportion of different mutations in total amount; ( B ) Average mutation number in MTB isolates resistant to four different antibiotics

SNV screening by Fisher’s exact test

After identifying mutations in MTB, we employed Fisher’s exact test to determine SNVs that distributed differently between resistant and susceptible strains. The detailed number of significant SNVs and the type of mutation for each antibiotic are presented in Table  3 . The number of non-synonymous (NS) mutations is larger than that of the synonymous mutations in all MTB isolates. The proportion of NS mutations is around 70% in three antibiotics (moxifloxacin, ofloxacin, amikacin) and 66.8% in capreomycin. A total of 3,536 and 3,541 NS mutations were found in MTB isolates resistant to moxifloxacin and ofloxacin, respectively. In addition, the frequency of screened SNVs showed significant differences in distribution between moxifloxacin-susceptible and moxifloxacin-resistant strains ( P  ≤ 0.001), but such differential distribution was not observed in the other three strains (Fig.  3 ). In moxifloxacin-resistant strains, the mutation frequency ranged from 0% to 20%, while in moxifloxacin-susceptible strains, it spanned from 5% to 15%. The median mutation frequency in susceptible strains surpassed that in resistant strains. These results indicated that moxifloxacin pressure induced more NS mutations and affected the evolution mechanism in MTB isolates.

Box plot of frequency rate of SNVs in susceptible and resistant MTB isolates of four drugs

Common SNVs related to four antibiotics

By overlap analysis of Venn diagram, a small set of 18 significant SNVs was found in MTB strains resistant to all four antibiotics, accounting for 0.2% of total identified SNVs (Fig.  4 A). The details of these 18 significant SNVs are detailed in Table  4 . Among these, three SNVs (A300922G, T3379432C, T3381356C) are located in non-coding regions. Another SNV T103849C located in the coding region of Rv0094c, which encoded a hypothetical protein (accession number NP_214608.1). Information of codon mutations and amino acid mutations was also obtained. The rest 14 significant SNVs were specifically enriched in the coding region of gene PE_PGRS4. Among them, nine SNVs were NS mutations that cause amino acid changes in the encoded protein and five SNVs were synonymous mutations. Of the nine NS mutations, five SNV mutations resulted in an amino acid change from hydrophobic (pho) AA to hydrophilic (phi) AA, while three SNVs led to a reverse mutation from phi AA to pho AA (Fig.  4 B). These results suggest that most SNVs could change the physiochemical characteristics of coding amino acids.

Common SNVs in MTB isolates are resistant to four antibiotics. ( A ) Venn diagram of common SNVs in MTB isolates resistant to different drugs; ( B ) Hydrophilic and hydrophobic analysis of translated amino acid of common SNVs.

Common genes in XDR-TB

The XDR-TB is defined as isolates that are resistant to first-line drugs (moxifloxacin and ofloxacin) and a least one second-line drug, i.e., the isolates resistant to three antibiotics (moxifloxacin, ofloxacin, capreomycin) or (moxifloxacin, ofloxacin, amikacin). In the case of XDR-TB resistant to moxifloxacin, ofloxacin, and capreomycin, we only found PE_PGRS4 and Rv0094c. In another case of XDR-TB resistant to moxifloxacin, ofloxacin, and amikacin, we identified more genes mutated, with a set of 60 significant SNVs (Fig.  5 A and Table S1 ). Of the 60 SNVs, 51 were located within the coding region while the remaining nine were found in non-coding regions. This result indicated that the drug-resistance-associated SNVs were mostly enriched in the coding region of genes. In addition, 20 SNVs were NS mutations corresponding to 20 genes (ctpI, desA3, mce1R, moeB1, ndhA, PPE18, rpsA, secF, Rv0311, Rv0347, Rv0654, Rv0698, Rv0888, Rv0923c, Rv1431, Rv1672c, Rv1692, Rv1836c, Rv2028c, Rv3630). The details of these 20 genes are shown in Table  5 . Out of these mutations, seven SNVs cause the coding AA to change from hydrophobic to hydrophilic, and other six SNVs do not change hydrophobic features (Fig.  5 B). These results suggest that MTB proteins tend to mutate from hydrophilic to evade the hydrolysis by the host enzymes.

Common SNVs in XDR-TB with resistance to three antibiotics (moxifloxacin, ofloxacin, and amikacin). ( A ) Venn diagram of common SNVs in three antibiotics; ( B ) Hydrophilic and hydrophobic analysis of translated amino acid of common SNVs

Three-dimensional protein structures of identified genes

Understanding three-dimensional (3D) structures of proteins is crucial for unraveling their functions and interactions in biological processes. We so far have identified 22 genes related to drug-resistance mechanisms in MTB, including 13 genes with only locus information (Rv0094c, Rv0311, Rv0347, Rv0654, Rv0698, Rv0888, Rv0923c, Rv1431, Rv1672c, Rv1692, Rv1836c, Rv2028c, Rv3630) and nine genes with more detailed information (ctpI, desA3, mce1R, moeB1, ndhA, PE_PGRS4, PPE18, rpsA, secF). We constructed the 3D structure of coding proteins for these nine genes by SWISS-MODEL and the parameters of these structures are shown in Table  6 . Results showed the sequence identity scores with all templates were larger than 70%, indicating a high similarity between the template and our proteins. In particular, the structural templates of six proteins (PE_PGRS4, ctpI, moeB1, rpsA, ndhA, and PPE18) were identical to the homologous protein. The Global Model Quality Estimation (GMQE) value, is common index used for model quality evaluation in SWISS-MODEL. The GMQE values of all proteins are larger than 0.6 in 3D model. Specifically, the GMQE values of five coding proteins (PE_PGRS4, desA3, mce1R, moeB1, and ndhA) are larger than 0.9, suggesting the robust and reliable of AlphaFold method in the prediction of the three-dimensional structures in SWISS-MODEL.

Our analysis revealed four important SNV cluster regions within the PE_PGRS4 gene, highlighting its key role in various antibiotic-resistant strains. The 3D structure analysis showed that the SNV mutations of PE_PGRS4 were exclusively located within its β-folding region (Fig.  6 ). Results showed that Arg at position 174 exhibited three types of mutations (Arg-> Gly, Arg-> Met, and Arg-> Ser), while Ala at position 188 had two types of mutations (Ala-> Asp and Ala-> Thr). Additionally, Thr, Thr, Ala, and Ala at positions 177, 179, 182, and 196 experienced mutations to Arg, Ile, Ser, and Gly, respectively. The active site residue Ala is at position 196. Taken together, the amino acid positions from 174 to 188 in β-folding region of PE_PGRS4 gene were speculated to serve as the important catalytic sites in the protein function. In addition, the other eight genes found in isolates resistant to the other three antibiotics testing also displayed a solitary NS mutation, as illustrated in Fig.  7 . Mutations in the five genes (ctpI, moeB1, rpsA, secF, PPE18) were situated within the α-helix structure, while mutations of rest genes (desA3, mce1R, and ndhA) located within the β-sheet structure.

The positions of the amino acid mutation are caused by all non-synonymous SNVs in the 3D structure of the PE_PGRS4 protein

The positions of the amino acid mutations were caused by non-synonymous SNVs in 3D structure of the coding proteins of identified genes

Identify critical active sites affected by SNVs

To uncover possible drug-resistance mechanisms, active sites of coding proteins of above genes were found by PrankWeb. The active sites affected by SNVs are shown in Table  7 . Among these proteins, six (PE_PGRS4, desA3, mce1R, moeB1, secF, ndhA) of them were found at active sites nearby the position of amino acid mutations translated by SNVs. In particular, the position of mutated amino acids of three genes (PE_PGRS4, DesA3, secF) were closely near the active sites. The active site 172 of PE_PGRS4 was only two residues away from the mutation site 174. The mutation site 196 of desA is also the active site. The mutation site 79 of secF was located between active sites 77 and 78. These findings underscored the structural alterations induced by genetic mutations in key MTB genes, shedding light on potential implications for protein function and, by extension, drug resistance mechanisms.

Mycobacterium tuberculosis is a bacterium that causes tuberculosis (TB), a potentially serious infectious disease that primarily affects the lungs. TB remains a significant global health concern, and efforts to control and eliminate the disease are ongoing. MTB is known for its slow growth rate compared to many other bacteria. However, MTB could be spread through the air when an infected person coughs or sneezes, releasing tiny droplets containing the bacteria [ 17 ]. Understanding the genome of MTB is essential for unraveling the mechanisms of its pathogenicity, drug resistance, and host interactions.

MTB have developed resistance to various drugs through a combination of genetic mutations and selective pressures [ 18 ]. The emergence of drug-resistant strains poses a significant challenge to TB control and treatment efforts. The drug resistance phenomenon could decrease the sensitivity of MTB strains and diminish the efficacy of available antibiotics [ 19 ]. This pathogen can carry antigenic mutation, allowing it to evade the immune response of host. Such mutations involve changes in surface antigens, making it more challenging for the immune system to recognize and eliminate the bacterium [ 20 ]. Understanding the mechanisms of drug resistance in MTB is crucial for developing new treatment strategies and improving the management of drug-resistant TB cases.

In this study, whole-genome sequencing (WGS) data of 716 clinical MTB isolates were analyzed for their drug-resistance mechanisms to two first-line drugs (moxifloxacin and ofloxacin) and two second-line drugs (amikacin and capreomycin). Moxifloxacin and ofloxacin are both fluoroquinolone antibiotics that target bacterial DNA synthesis and protein synthesis [ 21 ]. Amikacin inhibits translocation by binding peptide tRNA at the ribosomal A-site, thereby suppressing protein synthesis and rendering bacteria unable to survive [ 22 ]. Capreomycin, a ribosome-targeting peptide antibiotic, inhibits tRNA binding by interacting with the ribosome, thereby inhibiting protein synthesis [ 23 ]. We found a high proportion (94.2%) of testing isolates showed resistance to ofloxacin, indicating that this drug may not be suitable for the treatment of MTB. These MTB isolates were compared with H37Rv reference genome by MuMmer4 to identify SNVs. An average number of 3529 SNVs were observed per ofloxacin-resistant MTB isolate, with a mutation rate of around 0.08% under the selection pressure of ofloxacin.

As only a minority of SNVs influence MTB drug resistance mechanisms, Fisher’s exact test was employed to compare the mutation frequencies in the resistant and susceptible strains exposed to the four antibiotics (moxifloxacin, ofloxacin, amikacin, and capreomycin). At a significance threshold of p-value < 0.05, we found a total of 3536 and 3541 SNVs in moxifloxacin-resistant and ofloxacin-resistant MTB isolates respectively. The number of NS mutations is larger than that of the synonymous mutations in all four antibiotics.

To understand the resistance mechanisms in MTB, we examined 18 shared SNVs associated with these four antibiotics. Of the 18 SNVs, we found nine non-synonymous SNVs located in the coding region of PE_PGRS4 and Rv0094c. Previous study showed that the transcriptional expression profile of PE_PGRS4 was constitutively expressed and up-regulated under the circumstances of many antibiotics [ 24 ]. Besides, the mutations of PE_PGRS4 gene have been previously demonstrated in drug-resistant MTB [ 25 ]. Other SNVs in non-coding regions, such as A300922G, T3379432C, and T3381356C, may also have potential functional relevance. While these mutations may not directly impact protein coding, they could play a regulatory role in MTB. Regulatory variants have potential to exert a substantial influence on phenotype, emphasizing their significance in understanding the broader implications of these genetic mutations [ 26 ].

