questions to use for a research paper

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How to Write a Good Research Question (w/ Examples)

What is a Research Question?

A research question is the main question that your study sought or is seeking to answer. A clear research question guides your research paper or thesis and states exactly what you want to find out, giving your work a focus and objective. Learning  how to write a hypothesis or research question is the start to composing any thesis, dissertation, or research paper. It is also one of the most important sections of a research proposal . 

A good research question not only clarifies the writing in your study; it provides your readers with a clear focus and facilitates their understanding of your research topic, as well as outlining your study’s objectives. Before drafting the paper and receiving research paper editing (and usually before performing your study), you should write a concise statement of what this study intends to accomplish or reveal.

Research Question Writing Tips

Listed below are the important characteristics of a good research question:

A good research question should:

• Be clear and provide specific information so readers can easily understand the purpose.
• Be focused in its scope and narrow enough to be addressed in the space allowed by your paper
• Be relevant and concise and express your main ideas in as few words as possible, like a hypothesis.
• Be precise and complex enough that it does not simply answer a closed “yes or no” question, but requires an analysis of arguments and literature prior to its being considered acceptable. 
• Be arguable or testable so that answers to the research question are open to scrutiny and specific questions and counterarguments.

Some of these characteristics might be difficult to understand in the form of a list. Let’s go into more detail about what a research question must do and look at some examples of research questions.

The research question should be specific and focused 

Research questions that are too broad are not suitable to be addressed in a single study. One reason for this can be if there are many factors or variables to consider. In addition, a sample data set that is too large or an experimental timeline that is too long may suggest that the research question is not focused enough.

A specific research question means that the collective data and observations come together to either confirm or deny the chosen hypothesis in a clear manner. If a research question is too vague, then the data might end up creating an alternate research problem or hypothesis that you haven’t addressed in your Introduction section .

The research question should be based on the literature 

An effective research question should be answerable and verifiable based on prior research because an effective scientific study must be placed in the context of a wider academic consensus. This means that conspiracy or fringe theories are not good research paper topics.

Instead, a good research question must extend, examine, and verify the context of your research field. It should fit naturally within the literature and be searchable by other research authors.

References to the literature can be in different citation styles and must be properly formatted according to the guidelines set forth by the publishing journal, university, or academic institution. This includes in-text citations as well as the Reference section . 

The research question should be realistic in time, scope, and budget

There are two main constraints to the research process: timeframe and budget.

A proper research question will include study or experimental procedures that can be executed within a feasible time frame, typically by a graduate doctoral or master’s student or lab technician. Research that requires future technology, expensive resources, or follow-up procedures is problematic.

A researcher’s budget is also a major constraint to performing timely research. Research at many large universities or institutions is publicly funded and is thus accountable to funding restrictions. 

The research question should be in-depth

Research papers, dissertations and theses , and academic journal articles are usually dozens if not hundreds of pages in length.

A good research question or thesis statement must be sufficiently complex to warrant such a length, as it must stand up to the scrutiny of peer review and be reproducible by other scientists and researchers.

Research Question Types

Qualitative and quantitative research are the two major types of research, and it is essential to develop research questions for each type of study. 

Quantitative Research Questions

Quantitative research questions are specific. A typical research question involves the population to be studied, dependent and independent variables, and the research design.

In addition, quantitative research questions connect the research question and the research design. In addition, it is not possible to answer these questions definitively with a “yes” or “no” response. For example, scientific fields such as biology, physics, and chemistry often deal with “states,” in which different quantities, amounts, or velocities drastically alter the relevance of the research.

As a consequence, quantitative research questions do not contain qualitative, categorical, or ordinal qualifiers such as “is,” “are,” “does,” or “does not.”

Categories of quantitative research questions

Qualitative research questions.

In quantitative research, research questions have the potential to relate to broad research areas as well as more specific areas of study. Qualitative research questions are less directional, more flexible, and adaptable compared with their quantitative counterparts. Thus, studies based on these questions tend to focus on “discovering,” “explaining,” “elucidating,” and “exploring.”

Categories of qualitative research questions

Quantitative and qualitative research question examples.

Good and Bad Research Question Examples

Below are some good (and not-so-good) examples of research questions that researchers can use to guide them in crafting their own research questions.

Research Question Example 1

The first research question is too vague in both its independent and dependent variables. There is no specific information on what “exposure” means. Does this refer to comments, likes, engagement, or just how much time is spent on the social media platform?

Second, there is no useful information on what exactly “affected” means. Does the subject’s behavior change in some measurable way? Or does this term refer to another factor such as the user’s emotions?

Research Question Example 2

In this research question, the first example is too simple and not sufficiently complex, making it difficult to assess whether the study answered the question. The author could really only answer this question with a simple “yes” or “no.” Further, the presence of data would not help answer this question more deeply, which is a sure sign of a poorly constructed research topic.

The second research question is specific, complex, and empirically verifiable. One can measure program effectiveness based on metrics such as attendance or grades. Further, “bullying” is made into an empirical, quantitative measurement in the form of recorded disciplinary actions.

Steps for Writing a Research Question

Good research questions are relevant, focused, and meaningful. It can be difficult to come up with a good research question, but there are a few steps you can follow to make it a bit easier.

1. Start with an interesting and relevant topic

Choose a research topic that is interesting but also relevant and aligned with your own country’s culture or your university’s capabilities. Popular academic topics include healthcare and medical-related research. However, if you are attending an engineering school or humanities program, you should obviously choose a research question that pertains to your specific study and major.

Below is an embedded graph of the most popular research fields of study based on publication output according to region. As you can see, healthcare and the basic sciences receive the most funding and earn the highest number of publications. 

2. Do preliminary research  

You can begin doing preliminary research once you have chosen a research topic. Two objectives should be accomplished during this first phase of research. First, you should undertake a preliminary review of related literature to discover issues that scholars and peers are currently discussing. With this method, you show that you are informed about the latest developments in the field.

Secondly, identify knowledge gaps or limitations in your topic by conducting a preliminary literature review . It is possible to later use these gaps to focus your research question after a certain amount of fine-tuning.

3. Narrow your research to determine specific research questions

You can focus on a more specific area of study once you have a good handle on the topic you want to explore. Focusing on recent literature or knowledge gaps is one good option. 

By identifying study limitations in the literature and overlooked areas of study, an author can carve out a good research question. The same is true for choosing research questions that extend or complement existing literature.

4. Evaluate your research question

Make sure you evaluate the research question by asking the following questions:

Is my research question clear?

The resulting data and observations that your study produces should be clear. For quantitative studies, data must be empirical and measurable. For qualitative, the observations should be clearly delineable across categories.

Is my research question focused and specific?

A strong research question should be specific enough that your methodology or testing procedure produces an objective result, not one left to subjective interpretation. Open-ended research questions or those relating to general topics can create ambiguous connections between the results and the aims of the study. 

Is my research question sufficiently complex?

The result of your research should be consequential and substantial (and fall sufficiently within the context of your field) to warrant an academic study. Simply reinforcing or supporting a scientific consensus is superfluous and will likely not be well received by most journal editors.  

Editing Your Research Question

Your research question should be fully formulated well before you begin drafting your research paper. However, you can receive English paper editing and proofreading services at any point in the drafting process. Language editors with expertise in your academic field can assist you with the content and language in your Introduction section or other manuscript sections. And if you need further assistance or information regarding paper compositions, in the meantime, check out our academic resources , which provide dozens of articles and videos on a variety of academic writing and publication topics.

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How To Write a Research Question

Academic writing and research require a distinct focus and direction. A well-designed research question gives purpose and clarity to your research. In addition, it helps your readers understand the issue you are trying to address and explore.

Every time you want to know more about a subject, you will pose a question. The same idea is used in research as well. You must pose a question in order to effectively address a research problem. That's why the research question is an integral part of the research process. Additionally, it offers the author writing and reading guidelines, be it qualitative research or quantitative research.

In your research paper , you must single out just one issue or problem. The specific issue or claim you wish to address should be included in your thesis statement in order to clarify your main argument.

A good research question must have the following characteristics.

• Should include only one problem in the research question
• Should be able to find the answer using primary data and secondary data sources
• Should be possible to resolve within the given time and other constraints
• Detailed and in-depth results should be achievable
• Should be relevant and realistic.
• It should relate to your chosen area of research

While a larger project, like a thesis, might have several research questions to address, each one should be directed at your main area of study. Of course, you can use different research designs and research methods (qualitative research or quantitative research) to address various research questions. However, they must all be pertinent to the study's objectives.

What is a Research Question?

A research question is an inquiry that the research attempts to answer. It is the heart of the systematic investigation. Research questions are the most important step in any research project. In essence, it initiates the research project and establishes the pace for the specific research A research question is:

• Clear : It provides enough detail that the audience understands its purpose without any additional explanation.
• Focused : It is so specific that it can be addressed within the time constraints of the writing task.
• Succinct: It is written in the shortest possible words.
• Complex : It is not possible to answer it with a "yes" or "no", but requires analysis and synthesis of ideas before somebody can create a solution.
• Argumental : Its potential answers are open for debate rather than accepted facts.

A good research question usually focuses on the research and determines the research design, methodology, and hypothesis. It guides all phases of inquiry, data collection, analysis, and reporting. You should gather valuable information by asking the right questions.

Why are Research Questions so important?

Regardless of whether it is a qualitative research or quantitative research project, research questions provide writers and their audience with a way to navigate the writing and research process. Writers can avoid "all-about" papers by asking straightforward and specific research questions that help them focus on their research and support a specific thesis.

Types of Research Questions

There are two types of research: Qualitative research and Quantitative research . There must be research questions for every type of research. Your research question will be based on the type of research you want to conduct and the type of data collection.

The first step in designing research involves identifying a gap and creating a focused research question.

Below is a list of common research questions that can be used in a dissertation. Keep in mind that these are merely illustrations of typical research questions used in dissertation projects. The real research questions themselves might be more difficult.

Example Research Questions

The following are a few examples of research questions and research problems to help you understand how research questions can be created for a particular research problem.

Steps to Write Research Questions

You can focus on the issue or research gaps you're attempting to solve by using the research questions as a direction.

If you're unsure how to go about writing a good research question, these are the steps to follow in the process:

• Select an interesting topic Always choose a topic that interests you. Because if your curiosity isn’t aroused by a subject, you’ll have a hard time conducting research around it. Alos, it’s better that you pick something that’s neither too narrow or too broad.
• Do preliminary research on the topic Search for relevant literature to gauge what problems have already been tackled by scholars. You can do that conveniently through repositories like Scispace , where you’ll find millions of papers in one place. Once you do find the papers you’re looking for, try our reading assistant, SciSpace Copilot to get simple explanations for the paper . You’ll be able to quickly understand the abstract, find the key takeaways, and the main arguments presented in the paper. This will give you a more contextual understanding of your subject and you’ll have an easier time identifying knowledge gaps in your discipline.

     Also: ChatPDF vs. SciSpace Copilot: Unveiling the best tool for your research

• Consider your audience It is essential to understand your audience to develop focused research questions for essays or dissertations. When narrowing down your topic, you can identify aspects that might interest your audience.
• Ask questions Asking questions will give you a deeper understanding of the topic. Evaluate your question through the What, Why, When, How, and other open-ended questions assessment.
• Assess your question Once you have created a research question, assess its effectiveness to determine if it is useful for the purpose. Refine and revise the dissertation research question multiple times.

Additionally, use this list of questions as a guide when formulating your research question.

Are you able to answer a specific research question? After identifying a gap in research, it would be helpful to formulate the research question. And this will allow the research to solve a part of the problem. Is your research question clear and centered on the main topic? It is important that your research question should be specific and related to your central goal. Are you tackling a difficult research question? It is not possible to answer the research question with a simple yes or no. The problem requires in-depth analysis. It is often started with "How" and "Why."

Start your research Once you have completed your dissertation research questions, it is time to review the literature on similar topics to discover different perspectives.

Strong  Research Question Samples

Uncertain: How should social networking sites work on the hatred that flows through their platform?

Certain: What should social media sites like Twitter or Facebook do to address the harm they are causing?

This unclear question does not specify the social networking sites that are being used or what harm they might be causing. In addition, this question assumes that the "harm" has been proven and/or accepted. This version is more specific and identifies the sites (Twitter, Facebook), the type and extent of harm (privacy concerns), and who might be suffering from that harm (users). Effective research questions should not be ambiguous or interpreted.

Unfocused: What are the effects of global warming on the environment?

Focused: What are the most important effects of glacial melting in Antarctica on penguins' lives?

This broad research question cannot be addressed in a book, let alone a college-level paper. Focused research targets a specific effect of global heating (glacial  melting), an area (Antarctica), or a specific animal (penguins). The writer must also decide which effect will have the greatest impact on the animals affected. If in doubt, narrow down your research question to the most specific possible.

Too Simple: What are the U.S. doctors doing to treat diabetes?

Appropriately complex: Which factors, if any, are most likely to predict a person's risk of developing diabetes?

This simple version can be found online. It is easy to answer with a few facts. The second, more complicated version of this question is divided into two parts. It is thought-provoking and requires extensive investigation as well as evaluation by the author. So, ensure that a quick Google search should not answer your research question.

How to write a strong Research Question?

The foundation of all research is the research question. You should therefore spend as much time as necessary to refine your research question based on various data.

You can conduct your research more efficiently and analyze your results better if you have great research questions for your dissertation, research paper , or essay .

The following criteria can help you evaluate the strength and importance of your research question and can be used to determine the strength of your research question:

• Researchable
• It should only cover one issue.
• A subjective judgment should not be included in the question.
• It can be answered with data analysis and research.
• Specific and Practical
• It should not contain a plan of action, policy, or solution.
• It should be clearly defined
• Within research limits
• Complex and Arguable
• It shouldn't be difficult to answer.
• To find the truth, you need in-depth knowledge
• Allows for discussion and deliberation
• Original and Relevant
• It should be in your area of study
• Its results should be measurable
• It should be original

Conclusion - How to write Research Questions?

Research questions provide a clear guideline for research. One research question may be part of a larger project, such as a dissertation. However, each question should only focus on one topic.

Research questions must be answerable, practical, specific, and applicable to your field. The research type that you use to base your research questions on will determine the research topic. You can start by selecting an interesting topic and doing preliminary research. Then, you can begin asking questions, evaluating your questions, and start your research.

Now it's easier than ever to streamline your research workflow with SciSpace ResearchGPT . Its integrated, comprehensive end-to-end platform for research allows scholars to easily discover, read, write and publish their research and fosters collaboration.

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Research Question Examples 🧑🏻‍🏫

25+ Practical Examples & Ideas To Help You Get Started 

By: Derek Jansen (MBA) | October 2023

A well-crafted research question (or set of questions) sets the stage for a robust study and meaningful insights.  But, if you’re new to research, it’s not always clear what exactly constitutes a good research question. In this post, we’ll provide you with clear examples of quality research questions across various disciplines, so that you can approach your research project with confidence!

Research Question Examples

• Psychology research questions
• Business research questions
• Education research questions
• Healthcare research questions
• Computer science research questions

Examples: Psychology

Let’s start by looking at some examples of research questions that you might encounter within the discipline of psychology.

How does sleep quality affect academic performance in university students?

This question is specific to a population (university students) and looks at a direct relationship between sleep and academic performance, both of which are quantifiable and measurable variables.

What factors contribute to the onset of anxiety disorders in adolescents?

The question narrows down the age group and focuses on identifying multiple contributing factors. There are various ways in which it could be approached from a methodological standpoint, including both qualitatively and quantitatively.

Do mindfulness techniques improve emotional well-being?

This is a focused research question aiming to evaluate the effectiveness of a specific intervention.

How does early childhood trauma impact adult relationships?

This research question targets a clear cause-and-effect relationship over a long timescale, making it focused but comprehensive.

Is there a correlation between screen time and depression in teenagers?

This research question focuses on an in-demand current issue and a specific demographic, allowing for a focused investigation. The key variables are clearly stated within the question and can be measured and analysed (i.e., high feasibility).

Examples: Business/Management

Next, let’s look at some examples of well-articulated research questions within the business and management realm.

How do leadership styles impact employee retention?

This is an example of a strong research question because it directly looks at the effect of one variable (leadership styles) on another (employee retention), allowing from a strongly aligned methodological approach.

What role does corporate social responsibility play in consumer choice?

Current and precise, this research question can reveal how social concerns are influencing buying behaviour by way of a qualitative exploration.

Does remote work increase or decrease productivity in tech companies?

Focused on a particular industry and a hot topic, this research question could yield timely, actionable insights that would have high practical value in the real world.

How do economic downturns affect small businesses in the homebuilding industry?

Vital for policy-making, this highly specific research question aims to uncover the challenges faced by small businesses within a certain industry.

Which employee benefits have the greatest impact on job satisfaction?

By being straightforward and specific, answering this research question could provide tangible insights to employers.

Examples: Education

Next, let’s look at some potential research questions within the education, training and development domain.

How does class size affect students’ academic performance in primary schools?

This example research question targets two clearly defined variables, which can be measured and analysed relatively easily.

Do online courses result in better retention of material than traditional courses?

Timely, specific and focused, answering this research question can help inform educational policy and personal choices about learning formats.

What impact do US public school lunches have on student health?

Targeting a specific, well-defined context, the research could lead to direct changes in public health policies.

To what degree does parental involvement improve academic outcomes in secondary education in the Midwest?

This research question focuses on a specific context (secondary education in the Midwest) and has clearly defined constructs.

What are the negative effects of standardised tests on student learning within Oklahoma primary schools?

This research question has a clear focus (negative outcomes) and is narrowed into a very specific context.

Need a helping hand?

Examples: Healthcare

Shifting to a different field, let’s look at some examples of research questions within the healthcare space.

What are the most effective treatments for chronic back pain amongst UK senior males?

Specific and solution-oriented, this research question focuses on clear variables and a well-defined context (senior males within the UK).

How do different healthcare policies affect patient satisfaction in public hospitals in South Africa?

This question is has clearly defined variables and is narrowly focused in terms of context.

Which factors contribute to obesity rates in urban areas within California?

This question is focused yet broad, aiming to reveal several contributing factors for targeted interventions.

Does telemedicine provide the same perceived quality of care as in-person visits for diabetes patients?

Ideal for a qualitative study, this research question explores a single construct (perceived quality of care) within a well-defined sample (diabetes patients).

Which lifestyle factors have the greatest affect on the risk of heart disease?

This research question aims to uncover modifiable factors, offering preventive health recommendations.

Examples: Computer Science

Last but certainly not least, let’s look at a few examples of research questions within the computer science world.