To further analyze the drug-resistant mechanism in XDR-TB, we identified a set of 60 shared SNVs related to three antibiotics (moxifloxacin, ofloxacin, and capreomycin). In addition to the 18 shared SNVs identified in MTB resistant to four antibiotics, we also identified 42 SNVs located in the coding region of eight genes (ctpI, desA3, mce1R, moeB1, ndhA, PPE18, rpsA, and secF). Previous studies indicated that these genes were highly related to drug-resistant mechanisms in XDR-TB, for example, rpsA, secF, and desA3. The rpsA protein plays a crucial role in translation initiation and mRNA binding during protein synthesis. Due to its essential role in protein synthesis, protein rpsA is considered a potential target for the development of antimicrobial drugs [ 27 ]. The secF protein is involved in the process of protein secretion across the bacterial inner membrane. The disruptions in protein secretion processes can potentially impact the physiology of the bacterium, thus the secF mutation is considered to be related to drug resistance in MTB [ 28 ]. The desA3 gene is associated with the biosynthesis of oleic acids, which is essential for the formation of the cell wall of actively replicating bacteria [ 29 , 30 ]. The mutation of these genes may highly affect their function, which results in drug-resistant events in MTB.

We then constructed the 3D model of the coding protein of these nine genes. 3D structural analysis showed that SNV locations of three genes (PE_PGRS4, DesA3, and secF) were close to the active sites. These structural changes may have implications for protein structure and function, including protein stability, folding state, and interactions. For example, the mutations at position 174 are from arginine to glycine, resulting in the atom number also changed significantly. Specifically, arginine has six atoms on its side chain, while glycine has only one atom on its side chains. Previous studies showed that glycine-to-arginine mutations could cause conformational change and impaired transition metal transport in bacteria [ 31 ]. This result enhanced that the identified SNVs could be highly associated with drug-resistance mechanisms in MTB.

In this study, we obtained 716 MTB isolates tested by four antibiotics and identified the mutated SNVs by MuMmer4. Fisher’s exact test was applied to whole-genome sequencing data from clinically relevant MTB isolates to identify SNVs related to drug resistance. Non-synonymous mutations within the PE_PGRS4 gene were found in strains resistant to four antibiotics, highlighting the potential of PE_PGRS4 as a biomarker of broad-spectrum resistance. Our exploration of XDR-TB isolates revealed distinctive roles of another eight genes (ctpI, desA3, mce1R, moeB1, ndhA, PPE18, rpsA, and secF) in resisting second-line drugs. This study illustrates the complexity of drug-resistant tuberculosis and emphasizes the significance of non-synonymous SNVs in MTB drug resistance. These findings not only advance our comprehension of MTB resistance mechanisms but also offer insights for targeted intervention development.

Materials and methods

Wgs data retrieving and analysis.

Whole-genome sequencing (WGS) data of 716 MTB isolates (Table S2 ) were obtained from the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) database [ 32 ]. The MTB H37Rv reference genome was downloaded from the National Center for Biotechnology Information (NCBI) Genome database. The gene annotation file was also retrieved from the NCBI database.

SNV calling by MuMmer4

SNVs are the most common type of genetic mutation among pathogens and are often used as biomarkers to study the association between genetic mutations and bacterial traits. These SNVs represent mutations arising from point mutations at precise gene locations, resulting in resistance against certain antibiotics in bacteria [ 33 ]. The WGS data of MTB isolates resistant to antibiotics were compared against the H37Rv reference genome using MUMmer4 to identify possible SNVs by the script ‘nucmer reference_file.fasta query_file.fasta out-file.delta show-snps -Clr out.delta’. MUMmer4 presents a notable performance in processing large genomes, enhanced speed, compatibility with scripting languages, and suitability for SNV calling [ 34 ].

While most genomic mutations have minimal effects on phenotype, only a few SNVs were expected to strongly influence complex traits of bacteria [ 35 ]. In this study, a group of 716 strains were categorized as either resistant or sensitive to four antibiotics (moxifloxacin, ofloxacin, amikacin, and capreomycin). Subsequently, Fisher’s exact test was employed to compare the frequencies of resistant and non-resistant strains for each antibiotic and SNVs were screened at a threshold of statistical p-value < 0.05. Fisher’s exact test, which is recognized for its better accuracy with a small sample test, was chosen to assess the null hypothesis of SNVs in drug-resistant features [ 36 ]. For isolates resistant to moxifloxacin, ofloxacin, amikacin, and capreomycin, we identified 4996, 5026, 1730, and 2864 significant SNVs, respectively.

The formula for Fisher’s exact test is as follows:

• A: The number of strains with a given SNV in resistant strains.
• B: The number of strains without the given SNV in resistant strains.
• C: The number of strains with the given SNV in non-resistant strains.
• D: The number of strains without the given SNV in non-resistant strains.

SNV cluster regions, defined by the presence of multiple neighboring SNVs in the same or adjacent genomic regions, potentially influence MTB resistance [ 37 ]. We investigated the common resistance mechanism among the four antibiotic-resistant strains to identify shared SNVs among them. The genes associated with these SNVs were also identified based on their genomic location. Only PE_PGRS4 and Rv0094c were found in the SNVs related to four antibiotic-resistant isolates. Thus, the biological parameters of these two genes were further analyzed.

The XDR-TB was defined as those cases resistant to all first-line drugs (mainly fluoroquinolones) and a least one second-line drug [ 5 ]. To further investigate the resistance mechanisms associated with XDR-TB, we analyzed the shared significant SNVs for two fluoroquinolones (moxifloxacin and ofloxacin) and one of second-line drugs (amikacin and capreomycin). Thus, the two cases of XDR-TB (two fluoroquinolones with ofloxacin, and two fluoroquinolones with capreomycin) were further analyzed. The involved transcribed amino acids and proteins of these genes were identified to distinguish synonymous and non-synonymous (NS) mutations.

Construction of three-dimensional structures of identified proteins

Protein sequences, coupled with their three-dimensional (3D) structures, provide critical information for understanding protein functions, interactions, and biological processes [ 38 ]. In this study, three-dimensional structures of proteins with non-synonymous mutations were constructed by SWOSS-MODEL, the online server for automated protein homology modeling [ 39 ]. The SWISS-MODEL could incorporate features to predict the structure and stoichiometry parameters of complexes based on the amino acid sequences of interacting proteins. A novel modeling engine (AlphaFold) and a local model quality assessment method (QMEANDisCo) were applied to enhance the accuracy of protein modeling in SWISS-MODEL [ 40 ].

Mutations in the active site can lead to dramatic changes in protein activity and affect the efficiency of binding drugs, thus identification of the active site is necessary. In this study, protein active sites were predicted by PrankWeb, which adopted deep learning model to characterize the binding site of protein and the ligand [ 41 ]. PrankWeb is a user-friendly web tool that allows users to enter a UniProt accession number as the input. The predicted active sites were then compared with the locus of amino acid changes caused by SNVs to identify critical active sites.

Data availability

All genome sequencing data for this analysis are available in the BV-BRC database (https://www.bv-brc.org/).

Abbreviations

Tuberculosis

• Mycobacterium tuberculosis
• Whole-genome sequencing

Non-synonymous mutation

Synonymous mutation

• Single nucleotide variant

Hydrophilic

Hydrophobic

Extensively drug-resistant tuberculosis

Multidrug-resistant tuberculosis

National Center for Biotechnology Information

Bacterial and Viral Bioinformatics Resource Center

Three-dimensional

Global Model Quality Estimation

AlphaFold Protein Structure Database

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This research was funded by National Natural Science Foundation of China (grant number 61903107).

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Weiye Qian, Nan Ma & Zhiyuan Yang

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Mai Shi, Zhiyuan Yang & Stephen Kwok-Wing Tsui

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Zhiyuan Yang and Stephen Tsui conceptualized and designed the project. Weiye Qian, Nan Ma, Xi Zeng, Mai Shi, and Mingqiang Wang analyzed data and draw the figures. Weiye Qian wrote the draft manuscript and Zhiyuan Yang reviewed the manuscript. All authors have read and approved the final manuscript.

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Qian, W., Ma, N., Zeng, X. et al. Identification of novel single nucleotide variants in the drug resistance mechanism of Mycobacterium tuberculosis isolates by whole-genome analysis. BMC Genomics 25 , 478 (2024). https://doi.org/10.1186/s12864-024-10390-3

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M. Sholikhah Department of Mechanical Engineering, Universitas Sebelas Maret, Surakarta, Indonesia

R. Ridwan Department of Mechanical Engineering, Universitas Merdeka Madiun, Madiun, Indonesia

T. Ghanbari-Ghazijahani Faculty of Science and Engineering, School of Engineering, Macquarie University, New South Wales, Australia

I. Yaningsih Department of Mechanical Engineering, Universitas Sebelas Maret, Surakarta, Indonesia

N. Muhayat Department of Mechanical Engineering, Universitas Sebelas Maret, Surakarta, Indonesia

D. D. D. P. Tjahjana Department of Mechanical Engineering, Universitas Sebelas Maret, Surakarta, Indonesia

R. Adiputra Research Center for Hydrodynamics Technology, National Research and Innovation Agency (BRIN), Surabaya, Indonesia

J. M. Sohn Department of Naval Architecture and Marine Systems Engineering, Pukyong National University, Busan, Korea, Republic of

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• Published: 16 May 2024

Genome-wide identification and expression analysis of Ubiquitin-specific protease gene family in maize ( Zea mays L.)

• Weichao Fu 1 , 2 ,
• Delong Fan 1 , 2 ,
• Shenkui Liu 3 &
• Yuanyuan Bu   ORCID: orcid.org/0000-0002-7133-613X 1 , 2  

BMC Plant Biology volume  24 , Article number:  404 ( 2024 ) Cite this article

Metrics details

Ubiquitin-specific proteases (UBPs) are a large family of deubiquitinating enzymes (DUBs). They are widespread in plants and are critical for plant growth, development, and response to external stresses. However, there are few studies on the functional characteristics of the UBP gene family in the important staple crop, maize ( Zea mays L.).

In this study, we performed a bioinformatic analysis of the entire maize genome and identified 45 UBP genes. Phylogenetic analysis indicated that 45 ZmUBP genes can be divided into 15 subfamilies. Analysis of evolutionary patterns and divergence levels indicated that ZmUBP genes were present before the isolation of dicotyledons, were highly conserved and subjected to purifying selection during evolution. Most ZmUBP genes exhibited different expression levels in different tissues and developmental stages. Based on transcriptome data and promoter element analysis, we selected eight ZmUBP genes whose promoters contained a large number of plant hormones and stress response elements and were up-regulated under different abiotic stresses for RT-qPCR analysis, results showed that these genes responded to abiotic stresses and phytohormones to varying degrees, indicating that they play important roles in plant growth and stress response.

Conclusions

In this study, the structure, location and evolutionary relationship of maize UBP gene family members were analyzed for the first time, and the ZmUBP genes that may be involved in stress response and plant growth were identified by combining promoter element analysis, transcriptome data and RT-qPCR analysis. This study informs research on the involvement of maize deubiquitination in stress response.

Peer Review reports

Ubiquitin-specific proteases (UBPs), the largest subfamily of plant deubiquitinating enzymes (DUBs), are involved in diverse physiological processes such as plant growth and development [ 1 , 2 , 3 , 4 , 5 ], as well as stress response [ 6 , 7 , 8 ]. According to previous studies, eukaryotic DUBs can be classified into five subfamilies and two types based on their catalytic domains: cysteine proteases and metalloproteinases [ 9 , 10 ], with UBPs falling into the cysteine protease category [ 11 ]. UBP contains the Ub carboxy-terminal hydrolase (UCH) domain, which is mainly composed of two conserved motifs, cysteine cysteine (Cys) and histidine histidine (His) boxes, and plays an important role in the deubiquitination of plants [ 12 ]. Notably, the number of UBP genes varies significantly across species. For instance, the Arabidopsis ( Arabidopsis thaliana ) genome encodes 27 UBP genes [ 13 ], 25 genes in rice ( Oryza sativa ) [ 14 ], 48 genes in moso bamboo ( Phyllostachys edulis ) [ 15 ], and 97 in wheat ( Triticum aestivum ) [ 16 ]. The functions of Arabidopsis UBP genes have been extensively studied. Previous research has shown that AtUBP3 and AtUBP4 play crucial roles in male gametophyte development [ 1 ]. AtUBP14 plays a role in the early embryonic development of plants [ 17 ]. AtUBP26 , on the other hand, is involved in the ubiquitination modification process of histones and is essential for seed development [ 2 , 18 ]. Furthermore, a recent study has revealed that UBP15 plays a significant role in regulating seed development in both Arabidopsis and rice. UBP15 modulates organ development and seed size in an opposing manner to the ubiquitin receptor DA1, and it positively regulates seed size by promoting cell proliferation in the maternal bead tissues [ 19 ]. OsUBP15 directly interacts with OsDA1 to positively regulate the length and width of rice seeds [ 20 ]. Loss of AtUBP1 and AtUBP2 function results in hypersensitivity of plants to the amino acid analogue canavaline (CAN) and severe dwarfing, short root development, and yellowing of leaves [ 13 ]. Overexpression of UBP12 / UBP13 can increase the NAC domain transcription factor ORE1 level and positively regulate leaf senescence induced by nitrogen deficiency [ 4 ]. In addition, the deubiquitination enzymes UBP12 and UBP13 regulate the growth process of plants under nitrogen deficiency and positively regulate the recovery process after carbon starvation by regulating the stability of Arabidopsis BES1. In addition, UBP12 and UBP13 directly interact with RGF1 receptors to counteract RGF1-induced ubiquitination and promote root meristem development [ 21 ]. UBP12 and UBP13 also play an important role in the regulation of plant flowering time and the biological clock of plants [ 5 ]. UBPs have been shown to play an important role in stress responses, such as the plant immune response, drought response mediated by the ABA signaling pathway, salt response and other biological processes [ 6 , 8 , 22 , 23 , 24 ].

Maize is one of the world’s leading crops and is of considerable value to feed, food, pharmaceutical, and other industries [ 25 , 26 ]. However, the UBP gene family in maize has not been extensively studied. To date, only three ZmUBP genes ( ZmUBP15 , ZmUBP16 and ZmUBP19 ) have been characterized in maize [ 27 ]. ZmUBP15 , ZmUBP16 and ZmUBP19 are the three homologous genes of Arabidopsis UBP16 in maize, which play similar functions to Arabidopsis UBP16 in response to salt stress. ZmUBP15 , ZmUBP16 and ZmUBP19 expression levels are reduced under salt stress and partially rescue the salt-sensitive phenotype of Arabidopsis ubp16-1 mutants, significantly enhancing the tolerance of ubp16-1 mutants to salt stress [ 27 ]. In order to investigate the functions played by members of the maize UBP gene family in plant growth and development and stress response, we identified 45 ZmUBP genes in maize genome-wide, and their conserved motifs, gene structures, chromosome distributions, and expression patterns were analyzed. To understand their evolutionary relationship with other plants, a phylogenetic tree was constructed. Furthermore, the expression profiles of the ZmUBP genes under abiotic stresses and hormone conditions were assessed by using RT-qPCR. The findings of our study will help to understand the roles of ZmUBP genes in the stress response and to further identify the functions of this essential gene family in maize.

Identification of UBP genes in maize

The hidden Markov model (HMM) profile of the UCH domain (PF00443) obtained from the Pfam database ( http://pfam.xfam.org/ ) was used to blast the maize protein sequence file using the local HMMER 3.0 program [ 28 ]. The E-value was limited to less than 1 × 10 −18 . All the identified ZmUBP candidates were verified using the Pfam database ( http://pfam.xfam.org/ ). Proteins that did not have the UCH protein domain with highly conserved Cys residues (Cys-box) as well as His and Asp/Asn residues (His-box) were excluded. We then turned to the NCBI CD search ( https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi ) to further annotate these genes. With the essential information on hand, we bioinformatically analyzed the ZmUBP genes using ExPASy ( http://www.expasy.ch/tools/pi_tool.html ). This analysis allowed us to determine the molecular weight (MW) and isoelectric point (pI) of the ZmUBP proteins. To gain a deeper understanding of their structural features, we predicted transmembrane structural domains using TMHMM ( http://www.cbs.dtu.dk/services/TMHMM ). Additionally, we utilized Plant-Ploc ( http://www.csbio.sjtu.edu.cn/ ) to predict the hydrophobicity of the ZmUBP proteins.

Analysis of ZmUBP gene structure and protein structure

To analyze the structure of ZmUBP genes, we used TBtools [ 29 , 30 ] to compare CDS of the ZmUBP gene family with genomic DNA, mapped exon-intron structure, and predicted protein conserved motifs using MEME ( https://meme-suite.org/ ). These motifs were mapped with TBtools [ 29 , 30 ]. Finally, we combined the Gene Structure, protein conserved motifs, and protein domains based on the Gene Structure View (Advanced) function of Tbtools [ 29 , 30 ].

Sequence alignment and phylogenetic analysis

In this study, we retrieved 27 Arabidopsis UBP protein sequences from TAIR ( https://www.arabidopsis.org/ ), 25 rice UBP protein sequences from Phytozome ( https://phytozome.jgi.doe.gov/pz/portal.html ), and 97 wheat UBP protein sequences from a previous study [ 16 ]. To investigate the evolutionary relationships among these proteins, we constructed a phylogenetic tree using the neighbor-joining method in MEGA 6.0 and visualized by iTOL ( https://itol.embl.de/ ). To ensure the statistical reliability of our findings, we performed bootstrap testing with 1000 replicates.

Analysis of cis-acting elements of ZmUBP gene promoters

To gain insights into the cis-elements in the promoter region of ZmUBP genes, we extracted the 2000 bp sequence upstream of these genes. We utilized the PlantCARE ( http://www.dna.affrc.go.jp/PLACE/ ) to predict putative cis-regulatory elements in the promoter sequences. Notably, we identified functionally identical cis-acting elements that were uniquely named. To visualize the results of our analysis, we employed the Simple BioSequence Viewer function of TBtools [ 29 , 30 ].

Chromosome position, collinearity analysis and calculation of Ka/Ks ratios

We downloaded maize gff3 files from Ensembl Plants ( https://plants.ensembl.org/ ) to analyze the annotation of ZmUBP genes on the chromosome. Using the maize genome annotation information, we obtained the relative distance and location of the ZmUBP genes on the chromosome. To visualize this data, we utilized the Gene Locatin Visualize feature of TBtools [ 29 , 30 ].

The Dual Systeny Plot program in TBtools [ 29 , 30 ] was utilized to assess the homology of UBP genes among maize and other species, including Arabidopsis , sorghum, soybean, Kinnow Mandarin ( Citrus reticulata Blanco ), cotton, medicago ( Medicago sativa ), rice, and wheat. To further analyze the collinearity and Ka/Ks ratio, we employed the One Step MCScanX and simple Ka/Ks calculator (NJ) of TBtools [ 29 , 30 ], respectively. The results were visualized using the Microlife Letter ( http://www.bioinformatics.com.cn/ ), providing a comprehensive understanding of the homology relationships among these species.

KEGG and GO enrichment analysis

KEGG and GO enrichment analysis were performed using the microbiotics website ( http://www.bioinformatics.com.cn/ ) to investigate the signalling pathways, biological processes, cellular components and molecular functions involved in the 45 ZmUBP genes.

Temporal and spatial expression profiles of ZmUBP genes

We downloaded transcriptome data for different organs and stress responses of maize from the EMBL-EBI database ( https://www.ebi.ac.uk/ ). To integrate the transcriptome data of the same type, we utilized EXCEL. The results were visualized and presented as heatmaps using the Microbiology Letter website ( http://www.bioinformatics.com.cn/ ).

Plant material, growth conditions, and stress treatments

Hybrid Zhengdan 958 was provided by Grain Crops Research Institute, Henan Academy of Agricultural Sciences (Validation No.: Guoshiyu 20000009, Date of Validation: 2000, Selection and Breeding Unit: Grain Crops Research Institute, Henan Academy of Agricultural Sciences, Selected Breeder: Chunxin Du, Variety Source: Zheng 58/Chang 7 − 2). To investigate the response of maize to various stress conditions, uniform-sized maize seeds were selected and sterilized with 75% ethanol for 5 min. The seeds were then rinsed five times with sterile water and placed in an incubator containing double layers of filter paper. The incubator was set to vernalize the seeds under a 12-hour light (25 °C) and 12-hour dark (20 °C) cycle. After 1 day of germination, well-established and uniform seedlings were selected and transferred into pots containing a mixture of vermiculite and soil (3:1 ratio). After 10 days of growth, the roots of the maize seedlings were watered with 100 mL of a 12% PEG 2000 solution to simulate drought stress. The seedlings were then exposed to low temperature (4 °C) and high temperature (42 °C) stress conditions. Additionally, hormone treatments were applied by watering the roots of the seedlings with different types of plant hormone solutions. All experiments were sampled on the whole plant at 0, 6, and 12 h, with at least three replications for each set of experiments.

Reverse-transcription quantitative polymerase chain reaction

In order to study the expression characteristics of ZmUBP gene under different treatments, total RNA was extracted from the leaves of maize seedlings of control and treatment groups using OminiPlant RNA Kit from Beijing Kangwen Biotechnology Co. Subsequently, cDNA libraries were constructed using the Easy Script One-Step gDNA Removal and cDNA Synthesis Supermix from TransGen Biotech. Primers for the ZmUBP genes were designed using Primer 5.0 software. The cDNA from maize under different treatments was diluted 10-fold and used as a template, with the housekeeping gene actin serving as an internal reference. RT-qPCR was then performed to verify the gene expression characteristics under different treatments. The reaction system consisted of 1 µL cDNA, 1 µL Primer-FW, 1 µL Primer-RV, 10 µL 2× brilliant SYBR RT-qPCR master mix, and 7 µL ddH2O. The relative expression of the selected genes was calculated using the 2 -ΔΔCT method. RT-qPCR analysis was performed with three biological replicates.

Statistical analysis

GraphPad Prism 9 was used to draw column and line charts, and SPSS 17.0 was used to analyze the significant differences (among the averages, there was no significant difference when there was a same marked letter, and there was significant difference when there were different marked letters, and the screening condition was P  < 0.05). Pictures are mainly processed by Photoshop image processing software.

Identification and characterization of the UBP genes in maize

To identify members of the UBP gene family in maize, we first searched relevant databases using the 27 Arabidopsis UBP protein sequences as queries, and this analysis identified 48 putative ZmUBP genes. The Pfam database ( http://pfam.xfam.org ) and NCBI CD Search ( https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi ) were used to confirm the existence of the conserved domain UCH in these UBP proteins. After removing the unqualified sequences, a total of 45 ZmUBP genes were finally identified from the maize genome and named ZmUBP1 to ZmUBP45 according to their chromosomal locations (Table 1 ). All 45 ZmUBPs contained the UCH domain. Detailed information of ZmUBP genes were listed in Table 1 , including gene length, physical and chemical parameters, and subcellular location of all ZmUBP genes. The length of ZmUBP genes conding sequence ranged from 1393 to 4462 bp, and the protein length of ZmUBPs was between 368-1284 amino acids. The molecular weight (MW) ranged from 41.81 to 187.82 kDa with the predicted isoelectric point (pI) ranging from 4.72 to 9.35. The transmembrane (TM) domains prediction of ZmUBP protein showed that only ZmUBP26 and ZmUBP22 contained one transmembrane domains. Subcellular localization and protein stability prediction showed that all 45 ZmUBP proteins were located in the nucleus and were unstable proteins. The hydrophobicity of 45 ZmUBP proteins was predicted to be less than 0, all ZmUBP proteins were hydrophilic proteins (Table 1 ).