What are the perceived risks of cloud-based storage systems?

Highly relevant in our digital age, this research question would align well with a qualitative interview approach to better understand what users feel the key risks of cloud storage are.

Which factors affect the energy efficiency of data centres in Ohio?

With a clear focus, this research question lays a firm foundation for a quantitative study.

How do TikTok algorithms impact user behaviour amongst new graduates?

While this research question is more open-ended, it could form the basis for a qualitative investigation.

What are the perceived risk and benefits of open-source software software within the web design industry?

Practical and straightforward, the results could guide both developers and end-users in their choices.

Remember, these are just examples…

In this post, we’ve tried to provide a wide range of research question examples to help you get a feel for what research questions look like in practice. That said, it’s important to remember that these are just examples and don’t necessarily equate to good research topics . If you’re still trying to find a topic, check out our topic megalist for inspiration.

Psst… there’s more (for free)

This post is part of our dissertation mini-course, which covers everything you need to get started with your dissertation, thesis or research project. 

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Home » Research Paper – Structure, Examples and Writing Guide

Research Paper – Structure, Examples and Writing Guide

Table of Contents

Research Paper

Definition:

Research Paper is a written document that presents the author’s original research, analysis, and interpretation of a specific topic or issue.

It is typically based on Empirical Evidence, and may involve qualitative or quantitative research methods, or a combination of both. The purpose of a research paper is to contribute new knowledge or insights to a particular field of study, and to demonstrate the author’s understanding of the existing literature and theories related to the topic.

Structure of Research Paper

The structure of a research paper typically follows a standard format, consisting of several sections that convey specific information about the research study. The following is a detailed explanation of the structure of a research paper:

The title page contains the title of the paper, the name(s) of the author(s), and the affiliation(s) of the author(s). It also includes the date of submission and possibly, the name of the journal or conference where the paper is to be published.

The abstract is a brief summary of the research paper, typically ranging from 100 to 250 words. It should include the research question, the methods used, the key findings, and the implications of the results. The abstract should be written in a concise and clear manner to allow readers to quickly grasp the essence of the research.

Introduction

The introduction section of a research paper provides background information about the research problem, the research question, and the research objectives. It also outlines the significance of the research, the research gap that it aims to fill, and the approach taken to address the research question. Finally, the introduction section ends with a clear statement of the research hypothesis or research question.

Literature Review

The literature review section of a research paper provides an overview of the existing literature on the topic of study. It includes a critical analysis and synthesis of the literature, highlighting the key concepts, themes, and debates. The literature review should also demonstrate the research gap and how the current study seeks to address it.

The methods section of a research paper describes the research design, the sample selection, the data collection and analysis procedures, and the statistical methods used to analyze the data. This section should provide sufficient detail for other researchers to replicate the study.

The results section presents the findings of the research, using tables, graphs, and figures to illustrate the data. The findings should be presented in a clear and concise manner, with reference to the research question and hypothesis.

The discussion section of a research paper interprets the findings and discusses their implications for the research question, the literature review, and the field of study. It should also address the limitations of the study and suggest future research directions.

The conclusion section summarizes the main findings of the study, restates the research question and hypothesis, and provides a final reflection on the significance of the research.

The references section provides a list of all the sources cited in the paper, following a specific citation style such as APA, MLA or Chicago.

How to Write Research Paper

You can write Research Paper by the following guide:

• Choose a Topic: The first step is to select a topic that interests you and is relevant to your field of study. Brainstorm ideas and narrow down to a research question that is specific and researchable.
• Conduct a Literature Review: The literature review helps you identify the gap in the existing research and provides a basis for your research question. It also helps you to develop a theoretical framework and research hypothesis.
• Develop a Thesis Statement : The thesis statement is the main argument of your research paper. It should be clear, concise and specific to your research question.
• Plan your Research: Develop a research plan that outlines the methods, data sources, and data analysis procedures. This will help you to collect and analyze data effectively.
• Collect and Analyze Data: Collect data using various methods such as surveys, interviews, observations, or experiments. Analyze data using statistical tools or other qualitative methods.
• Organize your Paper : Organize your paper into sections such as Introduction, Literature Review, Methods, Results, Discussion, and Conclusion. Ensure that each section is coherent and follows a logical flow.
• Write your Paper : Start by writing the introduction, followed by the literature review, methods, results, discussion, and conclusion. Ensure that your writing is clear, concise, and follows the required formatting and citation styles.
• Edit and Proofread your Paper: Review your paper for grammar and spelling errors, and ensure that it is well-structured and easy to read. Ask someone else to review your paper to get feedback and suggestions for improvement.
• Cite your Sources: Ensure that you properly cite all sources used in your research paper. This is essential for giving credit to the original authors and avoiding plagiarism.

Research Paper Example

Note : The below example research paper is for illustrative purposes only and is not an actual research paper. Actual research papers may have different structures, contents, and formats depending on the field of study, research question, data collection and analysis methods, and other factors. Students should always consult with their professors or supervisors for specific guidelines and expectations for their research papers.

Research Paper Example sample for Students:

Title: The Impact of Social Media on Mental Health among Young Adults

Abstract: This study aims to investigate the impact of social media use on the mental health of young adults. A literature review was conducted to examine the existing research on the topic. A survey was then administered to 200 university students to collect data on their social media use, mental health status, and perceived impact of social media on their mental health. The results showed that social media use is positively associated with depression, anxiety, and stress. The study also found that social comparison, cyberbullying, and FOMO (Fear of Missing Out) are significant predictors of mental health problems among young adults.

Introduction: Social media has become an integral part of modern life, particularly among young adults. While social media has many benefits, including increased communication and social connectivity, it has also been associated with negative outcomes, such as addiction, cyberbullying, and mental health problems. This study aims to investigate the impact of social media use on the mental health of young adults.

Literature Review: The literature review highlights the existing research on the impact of social media use on mental health. The review shows that social media use is associated with depression, anxiety, stress, and other mental health problems. The review also identifies the factors that contribute to the negative impact of social media, including social comparison, cyberbullying, and FOMO.

Methods : A survey was administered to 200 university students to collect data on their social media use, mental health status, and perceived impact of social media on their mental health. The survey included questions on social media use, mental health status (measured using the DASS-21), and perceived impact of social media on their mental health. Data were analyzed using descriptive statistics and regression analysis.

Results : The results showed that social media use is positively associated with depression, anxiety, and stress. The study also found that social comparison, cyberbullying, and FOMO are significant predictors of mental health problems among young adults.

Discussion : The study’s findings suggest that social media use has a negative impact on the mental health of young adults. The study highlights the need for interventions that address the factors contributing to the negative impact of social media, such as social comparison, cyberbullying, and FOMO.

Conclusion : In conclusion, social media use has a significant impact on the mental health of young adults. The study’s findings underscore the need for interventions that promote healthy social media use and address the negative outcomes associated with social media use. Future research can explore the effectiveness of interventions aimed at reducing the negative impact of social media on mental health. Additionally, longitudinal studies can investigate the long-term effects of social media use on mental health.

Limitations : The study has some limitations, including the use of self-report measures and a cross-sectional design. The use of self-report measures may result in biased responses, and a cross-sectional design limits the ability to establish causality.

Implications: The study’s findings have implications for mental health professionals, educators, and policymakers. Mental health professionals can use the findings to develop interventions that address the negative impact of social media use on mental health. Educators can incorporate social media literacy into their curriculum to promote healthy social media use among young adults. Policymakers can use the findings to develop policies that protect young adults from the negative outcomes associated with social media use.

References :

• Twenge, J. M., & Campbell, W. K. (2019). Associations between screen time and lower psychological well-being among children and adolescents: Evidence from a population-based study. Preventive medicine reports, 15, 100918.
• Primack, B. A., Shensa, A., Escobar-Viera, C. G., Barrett, E. L., Sidani, J. E., Colditz, J. B., … & James, A. E. (2017). Use of multiple social media platforms and symptoms of depression and anxiety: A nationally-representative study among US young adults. Computers in Human Behavior, 69, 1-9.
• Van der Meer, T. G., & Verhoeven, J. W. (2017). Social media and its impact on academic performance of students. Journal of Information Technology Education: Research, 16, 383-398.

Appendix : The survey used in this study is provided below.

Social Media and Mental Health Survey

• How often do you use social media per day?
• Less than 30 minutes
• 30 minutes to 1 hour
• 1 to 2 hours
• 2 to 4 hours
• More than 4 hours
• Which social media platforms do you use?
• Others (Please specify)
• How often do you experience the following on social media?
• Social comparison (comparing yourself to others)
• Cyberbullying
• Fear of Missing Out (FOMO)
• Have you ever experienced any of the following mental health problems in the past month?
• Do you think social media use has a positive or negative impact on your mental health?
• Very positive
• Somewhat positive
• Somewhat negative
• Very negative
• In your opinion, which factors contribute to the negative impact of social media on mental health?
• Social comparison
• In your opinion, what interventions could be effective in reducing the negative impact of social media on mental health?
• Education on healthy social media use
• Counseling for mental health problems caused by social media
• Social media detox programs
• Regulation of social media use

Thank you for your participation!

Applications of Research Paper

Research papers have several applications in various fields, including:

• Advancing knowledge: Research papers contribute to the advancement of knowledge by generating new insights, theories, and findings that can inform future research and practice. They help to answer important questions, clarify existing knowledge, and identify areas that require further investigation.
• Informing policy: Research papers can inform policy decisions by providing evidence-based recommendations for policymakers. They can help to identify gaps in current policies, evaluate the effectiveness of interventions, and inform the development of new policies and regulations.
• Improving practice: Research papers can improve practice by providing evidence-based guidance for professionals in various fields, including medicine, education, business, and psychology. They can inform the development of best practices, guidelines, and standards of care that can improve outcomes for individuals and organizations.
• Educating students : Research papers are often used as teaching tools in universities and colleges to educate students about research methods, data analysis, and academic writing. They help students to develop critical thinking skills, research skills, and communication skills that are essential for success in many careers.
• Fostering collaboration: Research papers can foster collaboration among researchers, practitioners, and policymakers by providing a platform for sharing knowledge and ideas. They can facilitate interdisciplinary collaborations and partnerships that can lead to innovative solutions to complex problems.

When to Write Research Paper

Research papers are typically written when a person has completed a research project or when they have conducted a study and have obtained data or findings that they want to share with the academic or professional community. Research papers are usually written in academic settings, such as universities, but they can also be written in professional settings, such as research organizations, government agencies, or private companies.

Here are some common situations where a person might need to write a research paper:

• For academic purposes: Students in universities and colleges are often required to write research papers as part of their coursework, particularly in the social sciences, natural sciences, and humanities. Writing research papers helps students to develop research skills, critical thinking skills, and academic writing skills.
• For publication: Researchers often write research papers to publish their findings in academic journals or to present their work at academic conferences. Publishing research papers is an important way to disseminate research findings to the academic community and to establish oneself as an expert in a particular field.
• To inform policy or practice : Researchers may write research papers to inform policy decisions or to improve practice in various fields. Research findings can be used to inform the development of policies, guidelines, and best practices that can improve outcomes for individuals and organizations.
• To share new insights or ideas: Researchers may write research papers to share new insights or ideas with the academic or professional community. They may present new theories, propose new research methods, or challenge existing paradigms in their field.

Purpose of Research Paper

The purpose of a research paper is to present the results of a study or investigation in a clear, concise, and structured manner. Research papers are written to communicate new knowledge, ideas, or findings to a specific audience, such as researchers, scholars, practitioners, or policymakers. The primary purposes of a research paper are:

• To contribute to the body of knowledge : Research papers aim to add new knowledge or insights to a particular field or discipline. They do this by reporting the results of empirical studies, reviewing and synthesizing existing literature, proposing new theories, or providing new perspectives on a topic.
• To inform or persuade: Research papers are written to inform or persuade the reader about a particular issue, topic, or phenomenon. They present evidence and arguments to support their claims and seek to persuade the reader of the validity of their findings or recommendations.
• To advance the field: Research papers seek to advance the field or discipline by identifying gaps in knowledge, proposing new research questions or approaches, or challenging existing assumptions or paradigms. They aim to contribute to ongoing debates and discussions within a field and to stimulate further research and inquiry.
• To demonstrate research skills: Research papers demonstrate the author’s research skills, including their ability to design and conduct a study, collect and analyze data, and interpret and communicate findings. They also demonstrate the author’s ability to critically evaluate existing literature, synthesize information from multiple sources, and write in a clear and structured manner.

Characteristics of Research Paper

Research papers have several characteristics that distinguish them from other forms of academic or professional writing. Here are some common characteristics of research papers:

• Evidence-based: Research papers are based on empirical evidence, which is collected through rigorous research methods such as experiments, surveys, observations, or interviews. They rely on objective data and facts to support their claims and conclusions.
• Structured and organized: Research papers have a clear and logical structure, with sections such as introduction, literature review, methods, results, discussion, and conclusion. They are organized in a way that helps the reader to follow the argument and understand the findings.
• Formal and objective: Research papers are written in a formal and objective tone, with an emphasis on clarity, precision, and accuracy. They avoid subjective language or personal opinions and instead rely on objective data and analysis to support their arguments.
• Citations and references: Research papers include citations and references to acknowledge the sources of information and ideas used in the paper. They use a specific citation style, such as APA, MLA, or Chicago, to ensure consistency and accuracy.
• Peer-reviewed: Research papers are often peer-reviewed, which means they are evaluated by other experts in the field before they are published. Peer-review ensures that the research is of high quality, meets ethical standards, and contributes to the advancement of knowledge in the field.
• Objective and unbiased: Research papers strive to be objective and unbiased in their presentation of the findings. They avoid personal biases or preconceptions and instead rely on the data and analysis to draw conclusions.

Advantages of Research Paper

Research papers have many advantages, both for the individual researcher and for the broader academic and professional community. Here are some advantages of research papers:

• Contribution to knowledge: Research papers contribute to the body of knowledge in a particular field or discipline. They add new information, insights, and perspectives to existing literature and help advance the understanding of a particular phenomenon or issue.
• Opportunity for intellectual growth: Research papers provide an opportunity for intellectual growth for the researcher. They require critical thinking, problem-solving, and creativity, which can help develop the researcher’s skills and knowledge.
• Career advancement: Research papers can help advance the researcher’s career by demonstrating their expertise and contributions to the field. They can also lead to new research opportunities, collaborations, and funding.
• Academic recognition: Research papers can lead to academic recognition in the form of awards, grants, or invitations to speak at conferences or events. They can also contribute to the researcher’s reputation and standing in the field.
• Impact on policy and practice: Research papers can have a significant impact on policy and practice. They can inform policy decisions, guide practice, and lead to changes in laws, regulations, or procedures.
• Advancement of society: Research papers can contribute to the advancement of society by addressing important issues, identifying solutions to problems, and promoting social justice and equality.

Limitations of Research Paper

Research papers also have some limitations that should be considered when interpreting their findings or implications. Here are some common limitations of research papers:

• Limited generalizability: Research findings may not be generalizable to other populations, settings, or contexts. Studies often use specific samples or conditions that may not reflect the broader population or real-world situations.
• Potential for bias : Research papers may be biased due to factors such as sample selection, measurement errors, or researcher biases. It is important to evaluate the quality of the research design and methods used to ensure that the findings are valid and reliable.
• Ethical concerns: Research papers may raise ethical concerns, such as the use of vulnerable populations or invasive procedures. Researchers must adhere to ethical guidelines and obtain informed consent from participants to ensure that the research is conducted in a responsible and respectful manner.
• Limitations of methodology: Research papers may be limited by the methodology used to collect and analyze data. For example, certain research methods may not capture the complexity or nuance of a particular phenomenon, or may not be appropriate for certain research questions.
• Publication bias: Research papers may be subject to publication bias, where positive or significant findings are more likely to be published than negative or non-significant findings. This can skew the overall findings of a particular area of research.
• Time and resource constraints: Research papers may be limited by time and resource constraints, which can affect the quality and scope of the research. Researchers may not have access to certain data or resources, or may be unable to conduct long-term studies due to practical limitations.

About the author

Muhammad Hassan

Researcher, Academic Writer, Web developer

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• Developing a Research Question

by acburton | Mar 22, 2024 | Resources for Students , Writing Resources

Selecting your research question and creating a clear goal and structure for your writing can be challenging – whether you are doing it for the first time or if you’ve done it many times before. It can be especially difficult when your research question starts to look and feel a little different somewhere between your first and final draft. Don’t panic! It’s normal for your research question to change a little (or even quite a bit) as you move through and engage with the writing process. Anticipating this can remind you to stay on track while you work and that it’ll be okay even if the literature takes you in a different direction.

What Makes an Effective Research Question?

The most effective research question will usually be a critical thinking question and should use “how” or “why” to ensure it can move beyond a yes/no or one-word type of answer. Consider how your research question can aim to reveal something new, fill in a gap, even if small, and contribute to the field in a meaningful way; How might the proposed project move knowledge forward about a particular place or process? This should be specific and achievable!

The CEWC’s Grad Writing Consultant Tariq says, “I definitely concentrated on those aspects of what I saw in the field where I believed there was an opportunity to move the discipline forward.”

General Tips

Do your research.

Utilize the librarians at your university and take the time to research your topic first. Try looking at very general sources to get an idea of what could be interesting to you before you move to more academic articles that support your rough idea of the topic. It is important that research is grounded in what you see or experience regarding the topic you have chosen and what is already known in the literature. Spend time researching articles, books, etc. that supports your thesis. Once you have a number of sources that you know support what you want to write about, formulate a research question that serves as the interrogative form of your thesis statement.

Grad Writing Consultant Deni advises, “Delineate your intervention in the literature (i.e., be strategic about the literature you discuss and clear about your contributions to it).”

Start Broadly…. then Narrow Your Topic Down to Something Manageable

When brainstorming your research question, let your mind veer toward connections or associations that you might have already considered or that seem to make sense and consider if new research terms, language or concepts come to mind that may be interesting or exciting for you as a researcher. Sometimes testing out a research question while doing some preliminary researching is also useful to see if the language you are using or the direction you are heading toward is fruitful when trying to search strategically in academic databases. Be prepared to focus on a specific area of a broad topic.