To better elucidate the association between gene function and evolution, we explored the structural organization and conserved motifs of ZmUBP genes (Fig. 1 ). The largest number of exons was 30, and they were detected in ZmUBP25 from G5 subfamily, while the smallest number of exons was 2 detected in ZmUBP12 from G1 subfamily. The exon number in other ZmUBP genes was between 3-26. The ZmUBP genes from the same subfamily shared comparable gene structure, thereby suggesting functional conservation among the maize UBP gene family. Additionally, the exon variation in ZmUBP genes may indicate the functional diversity of the maize UBP gene family. The MEME program was used to predict the composition of the ZmUBP protein motifs. A total of twenty conserved motifs were detected (Fig. 1 ). With few exceptions, the motifs in most ZmUBP proteins are arranged in the same order as motif 1, motif 2, motif 3, motif 15, motif 2, motif 17, motif 5, and motif 13 (Fig. 1 ).

Gene and protein structures of 45 ZmUBPs. Phylogenetic relationships ( a ), protein motifs ( b ), protein structural domains ( c ) and gene structures ( d ) of 45 ZmUBPs. G1-G15 represent 45 ZmUBPs distributed in 15 different subfamilies, and the horizontal coordinates represent the length of genes/amino acid sequences

Phylogenetic analysis of the UBP genes in maize

To analyse the phylogenetic organisation of the UBP family, we performed phylogenetic analysis using the protein sequences of 45 maize UBPs, 48 rice UBPs, 27 Arabidopsis UBPs, and 97 wheat UBPs, and generated a phylogenetic tree based on the Neighbour Joining (NJ) method (Fig. 2 ). Based on their phylogenetic relationships, we divided these UBPs into 15 groups, namely G1 to G15 (Fig. 2 ). G5 consisted of the most members, namely 14 ZmUBP proteins, followed by G7 including seven ZmUBP proteins. G4, G6, G9, G10, and G15 possessed the fewest members, including only one ZmUBP protein. Similarly, the UBPs of rice, wheat, and Arabidopsis were distributed among 15 groups, indicating that the functions of UBP family members were preserved during species evolution.

Phylogenetic relationships of UBPs from maize, Arabidopsis , wheat and rice. The phylogenetic tree was generated using a neighborhood join method with 1000 duplicates. Group 1-Group 15 on the left indicate that the UBP family members of maize, as well as the UBP family members of rice, wheat and Arabidopsis , are distributed in 15 different subfamilies. The branches of each subfamily are represented by a specific color. The branches of different members in the same subfamily have the same color

Chromosomal location and evolution of ZmUBP genes

To examine the chromosomal distribution of ZmUBP genes, the chromosomal location of each ZmUBP gene was determined. The results showed that the 45 ZmUBP genes were mapped to 10 chromosomes and they were unevenly distributed within each chromosome (Fig. 3 ). We identified 8 ZmUBP genes on chromosome 2. On chromosomes 1, 3, and 4, 6 ZmUBP genes were identified. On chromosomes 7, 5, 6, 8, 9 and 10, 5, 4, 3, 3, 2, and 2 ZmUBP genes were identified. These results indicate that there is genetic variation in the evolution of maize. Similarly, UBPs were unevenly distributed on different chromosomes in wheat [ 16 ], rice [ 14 ] and moso bamboo [ 15 ]. Obviously, the distribution of UBP genes on different chromosomes of different species is different.

45 Distribution of ZmUBP genes on 10 chromosomes of maize. Each chromosome of maize has the distribution of ZmUBP genes, the vertical coordinate is the length of the chromosome, Zm1-Zm10 and the green columns indicate the 10 chromosomes of maize, and the red color represents 45 ZmUBP genes

Duplication is the major impetus underlying gene expansion during evolution. Five types of gene duplication may occur in evolution, including singleton, dispersed, tandem, proximal, and segmental duplication [ 31 ]. Based on the chromosomal distribution analysis, we conducted gene duplication analysis to reveal the expansion process of the maize UBP genes (Fig. 4 ). The duplicate gene pairs were identified by comparing the coding sequences of 45 ZmUBP genes, and a total of 10 segmental duplication events ( ZmUBP5 / ZmUBP16 , ZmUBP6 / ZmUBP37 , ZmUBP9 / ZmUBP29 , ZmUBP12 / ZmUBP21 , ZmUBP20 / ZmUBP41 , ZmUBP24 / ZmUBP30 , ZmUBP26 / ZmUBP28 , ZmUBP29 / ZmUBP45 , ZmUBP33 / ZmUBP40 , and ZmUBP13 / ZmUBP38 ) were found in the maize genome (Fig. 4 ). These results indicate that segmental duplication plays an important role in UBP gene expansion in the maize genome. These results are consistent with the reported UBP members of monocotyledons such as wheat [ 16 ] and bamboo [ 15 ]. To investigate the evolutionary patterns among ZmUBP genes, we calculated the rate of synonymous substitutions between duplicate gene pairs. Our findings indicate that ZmUBP genes were generally under purifying selection, as evidenced by the Ka/Ks ratios of all duplicate gene pairs being less than 1 (Table 2 ).

Duplication of 45 ZmUBP genes during evolution. Zm1-Zm10 in the innermost circle represents 10 chromosomes of maize, 0-150 represents the distance of genes on chromosomes, the yellow column represents the density of gene distribution on maize chromosomes, and the outermost circle is the distribution of 45 ZmUBP genes on chromosomes. The gray and red lines represent the gene duplication generated during the evolution of all genes in the maize genome and 45 ZmUBP genes, respectively

To elucidate the evolution of the UBP gene families in maize, comparative syntenic maps were constructed with six dicotyledons, Arabidopsis , sorghum ( Sorghum bicolor ), soybean ( Glycine max ), Kinnow Mandarin, cotton ( Gossypium spp.) and medicago, and two monocotyledons, rice and wheat (Fig. 5 ). The results of the comparison show that 20, 23, and 25 ZmUBP genes were covalently related to the UBP genes in sorghum, rice and wheat, respectively. This was followed by Kinnow mandarin, soybean, Arabidopsis and cotton, with three ZmUBP genes covalently related to their UBP genes, and only 2 ZmUBP genes were covalently related to the UBP genes of medicago. The ZmUBP genes had the largest number of covariate gene pairs with monocotyledons and much more than dicotyledons. These findings suggest that the UBP gene predates the evolution of dicotyledons and that genetic variation occurred during the transition from dicotyledons to monocotyledons.

Collinearity analysis of UBP genes among maize, Arabidopsis , sorghum, soybean, Kinnow Mandarin, cotton, medicago, rice and wheat. The gray lines indicate the gene duplication that occurred during evolution between all genes in the maize genome and all genes in the genomes of eight different species. The red lines indicate gene duplication between the maize UBP genes and the UBP genes of eight different species that arose during evolution. The green columns represent the chromosomes of different species

To investigate the evolutionary patterns among UBP genes, we calculated the rate of synonymous substitutions between the UBP family in monocotyledon rice/wheat and maize (Fig. 6 ). The results showed that the Ka/Ks values of 27 homologous genes pairs between the maize UBP family and rice UBP family, as well as 56 homologous genes pairs between the wheat UBP family and maize UBP family were all less than 1 (Fig. 6 ), indicating that UBP genes were generally under purifying selection during the evolution of monocotyledons. This is consistent with the close evolutionary relationship between monocotyledons, suggesting that the UBP gene may play an important role in the evolutionary process of species.

Ka/Ks ratios of homologous gene pairs in maize and rice (left) and wheat (right). The horizontal coordinates indicate the comparison of UBP genes between different species, and the vertical coordinates indicate Ka/Ks ratio ranging from 0-0.8. Red boxes and dots indicate homologous gene pairs between maize and rice, and blue boxes and triangles indicate homologous gene pairs between maize and wheat

Analysis of ZmUBP genes related to stress response and plant hormone response

KEGG enrichment analysis showed that ZmUBP was mainly involved in ubiquitin-mediated protein hydrolysis and secondary metabolite synthesis processes (Fig. 7 a). GO enrichment results showed that ZmUBP was mainly involved in biological processes such as protein deubiquitination, regulation of protein stability, autophagosome organization, jasmonic acid signaling pathway and immune response (Fig. 7 b). This suggests that ZmUBP is involved in plant growth and stress response by regulating protein deubiquitination.

KEGG ( a ) and GO ( b ) enrichment analysis of the ZmUBP genes. The horizontal coordinates in a indicate the ratio of the number of genes in the pathway to the number of ZmUBP family members, and the vertical coordinates indicate the function of the ZmUBP family members. The colour of the circle indicates the significance of the proportion of genes in the pathway to the total genes (Q-value < 0.05 is significant). The size of the black circle indicates the number of ZmUBP genes in the pathway. The horizontal coordinates in ss b indicate the ratio of the number of genes in the pathway to the number of members of the ZmUBP family,and the vertical coordinates indicate the functions of ZmUBP family members, including cellular components, molecular functions and biological processes. The color of the circle indicates the significance of the proportion of genes in the pathway to the total genes ( P < 0.05 is considered significant). After taking -log 10 ( P value), the higher the value is, the higher the significance. The size of the black circle indicates the number of ZmUBP genes in the pathway

The upstream regions of genes contain binding sites for promoters and transcription factors that regulate gene expression [ 32 ]. Therefore, the analysis of cis-acting elements contributes to the understanding of gene function and regulatory networks. To identify potential cy-acting elements in the promoter of ZmUBP genes, 2000 bp sequences upstream from the translation initiation codon of ZmUBP genes were retrieved and analyzed in the PlantCARE database. The promoters of the ZmUBP genes contain many elements or sites that respond to environmental factors such as light, temperature, and humidity. The presence of defense and stress response elements suggests that ZmUBPs may play an important role in the plant response to drought and low-temperature stress (Fig. 8 ). In addition, hormone response elements were found in the ZmUBP gene promoter, including abscisic acid (ABA), salicylic acid (SA), gibberellin (GA), auxin (IAA), and methyl jasmonate (MeJA) response elements. These results suggest that ZmUBP genes may be involved in the plant response to stress by regulating hormone signaling.