Writing Consultant Jessie recommends outlining: “I think some rough outlining with a research question in mind can be helpful for me. I’ll have a research question and maybe a working thesis that I feel may be my claim to the research question based on some preliminary materials, brainstorming, etc.” — Jessie, CEWC Writing Consultant

Try an Exercise

In the earliest phase of brainstorming, try an exercise suggested by CEWC Writing Specialist, Percival! While it is normally used in classroom or workshop settings, this exercise can easily be modified for someone working alone. The flow of the activity, if done within a group setting, is 1) someone starts with an idea, 2) three other people share their idea, and 3) the starting person picks two of these new ideas they like best and combines their original idea with those. The activity then begins again with the idea that was not chosen. The solo version of this exercise substitutes a ‘word bank,’ created using words, topics, or ideas similar to your broad, overarching theme. Pick two words or phrases from your word bank, combine it with your original idea or topic, and ‘start again’ with two different words. This serves as a replacement for different people’s suggestions. Ideas for your ‘word bank’ can range from vague prompts about mapping or webbing (e.g., where your topic falls within the discipline and others like it), to more specific concepts that come from tracing the history of an idea (its past, present, future) or mapping the idea’s related ideas, influences, etc. Care for a physics analogy? There is a particle (your topic) that you can describe, a wave that the particle traces, and a field that the particle is mapped on.

Get Feedback and Affirm Your Confidence!

Creating a few different versions of your research question (they may be the same topic/issue/theme or differ slightly) can be useful during this process. Sharing these with trusted friends, colleagues, mentors, (or tutors!) and having conversations about your questions and ideas with other people can help you decide which version you may feel most confident or interested in. Ask colleagues and mentors to share their research questions with you to get a lot of examples. Once you have done the work of developing an effective research question, do not forget to affirm your confidence! Based on your working thesis, think about how you might organize your chapters or paragraphs and what resources you have for supporting this structure and organization. This can help boost your confidence that the research question you have created is effective and fruitful.

Be Open to Change

Remember, your research question may change from your first to final draft. For questions along the way, make an appointment with the Writing Center. We are here to help you develop an effective and engaging research question and build the foundation for a solid research paper!

Example 1: In my field developing a research question involves navigating the relationship between 1) what one sees/experiences at their field site and 2) what is already known in the literature. During my preliminary research, I found that the financial value of land was often a matter of precisely these cultural factors. So, my research question ended up being: How do the social and material qualities of land entangle with processes of financialization in the city of Lahore. Regarding point #1, this question was absolutely informed by what I saw in the field. But regarding point #2, the question was also heavily shaped by the literature. – Tariq

Example 2: A research question should not be a yes/no question like “Is pollution bad?”; but an open-ended question where the answer has to be supported with reasons and explanation. The question also has to be narrowed down to a specific topic—using the same example as before—”Is pollution bad?” can be revised to “How does pollution affect people?” I would encourage students to be more specific then; e.g., what area of pollution do you want to talk about: water, air, plastic, climate change… what type of people or demographic can we focus on? …how does this affect marginalized communities, minorities, or specific areas in California? After researching and deciding on a focus, your question might sound something like: How does government policy affect water pollution and how does it affect the marginalized communities in the state of California? -Janella

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Formulating Strong Research Questions: Examples and Writing Tips

Abstract | Introduction | Literature Review | Research question | Materials & Methods | Results | Discussion | Conclusion

In this blog, we will see how to construct and present the research question in your research paper. We will also look at other components that make up the final paragraph of the introduction section of your paper.

1. What is a research question in a research paper?

The research questions are normally the aims and objectives of your work. The research question pinpoints exactly what it is you want to find out in your work. You can have a single research question or multiple research questions in your paper depending on the complexity of your research. Generally, it is a good idea to keep the number of research questions to less than four.

2. Research question examples

Let’s look at some examples of research questions. The research question is normally one of the major components of the final paragraph of the introduction section. We will look at the examples of the entire final paragraph of the introduction along with the research questions to put things into perspective.

2.1. Example #1 (Health sciences research paper)

Here is an example from a health sciences research paper. The passage starts with the research gap. The authors are saying that there is a need for a better understanding of the relationship between social media and mental health. Then, the authors explain the aims of their research and elaborate on what methodology they will be using to achieve their aims. The authors say that they will be using online surveys and face-to-face interviews to collect data to answer their research question. The passage flows very well and the author nicely lays out the research gap, the study aims, and the plan of action.

The effects of social media usage on mental health are poorly documented in the literature as research papers on the topic give contradictory conclusions. The present study aims to improve our understanding of the effects of social media usage on mental health. The data were collected from a variety of age-group over a period of two years in a structured manner. The methods of data collection involved online surveys and face-to-face interviews. _ Research gap  _  Research question _   _  Method summary

2.2. Example #2 (Hypothesis-driven research paper)

Here is a slightly different variant of the previous example. Here, the authors have formulated the research question in the form of a hypothesis. Same as before, the authors are establishing the research gap in the first statement. In the next couple of statements, they are defining a specific hypothesis that they will be testing in the paper. In this case, they are testing the link between social media and mental health. And in the final statement, they are explaining the research methodology they will be employing to either prove or disprove the hypothesis. This is a pretty good example to follow if your research work is hypothesis-driven.

Past research suggests that while social media use is correlated with levels of anxiety and depression, the evidence so far is limited [1-2]. Therefore, building on previous discussion, Hypothesis 1 proposes: The levels of anxiety and depression will be lower among those who use social media platforms less frequently compared to those who use social media more frequently. This hypothesis (H1) is tested in this study through surveys and face-to-face interviews. _  Research gap  _  Research question (Hypothesis)  _  Method summary

2.3. Example #3 (Computer sciences research paper)

Here is an example from a computer sciences research paper. The authors establish the research gap by saying that there aren’t many papers on the topic of stock price prediction. Then, they explain what they are proposing.  They are proposing a new method called the ‘Hybrid prediction model’. Then, they are providing a brief breakdown of their method by explaining how their method functions.  They are saying that in their approach they are combining multiple methods in a structured way to improve the overall prediction accuracy of stock prices.

Only a few papers have addressed the problem of accurately predicting stock prices. In this paper, we propose a method, called the Hybrid Prediction Method that combines a selection of existing methods in a structured way to improve on the results obtained by using any single method alone. This paper is organized as follows: In Section 2, we introduce the Hybrid Analysis. Section 3 presents a number of experiments and results, and these results are discussed in Section 6. Section 7 concludes the paper. _  Research gap  _  Research question  _  Paper outline

Finally, they finish off the section by providing the outline of the paper. Please note, providing the paper outline is optional. It depends on your personal preference and journal requirements.  This passage is a typical format you will see in engineering research papers that propose a new method to solve a particular problem.

2.4. Example #4 (Psychology research paper)

Here is an example from a psychology research paper. In the first line, the authors clearly state the research question, and the methodology they will be using to address it. The authors aim to test the impact of background music on the listener’s ability to remember words. They will be addressing this by performing a series of experiments in which observers will be shown words on the computer screen while playing different types of background music. Then, they are finishing off the section with a very brief summary of the results. This is a good idea because it will provide readers with a rough idea of what to expect from the rest of the paper.

In two experiments, we tested whether the presence of background music had an effect on memory recall. More precisely, we examined whether the type of music, either classical or pop, had an impact on the ability of people to remember a list of words. Observers viewed a list of words on a computer screen and listened to either classical or pop music in the background. The results of this study indicate significant differences between classical and pop music in terms of their effects on memory recall and cognition. _   _  Research questions  _  Methods summary  _  Results summary

3. Frequently Asked Questions

Your research question should align with your research gap and the problem statement. The research question should logically follow the problem statement and research gap you established in the previous sections of your paper. If your research objectives are misaligned with your problem statement and research gap, then reviewers will reject your paper. So make sure they are all tightly aligned with each other.

Look at the first example. We are saying that we are going to study the impact of social media on young people. The research question is too broad. As you can see there is no clear direction, and the study attempts to take on too much.

The research aims to find out the impact of social media on young people. Bad research question (Too broad)

Now, look at the second example. It is much more focused. We are very specific about our research questions.  We are saying that we are attempting to measure the average time spent by teenagers on social media. And, we are also trying to understand the exact nature of their interactions on social media. We will be using an online questionnaire to answer the questions and we will be choosing participants from England and Scotland. This is a good research question, because it clearly defines what you have set out to do and how you plan to achieve it.

The research aims to estimate the average time spent by 18-24 year-olds on social media, and investigate the nature of interactions and conversations they have on social media. We attempt to answer these questions by conducting an online questionnaire survey in England and Scotland. Good research question (Very specific and focussed)

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How to Write a Research Question: Types & Examples

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A research question is the main query that researchers seek to answer in their study. It serves as the basis for a scholarly project such as research paper, thesis or dissertation. A good research question should be clear, relevant and specific enough to guide the research process. It should also be open-ended, meaning that it allows for multiple possible answers or interpretations.

If you have located your general subject and main sources but still aren’t quite sure about the exact research questions for your paper, this guide will help you out. First, we will explore the concept of it together, so you could answer it in your work. Then some simple steps on composing your inquiry will be suggested. In the end, we will draw your attention to some specific details which can make your work good or bad. Sometimes it’s just easier to delegate all challenging tasks to a reliable research paper service . StudyCrumb is a trustable network of qualified writers ready to efficiently solve students’ challenges.

What Is a Good Research Question: Full Definition

Good research questions provide a concise definition of a problem. As a scholar, your main goal at the beginning is to select the main focus. It should be narrow enough so you could examine it within your deadline. Your work should be focused on something specific. Otherwise, it will require too much work and might not produce clear answers. At the same time your answer should be arguable and supported by data you’ve collected. Take a look at this example:

How to Write a Research Question: Step-By-Step Guide

In this section we will examine the process of developing a research question. We will guide you through it, step by step. Keep in mind that your subject should be important for your audience. So it requires some preliminary study and brainstorming. Let’s take a closer look at the main steps.

Step 1. Choose a Broad Topic for Your Research Paper Question

First, you need to decide on your general direction. When trying to identify your research paper questions, it is better to choose an area you are really interested in. You should be able to obtain enough data to write something about this topic. Therefore, do not choose something out of your reach. At the same time, your broad topic should not be too simple. Research paper questions that can be answered without any study would hardly make any sense for your project.

Step 2. Do Preliminary Reading Before Starting Your Research Question

Next, it is time we explore the context of the selected topic. You wouldn’t want to choose research questions that have already been examined and answered in detail. On the other hand, choosing a topic that is a complete ‘terra incognita’ might be a bridge too far for your project. Browse through available sources that are related to this topic. You should try and find out what has been discovered about it before. Do you see a gap that you can fill with your study? You can proceed with developing your exact inquiry! Have no time for in-depth topic exploration? Leave this task to professionals. Entrust your “ write my research paper ” order to StudyCrumb and get a top-notch work.

Step 3. Consider an Audience for Your Research Question

It is good to know your reader well to be able to convey your ideas and results to them in the best possible way. Before writing research questions for your projects, you might need to perform a brief analysis of your audience. That's how you'll be able to understand what is interesting for them and what is not. This will allow you to make better decisions when narrowing your broad topic down. Select a topic that is interesting for your reader! This would contribute much to the success for writing a research paper .

Step 4. Start Asking a Good Research Question

After you have considered your options, go ahead and compose the primary subject of your paper. What makes a good research question? It should highlight some problematic and relevant aspects of the general topic. So, after it is answered, you should have obtained some new valuable knowledge about the subject.  Typically scholars start narrowing down their general topic by asking ‘how’, ‘why’ or ‘what’s next’ questions. This approach might help you come up with a great idea quickly.

Step 5. Evaluate Your Research Question

Finally, after you have composed a research paper question, you should take a second look at it and see if it is good enough for your paper. It would be useful to analyze it from the following sides:

• Is it clear for your audience?
• Is it complex enough to require significant study?
• Is it focused on a certain aspect of your general topic?

You might use the help of your peers or your friends at this step. You can also show it to your tutor and ask for their opinion.

Types of Research Questions: Which to Choose

A number of research questions types are available for use in a paper. They are divided into two main groups:

Qualitative questions:

• Explanatory
• Ethnographic

Quantitative questions:

• Descriptive
• Comparative
• Relationship based.

Selecting a certain type would impact the course of your study. We suggest you think about it carefully. Below you can find a few words about each type. Also, you can seek proficient help from academic experts. Buy a research paper from real pros and forget about stress once and for all.

Qualitative Research Questions: Definition With Example

When doing qualitative research, you are expected to aim to understand the different aspects and qualities of your target problem. Therefore, your thesis should focus on analyzing people’s experience, ideas and reflections rather than on obtaining some statistical data and calculating trends. Thus, this inquiry typically requires observing people’s behavior, interacting with them and learning how they interpret your target problem.  Let’s illustrate this with an example:

What Is Contextual Research Questions

Contextual research revolves around examining your subject in its natural, everyday environment. It may be watching animals living in their usual habitats or people doing their normal activities in their familiar surroundings (at home, at school or at office). This academic approach helps to understand the role of the context. You'll be able to better explain connections between your problem, its environment and outcomes. This type of inquiry ought to be narrow enough. You shouldn’t have to examine each and every aspect of the selected problem in your paper. Consider this example:

Definition and Sample of Evaluative Research Questions

Evaluative research is performed in order to carefully assess the qualities of a selected object, individual, group, system or concept. It typically serves the purpose of collecting evidence that supports or contradicts solutions for a problem. This type of inquiry should focus on how useful a certain quality is for solving the problem.  To conduct such study, you need to examine selected qualities in detail. Then, you should assume whether they match necessary criteria. It might include some quantitative methods such as collecting statistics. Although, the most important part is analyzing the qualities. If you need some examples, here’s one for you:

Explanatory Research Questions: Definition With Example

Your paper can be dedicated to explaining a certain phenomenon, finding its reasons and important relationships between it and other important things. Your explanatory research question should aim to highlight issues, uncertainties and problematic aspects of your subject. So, your study should bring clarity about these qualities. It should show how and why they have developed this way. An explanation may include showing causes and effects of issues in question, comparing the selected phenomenon to other similar types and showing whether the selected qualities match some predefined criteria. If you need some examples, check this one:

Generative Research Questions

This type of research is conducted in order to better understand the subject. With its help, you can find some new solutions or opportunities for improvement. Therefore, its main purpose is to develop a theoretical basis for further actions. You need to compose your generative research questions in a way that facilitates obtaining new ideas. It would help to begin with asking ‘why’, ‘what is the relationship between the subject and the problems X, Y, and Z’, ‘what can be improved here’, ‘how we can prevent it’ and so on. Need relevant examples? We’ve got one for you:

Ethnographic Research Question

Ethnography research is focused on a particular group of people. The aim is to study their behavior, typical reactions to certain events or information, needs, preferences or habits. Important parameters of this group which are most relevant to your general subject are taken into consideration. These are age, sex, language, religion, ethnicity, social status and so on. Main method in this case is first-hand observation of people from the selected group during an extended period of time. If you need strong examples, here’s one:

Quantitative Research Questions: Full Definition With Examples

Quantitative research deals with data – first of all, it is numeric data. It involves mathematical calculations and statistical analysis. It helps to obtain knowledge which is mostly expressed in numbers, graphs and tables. Unlike the qualitative type, the purpose of quantitative research is finding patterns, calculating probabilities, testing causal relationships and making predictions. It is focused on testing theories and hypotheses. (We have the whole blog on what is a hypothesis .) It is mostly used in natural and social sciences. These are: chemistry, biology, psychology, economics, sociology, marketing, etc. Here are a couple of examples:

Descriptive Research Questions: Definition With Example

This is probably the most widespread type of quantitative research question. Such inquiries seek to explain when, where, why, or how something occurred. They describe it accurately and systematically. These inquiries typically start with ‘what’. You are expected to use various methods to investigate one or more variables and determine their dependencies. Note, however, that you cannot control or manipulate any of these variables. You can only observe and measure them. Looking for some interesting examples? Here is one:

Definition of Comparative Research Questions

Comparative research question is used to highlight different variables and provide numerical evidence. This type is based on comparing one object, parameter or issue with another one of a similar kind. It can help to discover the differences between two or more groups by examining their outcome variables.  Take a look at these two examples:

Relationship Research Questions

We conduct this type of research when we need to make it clear whether one parameter of a selected object causes another one. A relationship based quantitative research question should help us to explore and define trends and interactions between two or more variables. Are these two things mutually dependent? What kind of dependence is it? How has it developed? And what are possible outcomes of this connection? Here is an example of relationship-based quantitative research questions:

Research Questions Examples: Free

This section contains a number of helpful examples of research questions. Feel free to use them as inspiration to create your own questions and conduct productive study. Let’s start with two simple ones:

Are you interested in well written and inspiring questions? Do you want to learn what to avoid in your study? Just stay with us – there will be more of them below.

Examples of Good and Bad Research Questions

Everyone is interested in getting the best possible appraisal for their study. Choosing a topic which doesn't suit your specific situation may be discouraging. Thus, the quality of your paper might get affected by a poor choice. We have put together some good and bad examples so that you could avoid such mistakes.

Good Research Questions Examples

It is important to include clear terms into your questions. Otherwise, it would be difficult for you to plan your investigation properly. Also, they must be focused on a certain subject, not multiple ones. And finally, it should be possible to answer them. Let’s review several good examples:

Examples of Bad Research Questions

It is difficult to evaluate qualities of objects, individuals or groups if your purpose is not clear. This is why you shouldn’t create unclear research questions or try to focus on many problems at once. Some preliminary study might help to understand what you should focus on. Here are several bad examples:

In case you may need some information about the discussion section of a research paper example , find it in our blog.

Final Thoughts on Research Questions

In this article we have made a detailed review of the most popular types of research questions. We described peculiarities. We also provided some tips on conducting various kinds of study. Besides, a number of useful examples have been given for each category of questions.

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Frequently Asked Questions About Research Questions

1. what is an example of a weak research question.

Here is an example of the weakest research question: 

An answer would be simply making a list of species that inhabit the country. This subject does not require any actual study to be conducted. There is nothing to calculate or analyze here.

2. What is the most effective type of research question?

Most effective type of research question is the one that doesn't have a single correct answer. However, you should also pay close attention to your audience. If you need to create a strong effect, better choose a topic which is relevant for them.

3. What is a good nursing research question?

If you need an idea for a nursing research question, here are a few helpful examples you could use as a reference:

4. What are some sociological research questions?

Sociological questions are the ones that examine the social patterns or a meaning of a social phenomenon. They could be qualitative or quantitative. They should target groups of people with certain parameters, such as age or income level. Keep in mind that type of study usually requires collecting numerous data about your target groups.

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A Practical Guide to Writing Quantitative and Qualitative Research Questions and Hypotheses in Scholarly Articles

Edward barroga.