Cis-acting element analysis of 45 ZmUBP gene promoters. The 45 ZmUBP genes were classified according to phylogenetic relationships, and light, drought, gibberellin, jasmonic acid, abscisic acid, salicylic acid, auxin, stress, low-temperature, and seed growth response elements are indicated by 10 different colored columns. The horizontal coordinates represent gene length

Expression patterns of ZmUBP genes in different tissues

The expression profiles of 45 genes in different tissues were divided into four groups (Fig. 9 a). The first category includes ZmUBP42 . Except for the low expression in mature pollen, the expression level of ZmUBP42 in other maize parts was the highest among all ZmUBPs . This expression is similar to AtUBP23 (AT5G57990), which is in the same subfamily as ZmUBP42 in the evolutionary relationship (Fig. 2 ). AtUBP23 is highly expressed in all tissues of Arabidopsis [ 33 ]. The second category includes ZmUBP39 , ZmUBP15 , ZmUBP3 , ZmUBP2 , ZmUBP35 , ZmUBP25 , ZmUBP14 , ZmUBP1 and ZmUBP20 . Although the expression level of these genes is low in some plant tissues, the overall expression level is high (Fig. 9 a). In particular, ZmUBP39 , whose high-level expression during embryonic development is similar to AtUBP14 (AT3G20630), which is in the same subfamily as ZmUBP42 in the evolutionary relationship, further validates the conjecture in Fig. 2 that ZmUBP39 may play an important role in early embryonic development. In addition, the expression level of ZmUBP1 in reproductive organs was significantly higher than that in other parts, which was similar to AtUBP3 (AT4G39910) and AtUBP4 (AT2G22310) in Arabidopsis , which were in the same subfamily as ZmUBP1 in the evolutionary relationship. This is the same as the speculation in Fig. 2 that ZmUBP1 plays an important role in plant sexual reproduction. The third category includes ZmUBP30 , ZmUBP37 , ZmUBP7 , ZmUBP4 , ZmUBP21 , ZmUBP43 and ZmUBP8 . These genes have low expression levels in all tissues of plants and may not be involved in plant growth and development (Fig. 9 a). The expression levels of other genes in different plant tissues are quite different, ZmUBP41 and ZmUBP6 were highly expressed in mature pollen, ZmUBP19 and ZmUBP26 were highly expressed near the primordium, indicating that they may be involved in plant sexual reproduction. In addition, the expression levels of ZmUBP genes in maize roots and embryos were generally higher than those in other plant parts, indicating that ZmUBP -mediated deubiquitination may be crucial for nutrient uptake and sexual reproduction.

The temporal and spatial expression characteristics of ZmUBP genes.  a  Expression levels of 45 ZmUBP genes in 17 different tissues of maize. The horizontal coordinates represent different periods and different parts of the maize fetch, and the vertical coordinates indicate the clustering analysis of all ZmUBP genes according to the expression levels at different periods. The red and blue columns indicate the values after z-score normalization of the expression of ZmUBP genes, with red being expression above the mean and considered high expression, while blue being expression below the mean and considered low expression. Groups represent different parts of the plant, including root, stem, leaf, flower and embryo. b 8 ZmUBP genes that may play a role in plant growth and stress responses were selected based on the results of transcriptome analysis. Different colored columns represent different ZmUBP genes, and the horizontal coordinates represent different plant tissues, in which R stands for roots, S for stems, and L for leaves. The vertical coordinates represent the relative expression levels of the 8 ZmUBP genes in different plant tissues (using the expression in roots as control). Significant differences are indicated by the letters a, b, and c labeled at the top of the columns

Up to now, the only study on maize UBP genes is the identification of three Arabidopsis UBP gene homologs in maize, namely, ZmUBP15 , ZmUBP16 , and ZmUBP19 , which only analyzed their expression patterns in roots, leaves, spikes, and seeds, as well as in the presence of metal, salt, and osmotic stresses [ 25 ], but there is no in-depth study of the roles that all members of the maize UBP family play in plant growth and in response to high-temperature, low-temperature, and drought stresses. Therefore, we selected eight ZmUBP genes with high expression levels under abiotic stresses using RT-qPCR to investigate their roles in plant growth and abiotic stress response. The results showed that except for ZmUBP17 , which was highly expressed in stems and leaves, the other 7 ZmUBP genes were highly expressed in roots (Fig. 9 b), and the expression levels in roots were significantly higher than those in stems and leaves (except for ZmUBP14 ). This is consistent with the transcriptome data in Fig. 9 a (the expression levels of ZmUBP genes was significantly higher in roots than in stems and leaves), suggesting that ZmUBP genes were mainly involved in plant root growth.

Expression pattern of ZmUBP genes under abiotic stresses and phytohormone treatment

The expression levels of all ZmUBP genes were analyzed under abiotic stress (high-temperature, drought and low-temperature) based on transcriptomic data (Fig. 10 a). The expression levels of all ZmUBP genes except ZmUBP35 and ZmUBP17 decreased significantly when maize taproots were subjected to drought stress (Fig. 10 a), which is consistent with the results of promoter element analysis (Fig. 8 ), that is, most ZmUBP gene promoters contain drought response and ABA response elements, indicating that ZmUBP gene family members may be negative regulators of drought stress. The expression of ZmUBP37 , ZmUBP42 , ZmUBP7 , ZmUBP29 , ZmUBP31 and ZmUBP30 increased significantly after high-temperature stress was applied to different genotypes of maize (Fig. 10 a). The expression of other ZmUBP genes was significantly downregulated. The expression multiples of ZmUBP37 was most upregulated, which indicated that ZmUBP37 was important for maize to cope with high-temperature stress. ZmUBP16 , ZmUBP6 , ZmUBP33 , ZmUBP11 and ZmUBP17 were significantly induced in different varieties of maize after low-temperature stress. However, ZmUBP24 was only upregulated in the resistant genotype, so ZmUBP24 could be used as an important target for low-temperature resistance breeding of maize. In addition, the promoters of ZmUBP22 , ZmUBP1 , ZmUBP7 , ZmUBP10 , ZmUBP34 , ZmUBP27 , ZmUBP32 , ZmUBP35 and ZmUBP15 also contained low-temperature response elements, but their expression levels were significantly reduced under low-temperature stress, indicating that these genes may be negative regulators of low-temperature stress. In addition, no ZmUBP gene was resistant to all three abiotic stresses.

Expression analysis of ZmUBP genes in different tissues by RT-qPCR. a Expression levels of 45 ZmUBP genes under drought, low-temperature and high-temperature treatments. The horizontal coordinates represent the different treatments and treatment times of maize materials, and the vertical coordinates indicate the clustering analysis of all ZmUBP genes according to the expression levels of different treatments. The red and blue columns indicate the multiples of upregulated or downregulated expression of the treatment group compared with the untreated control group (red is upregulated expression, blue is downregulated expression). Group is different treatment methods of maize materials. b- g 8 ZmUBP genes that may play a role in plant growth and stress responses were selected based on the results of transcriptome analysis. Different colored columns represent different ZmUBP genes, and the horizontal coordinates represent abiotic stresses and phytohormones treatments for 0, 6 and 12 hours. The vertical coordinates represent the relative expression levels of the 8 ZmUBP genes under abiotic stresses and phytohormones treatments (using the expression under 0 hour as control). Significant differences are indicated by the letters a, b, and c labeled at the top of the columns

RT-qPCR results showed that ZmUBP14 was significantly induced after low-temperature, high-temperature and drought stresses, and the expression level showed a trend of decreasing and then increasing after IAA and SA treatments (Fig. 10 b-d, f and g), suggesting that ZmUBP14 may respond to abiotic stresses by cross-linking multiple phytohormone signaling pathways. In addition, the expression level of ZmUBP35 was significantly increased after high-temperature, low-temperature and ABA treatments (Fig. 10 b, c and e), suggesting that ZmUBP35 may be an ABA-mediated temperature regulator. ZmUBP37 was significantly induced by all three phytohormone treatments but did not respond to abiotic stresses (Fig. 10 b-g), suggesting that ZmUBP37 may regulate plant growth and development through a cross-linked phytohormone signaling pathway. Interestingly, ZmUBP17 , which was highly expressed in roots, stems and leaves, did not respond to to either abiotic stress or phytohormones (Figs. 9 and 10 ), suggesting that ZmUBP17 may affect plant growth and development through the regulation of other phytohormones. It is well known that ABA is a key phytohormone that regulates drought tolerance in plants, but no ZmUBP genes were found in this study that existed in response to both ABA and drought, suggesting that ZmUBP genes may regulate drought tolerance in plants through other pathways.

Identification of UBP gene family members in maize

The eukaryote-specific UBP family is one of the largest DUB families identified to date and plays an important role in plant growth and development [ 34 ]. The plant UBP family has different types and amounts of UBP genes. There are 27 UBP members in Arabidopsis [ 13 ]. There are 48 UBP members in moso bamboo [ 15 ], and they are divided into two major groups (G13-G15, G1). There are 97 UBP members in wheat distributed in 15 subfamilies [ 16 ], and 48 UBP members in rice are also distributed in 15 subfamilies [ 14 ]. However, the UBP gene family members have not been identified in the maize genome. In this study, 45 putative UBP genes were identified in maize using genome-wide analysis, and were unevenly distributed on 10 chromosomes. According to phylogenetic analysis, the presumed ZmUBP gene family members were divided into 15 subfamilies as in wheat and rice, indicating that no UBP members were deleted during maize evolution. In addition, different members of the same subfamily are generally considered to have similar functions. AtUBP3 (AT4G39910) and AtUBP4 (AT2G22310) in Arabidopsis regulate male gamete development and affect plant sexual reproduction [ 1 ]. Therefore, ZmUBP29 , ZmUBP1 , ZmUBP9 and ZmUBP45 , which are in group 2, may also have the same function as AtUBP3 and AtUBP4 . Similarly, ZmUBP39 in group 6 may be able to regulate plant early embryonic development like AtUBP14 (AT3G20630). AtUBP12 (AT5G06600) and AtUBP13 (AT3G11910) have been shown to enhance plant drought tolerance through activation of the ABA signalling pathway and to play a role in plant histone debuquitination and organ development [ 35 ]. Therefore, ZmUBP15 , ZmUBP44 , ZmUBP25 , ZmUBP35 , ZmUBP14 , ZmUBP19 , ZmUBP32 , ZmUBP41 , ZmUBP18 , ZmUBP17 , ZmUBP5 , ZmUBP4 , ZmUBP23 and ZmUBP8 were also speculated to enhance plant drought resistance and to play a role in plant histone debuquitination and organ development because they also belong to the group 5. A recent study showing that The UBP5 histone H2A deubiquitinase counteracts PRCs-mediated repression to regulate Arabidopsis development informs the study of the histone deubiquitination function of the homologue ZmUBP5 in maize [ 36 ]. In addition, OsUBP2 (Os09g0505100) in rice and TaUBP1A.1 in wheat were proved to be resistant to leaf blight and Chinese wheat mosaic virus (CWMV), and both belonged to group 1 [ 6 , 16 ], indicating that ZmUBP12 and ZmUBP21 belonging to group 1 subfamily may be resistant to biotic stress. In addition, UBP genes of wheat and rice were evolutionarily closer to maize UBP genes than Arabidopsis UBP genes (Fig. 2 ), as were interspecies covariance analyses (Fig. 5 ), confirming previously reported relationships between dicotyledons and monocotyledons during evolution [ 37 ]. These results indicated that ZmUBP genes existed before the isolation of monocotyledon plants and were highly conserved during plant evolution.

Duplication events of ZmUBP genes during evolution

Gene duplication helps organisms adapt to environmental changes during development and growth and is essential for gene evolution and amplification [ 38 , 39 ]. Among them, the tandem duplication of genes in the process of genomic DNA duplication and recombination is the key driver of gene family amplification [ 40 ]. In the genomes of Arabidopsis and rice, 15–20% of genes consist of tandem repeats of gene clusters thought to be critical for evolution, plant disease resistance, and abiotic stress responses [ 41 ]. Two tandem repeat clusters were found in the TaUBP gene family of wheat. The two tandem repeat genes were located on chromosomes 1D and 7D, accounting for only 3.7% of the 54 TaUBP collinear gene pairs, suggesting that tandem repetition may not be the main amplification method during the evolution of the UBP gene family.In the present study, chromosomal localization and gene structure revealed that gene duplication events occurred during genome expansion and evolution in maize. Forty-five members of the ZmUBP gene family produced 10 pairs of segmental duplicated genes without tandem duplication, with the same results as in wheat. It is further suggested that segmental duplication may be the primary method of gene amplification during the evolution of the UBP gene family, rather than tandem duplication. In addition, the Ka/Ks ratio can be used to determine whether selective pressure acts on protein-coding genes. In this study, the Ka/Ks ratio was significantly less than 1 in the intraspecific and interspecific repeat gene pairs of all maize UBP genes, indicating that strong purifying selection plays an important role in the constraint of UBP gene function, which is consistent with the conservation of the UBP gene in the evolutionary process.