1 Department of General Education, Graduate School of Nursing Science, St. Luke’s International University, Tokyo, Japan.

Glafera Janet Matanguihan

2 Department of Biological Sciences, Messiah University, Mechanicsburg, PA, USA.

The development of research questions and the subsequent hypotheses are prerequisites to defining the main research purpose and specific objectives of a study. Consequently, these objectives determine the study design and research outcome. The development of research questions is a process based on knowledge of current trends, cutting-edge studies, and technological advances in the research field. Excellent research questions are focused and require a comprehensive literature search and in-depth understanding of the problem being investigated. Initially, research questions may be written as descriptive questions which could be developed into inferential questions. These questions must be specific and concise to provide a clear foundation for developing hypotheses. Hypotheses are more formal predictions about the research outcomes. These specify the possible results that may or may not be expected regarding the relationship between groups. Thus, research questions and hypotheses clarify the main purpose and specific objectives of the study, which in turn dictate the design of the study, its direction, and outcome. Studies developed from good research questions and hypotheses will have trustworthy outcomes with wide-ranging social and health implications.

INTRODUCTION

Scientific research is usually initiated by posing evidenced-based research questions which are then explicitly restated as hypotheses. 1 , 2 The hypotheses provide directions to guide the study, solutions, explanations, and expected results. 3 , 4 Both research questions and hypotheses are essentially formulated based on conventional theories and real-world processes, which allow the inception of novel studies and the ethical testing of ideas. 5 , 6

It is crucial to have knowledge of both quantitative and qualitative research 2 as both types of research involve writing research questions and hypotheses. 7 However, these crucial elements of research are sometimes overlooked; if not overlooked, then framed without the forethought and meticulous attention it needs. Planning and careful consideration are needed when developing quantitative or qualitative research, particularly when conceptualizing research questions and hypotheses. 4

There is a continuing need to support researchers in the creation of innovative research questions and hypotheses, as well as for journal articles that carefully review these elements. 1 When research questions and hypotheses are not carefully thought of, unethical studies and poor outcomes usually ensue. Carefully formulated research questions and hypotheses define well-founded objectives, which in turn determine the appropriate design, course, and outcome of the study. This article then aims to discuss in detail the various aspects of crafting research questions and hypotheses, with the goal of guiding researchers as they develop their own. Examples from the authors and peer-reviewed scientific articles in the healthcare field are provided to illustrate key points.

DEFINITIONS AND RELATIONSHIP OF RESEARCH QUESTIONS AND HYPOTHESES

A research question is what a study aims to answer after data analysis and interpretation. The answer is written in length in the discussion section of the paper. Thus, the research question gives a preview of the different parts and variables of the study meant to address the problem posed in the research question. 1 An excellent research question clarifies the research writing while facilitating understanding of the research topic, objective, scope, and limitations of the study. 5

On the other hand, a research hypothesis is an educated statement of an expected outcome. This statement is based on background research and current knowledge. 8 , 9 The research hypothesis makes a specific prediction about a new phenomenon 10 or a formal statement on the expected relationship between an independent variable and a dependent variable. 3 , 11 It provides a tentative answer to the research question to be tested or explored. 4

Hypotheses employ reasoning to predict a theory-based outcome. 10 These can also be developed from theories by focusing on components of theories that have not yet been observed. 10 The validity of hypotheses is often based on the testability of the prediction made in a reproducible experiment. 8

Conversely, hypotheses can also be rephrased as research questions. Several hypotheses based on existing theories and knowledge may be needed to answer a research question. Developing ethical research questions and hypotheses creates a research design that has logical relationships among variables. These relationships serve as a solid foundation for the conduct of the study. 4 , 11 Haphazardly constructed research questions can result in poorly formulated hypotheses and improper study designs, leading to unreliable results. Thus, the formulations of relevant research questions and verifiable hypotheses are crucial when beginning research. 12

CHARACTERISTICS OF GOOD RESEARCH QUESTIONS AND HYPOTHESES

Excellent research questions are specific and focused. These integrate collective data and observations to confirm or refute the subsequent hypotheses. Well-constructed hypotheses are based on previous reports and verify the research context. These are realistic, in-depth, sufficiently complex, and reproducible. More importantly, these hypotheses can be addressed and tested. 13

There are several characteristics of well-developed hypotheses. Good hypotheses are 1) empirically testable 7 , 10 , 11 , 13 ; 2) backed by preliminary evidence 9 ; 3) testable by ethical research 7 , 9 ; 4) based on original ideas 9 ; 5) have evidenced-based logical reasoning 10 ; and 6) can be predicted. 11 Good hypotheses can infer ethical and positive implications, indicating the presence of a relationship or effect relevant to the research theme. 7 , 11 These are initially developed from a general theory and branch into specific hypotheses by deductive reasoning. In the absence of a theory to base the hypotheses, inductive reasoning based on specific observations or findings form more general hypotheses. 10

TYPES OF RESEARCH QUESTIONS AND HYPOTHESES

Research questions and hypotheses are developed according to the type of research, which can be broadly classified into quantitative and qualitative research. We provide a summary of the types of research questions and hypotheses under quantitative and qualitative research categories in Table 1 .

Research questions in quantitative research

In quantitative research, research questions inquire about the relationships among variables being investigated and are usually framed at the start of the study. These are precise and typically linked to the subject population, dependent and independent variables, and research design. 1 Research questions may also attempt to describe the behavior of a population in relation to one or more variables, or describe the characteristics of variables to be measured ( descriptive research questions ). 1 , 5 , 14 These questions may also aim to discover differences between groups within the context of an outcome variable ( comparative research questions ), 1 , 5 , 14 or elucidate trends and interactions among variables ( relationship research questions ). 1 , 5 We provide examples of descriptive, comparative, and relationship research questions in quantitative research in Table 2 .

Hypotheses in quantitative research

In quantitative research, hypotheses predict the expected relationships among variables. 15 Relationships among variables that can be predicted include 1) between a single dependent variable and a single independent variable ( simple hypothesis ) or 2) between two or more independent and dependent variables ( complex hypothesis ). 4 , 11 Hypotheses may also specify the expected direction to be followed and imply an intellectual commitment to a particular outcome ( directional hypothesis ) 4 . On the other hand, hypotheses may not predict the exact direction and are used in the absence of a theory, or when findings contradict previous studies ( non-directional hypothesis ). 4 In addition, hypotheses can 1) define interdependency between variables ( associative hypothesis ), 4 2) propose an effect on the dependent variable from manipulation of the independent variable ( causal hypothesis ), 4 3) state a negative relationship between two variables ( null hypothesis ), 4 , 11 , 15 4) replace the working hypothesis if rejected ( alternative hypothesis ), 15 explain the relationship of phenomena to possibly generate a theory ( working hypothesis ), 11 5) involve quantifiable variables that can be tested statistically ( statistical hypothesis ), 11 6) or express a relationship whose interlinks can be verified logically ( logical hypothesis ). 11 We provide examples of simple, complex, directional, non-directional, associative, causal, null, alternative, working, statistical, and logical hypotheses in quantitative research, as well as the definition of quantitative hypothesis-testing research in Table 3 .

Research questions in qualitative research

Unlike research questions in quantitative research, research questions in qualitative research are usually continuously reviewed and reformulated. The central question and associated subquestions are stated more than the hypotheses. 15 The central question broadly explores a complex set of factors surrounding the central phenomenon, aiming to present the varied perspectives of participants. 15

There are varied goals for which qualitative research questions are developed. These questions can function in several ways, such as to 1) identify and describe existing conditions ( contextual research question s); 2) describe a phenomenon ( descriptive research questions ); 3) assess the effectiveness of existing methods, protocols, theories, or procedures ( evaluation research questions ); 4) examine a phenomenon or analyze the reasons or relationships between subjects or phenomena ( explanatory research questions ); or 5) focus on unknown aspects of a particular topic ( exploratory research questions ). 5 In addition, some qualitative research questions provide new ideas for the development of theories and actions ( generative research questions ) or advance specific ideologies of a position ( ideological research questions ). 1 Other qualitative research questions may build on a body of existing literature and become working guidelines ( ethnographic research questions ). Research questions may also be broadly stated without specific reference to the existing literature or a typology of questions ( phenomenological research questions ), may be directed towards generating a theory of some process ( grounded theory questions ), or may address a description of the case and the emerging themes ( qualitative case study questions ). 15 We provide examples of contextual, descriptive, evaluation, explanatory, exploratory, generative, ideological, ethnographic, phenomenological, grounded theory, and qualitative case study research questions in qualitative research in Table 4 , and the definition of qualitative hypothesis-generating research in Table 5 .

Qualitative studies usually pose at least one central research question and several subquestions starting with How or What . These research questions use exploratory verbs such as explore or describe . These also focus on one central phenomenon of interest, and may mention the participants and research site. 15

Hypotheses in qualitative research

Hypotheses in qualitative research are stated in the form of a clear statement concerning the problem to be investigated. Unlike in quantitative research where hypotheses are usually developed to be tested, qualitative research can lead to both hypothesis-testing and hypothesis-generating outcomes. 2 When studies require both quantitative and qualitative research questions, this suggests an integrative process between both research methods wherein a single mixed-methods research question can be developed. 1

FRAMEWORKS FOR DEVELOPING RESEARCH QUESTIONS AND HYPOTHESES

Research questions followed by hypotheses should be developed before the start of the study. 1 , 12 , 14 It is crucial to develop feasible research questions on a topic that is interesting to both the researcher and the scientific community. This can be achieved by a meticulous review of previous and current studies to establish a novel topic. Specific areas are subsequently focused on to generate ethical research questions. The relevance of the research questions is evaluated in terms of clarity of the resulting data, specificity of the methodology, objectivity of the outcome, depth of the research, and impact of the study. 1 , 5 These aspects constitute the FINER criteria (i.e., Feasible, Interesting, Novel, Ethical, and Relevant). 1 Clarity and effectiveness are achieved if research questions meet the FINER criteria. In addition to the FINER criteria, Ratan et al. described focus, complexity, novelty, feasibility, and measurability for evaluating the effectiveness of research questions. 14

The PICOT and PEO frameworks are also used when developing research questions. 1 The following elements are addressed in these frameworks, PICOT: P-population/patients/problem, I-intervention or indicator being studied, C-comparison group, O-outcome of interest, and T-timeframe of the study; PEO: P-population being studied, E-exposure to preexisting conditions, and O-outcome of interest. 1 Research questions are also considered good if these meet the “FINERMAPS” framework: Feasible, Interesting, Novel, Ethical, Relevant, Manageable, Appropriate, Potential value/publishable, and Systematic. 14

As we indicated earlier, research questions and hypotheses that are not carefully formulated result in unethical studies or poor outcomes. To illustrate this, we provide some examples of ambiguous research question and hypotheses that result in unclear and weak research objectives in quantitative research ( Table 6 ) 16 and qualitative research ( Table 7 ) 17 , and how to transform these ambiguous research question(s) and hypothesis(es) into clear and good statements.

a These statements were composed for comparison and illustrative purposes only.

b These statements are direct quotes from Higashihara and Horiuchi. 16

a This statement is a direct quote from Shimoda et al. 17

The other statements were composed for comparison and illustrative purposes only.

CONSTRUCTING RESEARCH QUESTIONS AND HYPOTHESES

To construct effective research questions and hypotheses, it is very important to 1) clarify the background and 2) identify the research problem at the outset of the research, within a specific timeframe. 9 Then, 3) review or conduct preliminary research to collect all available knowledge about the possible research questions by studying theories and previous studies. 18 Afterwards, 4) construct research questions to investigate the research problem. Identify variables to be accessed from the research questions 4 and make operational definitions of constructs from the research problem and questions. Thereafter, 5) construct specific deductive or inductive predictions in the form of hypotheses. 4 Finally, 6) state the study aims . This general flow for constructing effective research questions and hypotheses prior to conducting research is shown in Fig. 1 .

Research questions are used more frequently in qualitative research than objectives or hypotheses. 3 These questions seek to discover, understand, explore or describe experiences by asking “What” or “How.” The questions are open-ended to elicit a description rather than to relate variables or compare groups. The questions are continually reviewed, reformulated, and changed during the qualitative study. 3 Research questions are also used more frequently in survey projects than hypotheses in experiments in quantitative research to compare variables and their relationships.

Hypotheses are constructed based on the variables identified and as an if-then statement, following the template, ‘If a specific action is taken, then a certain outcome is expected.’ At this stage, some ideas regarding expectations from the research to be conducted must be drawn. 18 Then, the variables to be manipulated (independent) and influenced (dependent) are defined. 4 Thereafter, the hypothesis is stated and refined, and reproducible data tailored to the hypothesis are identified, collected, and analyzed. 4 The hypotheses must be testable and specific, 18 and should describe the variables and their relationships, the specific group being studied, and the predicted research outcome. 18 Hypotheses construction involves a testable proposition to be deduced from theory, and independent and dependent variables to be separated and measured separately. 3 Therefore, good hypotheses must be based on good research questions constructed at the start of a study or trial. 12

In summary, research questions are constructed after establishing the background of the study. Hypotheses are then developed based on the research questions. Thus, it is crucial to have excellent research questions to generate superior hypotheses. In turn, these would determine the research objectives and the design of the study, and ultimately, the outcome of the research. 12 Algorithms for building research questions and hypotheses are shown in Fig. 2 for quantitative research and in Fig. 3 for qualitative research.

EXAMPLES OF RESEARCH QUESTIONS FROM PUBLISHED ARTICLES

• EXAMPLE 1. Descriptive research question (quantitative research)
• - Presents research variables to be assessed (distinct phenotypes and subphenotypes)
• “BACKGROUND: Since COVID-19 was identified, its clinical and biological heterogeneity has been recognized. Identifying COVID-19 phenotypes might help guide basic, clinical, and translational research efforts.
• RESEARCH QUESTION: Does the clinical spectrum of patients with COVID-19 contain distinct phenotypes and subphenotypes? ” 19
• EXAMPLE 2. Relationship research question (quantitative research)
• - Shows interactions between dependent variable (static postural control) and independent variable (peripheral visual field loss)
• “Background: Integration of visual, vestibular, and proprioceptive sensations contributes to postural control. People with peripheral visual field loss have serious postural instability. However, the directional specificity of postural stability and sensory reweighting caused by gradual peripheral visual field loss remain unclear.
• Research question: What are the effects of peripheral visual field loss on static postural control ?” 20
• EXAMPLE 3. Comparative research question (quantitative research)
• - Clarifies the difference among groups with an outcome variable (patients enrolled in COMPERA with moderate PH or severe PH in COPD) and another group without the outcome variable (patients with idiopathic pulmonary arterial hypertension (IPAH))
• “BACKGROUND: Pulmonary hypertension (PH) in COPD is a poorly investigated clinical condition.
• RESEARCH QUESTION: Which factors determine the outcome of PH in COPD?
• STUDY DESIGN AND METHODS: We analyzed the characteristics and outcome of patients enrolled in the Comparative, Prospective Registry of Newly Initiated Therapies for Pulmonary Hypertension (COMPERA) with moderate or severe PH in COPD as defined during the 6th PH World Symposium who received medical therapy for PH and compared them with patients with idiopathic pulmonary arterial hypertension (IPAH) .” 21
• EXAMPLE 4. Exploratory research question (qualitative research)
• - Explores areas that have not been fully investigated (perspectives of families and children who receive care in clinic-based child obesity treatment) to have a deeper understanding of the research problem
• “Problem: Interventions for children with obesity lead to only modest improvements in BMI and long-term outcomes, and data are limited on the perspectives of families of children with obesity in clinic-based treatment. This scoping review seeks to answer the question: What is known about the perspectives of families and children who receive care in clinic-based child obesity treatment? This review aims to explore the scope of perspectives reported by families of children with obesity who have received individualized outpatient clinic-based obesity treatment.” 22
• EXAMPLE 5. Relationship research question (quantitative research)
• - Defines interactions between dependent variable (use of ankle strategies) and independent variable (changes in muscle tone)
• “Background: To maintain an upright standing posture against external disturbances, the human body mainly employs two types of postural control strategies: “ankle strategy” and “hip strategy.” While it has been reported that the magnitude of the disturbance alters the use of postural control strategies, it has not been elucidated how the level of muscle tone, one of the crucial parameters of bodily function, determines the use of each strategy. We have previously confirmed using forward dynamics simulations of human musculoskeletal models that an increased muscle tone promotes the use of ankle strategies. The objective of the present study was to experimentally evaluate a hypothesis: an increased muscle tone promotes the use of ankle strategies. Research question: Do changes in the muscle tone affect the use of ankle strategies ?” 23

EXAMPLES OF HYPOTHESES IN PUBLISHED ARTICLES

• EXAMPLE 1. Working hypothesis (quantitative research)
• - A hypothesis that is initially accepted for further research to produce a feasible theory
• “As fever may have benefit in shortening the duration of viral illness, it is plausible to hypothesize that the antipyretic efficacy of ibuprofen may be hindering the benefits of a fever response when taken during the early stages of COVID-19 illness .” 24
• “In conclusion, it is plausible to hypothesize that the antipyretic efficacy of ibuprofen may be hindering the benefits of a fever response . The difference in perceived safety of these agents in COVID-19 illness could be related to the more potent efficacy to reduce fever with ibuprofen compared to acetaminophen. Compelling data on the benefit of fever warrant further research and review to determine when to treat or withhold ibuprofen for early stage fever for COVID-19 and other related viral illnesses .” 24
• EXAMPLE 2. Exploratory hypothesis (qualitative research)
• - Explores particular areas deeper to clarify subjective experience and develop a formal hypothesis potentially testable in a future quantitative approach
• “We hypothesized that when thinking about a past experience of help-seeking, a self distancing prompt would cause increased help-seeking intentions and more favorable help-seeking outcome expectations .” 25
• “Conclusion
• Although a priori hypotheses were not supported, further research is warranted as results indicate the potential for using self-distancing approaches to increasing help-seeking among some people with depressive symptomatology.” 25
• EXAMPLE 3. Hypothesis-generating research to establish a framework for hypothesis testing (qualitative research)
• “We hypothesize that compassionate care is beneficial for patients (better outcomes), healthcare systems and payers (lower costs), and healthcare providers (lower burnout). ” 26
• Compassionomics is the branch of knowledge and scientific study of the effects of compassionate healthcare. Our main hypotheses are that compassionate healthcare is beneficial for (1) patients, by improving clinical outcomes, (2) healthcare systems and payers, by supporting financial sustainability, and (3) HCPs, by lowering burnout and promoting resilience and well-being. The purpose of this paper is to establish a scientific framework for testing the hypotheses above . If these hypotheses are confirmed through rigorous research, compassionomics will belong in the science of evidence-based medicine, with major implications for all healthcare domains.” 26
• EXAMPLE 4. Statistical hypothesis (quantitative research)
• - An assumption is made about the relationship among several population characteristics ( gender differences in sociodemographic and clinical characteristics of adults with ADHD ). Validity is tested by statistical experiment or analysis ( chi-square test, Students t-test, and logistic regression analysis)
• “Our research investigated gender differences in sociodemographic and clinical characteristics of adults with ADHD in a Japanese clinical sample. Due to unique Japanese cultural ideals and expectations of women's behavior that are in opposition to ADHD symptoms, we hypothesized that women with ADHD experience more difficulties and present more dysfunctions than men . We tested the following hypotheses: first, women with ADHD have more comorbidities than men with ADHD; second, women with ADHD experience more social hardships than men, such as having less full-time employment and being more likely to be divorced.” 27
• “Statistical Analysis
• ( text omitted ) Between-gender comparisons were made using the chi-squared test for categorical variables and Students t-test for continuous variables…( text omitted ). A logistic regression analysis was performed for employment status, marital status, and comorbidity to evaluate the independent effects of gender on these dependent variables.” 27