ZmUBP genes play an important role in plant growth and stress response

Transcriptional regulation of genes may be influenced by cis-elements in promoter regions that control responses to different stimuli. To investigate the biological function of ZmUBPs , we predicted cis-acting elements in the ZmUBP gene promoter. The results showed that the type of cis-acting element was different for each ZmUBP gene. Therefore, ZmUBPs may be involved in various specific regulatory mechanisms related to the stress response. Cis-acting regulatory elements largely determine tissue-specific gene expression patterns. In this study, we explored the expression profile of ZmUBPs in different tissues (root, stem, leaf, flower and embryo). The expression pattern of ZmUBPs showed that the expression levels of ZmUBPs were different in different plant tissues. The expression levels of ZmUBPs were the highest in maize roots and embryos and the lowest in leaves. This indicates that ZmUBP -mediated deubiquitination may be essential for the development of maize roots and embryos. In addition, some TaUBPs showed tissue-specific expression in maize, such as ZmUBP42 , which was not expressed in mature pollen and was expressed at very high levels in all other sites, while ZmUBP1 was only expressed at high levels in the root cortex and was expressed at low levels in other sites. These results indicate that ZmUBPs play different roles in plant growth and development. Cis-acting regulatory elements also largely determine the expression patterns of stress response genes. We found drought, low-temperature, GA, MeJA, ABA, and SA response elements in the promoter region of the ZmUBP genes, suggesting that ZmUBPs play an important role in the hormone-mediated stress response. This was confirmed by transcriptomic results. Except for ZmUBP35 and ZmUBP17 , the expression levels of all ZmUBP genes were significantly downregulated after drought treatment, indicating that ZmUBPs responded negatively to drought stress. The expression of ZmUBPs was different under low and high-temperature stress but showed a downward trend, indicating that ZmUBPs negatively regulated the plant stress response to abiotic stress.

Up to now, few studies have been conducted on the function of maize UBP genes. Kong et al. identified three Arabidopsis UBP gene homologs in maize and analyzed their expression patterns in roots, leaves, spikes, and seeds, as well as under metal, salt, and osmotic stresses [ 25 ]. However, the functions of ZmUBP genes in phytohormone, high-temperature, low-temperature, and drought responses have not been thoroughly investigated. In present study, we presented a comprehensive investigation of ZmUBP genes expression profiles in different plant tissues and abiotic stresses based on RT-qPCR and transcriptome data. Among them, ZmUBP17 showed high expression levels in roots, stems and leaves, implying that it plays an important role in the growth and development of different parts of plants. Except for ZmUBP17 , all other ZmUBP genes were expressed at high levels in roots and lower levels in stems and leaves. The process of deubiquitination has been shown to regulate nutrient uptake and transport in plant roots to maintain optimal root function, so we hypothesised that these seven ZmUBP genes also have the function of regulating nutrient uptake and transport in plant roots [ 42 , 43 ]. In addition, we found that ZmUBP14 was significantly induced by high-temperature, low-temperature, and drought treatments, and there was a positive response to IAA and SA, suggesting that ZmUBP14 may regulate plant tolerance to abiotic stresses through an ABA-independent pathway. Interestingly, ZmUBP17 showed a negative response to both abiotic stresess and phytohormones despite its high expression level in roots, stems and leaves, suggesting that ZmUBP14 may regulate plant growth and development through other phytohormones besides ABA, IAA, and SA. ABA is a key phytohormone for regulating drought tolerance in plants, and ABA accumulation in the tissues can significantly enhance plant drought tolerance. However, this study did not find any ZmUBP genes that were responsive to both ABA and drought stress, suggesting that ZmUBP genes may regulate plant drought tolerance through an ABA-independent pathway. In addition, ZmUBP37 was responsive to all three phytohormones, ABA, IAA, and SA, and was highly expressed in roots, suggesting that ZmUBP37 may regulate the growth and development of plant roots by cross-linking multiple phytohormone signaling pathways. Combining the transcriptome and RT-qPCR results, we hypothesized that ZmUBP14 may be a core member of the ZmUBP gene family that regulates plant growth and development and stress response.

As the largest subfamily of plant DUBs , UBP is essential for deubiquitination and plays an important role in plant development and stress response. Here, we have conducted a genome-wide analysis of the UBP gene family in maize for the first time. A total of 45 ZmUBP genes were comprehensively identified from the maize genome, and all the maize UBP genes were randomly distributed on the 10 chromosomes of maize and produced 10 duplicate gene pairs in the evolutionary process. Phylogenetic analysis revealed that these maize UBP genes were divided into 15 subfamilies. The protein motifs and gene structures of the ZmUBPs were highly conserved in each group, reflecting their functional conservation. Collinearity analysis showed that a high proportion of the ZmUBP genes might be derived from tandem duplications with purifying selection, providing insights into possible functional divergence among members of the ZmUBP gene family. Furthermore, a large number of stress and hormone response elements are raised on the ZmUBP promoters. ZmUBP14 was highly expressed in roots, stems, and leaves, and there was a positive response to drought, low-temperature, high-temperature, IAA, and SA, which has certain guiding significance for studying the mechanism of ZmUBP genes in response to abiotic stresses and hormones during development mediated by deubiquitination.

Availability of data and materials

Hybrid Zhengdan 958 provided by Grain Crops Research Institute, Henan Academy of Agricultural Sciences (Validation No.: Guoshiyu 20000009, Date of Validation: 2000, Selection and Breeding Unit: Grain Crops Research Institute, Henan Academy of Agricultural Sciences, Selected Breeder: Chunxin Du, Variety Source: Zheng 58/Chang 7-2). The genomic data analyzed in this study are available in the NCBI repository (the accession number of the maize genomic data is CABHLF000000000, the accession number of rice genomic data is JACJVL000000000, the accession number of sorghum genomic data is ABXC00000000, the accession number of soybean genomic data is ACUP00000000, the accession number of Kinnow Mandarin genomic data is NIHA00000000, the accession number of cotton genomic data is VKGJ00000000, the accession number of medicago genomic data is PSQE00000000, the accession number of wheat genomic data is NMPL00000000, The accession number of Arabidopsis genomic data is JAEFBJ000000000). All transcriptome data used in this study are available in the EMBL-EBI database ( https://www.ebi.ac.uk/ ). Website links for all data used in this study are as follows.

Abbreviations

Zea mays L.

Ubiquitin-specific proteases

Deubiquitinating enzymes

Abscisic acid

Ub carboxy-terminal hydrolase

Cysteine cysteine

Histidine histidine

Arabidopsis thaliana

Oryza sativa

Phyllostachys edulis

Triticum aestivum

Chinese wheat mosaic virus

Sorghum bicolor

Glycine max

Citrus reticulata Blanco

Gossypium spp.

Medicago sativa

Nonsynonymous substitution rates

Synonymous substitution rates

Salicylic acid

Gibberellin

Methyl jasmonate

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We thank the editors and reviewers for their careful reading and valuable comments. We apologize to researchers whose studies are not cited due to space limitations.

This work was supported by the Program for Changjiang Scholars and Innovative Research Team in University (grant number: No. IRT17R99) and by Heilongjiang Province Government Postdoctoral Science Foundation (grant number: LBH-Q18008).

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W.C.F. and Y.Y.B. designed the experiments, analyzed the data and wrote the manuscript. W.C.F. and D.L.F. performed the experiments and bioinformatics analysis. S.K.L. supervised the study and critically reviewed the manuscript. All authors read and approved the manuscript.

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Fu, W., Fan, D., Liu, S. et al. Genome-wide identification and expression analysis of Ubiquitin-specific protease gene family in maize ( Zea mays L.). BMC Plant Biol 24 , 404 (2024). https://doi.org/10.1186/s12870-024-04953-5

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Effects of porous structure and oxygen functionalities on electrochemical synthesis of hydrogen peroxide on ordered mesoporous carbon

• Abdalazeez Ismail Mohamed Albashir 1 , 2 ,
• Xingyu Lu 1 , 2 ,
• Xueya Dai 1 , 2 &
• Wei Qi 1 , 2  

Communications Chemistry volume  7 , Article number:  111 ( 2024 ) Cite this article

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• Electrocatalysis
• Electrochemistry
• Porous materials

Two-electron oxygen reduction reaction (2e − ORR) is a promising alternative to energy-intensive anthraquinone process for hydrogen peroxide (H 2 O 2 ) production. Metal-free nanocarbon materials have garnered intensive attention as highly prospective electrocatalysts for H 2 O 2 production, and an in-depth understanding of their porous structure and active sites have become a critical scientific challenge. The present research investigates a range of porous carbon catalysts, including non-porous, microporous, and mesoporous structures, to elucidate the impacts of porous structures on 2e − ORR activity. The results highlighted the superiority of mesoporous carbon over other porous materials, demonstrating remarkable H 2 O 2 selectivity. Furthermore, integration of X-ray photoelectron spectroscopy (XPS) data analysis with electrochemical assessment results unravels the moderate surface oxygen content is the key to increase 2e − ORR activity. These results not only highlight the intricate interplay between pore structure and oxygen content in determining catalytic selectivity, but also enable the design of carbon catalysts for specific electrochemical reactions.

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Introduction.

Hydrogen peroxide (H 2 O 2 ) is a crucial and versatile chemical with wide-ranging applications, including chemical industry, environmental protection and medical field 1 , 2 , 3 . The anthraquinone oxidation/reduction process currently stands as the predominant method for large-scale H 2 O 2 production 4 . However, despite its massive scale applications, the high energy consumption and hazardous waste generation make it non-environmentally friendly 5 . Consequently, there is an urgent need to develop efficient and environmentally friendly methods for H 2 O 2 production, particularly for on-site applications. In this regard, electrochemical two-electron oxygen reduction reaction (2e − ORR) has been considered as a potentially advantageous strategy for generating H 2 O 2 due to its mild operation condition, green reactants such as air and water, and the capability of being powered by green electricity 6 , 7 , 8 .

Despite the advantages offered by the 2e − ORR for H 2 O 2 electrosynthesis, a significant challenge remains due to the lack of an efficient catalyst that could effectively enhance the selectivity towards H 2 O 2 . Recent investigations have demonstrated the 2e − ORR activity of precious-metal electrocatalysts, such as Pt 9 and Pd 10 , as well as transition metals like Co 11 , Fe 12 , Ni 13 etc. However, their high cost and limited availability of resources hinder the extensive utilization in large-scale applications. Metal-free carbon nanomaterials have attracted significant attention as potential electrocatalysts for H 2 O 2 production due to their sustainability, abundance, cost-effective, and tunable surface properties 14 , 15 . As pristine pure carbon catalysts typically exhibit poor catalytic performance, modification strategies such as porous construction 16 , defect engineering 17 surface modification 18 , and heteroatom doping, including oxygen 19 , nitrogen 20 , fluorine 21 , sulfur 22 , phosphorous 23 , and boron 24 have emerged as beneficial approaches to enhance the catalytic efficiency of carbon materials. These modifications not only improve catalyst efficiency but also maintain the principles of sustainability and cost-effective, which both crucial for advancing H 2 O 2 electrosynthesis technologies.

Two critical factors have been found to significantly impact the 2e − ORR catalytic activity of carbon materials, including porous structure and the oxygen functional groups 25 , 26 . First, well-ordered micro-/mesoporous structures provide a larger surface area for the reaction interface, more accessible active sites, and improved electron transfer paths during 2e − ORR 27 , 28 . Assessing the impact of each pore size in porous carbon (microporous and mesoporous) is crucial in the electrochemical production of H 2 O 2 29 , 30 , 31 , 32 . Related research has shown that mesoporous structures are preferable to microporous ones, as the former facilitates faster mass transfer and promotes the formation of H 2 O 2 . For instance, it has been reported that a mesoporous-dominant nitrogen-doped carbon material exhibited higher H 2 O 2 selectivity than a microporous-dominant nitrogen-doped carbon 33 . However, the role of porous structure in enhancing H 2 O 2 activity is debated, and it has also been reported that carbon materials with greater microporosity content demonstrated a relatively high H 2 O 2 partial current due to the enhanced density of defect sites on the surface 32 . Consequently, by comprehensively assessing the effects of each pore size in porous materials, scientists can gain a deeper understanding of the fundamental principles governing the electrochemical synthesis of H 2 O 2 and enhance the efficiency and selectivity of the overall process.

Oxygen functional groups also play critical roles in the 2e − ORR process, highlighting their potential to fine-tune the electrocatalytic properties and kinetics of the H 2 O 2 synthesis reaction 34 , 35 . Some early conclusive evidence has demonstrated that some oxygen functional groups, such as carboxyl (-COOH) and ether (-C-O-C) moieties, may serve as active sites in the 2e − ORR process through experimental study or density functional theory (DFT) calculation methods 25 . Recent studies on successful functionalization of carbon nanotubes with specific oxygen groups highlighted the crucial role of the -C = O group in achieving excellent 2e − ORR selectivity 36 . However, understanding the intricate interplay between oxygen content and catalytic behavior is crucial for rational designing effective and selective carbon catalysts for the 2e − ORR process.

In this comprehensive study, we systematically assessed the influence of pore size and oxygen content on the catalytic activity of carbon materials for electrochemical synthesis of H 2 O 2 . Among the array of carbon catalysts examined, mesoporous carbon exhibits outstanding electrocatalytic activity, demonstrating significantly higher H 2 O 2 selectivity up to 88% at 0.4 V RHE in alkaline media (0.1 M KOH) compared to its other porous carbon counterparts. This elevated selectivity of mesoporous carbon can be attributed to its precisely tailored pore structure, which facilitated the mass transportation of reactants and products, coupled with efficient confinement of reaction intermediates, ultimately favoring the preferred formation of H 2 O 2 over competing side reactions. In addition, analyses from X-ray photoelectron spectroscopy (XPS) and electrochemical activity assessments unveiled that the C = O may be the main active site of 2e − ORR, and appropriate oxygen content could benefit the selective synthesis of H 2 O 2 . Furthermore, we explored the synergistic interplay between porous structure and oxygen species, and an optimized activity at 100% H 2 O 2 selectivity highlighted the significant roles of both factors in determining the catalytic activity. This study serves as a foundational platform for comprehending the intricate relations governing catalyst activity, and it contributes not only to the fundamental understanding of structure-function correlations but also offers valuable insights for the development of improved electrocatalysts tailored for efficient and selective H 2 O 2 electrosynthesis.

Results and discussion

Insights into effect of porous structure on catalytic activity.

To systematically explore the influence of porous structure on the efficiency of electrochemical synthesis of hydrogen peroxide, a series of porous carbon (PC) materials spanning a wide range of porosities, from micropores to macropores, were synthesized utilizing ZnCl 2 salts, Y-zeolite (SiO 2 /Al 2 O 3 ) and SBA-15 hard templates as pore-forming agents. In a typical procedure, the synthesis involved impregnating glucose and ZnCl 2 into the pores of SBA-15 hard templates, followed by subsequent polymerization, carbonization, and template removal, as illustrated in Fig.  1 . To precisely assess the impact of the porous structure on the 2e − ORR performance, hierarchical porous carbon (Hiera-PC), microporous carbon (micro-PC), and non-porous carbon (template-free-C) were also fabricated for comparisons (the detailed fabrication process was provided in the Methods section).

The schematic diagram depicts the synthesis process of Micro/meso-PC, illustrating the key steps involved, impregnating, polymerization, carbonization, and template Removal.

As illustrated in SEM images in Supplementary Fig.  1a, b , the Template-free-C exhibits a glassy-like structure, indicative of its limited porosity and low specific surface area. Upon impregnating glucose into the pores of Y-zeolite or SBA-15 templates, a significant increase in surface roughness could be observed in the Meso-PC (Fig.  2a, b ) and micro-PC (Fig.  2c, d ) catalysts, signifying a substantial increase in specific surface area. The SEM images of Hiera-PC (Supplementary Fig.  1c, d ) and micro/meso-PC (Supplementary Fig.  1e, f ) reveal irregular pore sizes attributable to the introducing of ZnCl 2 as a pore-forming agent. The transmission electron microscopy (TEM) images show that the density of mesoporous channels in pure SBA-15 are evident (Supplementary Fig.  2a, b ). These mesoporous channels are completely filled with the carbon precursors in the following impregnation procedure, resulting in mesoporous features within the carbon material after the template removal process (Fig.  2e ) 37 . Furthermore, the micro-PC catalyst displays a significant number of microporous channels, as shown in Fig.  2f . X-ray diffraction (XRD) measurements (Supplementary Fig.  3 ) reveal two diffraction peaks centered at around 24° and 44°, corresponding to the (002) and (100) faces of hexagonal carbon 38 .

a , b SEM and c TEM images of meso-PC. d , e SEM and f TEM images of micro-PC.

N 2 -adsorption-desorption isotherm measurements (Fig.  3a, b ) reveal that the micro-PC catalyst exhibits an absence of hysteresis loops in the medium-pressure region (adsorption-desorption region for mesopores), indicating a higher volume of micropores. In contrast, the isotherms of meso-PC displayed large hysteresis loops in the medium-pressure region, signifying the presence of a higher volume content of mesopores. The absence of N 2 uptake at both lower and higher-pressure regions for Template-free-C indicates its poor porous structure. Additionally, the increased loops observed in micro/meso-PC at higher pressure indicates greater microporous volume, which can be ascribed to the addition of ZnCl 2 . During carbonization process, ZnCl 2 acts as a chemical activation agent, decomposing into volatile compounds under high temperatures 39 . This decomposition is critical to forming voids within the carbon matrix and leads to the emergence of a hierarchical pore structure, which includes both micro-and mesopores. The BET surface area of template-free-C, hiera-PC, micro-PC, micro/meso-C, and meso-PC catalysts are determined at 169, 1571, 858, 705, and 594 m 2  g −1 , respectively (Supplementary Table  1 ).

a N 2 -adsorption-desorption isotherms, and b pore-size distributions of Template-free-C (Black), Hiera-PC (purpule), micro/meso-PC(Blue), micro-PC (Red) and Meso-PC (Green).

The electrochemical ORR process was evaluated using a rotating ring-disk electrode (RRDE) in an oxygen-saturated 0.1 M KOH electrolyte solution. From LSV curves it is observed that the Meso-PC exhibited a higher ring current density ( j D ) compared to all porous carbon and Template-free-C catalysts (Fig.  4a ), indicating its high catalytic selectivity toward 2e − ORR. The calculated values for the ring current density ( j R ) and disk current density ( j D ) for Template-free-C, Hiera-PC, micro-PC, micro/meso-PC, and Meso-PC were 0.12, 0.0.49, 0.0.44, 0.0.33 and 0.0.52 jmAcm −2 , and −4.1, −3.4, −3.6, −2.59 and 3.09 jmAcm −2 , respectively. The H 2 O 2 selectivity for template-free-C, hiera-PC, micro-PC, micro/meso-PC and meso-PC were calculated at 28%, 69%, 58%, 60%, and 88%, respectively (Fig.  4b ). Moreover, the transferred electron numbers for template-free-C, hiera-PC, micro-PC, micro/meso-PC and meso-PC are calculated to be 3.43, 2.60, 2.82, 2.80, and 2.24, respectively (Fig.  4c ), suggesting that porous carbon catalysts exhibit higher 2e − ORR selectivity than non-porous carbon. Notably, the meso-PC electrocatalyst exhibits superior catalytic activity and H 2 O 2 selectivity, which is attributed to its improved mass transfer during the ORR process, facilitating the rapid release of generated H 2 O 2 and preventing its complete 4e − reduction to water 40 . The lower H 2 O 2 selectivity of micro-PC should be attributed to its microporous structure, which confines H 2 O 2 within small pores and leading to its further 4e − reduction to H 2 O as the final product 30 .

a SCV-RRDE profiles, b hydrogen peroxide selectivity (H 2 O 2 %), and c average number of electron transferred ( n av ) of template-free-C (Black), hiera-PC (purpule), micro/meso-PC(Blue), micro-PC (Red) and meso-PC (Green) in O 2 -saturated 0.1 M KOH solution, respectively. d H 2 O 2 % selectivity of Meso-PC comparing with previously reported carbon-based or metal-based electrocatalysts.

Interestingly, we find that the specific surface area may not significantly affect H 2 O 2 selectivity, as observed in the comparison between Hiera-PC (higher specific surface area of 1571 m 2  g −1 ) and Meso-PC (lower surface area of 594 m 2  g −1 ). While there is typically a correlation between specific surface area and catalytic activity because of the increased number of exposed active sites 41 , but it is not the only factor determining catalytic activity. The nature, distribution, and accessibility of active sites, along with the diffusion and confinement of species involved in the H 2 O 2 synthesis within the porous structure, also play critical roles 33 . In this case, mesoporous carbon possesses a more favorable pore architecture for facilitating the necessary mass transportation and timely release of reaction intermediates, thereby enhancing H 2 O 2 selectivity despite having a lower overall surface area compared with all other porous carbon catalysts (Supplementary Table  1 ). Compared with previously reported electrocatalysts, the H 2 O 2 selectivity of the mesoporous carbon catalyst (meso-PC) is found to be significantly higher than most carbon-based materials and even comparable to some metal-based catalysts (Fig.  4d ). To evaluate the 2e − ORR performance across different pH conditions, all porous materials and Template-free-C, were also assessed in 0.1 M H 2 SO 4 (Supplementary Fig.  4a–c ). We observed a reduction in H 2 O 2 selectivity in acidic media compared to alkaline conditions. This decrease can be attributed to a shift in the ORR pathway from 2e − ORR to four-electron oxygen reduction reaction (4e − ORR), resulting in H 2 O production. Typically, alkaline solutions facilitate the 2e − ORR due to the presence of hydroxide ions (OH − ), whereas acidic conditions are rich in protons (H + ) tend to favor the four-electron oxygen reduction reaction (4e − ORR) process. To enhance selectivity in acidic media, future work would focus on modifying the surface chemistry of carbon-based catalysts with specific functional groups or composite materials that leverage synergistic effects between carbon and other catalytically active sites optimized for acidic media.