EXAMPLES OF HYPOTHESIS AS WRITTEN IN PUBLISHED ARTICLES IN RELATION TO OTHER PARTS

• EXAMPLE 1. Background, hypotheses, and aims are provided
• “Pregnant women need skilled care during pregnancy and childbirth, but that skilled care is often delayed in some countries …( text omitted ). The focused antenatal care (FANC) model of WHO recommends that nurses provide information or counseling to all pregnant women …( text omitted ). Job aids are visual support materials that provide the right kind of information using graphics and words in a simple and yet effective manner. When nurses are not highly trained or have many work details to attend to, these job aids can serve as a content reminder for the nurses and can be used for educating their patients (Jennings, Yebadokpo, Affo, & Agbogbe, 2010) ( text omitted ). Importantly, additional evidence is needed to confirm how job aids can further improve the quality of ANC counseling by health workers in maternal care …( text omitted )” 28
• “ This has led us to hypothesize that the quality of ANC counseling would be better if supported by job aids. Consequently, a better quality of ANC counseling is expected to produce higher levels of awareness concerning the danger signs of pregnancy and a more favorable impression of the caring behavior of nurses .” 28
• “This study aimed to examine the differences in the responses of pregnant women to a job aid-supported intervention during ANC visit in terms of 1) their understanding of the danger signs of pregnancy and 2) their impression of the caring behaviors of nurses to pregnant women in rural Tanzania.” 28
• EXAMPLE 2. Background, hypotheses, and aims are provided
• “We conducted a two-arm randomized controlled trial (RCT) to evaluate and compare changes in salivary cortisol and oxytocin levels of first-time pregnant women between experimental and control groups. The women in the experimental group touched and held an infant for 30 min (experimental intervention protocol), whereas those in the control group watched a DVD movie of an infant (control intervention protocol). The primary outcome was salivary cortisol level and the secondary outcome was salivary oxytocin level.” 29
• “ We hypothesize that at 30 min after touching and holding an infant, the salivary cortisol level will significantly decrease and the salivary oxytocin level will increase in the experimental group compared with the control group .” 29
• EXAMPLE 3. Background, aim, and hypothesis are provided
• “In countries where the maternal mortality ratio remains high, antenatal education to increase Birth Preparedness and Complication Readiness (BPCR) is considered one of the top priorities [1]. BPCR includes birth plans during the antenatal period, such as the birthplace, birth attendant, transportation, health facility for complications, expenses, and birth materials, as well as family coordination to achieve such birth plans. In Tanzania, although increasing, only about half of all pregnant women attend an antenatal clinic more than four times [4]. Moreover, the information provided during antenatal care (ANC) is insufficient. In the resource-poor settings, antenatal group education is a potential approach because of the limited time for individual counseling at antenatal clinics.” 30
• “This study aimed to evaluate an antenatal group education program among pregnant women and their families with respect to birth-preparedness and maternal and infant outcomes in rural villages of Tanzania.” 30
• “ The study hypothesis was if Tanzanian pregnant women and their families received a family-oriented antenatal group education, they would (1) have a higher level of BPCR, (2) attend antenatal clinic four or more times, (3) give birth in a health facility, (4) have less complications of women at birth, and (5) have less complications and deaths of infants than those who did not receive the education .” 30

Research questions and hypotheses are crucial components to any type of research, whether quantitative or qualitative. These questions should be developed at the very beginning of the study. Excellent research questions lead to superior hypotheses, which, like a compass, set the direction of research, and can often determine the successful conduct of the study. Many research studies have floundered because the development of research questions and subsequent hypotheses was not given the thought and meticulous attention needed. The development of research questions and hypotheses is an iterative process based on extensive knowledge of the literature and insightful grasp of the knowledge gap. Focused, concise, and specific research questions provide a strong foundation for constructing hypotheses which serve as formal predictions about the research outcomes. Research questions and hypotheses are crucial elements of research that should not be overlooked. They should be carefully thought of and constructed when planning research. This avoids unethical studies and poor outcomes by defining well-founded objectives that determine the design, course, and outcome of the study.

Disclosure: The authors have no potential conflicts of interest to disclose.

Author Contributions:

• Conceptualization: Barroga E, Matanguihan GJ.
• Methodology: Barroga E, Matanguihan GJ.
• Writing - original draft: Barroga E, Matanguihan GJ.
• Writing - review & editing: Barroga E, Matanguihan GJ.

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Develop Your Topic

What are you interested in? Start by brainstorming topics of interest.  Once you’ve landed on a few, do some critical background research.  What questions does your reading raise?

Once you’ve done a little digging, write down everything you know about your potential topic.  Then, write down the questions you thought of while reading.  Can you answer these questions easily, using only the background research you’ve done?

If so, then do some more digging, and write probing questions, beginning with “Why…? What if…?  How…?”  The goal is to have detailed answers, not a simple yes or no.

Sample Topic and Probing Questions

Undergraduate class: POL 370--Political Violence

Topic of interest: terrorism

Background information: “ Terrorism is  the systematic use of terror, especially as a means of coercion. In the international community, however, terrorism has no universally agreed, legally binding, criminal law definition. Common definitions of terrorism refer only to those violent acts which are intended to create fear (terror), are perpetrated for a religious, political or ideological goal, and deliberately target or disregard the safety of non-combatants (civilians).”

My questions upon reading the background information:

• What motivates someone to engage in terrorist activity? 
• How can we find out what motivates people to engage in terrorist activity?

Develop Your Research Question

It’s time to move beyond background research into the literature.  Start by first identifying the key scholars within your topic area, and read what they have to say.  What questions do they have?  How do they go about answering them?

Go back to your original questions and add to them or refine them based on the latest research you’ve done.  Write down some persuasive claims that you’d like to make about your topic.  Keep in mind that a good research question will have more than one possible answer.  Be careful not to ask the exact same questions or statements as the scholars you’ve read; you want to take an original stance on the topic.

Bad Sample Question:   What motivates terrorists to engage in terrorist activity?  (too broad)

Good Sample Question:   Why did the Northern Irish who joined the IRA during the Troubles choose to employ terrorist tactics in their fight for independence?  (narrow enough, places the topic in context)

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How to Write a Research Paper Introduction (with Examples)

The research paper introduction section, along with the Title and Abstract, can be considered the face of any research paper. The following article is intended to guide you in organizing and writing the research paper introduction for a quality academic article or dissertation.

The research paper introduction aims to present the topic to the reader. A study will only be accepted for publishing if you can ascertain that the available literature cannot answer your research question. So it is important to ensure that you have read important studies on that particular topic, especially those within the last five to ten years, and that they are properly referenced in this section. 1 What should be included in the research paper introduction is decided by what you want to tell readers about the reason behind the research and how you plan to fill the knowledge gap. The best research paper introduction provides a systemic review of existing work and demonstrates additional work that needs to be done. It needs to be brief, captivating, and well-referenced; a well-drafted research paper introduction will help the researcher win half the battle.

The introduction for a research paper is where you set up your topic and approach for the reader. It has several key goals:

• Present your research topic
• Capture reader interest
• Summarize existing research
• Position your own approach
• Define your specific research problem and problem statement
• Highlight the novelty and contributions of the study
• Give an overview of the paper’s structure

The research paper introduction can vary in size and structure depending on whether your paper presents the results of original empirical research or is a review paper. Some research paper introduction examples are only half a page while others are a few pages long. In many cases, the introduction will be shorter than all of the other sections of your paper; its length depends on the size of your paper as a whole.

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Table of Contents

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The introduction in a research paper is placed at the beginning to guide the reader from a broad subject area to the specific topic that your research addresses. They present the following information to the reader

• Scope: The topic covered in the research paper
• Context: Background of your topic
• Importance: Why your research matters in that particular area of research and the industry problem that can be targeted

The research paper introduction conveys a lot of information and can be considered an essential roadmap for the rest of your paper. A good introduction for a research paper is important for the following reasons:

• It stimulates your reader’s interest: A good introduction section can make your readers want to read your paper by capturing their interest. It informs the reader what they are going to learn and helps determine if the topic is of interest to them.
• It helps the reader understand the research background: Without a clear introduction, your readers may feel confused and even struggle when reading your paper. A good research paper introduction will prepare them for the in-depth research to come. It provides you the opportunity to engage with the readers and demonstrate your knowledge and authority on the specific topic.
• It explains why your research paper is worth reading: Your introduction can convey a lot of information to your readers. It introduces the topic, why the topic is important, and how you plan to proceed with your research.
• It helps guide the reader through the rest of the paper: The research paper introduction gives the reader a sense of the nature of the information that will support your arguments and the general organization of the paragraphs that will follow. It offers an overview of what to expect when reading the main body of your paper.

What are the parts of introduction in the research?

A good research paper introduction section should comprise three main elements: 2

• What is known: This sets the stage for your research. It informs the readers of what is known on the subject.
• What is lacking: This is aimed at justifying the reason for carrying out your research. This could involve investigating a new concept or method or building upon previous research.
• What you aim to do: This part briefly states the objectives of your research and its major contributions. Your detailed hypothesis will also form a part of this section.

How to write a research paper introduction?

The first step in writing the research paper introduction is to inform the reader what your topic is and why it’s interesting or important. This is generally accomplished with a strong opening statement. The second step involves establishing the kinds of research that have been done and ending with limitations or gaps in the research that you intend to address. Finally, the research paper introduction clarifies how your own research fits in and what problem it addresses. If your research involved testing hypotheses, these should be stated along with your research question. The hypothesis should be presented in the past tense since it will have been tested by the time you are writing the research paper introduction.

The following key points, with examples, can guide you when writing the research paper introduction section:

• Highlight the importance of the research field or topic
• Describe the background of the topic
• Present an overview of current research on the topic

Example: The inclusion of experiential and competency-based learning has benefitted electronics engineering education. Industry partnerships provide an excellent alternative for students wanting to engage in solving real-world challenges. Industry-academia participation has grown in recent years due to the need for skilled engineers with practical training and specialized expertise. However, from the educational perspective, many activities are needed to incorporate sustainable development goals into the university curricula and consolidate learning innovation in universities.

• Reveal a gap in existing research or oppose an existing assumption
• Formulate the research question

Example: There have been plausible efforts to integrate educational activities in higher education electronics engineering programs. However, very few studies have considered using educational research methods for performance evaluation of competency-based higher engineering education, with a focus on technical and or transversal skills. To remedy the current need for evaluating competencies in STEM fields and providing sustainable development goals in engineering education, in this study, a comparison was drawn between study groups without and with industry partners.

• State the purpose of your study
• Highlight the key characteristics of your study
• Describe important results
• Highlight the novelty of the study.
• Offer a brief overview of the structure of the paper.

Example: The study evaluates the main competency needed in the applied electronics course, which is a fundamental core subject for many electronics engineering undergraduate programs. We compared two groups, without and with an industrial partner, that offered real-world projects to solve during the semester. This comparison can help determine significant differences in both groups in terms of developing subject competency and achieving sustainable development goals.

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Paperpal Copilot is a generative AI-powered academic writing assistant. It’s trained on millions of published scholarly articles and over 20 years of STM experience. Paperpal Copilot helps authors write better and faster with:

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With Paperpal Copilot, create a research paper introduction effortlessly. In this step-by-step guide, we’ll walk you through how Paperpal transforms your initial ideas into a polished and publication-ready introduction.

How to use Paperpal to write the Introduction section

Step 1: Sign up on Paperpal and click on the Copilot feature, under this choose Outlines > Research Article > Introduction

Step 2: Add your unstructured notes or initial draft, whether in English or another language, to Paperpal, which is to be used as the base for your content.

Step 3: Fill in the specifics, such as your field of study, brief description or details you want to include, which will help the AI generate the outline for your Introduction.

Step 4: Use this outline and sentence suggestions to develop your content, adding citations where needed and modifying it to align with your specific research focus.

Step 5: Turn to Paperpal’s granular language checks to refine your content, tailor it to reflect your personal writing style, and ensure it effectively conveys your message.

You can use the same process to develop each section of your article, and finally your research paper in half the time and without any of the stress.

The purpose of the research paper introduction is to introduce the reader to the problem definition, justify the need for the study, and describe the main theme of the study. The aim is to gain the reader’s attention by providing them with necessary background information and establishing the main purpose and direction of the research.

The length of the research paper introduction can vary across journals and disciplines. While there are no strict word limits for writing the research paper introduction, an ideal length would be one page, with a maximum of 400 words over 1-4 paragraphs. Generally, it is one of the shorter sections of the paper as the reader is assumed to have at least a reasonable knowledge about the topic. 2 For example, for a study evaluating the role of building design in ensuring fire safety, there is no need to discuss definitions and nature of fire in the introduction; you could start by commenting upon the existing practices for fire safety and how your study will add to the existing knowledge and practice.

When deciding what to include in the research paper introduction, the rest of the paper should also be considered. The aim is to introduce the reader smoothly to the topic and facilitate an easy read without much dependency on external sources. 3 Below is a list of elements you can include to prepare a research paper introduction outline and follow it when you are writing the research paper introduction. Topic introduction: This can include key definitions and a brief history of the topic. Research context and background: Offer the readers some general information and then narrow it down to specific aspects. Details of the research you conducted: A brief literature review can be included to support your arguments or line of thought. Rationale for the study: This establishes the relevance of your study and establishes its importance. Importance of your research: The main contributions are highlighted to help establish the novelty of your study Research hypothesis: Introduce your research question and propose an expected outcome. Organization of the paper: Include a short paragraph of 3-4 sentences that highlights your plan for the entire paper

Cite only works that are most relevant to your topic; as a general rule, you can include one to three. Note that readers want to see evidence of original thinking. So it is better to avoid using too many references as it does not leave much room for your personal standpoint to shine through. Citations in your research paper introduction support the key points, and the number of citations depend on the subject matter and the point discussed. If the research paper introduction is too long or overflowing with citations, it is better to cite a few review articles rather than the individual articles summarized in the review. A good point to remember when citing research papers in the introduction section is to include at least one-third of the references in the introduction.

The literature review plays a significant role in the research paper introduction section. A good literature review accomplishes the following: Introduces the topic – Establishes the study’s significance – Provides an overview of the relevant literature – Provides context for the study using literature – Identifies knowledge gaps However, remember to avoid making the following mistakes when writing a research paper introduction: Do not use studies from the literature review to aggressively support your research Avoid direct quoting Do not allow literature review to be the focus of this section. Instead, the literature review should only aid in setting a foundation for the manuscript.

Remember the following key points for writing a good research paper introduction: 4

• Avoid stuffing too much general information: Avoid including what an average reader would know and include only that information related to the problem being addressed in the research paper introduction. For example, when describing a comparative study of non-traditional methods for mechanical design optimization, information related to the traditional methods and differences between traditional and non-traditional methods would not be relevant. In this case, the introduction for the research paper should begin with the state-of-the-art non-traditional methods and methods to evaluate the efficiency of newly developed algorithms.
• Avoid packing too many references: Cite only the required works in your research paper introduction. The other works can be included in the discussion section to strengthen your findings.
• Avoid extensive criticism of previous studies: Avoid being overly critical of earlier studies while setting the rationale for your study. A better place for this would be the Discussion section, where you can highlight the advantages of your method.
• Avoid describing conclusions of the study: When writing a research paper introduction remember not to include the findings of your study. The aim is to let the readers know what question is being answered. The actual answer should only be given in the Results and Discussion section.

To summarize, the research paper introduction section should be brief yet informative. It should convince the reader the need to conduct the study and motivate him to read further. If you’re feeling stuck or unsure, choose trusted AI academic writing assistants like Paperpal to effortlessly craft your research paper introduction and other sections of your research article.

1. Jawaid, S. A., & Jawaid, M. (2019). How to write introduction and discussion. Saudi Journal of Anaesthesia, 13(Suppl 1), S18.

2. Dewan, P., & Gupta, P. (2016). Writing the title, abstract and introduction: Looks matter!. Indian pediatrics, 53, 235-241.

3. Cetin, S., & Hackam, D. J. (2005). An approach to the writing of a scientific Manuscript1. Journal of Surgical Research, 128(2), 165-167.

4. Bavdekar, S. B. (2015). Writing introduction: Laying the foundations of a research paper. Journal of the Association of Physicians of India, 63(7), 44-6.

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Research Paper Guide

Ethical Research Paper Topics

200+ Ethical Research Paper Topics to Begin With (2024)

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When writing a research paper, the tricky part is choosing a topic that's not just interesting but also follows the rules about what's right and fair in research. 

Ethical problems are always changing, and it's important to face them directly. Ignoring these issues can cause big problems, making it hard for people to trust your research.

Whether you're a student or a scholar, our blog has ethics research paper ideas that will inspire you. 

With this blog, you'll have a comprehensive guide to find a topic that not only captivates your interest but also addresses the ethical considerations surrounding research participants. 

Keep reading to find the best ethical topics for your research paper .

• 1. Ethical Topics for Students
• 2. Ethical Hacking Research Paper Topics 
• 3. Ethical Leadership Research Paper Topics 
• 4. Ethical Issue Research Paper Topics
• 5. Business Ethics Research Paper Topics
• 6. Medical Ethics Research Paper Topics
• 7. Accounting Ethics Research Paper Topics
• 8. Legal Ethics Research Paper Topics
• 9. Environmental Ethics Research Paper Topics
• 10. Bioethics Research Paper Topics
• 11. Research Paper Topics in Sports Ethics
• 12. Criminal Justice Research Topics in Ethics
• 13. Research Paper Topics About Ethical Behavior
• 14. Research Paper Topics Related To Ethics And Values
• 15. Research Project Topics On Ethics And Morality
• 16. Research Paper Topics about Health Care Ethics
• 17. Biomedical Ethics Research Paper Topics
• 18. Ethical Egoism Research Paper Topics
• 19. Controversial Ethical Research Topics
• 20. Social Responsibility Ethics Topics
• 21. Ethical Conflicts Topics
• 22. How to Select the Best Ethical Research Paper Topics? 

Ethical Topics for Students

If you're starting to explore ethical paper topics, check out this list for some interesting topics. These ideas will help you with your research while keeping your work fair and ethical.

Ethics Topics for College Students

• Academic Integrity and Plagiarism
• The Ethical Implications of Social Media Use
• Impact of Job Automation on Employment
• Ethical Challenges in Group Projects
• Academic Honesty in the Digital Age
• The Intersection of Ethics and Student Mental Health
• Ethical Dilemmas in Research Involving Student Participants
• Cheating Prevention Strategies: Balancing Security and Trust
• Ethical Decision-Making in Internships and Work Placements
• Addressing Bias and Diversity in Academic Settings
• Euthanasia pros and cons and its ethical considerations

Ethical Hacking Research Paper Topics 

• Evolution of Ethical Hacking: Past, Present, and Future Trends
• Ethical Hacking vs. Cybersecurity: Exploring the Intersections and Distinctions
• Ethical Hacking in Critical Infrastructure Protection
• Penetration Testing: Ethical Hacking in Corporate Environments
• The Role of Ethical Hacking in National Security
• Ethical Hacking in Cloud Computing Environments
• Challenges and Ethical Considerations in Bug Bounty Programs
• Ethical Hacking in Healthcare: Safeguarding Patient Data
• The Human Element in Ethical Hacking: Social Engineering and Awareness
• Should tech companies need ethics boards?
• Analyze the ethics behind cryptocurrency and hacking

Ethical Leadership Research Paper Topics 

• The Impact of Ethical Leadership on Organizational Culture
• Ethical Decision-Making in Leadership: Models and Approaches
• The Role of Emotional Intelligence in Ethical Leadership
• Cross-Cultural Perspectives on Ethical Leadership
• Ethical Leadership in Times of Crisis: Lessons from Case Studies
• The Influence of Ethical Leadership on Employee Morale and Productivity
• Ethical Leadership and Corporate Social Responsibility
• The Relationship Between Ethical Leadership and Employee Well-being
• Leadership Integrity: Building Trust through Ethical Behavior
• Gender and Ethical Leadership: Analyzing Disparities and Opportunities

Ethical Issue Research Paper Topics

• The Ethics of Animal Testing in Scientific Research
• Social Media and Ethical Considerations: From Privacy to Online Harassment
• Ethical Challenges in Human Cloning and Reproductive Technologies
• Corporate Social Responsibility: Examining Business Ethics
• Ethical Dilemmas in Autonomous Vehicles and Technology
• Cybersecurity Ethics: Defending Against Threats Responsibly
• Surveillance and Civil Liberties: Ethical Perspectives
• The Morality of Capital Punishment: An Ongoing Debate
• Bioethics: Ethical Issues in Healthcare and Medical Research
• Ethical Considerations in Artificial Intelligence and Education

Business Ethics Research Paper Topics

• Ethical Considerations in Marketing and Advertising Practices
• The Role of Business Ethics in Supply Chain Management
• Fair Trade Practices: Balancing Profitability and Ethical Standards
• The Influence of Organizational Culture on Business Ethics
• Corporate Governance: Ethics in Decision-Making and Accountability
• Ethics in the Digital Economy: Privacy, Data, and E-Commerce
• Diversity and Inclusion: Ethical Imperatives in the Workplace
• Ethics of Outsourcing: Examining Global Business Practices
• The Intersection of Technology and Business Ethics
• Ethical Challenges in International Business: Cultural Perspectives

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Medical Ethics Research Paper Topics

• The Role of Ethics in Clinical Trials and Experimental Treatments
• Allocation of Scarce Medical Resources: Ethical Decision-Making
• Patient Autonomy and Decision-Making: Striking a Balance in Healthcare
• Cultural Competence in Healthcare: Ethical Considerations
• Medical Research Involving Vulnerable Populations: Ethical Guidelines
• Ethics of Stem Cell Research: Balancing Potential and Moral Concerns
• Access to Healthcare: Ethical Issues in Global Health Disparities
• The Impact of Emerging Technologies on Medical Ethics
• Conflicts of Interest in Medical Practice: Transparency and Accountability
• The Role of Ethics Committees in Healthcare Institutions

Accounting Ethics Research Paper Topics

• Ethical Issues in Taxation: Balancing Compliance and Strategic Planning
• Corporate Social Responsibility Reporting: Ethical Perspectives
• The Impact of Technology on Accounting Ethics: Privacy and Security Concerns
• Ethics in Forensic Accounting: Investigating Financial Irregularities
• Environmental Accounting: Ethical Dimensions in Sustainability Reporting
• Earnings Management and Ethics: Manipulating Financial Statements
• Insider Trading: Unraveling Ethical Issues in Capital Markets
• Ethical Considerations in Nonprofit Accounting and Financial Reporting
• International Financial Reporting Standards (IFRS) and Ethical Decision-Making
• Accounting for Social Justice: Ethical Implications in Resource Allocation

Legal Ethics Research Paper Topics

• Legal Ethics and Access to Justice: Addressing Disparities
• The Role of Ethics in Alternative Dispute Resolution (ADR)
• Whistleblowing in the Legal Profession: Challenges and Protections
• Ethics of Legal Advertising: Balancing Professionalism and Marketing
• The Intersection of Legal Ethics and Diversity in Law Firms
• Confidentiality and Social Media: Ethical Implications for Lawyers
• Judicial Ethics: Impartiality and Independence in the Legal System
• Ethical Challenges in International Law Practice
• Legal Ethics and Pro Bono Service: Serving the Public Interest
• Corporate Lawyers and Ethical Decision-Making: Balancing Duties

Environmental Ethics Research Paper Topics

• Biodiversity Conservation: Ethical Dilemmas in Species Preservation
• Ecofeminism and Environmental Ethics: Exploring Intersections
• Sustainable Development and Environmental Ethics
• Land Ethics: Balancing Human Needs and Ecological Integrity
• Animal Rights and Environmental Ethics: The Moral Status of Non-Human Entities
• The Ethics of Environmental Restoration and Rehabilitation
• Environmental Virtue Ethics: Fostering Ecological Virtues
• Corporate Environmental Responsibility: Ethical Considerations in Business Practices
• Ethical Dimensions of Wilderness Preservation and Management
• Environmental Education: Fostering Ethical Relationships with Nature

Bioethics Research Paper Topics

• The Ethics of Genetic Engineering and Designer Babies
• Human Cloning: Bioethical Considerations and Moral Implications
• End-of-Life Decision-Making: Euthanasia and Physician-Assisted Suicide
• Ethical Issues in Organ Transplantation and Donor Allocation
• Research Ethics in Biomedical Studies: Balancing Innovation and Patient Safety
• Animal Rights in Biomedical Research: Ethical Treatment and Alternatives
• Genomic Privacy and Confidentiality: Challenges in the Genomic Era
• The Ethics of Neuroenhancement and Cognitive Enhancement
• Environmental Ethics in Bioethics: Sustainable Practices in Biomedical Research
• Global Health Ethics: Challenges and Responsibilities in a Connected World

Research Paper Topics in Sports Ethics

• Performance-Enhancing Drugs: Ethical Dilemmas in Sports
• The Role of Fair Play and Sportsmanship in Competitive Sports
• Ethical Issues in Sports Medicine: Player Health and Treatment
• Doping in Professional Sports: Consequences and Prevention
• Gender Equality and Ethical Considerations in Sports
• Sports Betting and Match-Fixing: Ethical Challenges in the Industry
• The Impact of Technology on Sports Ethics: Video Review, Wearable Tech, and Beyond
• Youth Sports and Ethical Coaching Practices
• Social Justice in Sports: Athlete Activism and Ethical Responsibilities
• Sports Sponsorship and Ethical Considerations: Aligning Values with Partnerships

Criminal Justice Research Topics in Ethics

• Police Ethics: Use of Force, Racial Profiling, and Accountability
• Ethical Issues in Criminal Investigations: Privacy, Surveillance, and Informants
• Capital Punishment: Morality and Legality in the Criminal Justice System
• The Ethics of Plea Bargaining: Balancing Justice and Expediency
• Juvenile Justice: Ethical Considerations in the Treatment of Youth Offenders
• Ethics of Criminal Defense: Zealous Representation vs. Ethical Boundaries
• Corrections Ethics: Rehabilitation, Punishment, and Inmate Rights
• Criminal Justice and Mental Health: Ethical Approaches to Offenders with Mental Illness
• Community Policing and Ethical Engagement with Communities
• Corruption in Law Enforcement: Causes, Effects, and Ethical Solutions

Research Paper Topics About Ethical Behavior

• The Influence of Organizational Culture on Ethical Behavior
• The Role of Ethics Training in Promoting Ethical Behavior in the Workplace
• Cultural Variations in Ethical Behavior: A Cross-Cultural Analysis
• The Impact of Technology on Ethical Behavior in Business
• Professional Codes of Ethics: A Comparative Analysis Across Industries
• The Relationship Between Personal Values and Ethical Behavior
• Ethical Behavior in Healthcare: Patient-Centered Approaches
• Ethics in Research: Ensuring Ethical Behavior in Scientific Inquiry
• The Effectiveness of Ethics Hotlines in Encouraging Ethical Reporting
• Ethical Behavior in Education: Fostering Integrity in Academic Settings

Research Paper Topics Related To Ethics And Values

• The Intersection of Cultural Values and Ethical Decision-Making
• Religious Perspectives on Ethical Issues: A Comparative Analysis
• Moral Development Theories: Understanding the Formation of Values
• The Impact of Family Values on Ethical Behavior in Individuals
• Corporate Values Statements: Alignment with Ethical Business Practices
• The Role of Education in Instilling Ethical Values in Students
• Ethics and Values in the Digital Age: Challenges and Opportunities
• Professional Ethics Codes: Examining Values Across Professions
• Environmental Ethics: Values-Based Approaches to Conservation
• The Influence of Personal Values on Career Choices and Workplace Behavior

Research Project Topics On Ethics And Morality

• Ethics of Artificial Intelligence and Machine Learning: Challenges and Solutions
• Moral Dilemmas in Healthcare: Balancing Patient Autonomy and Beneficence
• Moral Psychology: Understanding the Cognitive Basis of Ethical Decision-Making
• The Morality of Capital Punishment: Examining Ethical Arguments
• Ethics in Global Governance: Addressing Moral Issues in International Relations
• Environmental Ethics: Moral Considerations in Conservation and Sustainability
• Morality in Business: Corporate Social Responsibility and Ethical Practices
• The Impact of Religion on Moral Values: A Comparative Study
• Ethics of Genetic Engineering and Biotechnology: A Moral Inquiry
• Morality and Technology Addiction: Exploring the Ethical Dimensions

Research Paper Topics about Health Care Ethics

• Ethical Issues in Organ Transplantation and Allocation
• Medical Research Ethics: Ensuring the Welfare of Research Participants
• The Ethics of Genetic Testing and Counseling in Healthcare
• Equity in Healthcare: Addressing Disparities and Access Issues
• The Impact of Technology on Healthcare Ethics: Telemedicine, AI, and Data Security
• Healthcare and Social Media: Ethical Considerations for Professionals
• Explore the Grounds of Surrogacy. Is it Ethical?
• Ethical Considerations in Prenatal Invasive Testing
• Health Information Exchange: Ethical Concerns in Electronic Health Records
• The Role of Healthcare Ethics Committees in Decision-Making

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Biomedical Ethics Research Paper Topics

• Autonomy in Medical Decision-Making: Examining Patient Rights
• Informed Consent: Ethical Considerations in Medical Research and Treatment
• The Morality of Human Genetic Editing: Implications and Limits
• End-of-Life Care Ethics: Euthanasia, Palliative Care, and Assisted Suicide
• Clinical Trials and Research Ethics: Balancing Innovation and Patient Welfare
• Organ Transplantation Ethics: Allocation, Donor Rights, and Fair Practices
• Ethics of Human Cloning: Scientific Advances and Moral Dilemmas
• Healthcare Access and Equity: Addressing Disparities in Medical Treatment
• Ethical Implications of Precision Medicine and Personalized Healthcare
• Neuroethics: Ethical Considerations in Brain Research and Neurological Interventions

Ethical Egoism Research Paper Topics

• Foundations of Ethical Egoism: A Comprehensive Analysis
• Psychological Egoism vs. Ethical Egoism: Distinguishing Self-Interest Theories
• Ethical Egoism and Altruism: Examining the Tension between Self-Interest and Concern for Others
• The Role of Rationality in Ethical Egoism: A Philosophical Inquiry
• Ethical Egoism in Business: Balancing Individual Interests and Corporate Ethics
• Criticisms and Challenges to Ethical Egoism: Addressing Counter Arguments
• Egoism and Morality: Exploring the Compatibility of Self-Interest with Moral Principles
• Personal Responsibility and Ethical Egoism: Individual Accountability in Decision-Making
• Ethical Egoism in Professional Ethics: Implications for Various Professions
• Ethical Egoism and Social Contracts: Assessing the Role of Self-Interest in Social Cooperation

Controversial Ethical Research Topics

• Climate Change Policies: Ethical Dilemmas in Balancing Economic Growth and Environmental Conservation
• Gene Editing and Designer Babies: Ethical Boundaries in Genetic Modification
• End-of-life Decision-making: Examining the Ethics of Euthanasia and Physician-Assisted Suicide
• Bioethics and stem cell research and its legality
• Can war be ethical? What are its after-effects?
• Surveillance Capitalism: Balancing Corporate Interests with User Privacy Rights
• Data Privacy in the Digital Age: Analyzing the Ethical Dimensions of Big Data Collection
• Criminal Justice and Racial Profiling: Unraveling Ethical Issues in Law Enforcement
• Ethics of Human Cloning: Examining the Moral and Scientific Frontiers

Social Responsibility Ethics Topics

• Corporate Social Responsibility: Integrating Ethics into Business Practices
• Sustainable Business Practices: Balancing Profitability and Social Impact
• Ethics in Philanthropy: Ensuring Transparency and Accountability
• Socially Responsible Investing: Aligning Financial Goals with Ethical Values
• Community Engagement and Ethical Responsibility of Businesses
• The Role of Government in Promoting Social Responsibility
• Environmental Ethics in Corporate Decision-Making: Green Initiatives and Sustainability
• Ethics in Supply Chain Management: Ensuring Fair Labor Practices
• Measuring and Reporting Social Impact: Ethical Standards in Evaluation
• Ethics of Animal Testing: Scientific Advancements vs. Animal Welfare

Ethical Conflicts Topics

• Ethical Dilemmas in Healthcare: Balancing Patient Autonomy and Beneficence
• The Tension Between Freedom of Speech and Hate Speech: Ethical Considerations
• Military Ethics: Navigating Conflicts Between Duty and Morality
• Ethics of Whistleblowing: Balancing Loyalty and Accountability
• Religious Freedom vs. LGBTQ+ Rights: Ethical Debates and Legal Conflicts
• Surveillance Ethics: Individual Privacy vs. National Security
• The Ethics of Artificial Intelligence: Addressing Bias and Autonomy
• Political Ethics: Balancing Public Service and Personal Interests
• The Right to Die: Ethical Debates Surrounding Assisted Suicide
• Ethics of Journalism: Objectivity, Sensationalism, and Media Integrity

How to Select the Best Ethical Research Paper Topics? 

Selecting the best ethical research paper topics requires thoughtful consideration and a strategic approach. 

Here are some guidelines to help you choose the most suitable and engaging research paper topics :

1. Identify Your Interests

Start by reflecting on your personal interests and passions. Choosing a topic that genuinely intrigues you will make the research process more enjoyable and rewarding.

2. Consider the Assignment Guidelines

Review any guidelines or requirements provided by your instructor. Ensure that your chosen topic aligns with the scope and objectives of the assignment.

3. Explore Current Issues

Stay updated on current events and trends in the field of ethics. Selecting a topic that addresses contemporary ethical issues can make your research more relevant and impactful.

4. Research Existing Literature

Conduct a preliminary review of existing literature in the chosen area. This will help you identify gaps, controversies, and potential research questions that can guide your exploration.

5. Consider Multiple Perspectives

Choose a topic that allows you to explore different perspectives and viewpoints. Ethical issues are often complex, and a well-rounded analysis can provide a more comprehensive understanding.

6. Ethical Considerations

Ensure that your chosen topic aligns with ethical standards. If your research involves human subjects or sensitive issues, be mindful of ethical guidelines and obtain any necessary approvals.

In conclusion, this blog consists of a wide range of ethical research paper topics for researchers and scholars. It helps you understand and choose ethical topics for your research. 

So, as you start your own research, remember that ethical questions can lead to important discoveries and a better understanding of the world around us.

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What are some ethical research questions examples.

• How does social media usage impact individual privacy, and what ethical concerns arise?
• What are the ethical implications of AI in healthcare decision-making for patient treatment?
• How can businesses balance profitability with environmental sustainability, and what ethical challenges does this pose?
• In what ways do cultural differences influence ethical decisions in multinational corporations?
• What ethical considerations surround the use of CRISPR technology in human genetic editing?
• How does social media contribute to online harassment, and what ethical safeguards can be implemented?
• What are the ethical dilemmas associated with autonomous vehicles and technology in transportation?
• How can organizations ensure fair and unbiased AI algorithms, addressing ethical concerns in technology?
• In healthcare, how do we navigate the ethical challenges of gene editing and its potential consequences?
• What ethical considerations arise in the use of big data analytics, particularly in terms of individual privacy and consent?

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Research papers are an essential part of academic life, but one of the most challenging aspects can be finding credible sources to support your arguments. 

With the vast amount of information available online, it's easy to feel overwhelmed. However, by following some simple steps, you can streamline the process of finding reliable sources for your research paper . 

In this guide, we'll break down the process into easy-to-follow steps to help you find the best sources for your paper.