Insights into effect of oxygen functionalities on catalytic activity

To get an in-depth understanding on the oxygen species responsible for electrochemical H 2 O 2 production, a series of oxidized samples with varying oxygen contents are synthesized undergoing nitric acid oxidation with varying duration time. The micro/meso-PC catalyst material is chosen as an ideal catalyst for identifying surface oxygen functional groups due to its intermediate selectivity and diverse pore sizes. The XPS survey spectra (Supplementary Fig.  5a ) shows that micro/meso-OPC-x samples comprise carbon, nitrogen, and oxygen elements with slight silicon residue (0.37–0.4%) remaining in the catalysts structure after exclusive acid etching (Supplementary Fig.  5b ). As indicated by the XPS results, the Zn element was completely evaporated during the carbonization process (Supplementary Fig.  5c ), resulting in a porous structure in carbon material. The oxygen contents strengthen from 5.3% to 32.5% with increasing the oxidation time (Supplementary Table  2 ). The high-resolution O1s XPS spectra of micro/meso-PC-x (Fig.  5a–f ) could be deconvoluted into three peaks located at 531.4 eV, 532.7 eV, and 534.0 eV, corresponding to carbonyl (-C = O), carboxyl (-COOH), and hydroxyl (-C-OH) surface functionalities, respectively 35 . The high-resolution C1s XPS spectra further reveals four peaks at 284.6 eV, 286.0 eV, 287.7 eV, and 289.4 eV, corresponding to the binding energy signals belonging to -C-C, -C-OH, -C = O, and -COOH groups, respectively (Supplementary Fig.  6a–f ). Notably, the micro/meso-PC-x catalysts exhibit various quantities of oxygen functional groups (Supplementary Table  3 ), which are expected to fine-tune ORR activity for efficient H 2 O 2 production.

a Micro/meso-PC, b micro/meso-OPC-20, c micro/meso-OPC-40, d micro/meso-OPC-60, e micro/meso-OPC-180 and f micro/meso-OPC-300, respectively.

The H 2 O 2 selectivity and the numbers of transferred electrons of the Micro/meso-OPC-x catalysts could be calculated from SCV-RRDE profiles as shown in Fig.  6a . The selectivity initially increases and subsequently decreases (from 60.0% to 95.5% and then 84.9%) with increased oxygen content, suggesting that moderate and proper surface content of oxygen is the key to the improved H 2 O 2 electrochemical synthesis activity. The micro/meso-OPC-60 sample exhibits the best H 2 O 2 selectivity among all micro/meso-OPC-x catalysts. The observed trend in H 2 O 2 selectivity as a function of oxygen contents in micro/meso-OPC-x catalysts demonstrates a critical balance in surface oxygen functionalization for optimal electrocatalytic performance (Supplementary Fig.  7 ) 27 , 35 . This balance is further demonstrated by the remarkable selectivity of micro/meso-OPC-60, which can be attributed to the higher content of the -C = O group (Supplementary Fig.  8 ), highlighting its pivotal role as a vital active site in the electrochemical production of H 2 O 2 . The finding agrees well with previous research 36 and provides additional experimental evidence that targeted functionalization can significantly contribute to superior catalytic performance. Notably, the decline in selectivity may arise from the excessive oxygen content within the catalyst structure, which may result in poor electron transportation 42 , 43 . In addition, over-high oxygen content may lead to strong interactions between oxygen-active sites and reaction intermediates, leading to the unwanted breaking of the O–O bond and thus the over-reduction yielding H 2 O 3 . Therefore, an in-depth understanding and precise control of oxygen content is essential for tailoring and optimizing the H 2 O 2 selectivity in oxygen-functionalized carbon catalysts for efficient 2e − ORR applications. To further elucidate the synergistic effect between the porous structure and oxygen function groups on H 2 O 2 selectivity, we conducted a comprehensive analysis comparing catalysts featuring porous structure and oxygen functional groups and their integration. All porous materials and Template-free-C were oxidized in nitric acid for 60 min and subsequently analyzed via XPS, as depicted in Supplementary Fig.  9a–f and Supplementary Table  4 . The results revealed distinct trend, the oxidized non-porous carbon (Template-free-OC-60) exhibited higher transferred electron numbers and the lowest H 2 O 2 selectivity at 0.4 V RHE in 0.1 M KOH solution as compared to its counterparts featuring both oxygen function groups and porous structure, irrespective of their specific porous structure (Fig.  6b ). In particular, introducing oxygen to the non-porous carbon resulted in significant enhancement of H 2 O 2 selectivity, from 28% to 76.7%. Furthermore, the construction of diverse porous structures within certain oxygen functionalities boosted selectivity to a range of 84.9–100%, emphasizing the significant roles of both factors in determining the catalytic activity. Mechanistic insights into this synergistic effect can be explained from a two-step promotional mechanism where the porous structure ensures efficient accessibility, mass transfer and product desorption 28 , while the oxygen functional groups act as catalytic active sites that preferentially drive the formation of H 2 O 2 by altering the electronic structure of the adjacent carbon matrix 25 , 44 . This synergy between structural and functional groups highlights the essential of beneficial material design in optimizing the catalyst performance for specific reactions.

a Hydrogen peroxide selectivity (H 2 O 2 %) and average number of electron transferred ( n av ) of micro/meso-OPC-x in O 2 -saturated 0.1 M KOH solution, b hydrogen peroxide selectivity (H 2 O 2 %) and average number of electron transferred ( n av ) of Template-free-OC-60, Hiera-OPC-60, micro/meso-OPC-60, micro-OPC-60 and Meso-OPC-60 in O 2 -saturated 0.1 M KOH solution, c SCV-RRDE profiles of Meso-OPC-60 catalyst in an O 2 -saturated 0.1 M KOH electrolyte at 1600 rpm before and after reaction for 10 h. d Current density as function of reaction time at the constant potential of 0.2 V RHE for long-term stability test of Meso-OPC-60 catalyst.

Following above structure-function relation guidance and combining the synergistic effect between porous structure and oxygen functionalities, we have fabricated Meso-OPC-60 carbon catalyst, which contains both mesopores and proper content of oxygen functionalities (Supplementary Table  3 ), and the optimized Meso-OPC-60 exhibits ~100% H 2 O 2 selectivity at the potential of 0.4 V RHE , marking the highest recorded value compared to all the control samples in the present work or any other recently reported cutting-edge electrocatalysts 9 , 40 , 45 , 46 (Supplementary Fig.  10 ). In addition, Meso-OPC-60 material also exhibits remarkable stability without significant change in ring and disk current densities after 10 h of continuous reaction (Fig.  6c, d ), indicating that the catalyst has excellent durability.

Conclusions

In conclusion, the present research has successfully synthesized various carbon materials with diverse porous structures and oxygen content. The mesoporous material (Meso-PC) displays outstanding 2e − ORR selectivity, which can be attributed to its precisely tailored pore structure, facilitating the mass transfer of reactants and products. Furthermore, it is observed that the ketonic carbonyl (-C = O) group may be the main active site for 2e − ORR and a moderate surface content of oxygen functionalities is the key to improved H 2 O 2 electrochemical synthesis activity. Notably, the Meso-OPC-60 catalyst achieved 100% H 2 O 2 selectivity at a potential of 0.4 V RHE . These findings provide a theoretical basis for improving the oxygen reduction performance of carbon nanomaterials for the highly efficient electrochemical synthesis of H 2 O 2 .

Synthesis of micro/mesoporous carbon (micro/meso-PC)

In a typical procedure for synthesizing micro/meso-PC, 1.25 g glucose was dispersed in 5 ml of purified water and stirred for approximately 30 min. Subsequently, a mixture of 1.0 g SBA-15, 1.0 g ZnCl 2 and 0.14 g H 2 SO 4 was added to the glucose solution and stirred for 6 h. The resulting product was placed to a Teflon-lined autoclave and kept at 100 °C for 6 h, followed by an additional 6 h at 160 °C. Afterward, the mixture was impregnated again with 5 ml H 2 O, 0.8 g glucose, and 0.14 g H 2 SO 4 , followed by heating at 100 °C for 6 h and 160 °C for 6 h. The obtained solid product was carbonized at 900 °C for 2 h under Ar flow. To remove the silica template, the SBA-15/carbon composite was dispersed in 5.0 M HF at room temperature, and the mixture was stirred for two days. The resulting suspension underwent centrifugation, raised with water, and dried at 60 °C, to obtain the micro/meso-PC.

Synthesis of mesoporous carbon (meso-PC)

Meso-PC catalyst was synthesized using the same method described above, except for the absence of zinc chloride.

Synthesis of microporous carbon (micro-PC)

Micro-PC catalyst was synthesized using the same method described above, except for the absence of zinc chloride and using Y-zeolite (SiO 2 /Al 2 O 3 ) as a microporous template instead of mesoporous SBA-15.

Synthesis of hierarchical porous carbon (hiera-PC)

Hiera-PC catalyst was synthesized by direct carbonization of the glucose and zinc chloride mixture with the mass ratio of 1:1 at 900 °C for 2 h under Ar flow.

Synthesis of template-free carbon

Template-free-C catalyst was synthesized by direct carbonization of glucose at 900 °C for 2 h under Ar flow.

HNO 3 -oxidation

200 mg above synthesized carbon materials were placed in a 100 mL one-neck flask containing 50 mL of 68% HNO 3 and were refluxed at 120 °C. The suspension was centrifuged, rinsed with water, and dried at 60 °C to obtain micro/meso-OPC-x, where x represents the oxidation time, and x  = 20, 40, 60, 180, and 300 min, respectively.

Structure characterization

The morphological and microstructural properties of the catalyst materials were analyzed using a field emission scanning electron microscope (SEM) and a transmission electron microscope (TEM, FEI Tecnai G2 F20). The surface chemical compositions were quantified by X-ray photoelectron spectroscopy (XPS, ESCALAB250). Powder X-ray diffraction analysis was used to determine the crystallinity of the samples. The porosity of catalyst materials and pore size distribution were analyzed using the Brunauer-Emmett-Teller (BET) technique.

Electrochemical measurements

The ORR studies were carried out at ambient temperature using a rotating ring disk electrode (RRDE) as the working electrode, a Pt wire as the counter electrode, and Hg/HgO (0.1 M KOH) as the reference electrode. The catalyst ink was obtained by combining 6 mg of the appropriate powder with 2 ml of a 0.3 wt% Nafion solution. The mixture was then subjected to ultrasonic treatment for 30 min. Next, 10 μL of the ink was deposited onto the working electrode. The SCV curves were obtained by employing a GC (glassy carbon) disk electrode surrounded by a Pt ring (with an inner diameter of 4 mm) in an O 2 -saturated solution of 0.1 M KOH. The scan rate was set at 10 mV/s, and the rotation rate was maintained at 1600 rpm. The H 2 O 2 selectivity and number of electrons transferred were calculated using Eqs. ( 1 ) and ( 2 ), respectively.

where σ represents the ratio of the current densities of the disk ( j D) and the ring ( j R), and N is the collecting efficiency ( N  = 0.276).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

The authors acknowledge the financial support from the NSFC of China (22072163, U23A20545), Shccig-Qinling Program, and China Baowu Low Carbon Metallurgy Innovation Foundation-BWLCF202113, IMR Innovation Fund (2023-PY13).

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Abdalazeez Ismail Mohamed Albashir, Xingyu Lu, Xueya Dai & Wei Qi

Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences, Shenyang, Liaoning, People’s Republic of China

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Abdalazeez Ismail Mohamed Albashir: Experiments, Methodology, & Writing. Xingyu Lu: Writing—review & editing. Xueya Dai: Methodology & investigation. Wei Qi: Conceptualization, Writing—review & editing.

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Albashir, A.I.M., Lu, X., Dai, X. et al. Effects of porous structure and oxygen functionalities on electrochemical synthesis of hydrogen peroxide on ordered mesoporous carbon. Commun Chem 7 , 111 (2024). https://doi.org/10.1038/s42004-024-01194-3

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Received : 28 January 2024

Accepted : 30 April 2024

Published : 13 May 2024

DOI : https://doi.org/10.1038/s42004-024-01194-3

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