On This Page On This Page -->

Step 1: Define Your Topic and Research Questions

Before you venture into your quest for sources, it's essential to have a clear understanding of your research topic and the specific questions you aim to address. Define the scope of your paper and identify keywords and key concepts that will guide your search for relevant sources.

Step 2: Utilize Academic Databases

Academic databases are treasure troves of scholarly articles, research papers, and academic journals covering a wide range of subjects. Institutions often provide access to these databases through their libraries. Some popular academic databases include:

• IEEE Xplore
• Google Scholar

These databases allow you to search for peer-reviewed articles and academic papers related to your topic. 

Use advanced search features to narrow down your results based on publication date, author, and keywords .

Academic Resources Classified by Discipline

Here's a breakdown of prominent databases categorized by academic discipline:

Step 3: Explore Library Catalogs

Your university or local library's catalog is another valuable resource for finding sources. Library catalogs contain books, periodicals, and other materials that may not be available online. 

Use the catalog's search function to locate relevant books, journals, and other materials that can contribute to your research.

Step 4: Consult Bibliographies and References

When you find a relevant source, take note of its bibliography or make a list of sources for the research paper. These lists often contain citations to other works that may be useful for your research. 

By exploring the references cited in a particular source, you can uncover additional resources and expand your understanding of the topic.

Step 5: Boolean Operators for Effective Searches

Boolean operators are words or symbols used to refine search queries by defining the relationships between search terms. The three primary operators include "AND," which narrows searches by requiring all terms to be present; "OR," which broadens searches by including either term or both; and "NOT," which excludes specific terms to refine results further. 

Most databases provide advanced search features for seamless application of Boolean logic.

Step 6: Consider Primary Sources 

Depending on your research topic, primary sources such as interviews, surveys, archival documents, and original data sets can provide valuable insights and support for your arguments. 

Primary sources offer firsthand accounts and original perspectives on historical events, social phenomena, and scientific discoveries.

Step 7: Evaluate the Credibility of Sources

Not all sources are created equal, and it's crucial to evaluate the credibility and reliability of the information you encounter. 

Consider the author's credentials, the publication venue, and whether the source is peer-reviewed. Look for evidence of bias or conflicts of interest that may undermine the source's credibility.

Step 8: Keep Track of Your Sources

As you gather sources for your research paper, maintain a systematic record of the materials you consult.  Keep track of bibliographic information, including author names, publication dates, titles, and page numbers . This information will be invaluable when citing your sources and creating a bibliography or works cited page.

Other Online Sources

In addition to academic databases and library catalogs, exploring popular online sources can provide valuable insights and perspectives on your research topic.  Here are some types of online sources you can consider:

Websites hosted by reputable organizations, institutions, and experts (such as the New York Times) can offer valuable information and analysis on a wide range of topics. Look for websites belonging to universities, research institutions, government agencies, and established non-profit organizations.

Crowdsourced Encyclopedias like Wikipedia

While Wikipedia can provide a broad overview of a topic and lead you to other sources, it's essential to verify the information found there with more authoritative sources. 

Use Wikipedia as a starting point for your research, but rely on peer-reviewed journal articles and academic sources for in-depth analysis and evidence.

Tips for Assessing the Credibility of Online Sources

When using online sources, it's important to exercise caution and critically evaluate the credibility and reliability of the information you find. Here are some tips for assessing the credibility of online sources:

• Check the Domain Extension: Look for websites with domain extensions that indicate credibility. URLs ending in .edu are educational resources, while URLs ending in .gov are government-related resources. These sites often provide reliable and authoritative information.
• Look for DOIs (Digital Object Identifiers): DOIs are unique alphanumeric strings assigned to scholarly articles and indicate that the article has been published in a peer-reviewed, scientific journal. Finding a DOI can help you assess the scholarly rigor of the source.
• Evaluate the Authorship and Credentials: Consider the qualifications and expertise of the author or organization behind the website or blog. Look for information about the author's credentials, affiliations, and expertise in the subject matter.
• Consider the Currency and Relevance: Assess how up-to-date the information is and whether it aligns with the scope and focus of your research. Look for recent publications and timely analyses that reflect current trends and developments in the field.

Wrapping it up!

Finding sources for your research paper may seem like a challenge, but by following these steps, you can locate credible sources to support your arguments and enhance the quality of your paper. 

By approaching the research process systematically and critically evaluating the information you encounter, you can produce a well-researched and compelling research paper.

If you are struggling with finding credible sources or have time constraints, do not hesitate to seek writing help for your research papers . CollegeEssay.org has professional writers ready to assist you. 

Connect with our essay writing service now and receive expert guidance and support to elevate your research paper to the next level.

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Please note you do not have access to teaching notes, homelessness: challenges and opportunities in the “new normal”.

Mental Health and Social Inclusion

ISSN : 2042-8308

Article publication date: 29 March 2024

This paper – the final paper of a series of three – aims to discuss the implications of the findings from a service user needs assessment of people experiencing homelessness in the Northwest of England. It will expand on the previous paper by offering a more detailed analysis and discussion of the identified key themes and issues. The service user needs assessment was completed as part of a review of local service provision in the Northwest of England against the backdrop of the COVID-19 pandemic.

Design/methodology/approach

Semi-structured questionnaires were administered and used by health-care professionals to collect data from individuals accessing the Homeless and Vulnerable Adults Service (HVAS) in Bolton. The questionnaires included a section exploring Adverse Childhood Experiences. Data from 100 completed questionnaires were analysed to better understand the needs of those accessing the HVAS.

Multiple deprivations including extensive health and social care needs were identified within the cohort. Meeting these complex needs was challenging for both service users and service providers. This paper will explore key themes identified by the needs assessment and draw upon further comments from those who participated in the data-gathering process. The paper discusses the practicalities of responding to the complex needs of those with lived experience of homelessness. It highlights how a coordinated partnership approach, using an integrated service delivery model can be both cost-effective and responsive to the needs of those often on the margins of our society.

Research limitations/implications

Data collection during the COVID-19 pandemic presented a number of challenges. The collection period had to be extended whilst patient care was prioritised. Quantitative methods were used, however, this limited the opportunity for service user involvement and feedback. Future research could use qualitative methods to address this balance and use a more inclusive approach.

Practical implications

This study illustrates that the needs of the homeless population are broad and varied. Although the population themselves have developed different responses to their situations, their needs can only be fully met by a co-ordinated, multi-agency, partnership response. An integrated service model can help identify, understand, and meet the needs of the whole population and individuals within it to improve healthcare for a vulnerable population.

Social implications

This study highlighted new and important findings around the resilience of the homeless population and the significance of building protective factors to help combat the multiplicity of social isolation with both physical and mental health problems.

Originality/value

The discussion provides an opportunity to reflect on established views in relation to the nature and scope of homelessness. The paper describes a contemporary approach to tackling current issues faced by those experiencing homelessness in the current context of the COVID-19 pandemic. Recommendations for service improvements will include highlighting established good practices including embedding a more inclusive/participatory approach.

• Homelessness
• Social exclusion
• Health inequalities
• Mental health
• Partnerships

Acknowledgements

The authors received no financial support for the research, authorship and/or publication of this article. The authors wish to acknowledge the contributions made by those with lived experience who completed the survey. Recognition and thanks are also given to those involved in the delivery of services that seek to improve the lives of those who are homeless.

Woods, A. , Lace, R. , Dickinson, J. and Hughes, B. (2024), "Homelessness: challenges and opportunities in the “new normal”", Mental Health and Social Inclusion , Vol. ahead-of-print No. ahead-of-print. https://doi.org/10.1108/MHSI-02-2024-0032

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'Pretending to grow forests in the desert': New research questions integrity in safeguard mechanism scheme

A major Australian study has found some of the nation's biggest polluters are meeting their emissions obligations using carbon credits that have not actually resulted in emissions reductions.

Almost a third of projects under Australia's carbon credit scheme did little to nothing to reduce emissions despite costing taxpayers hundreds of millions of dollars, the researchers found.

Andrew Macintosh, one of the lead authors of the paper and an environment law and policy professor at the Australian National University first sounded the alarm two years ago, calling the carbon market "largely a sham".

His calls were rejected by a government-commissioned review, but Professor Macintosh said the new research shows further evidence that human-induced regeneration – a core part of the Australian Carbon Credit Unit (ACCU) scheme – hasn't worked.

"The data is telling us very clearly that the credits that are being issued are in no way shape or form being matched by the abatement that these projects are actually generating," He told the ABC.

Researchers monitored 182 Human Induced Regeneration (HIR) projects, which make up about 30 per cent of all ACCUs and have cost taxpayers nearly $300 million over their lifetime.

They found many of the projects to grow native forests were claiming to be regenerating them in uncleared desert and semi-desert areas.

According to the study, 80 per cent of those projects experienced either no change, or negative change to their tree cover between 2013 when first registered, and June 2022, despite receiving nearly 23 million credits.

Researchers say study calls into question emissions results of biggest emitters

In October 2023 the government banned the registration of new HIR projects, but existing projects are still operating and generating carbon credits.

The research group's claims would have significant implications for the government's safeguard mechanism, the emissions cap placed on heavy emitters that forces them to either lower their emissions or buy credits to abate them.

The safeguard mechanism applies to facilities that emit more than 100,000 tonnes of carbon dioxide emissions a year in the mining, oil and gas, manufacturing, transport and waste sectors.

Professor Macintosh said the ACCU scheme was "severely lacking in integrity" and jeopardised the integrity of the safeguard mechanism.

"Most of the [safeguard mechanism] entities that are covered by the [ACCU] scheme will meet their obligations by buying carbon credits and surrendering them, rather than reducing their own emissions," Professor Macintosh said.

"That would be fine if the carbon credits reflected real, additional and permanent abatement but what our results are saying is they're not."

HIR projects received 37 million credits to June 2023, nearing a third of the issuances under the scheme, and covering a total area of around 42 million hectares.

The researchers found an increase of just 1.8 per cent in tree cover across those projects, based on satellite data since the projects were registered.

Professor Macintosh said those projects also didn't show a marked difference in regeneration from other nearby areas outside the credit scheme.

"We also compared the trends of tree cover inside the projects that have been credited to what's happening in comparison areas," he said.

"The primary driver is not the controlling of grazing areas but something else, and that something else is very likely to be rainfall."

Recommendations for ACCU monitor progressing

In 2022 after Professor Macintosh blew the whistle on the carbon credit scheme, the government ordered a review led by the former national chief scientist Ian Chubb.

That review, released in January last year, dismissed claims the scheme lacked integrity . 

It said it disagreed with Professor Macintosh's claims the level of emissions reduction was overstated, and that the carbon credits scheme was not effective.

But the review did make a suite of recommendations, including a new integrity committee to monitor the scheme, which the government has agreed to establish.

Professor Macintosh has always questioned the methodology of that review.

"The main problem with the Chubb review is that they didn't analyse the performance of a single project to inform their decision or their conclusion that there wasn't a problem with over-crediting," he said.

Professor Macintosh has declared he has a competing interest as a non-executive director of Paraway Pastoral Company, which has projects that use Australia's offset scheme but does not have any HIR projects.

Climate Change and Energy Minister Chris Bowen reiterated that while he understood Professor Macintosh's disagreement with Professor Chubb's findings, he emphasised that Professor Macintosh's own findings were not supported by Professor Chubb.

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• Open access
• Published: 14 March 2024

Edible mycelium bioengineered for enhanced nutritional value and sensory appeal using a modular synthetic biology toolkit

• Vayu Maini Rekdal 1 , 2 , 3 ,
• Casper R. B. van der Luijt   ORCID: orcid.org/0000-0001-5978-7731 3 , 4 , 5 , 6 ,
• Yan Chen 3 , 6 ,
• Ramu Kakumanu 3 , 6 ,
• Edward E. K. Baidoo 3 , 6 ,
• Christopher J. Petzold   ORCID: orcid.org/0000-0002-8270-5228 3 , 6 ,
• Pablo Cruz-Morales 4 &
• Jay D. Keasling   ORCID: orcid.org/0000-0003-4170-6088 1 , 3 , 4 , 6 , 7 , 8  

Nature Communications volume  15 , Article number:  2099 ( 2024 ) Cite this article

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• Food microbiology
• Genetic engineering
• Metabolic engineering
• Synthetic biology

Filamentous fungi are critical in the transition to a more sustainable food system. While genetic modification of these organisms has promise for enhancing the nutritional value, sensory appeal, and scalability of fungal foods, genetic tools and demonstrated use cases for bioengineered food production by edible strains are lacking. Here, we develop a modular synthetic biology toolkit for Aspergillus oryzae , an edible fungus used in fermented foods, protein production, and meat alternatives. Our toolkit includes a CRISPR-Cas9 method for gene integration, neutral loci, and tunable promoters. We use these tools to elevate intracellular levels of the nutraceutical ergothioneine and the flavor-and color molecule heme in the edible biomass. The strain overproducing heme is red in color and is readily formulated into imitation meat patties with minimal processing. These findings highlight the promise of synthetic biology to enhance fungal foods and provide useful genetic tools for applications in food production and beyond.

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Introduction

The global food system has been identified as one of the major contributors to climate change. Food production is responsible for an estimated one-third of global greenhouse gas emissions and contributes to widespread environmental degradation, biodiversity loss, and the emergence of new diseases 1 , 2 , 3 . Transitioning food production away from resource-intensive industrial animal agriculture toward alternative methods, including microbial processes, is critical for mitigating these negative planetary impacts and sustainably feeding a growing global population that is estimated to reach over 9 billion by 2050 1 , 2 , 4 , 5 , 6 , 7 . Among other applications, microbes can be used for upcycling byproducts 8 , as hosts for production of environmentally taxing small molecules and proteins 9 , 10 , and for producing nutritious biomass that can be consumed directly 11 . Compared to animal agriculture, microbial food production can offer increased resource efficiency and safety, more precise control of production, reduced animal suffering, and a reduced environmental footprint 5 .

Filamentous fungi, a diverse group of microorganisms that includes molds and mushrooms, have several advantages compared to other hosts for microbially based food production 12 (Fig.  1A ). In addition to the historical use of many fungi for safe and delicious fermented foods 13 , the naturally high secretory capacity of these organisms makes them powerful hosts for production of proteins for food and other uses 14 . Additionally, many fungi rapidly degrade and grow on complex substrates such as food byproducts or lignocellulose, which can alleviate the cost and environmental burden associated with highly purified substrates such as glucose 9 , 15 . Finally, owing to its filamentous morphology which mimics the structure of animal muscle, fungal biomass (mycelium) can be formulated into meat alternatives with convincing textures (mycoprotein), and used as scaffolds for adherent animal cells in cellular agriculture 11 . A recent Life Cycle Assessment revealed that substituting 20% of animal protein with mycoprotein by 2050 could lower methane emissions as well as reduce deforestation and associated CO 2 emissions by half, underscoring the concrete environmental benefits of fungal foods 6 .

A Fungal applications in sustainable food production. The figure was created was created using BioRender ( http://BioRender.com ). B Strategy for RNP-based CRISPR-Cas9 editing. Upon integration at the correct locus, the pyrG selection marker becomes flanked by two identical 300-bp sequences. Counter-selection using 5-fluoroorotic acid (5-FOA) allows locus-specific marker excision. C Strategy for integration of a GFP-expression cassette (pAmyB promoter) at the wA locus, which controls spore pigmentation, in A. oryzae RIB40. The two squiggly lines indicate that not the whole 6.7-kb wA gene is shown. Primers TamyB-DC-F (on fixing template) and wA-in-1R (on chromosome) were used to confirm successful insertion. Ladder is Generuler 1 kb ladder (Thermo Scientific). D The wA : gfp transformant has white conidia instead of the yellow-green conidial pigmentation of the RIB40 strain (grown on PDA medium). E Efficiency of gene integration at the wA locus as a function of the homology arm length in the fixing template. 10 colonies were randomly selected at each length and subjected to colony PCR. F Following pyrG marker recycling from the wA locus the strain displays uracil/uridine auxotrophy when grown on CDA medium. UU = uracil and uridine supplementation G The auxotrophy was accompanied by clear marker loop out from the wA locus. Ladder is Generuler 1 kb ladder (Thermo Scientific). H The looped-out wA:gfp strain (Δ pyrG) was transformed with an mCherry expression cassette targeted at the niaD locus, which controls nitrate assimilation. Microscopy in the wA:gfp and niaD:mCherry strain confirmed the expected pattern of protein expression. Scale bar = 25 µm. I The resulting transformant only grows with leucine (CDA-Leu) as the nitrogen source instead of nitrate (CDA), as indicated by the colony radiating from the center of the plate. J The wA:gfp and niaD:mCherry strain was subjected to marker recycling from the niaD locus. PCR confirmation indicates that marker recycling was successful from the niaD locus (see Supplementary Fig.  3 for details), while the wA locus remained unchanged. Ladder is Generuler 1 kb ladder (Thermo Scientific).

Fungal food production is a rapidly growing area with vast commercial interest and potential, and a growing number of meat and dairy substitutes based on fungi are now available on the market across Europe, U.S., and Asia 12 , 16 . While these products showcase the astonishing versatility and commercial promise of fungi for sustainable food production, most current products are based on a limited group of non-engineered strains, which have inherent limitations in their metabolism, structure, and industrial capacity. Genetic engineering could overcome these limitations and further expand beyond naturally occurring biodiversity, allowing new uses and applications of fungi in human food production 17 . For instance, a synthetic gene expression tool in Trichoderma reesei , an industrial fungus traditionally used for enzyme production, recently enabled production of gram-scale quantities of egg white and milk protein 14 , 18 . However, like many other industrial fungi, T. reesei has no history of safe or palatable consumption by humans, which limits the possible food contexts in which the fungus can be utilized, such as the highly efficient and sustainable production of fungal biomass for human food. Extending such synthetic biology tools and approaches to historically consumed, food-safe, edible fungi could expand the engineering possibilities for fungal food production, including enhancing fermented foods and altering the properties of mycoprotein to better suit human dietary needs and preferences. However, synthetic biology tools and demonstrated use cases for bioengineered food production by historically consumed food-safe filamentous fungi are lacking.

Here, we develop a modular synthetic biology toolkit for Aspergillus oryzae , an edible fungus with a long history of safe and palatable human consumption, and demonstrate its applicability for enhancing fungal foods. Our toolkit includes a CRISPR-Cas9 method for precise and efficient gene modification, neutral loci for targeted gene insertion, and tunable promoters, including bidirectional promoters as well as a synthetic expression system that offers strong gene expression independent of the medium composition. We use these tools to engineer the nutritional value and sensory appeal of the edible fungal biomass for alternative meat applications. We overproduce ergothioneine, a potent antioxidant, at levels that are higher than in mushrooms, the largest source of this molecule in the human diet. Additionally, we engineer the eight-step heme biosynthetic pathway to create an edible biomass that contains heme at levels approaching those found in leading plant-based meats incorporating heme for flavor and color. In contrast to plant-based protein, the engineered fungal biomass can be readily formulated into meat-like patties without the need for extensive processing, protein purification, or ingredient addition. In addition to demonstrating the potential of bioengineering edible fungi, this work provides synthetic biology tools and approaches that could be useful for fungi across diverse applications and industries.

A recyclable CRISPR-Cas9 method for efficient gene integration and expression

We selected Aspergillus oryzae (koji mold) as our model edible fungus and engineering target, as this fungus has a long history of safe use and human acceptance in fermented foods 13 , has a biomass with a palatable and umami-rich flavor that is commercially available as mycoprotein 19 , 20 , secretes high amounts of protein 21 , is used industrially for enzyme production 22 , and has demonstrated promise as a scaffold for animal cells in cellular agriculture 23 . To enable synthetic biology efforts across the diverse food applications of this edible fungus, we first set out to create a comprehensive genetic toolkit, including a method for efficient gene integration, neutral loci for high expression, and tunable promoters.

In designing our toolkit, we first considered the challenge of efficiently integrating heterologous genes in desired genomic locations. Filamentous fungi are notoriously poor at homology-based recombination and thus transformation with linear DNA templates often results in off-target, ectopic integrations 24 . To overcome this, strains deficient in non-homologous end-joining (NHEJ) have historically been used 25 . However, disruption of NHEJ presents potential issues with genomic instability or increased risk of DNA damage 26 . Recently, CRISPR-Cas9 has revolutionized the ability to transform and genetically modify fungi, including A. oryzae 18 , 27 , 28 , 29 . For example, the recently developed state-of-the-art CRISPR-Cas9 method for A. oryzae allows high-efficiency modification of strains proficient in non-homologous end-joining (NHEJ) 28 . The method uses plasmids to drive constitutive Cas9 and sgRNA expression, and the plasmid can be readily removed using selection, which enables sequential rounds of transformation and genome modification 28 .

To efficiently engineer A. oryzae , we sought to develop an alternative, easy-to-use CRISPR-Cas9 approach that is compatible with readily available commercial reagents 26 , minimizes the possibility of off-target effects and toxicity resulting from constitutive Cas9 expression 30 , 31 , 32 and incorporates a straight-forward phenotypic screen to verify that integration at the locus of interest has taken place 23 . Rather than encoding the Cas9 and sgRNAs from a plasmid, our method involves direct transformation of CRISPR-Cas9 Ribonucleoprotein complexes (RNPs), which can be formed in vitro from commercially available Cas9 protein and sgRNAs. At the start of our study, the RNP-based approach had been demonstrated as a strategy for rapid and precise genome editing compatible with high-throughput screening in diverse filamentous fungi 29 , 33 , 34 , 35 , 36 but had not been experimentally validated for gene integration in A. oryzae .

In our method, the DNA template introduced to fix the Double-Stranded Breaks (DSBs) harbors a pyrG marker to allow for both positive selection using uracil/uridine auxotroph and negative selection using media with 5-fluoroorotic acid (5-FOA). Although pyrG marker recycling has been demonstrated in A. oryzae , previous approaches did not incorporate CRISPR-Cas9 and required an NHEJ-deficient strain to avoid off-target integrations 37 . To overcome potential issues with ectopic integrations in wild-type A. oryzae , we designed the system such that a successful loop out of the pyrG marker can only occur if the fixing template is integrated at the locus of interest, in which case it will be flanked by two identical 300 bp sequences. In this system, ectopic integrations resulting from NHEJ are unable to loop out and survive on media supplemented with 5-FOA (Fig.  1B ). This phenotypic screening approach to locus-specific gene integration and pyrG marker recycling was first established using a plasmid-based CRISPR-Cas9 system in the related A. niger , where it allowed precise and efficient genome modification 27 . In A. niger , all colonies surviving on 5-FOA had the expected genome modification, highlighting the robustness of this approach. The A. niger method was used for mutation and gene deletion but was not utilized for integration and expression of proteins 27 .

To evaluate our RNP-based method for integration and expression in A. oryzae , we first targeted a GFP-expression cassette to the wA locus, which controls spore pigmentation, into a Δ pyrG mutant of the common laboratory strain RIB40 28 (Supplementary Table  1 ). This experiment yielded strains displaying the expected white spore phenotype, GFP expression, and fixing template insert (Fig.  1C, D , and Supplementary Fig.  1A, B ). To explore whether this method works beyond the model laboratory strain RIB40, we collected a group of A. oryzae strains with distinct industrial uses and geographical origins (Supplementary Fig.  2 ). Whole-genome sequencing of these strains revealed that these strains are phylogenetically distinct from one another and display minor variations in both the number of coding genes and biosynthetic gene clusters (Supplementary Table  2 ). To enable gene editing, we first generated Δ pyrG strains by targeting two RNPs to the pyrG locus and plating on agar supplemented with 5-FOA, uracil, and uridine to select mutants. PCR amplification of the region revealed clear mutations at the predicted sgRNA cut sites, including deletions and insertions, likely resulting from erroneous fixing by A. oryae . We observed no off-target effect on the surrounding genes despite not providing a fixing template, highlighting the precision of the RNP-based editing method (Supplementary Fig.  3 ). We then successfully introduced a GFP-expression cassette at the wA locus to alter the spore phenotype and establish heterologous protein production across the strain collection (Supplementary Fig.  4 ). These strains had not previously been genome sequenced or edited, suggesting that “wild” A. oryzae strains with potentially favorable phenotypes could be efficiently modified using our method. Moreover, these results indicate that the Δ pyrG mutants required for our method are readily generated in a single-step transformation.

The RNP-based CRISPR-Cas9 method was highly efficient. PCR amplification of RIB40 transformants at the wA locus revealed a 90% integration efficiency with 950 bp homology arms (Fig.  1E ), similar to the high targeting efficiency of the previously developed plasmid-based CRISPR-Cas9 system 28 . Even with homology arms as short as 25 bp, the method proved highly efficient (70%), suggesting that PCR primer overhangs could be used to specify the integration locus of interest (Fig.  1E ). The high efficiency with such small homology arms is consistent with findings from other filamentous fungi 34 , 38 . We also found that we could miniaturize the transformation to smaller volumes and remove the final top agar step without major decreases in integration efficiency, making the transformation process quicker and easier compared to the standard A. oryzae protoplast transformation protocol and compatible with a microplate format 28 , 35 (Supplementary Fig.  1E ). Although our method utilizes two RNP complexes for each locus to maximize the likelihood of DSB based on previous successes in fungi 27 , 39 , 40 , 41 , we found no major difference in integration efficiency between using one and two RNP complexes across two distinct genomic loci in A. oryzae ( wA and niaD ) (Supplementary Table  3 ).

A core design feature of our method is the ability to recycle the pyrG marker upon insertion to the correct locus, as transformants should undergo marker excision in the presence of 5-FOA. We first confirmed successful recycling from the wA locus. Consistent with previous results in A. niger 27 , surviving colonies displayed uracil-uridine auxotrophy and marker excision, indicating successful pyrG removal. We observed this across three out of three colonies analyzed, highlighting the robustness of the growth-based method to assess locus-specific marker recycling (Fig.  1F, G , and Supplementary Fig.  1C, D ). Finally, we successfully integrated GFP-expression cassettes and excised pyrG markers at the niaD locus, which controls nitrate assimilation, and the yA locus, which contributes to spore coloration (Supplementary Fig.  5 and Supplementary Table  1 ) 28 .

The recyclability of the pyrG marker allows for potentially endless rounds of sequential engineering. To evaluate this possibility, we transformed the looped-out wA : gfp strains with an mCherry cassette targeted to the niaD locus. Positive transformants demonstrated the expected phenotype and protein expression (Fig.  1H, I ). We then successfully recycled the pyrG marker from the niaD locus, enabling sequential engineering using our marker recycling approach (Fig.  1J ). Finally, we also targeted the wA and niaD loci simultaneously in a single experiment. However, in contrast to the high efficiency observed with single integration at the wA locus, simultaneous modification at the niaD and wA loci was only 30% efficient with 950 bp homology arms (Supplementary Fig.  6 ). This is consistent with previous findings of reduced efficiency with multiple RNP complexes and fixing templates in fungi 35 . Overall, these results establish the RNP-based method as a method for genome modification and protein expression in diverse strains of the edible fungus A. oryzae . This method displays a comparable high efficiency and scope as the plasmid-based method for genetically engineering this fungus 28 . The use of commercially available reagents and the ability to phenotypically screen for insertion at the locus of interest makes the protocol easy to use.

Identification and evaluation of neutral loci for gene expression

After establishing the RNP-based CRISPR-Cas9 method for gene modification, we considered another challenge in genetic engineering of filamentous fungi: where to integrate genes for overexpression. While multi-gene expression has been achieved in A. oryzae for natural products biosynthesis, the historically preferred method involves plasmids that integrate randomly throughout the genome 42 . These can cause unintended pleiotropic effects or genomic instability and make it challenging to compare phenotypes between constructs and strains 43 . In contrast, neutral loci, intergenic regions, and genomic safe havens that allow targeted expression without interfering with host physiology, is a standard feature of engineering for many bacteria and yeasts such as S. cerevisae 44 , 45 . Recently, genome sequencing of A. oryzae transformed with randomly integrated plasmids revealed two intergenic regions (called “hot-spots”) that were successfully targeted with CRISPR-Cas9 for expression of natural products genes 46 . However, to this date, neutral loci have not been systematically identified and evaluated for the efficiency of gene integration and level of protein expression across the A. oryzae genome. This information, along with readily available plasmids and DNA parts targeted to characterized loci, is critical to advance engineering efforts, as has been shown in S. cerevisae 45 .

We took a computational approach to identify candidate-neutral loci in A. oryzae . We first identified intergenic regions in the A. oryzae RIB40 genome. Using publicly available RNA-sequencing data across diverse conditions and growth stages, we ranked the expression level of the two genes immediately surrounding the intergenic region, thus generating a list of candidate loci predicted to enable high gene expression (Fig.  2A and Supplementary Data file  1 ). From this set, we selected 10 promising high-expression regions (>4.8 kb) spread across A. oryza e chromosomes for further evaluation (Fig.  2B , Supplementary Tables  4 and 5 ). We then integrated cassettes harboring GFP under control of the strong, constitutive pTEF1 promoter, and assessed fluorescence using flow cytometry on the conidia of looped-out strains 47 (Fig.  2B, C and Supplementary Fig.  7 ).

A A computational approach was used to identify intergenic regions with high expression of surrounding genes. The highest expressing regions were selected as promising neutral loci for further experimental evaluation. B Targeting plasmids were designed with the 5′ and 3′ homology arms as well as the specific 300 bp sequence for the locus of interest. The plasmid harbors a GFP-expression cassette driven by the constitutive pTEF1 promoter and terminated by the commonly used TamyB terminator, both from A. oryzae . The plasmids were cloned in E. coli and were linearized using PCR to create linear fixing templates that target the locus of interest. C Flow cytometry of conidia constitutively expressing pTEF1 was used to evaluate expression strength at the neutral locus of interest and determine their suitability for engineering efforts. A representative microscopy image showing GFP expression from A. oryzae conidia is shown. Scale bar = 25 µm. The flow cytometry figure was created was created using BioRender ( http://BioRender.com ). D Integration efficiency of GFP-expression cassette at neutral loci. All loci except for chro3_1 displayed a high efficiency of integration (>50%), as assessed by PCR. We could not detect insertion at chro3_1 by PCR. E GFP expression (expressed as Mean Equivalents of Fluorescein, or MEFL) across neutral loci. All loci except for chro4_2 displayed expression levels above the background strain (RIB40) and were higher than the amyA locus, which was included as a positive control to validate the method. Results are average and standard error of the mean (SEM) of three biological replicates.

Out of 10 tested loci, 9 showed high-efficiency integration (>50%) (Fig.  2D ). We could not detect successful gene insertion at chro3_1 by PCR amplification, suggesting issues with PCR amplification or the integration itself. Following marker loop out, we detected GFP expression above background levels from 8 of the remaining 9 loci, with chro4_2 displaying no clear GFP expression (Fig.  2E ). Expression levels were largely consistent across the loci, with the highest (chro6-1) showing ~25% higher expression than the lowest (chro7-1) (Fig.  2E ). All loci displayed higher expression than the amyA locus, which was included as a positive control to validate the method. Finally, colony growth and morphology were consistent between all strains and similar to those of the background strain, suggesting no gross effects of gene integration and expression on fungal growth (Supplementary Fig.  8 ). Overall, these efforts identified not only neutral loci, but also a set of plasmids and sgRNAs that facilitates easy transformation and expression for diverse purposes (Supplementary Table  5 ). Our computational identification and experimental evaluation using flow cytometry provides a framework for how to identify and evaluate promising candidate loci across fungal hosts.

Expansion of the promoter toolkit using a synthetic expression system and bidirectional promoters

An additional challenge in engineering edible filamentous fungi is the narrow set of characterized parts available for gene regulation, as fungal promoters remain limited in both sophistication and scope. For example, only a handful of endogenous promoters have been used for gene expression in A. oryzae , and these are either regulated by the nutrient source (such as the amylase or glucoamylase promoters), have a limited dynamic range, or a poorly understood mode of regulation 48 , 49 , 50 . Synthetic expression systems (SES), which are widely available in yeast and bacteria and increasingly in mammalian cells and plants, could address the technical limitations of current fungal expression tools and expand engineering opportunities in A. oryzae 51 , 52 , 53 , 54 , 55 , 56 , 57 . SES couple synthetic transcription factors (sTF) with minimal core promoters (Cp) and DNA binding sites (UAS) and offer an orthogonal and highly programmable mode of gene expression 18 (Fig.  3A ). The Tet-On SES has shown promise in A. niger and A. fumigatus for inducible and titratable gene expression 58 , 59 , but food applications are limited by the cost and potential food incompatibility of the small molecule inducer. In contrast, a constitutive SES based on the Bm3R1 DNA binding domain and the VP16 activation domain was recently established in the two non-edible, industrial filamentous fungi A. niger and T. reesei . The highly modular SES showed high programmability and stability across the two hosts and afforded high secreted protein expression independent of the composition of the growth medium 18 .

A Design of synthetic expression system (SES). Coupling a synthetic transcription factor (sTF, composed of an Activating Domain = AD and DNA binding domain = DBD) and Upstream Activating Sequences (UAS) enables orthogonal and highly programmable gene expression from core promoters (Cp). B Confirmation of the Bm3R1-VP16-based SES in A. oryzae using the An_201205 core promoter. Fluorescence imaging that the SES in A. oryzae requires both the sTF and the UAS for expression. Scale bar = 50 µm for −UAS, 25 µm for other strains. C Conidia from strains shown in ( B ) were subjected to flow cytometry for fluorescence quantification of the constitutively expressed mCherry (expressed as Mean Equivalents of Texas Red, or METR). Results are average and SEM of three biological replicates. D Core promoter screen using the SES in A. oryzae . 200-bp sequences were cloned upstream of mCherry and fluorescence intensity was quantified using flow cytometry of conidia. The full-length, constitutively expressed promoter pTEF1 was included as a benchmark for promoter strength. Results are average and SEM of three biological replicates. E Proteomic comparison of intracellular mCherry abundance between the core promoter, Ao_0583, and the full-length starch-inducible endogenous promoter pAmyB from A. oryzae . Proteomics was conducted on lyophilized mycelia grown in liquid cultures (CDA medium with dextrin or glucose as the sole carbon source). Results are average and SEM of three biological replicates. F A minimal bidirectional promoter (Syn-BD) constructed of 2x UAS binding sites and the gpdA and hhfA core promoters can drive dual mCherry and GFP expression. Scale bar = 25 µm for RIB40, 50 µm for engineered strain. G Identification and evaluation of endogenous bidirectional promoters from A. oryzae . Flow cytometry quantification indicated that two promoters, p2-1 and p4-2, could drive bidirectional gene expression at varying levels. p4-2 was similar to Syn-BD in terms of expression. A concatenated sequence of pAmyB and pTEF1 pointing in opposite directions was included as a positive control. MEFL = mean equivalents of fluorescein. METR = mean equivalents of Texas red. Results are average and SEM of three biological replicates.

We sought to expand the engineering possibilities in the edible A. oryzae by building on these advances in the industrial workhorses A. niger and T. reesei . To first establish SES as a mode for gene regulation in A. oryzae , we initially evaluated the ability of the previously characterized Bm3R1-NLS-VP16 sTF to drive mCherry expression from a core promoter. Using the RNP-based CRISPR-Cas9 integration tools and neutral loci, we genetically integrated the sTF and drove low levels of basal expression of this transcription factor using a characterized core promoter from A. niger (An008). In a separate genomic location, we integrated an mCherry cassette harboring 6x UAS upstream of the A. niger An201205 core promoter. There was clear expression of mCherry in mycelia and conidia using the full system. The UAS and the sTF were both necessary for activity, validating the predicted function of the SES in A. oryzae 18 (Fig.  3B, C ).

To explore the programmability of this modular SES in A. oryzae , we initially focused on core promoters, as the identity of these short 200-bp sequences influences the level of gene expression upon sTF binding 18 , 57 . Using available transcriptome data and a curated list of promoters from highly expressed A. oryzae and Aspergillus flavus genes, we first assembled an initial library of twelve 200-bp core promoters and evaluated their ability to drive mCherry expression in the SES (Supplementary Table  6 ). We used flow cytometry of conidia 47 as an initial screening approach to assess expression and used the strong, constitutive pTEF1 promoter as a benchmark for comparison. Three of twelve selected core promoters did not drive mCherry expression at levels above the background strain (Fig.  3D ). However, across strains producing detectable mCherry, mean expression across the core promoter library displayed a 14-fold expression range, from 0.25 to more than 5-fold pTEF1. These results indicate that, like full-length promoters, core promoter sequences can drive divergent transcriptional outputs in A. oryzae (Fig.  3D ). Proteomics analysis of mCherry in biomass grown in submerged fermentations confirmed that core promoters could also drive protein expression in mycelia, including at levels that were several-fold higher than pTEF1 (Supplementary Fig.  9A ). There was a significant correlation between the flow cytometry and proteomics data ( r  = 0.82, R 2  = 0.67, p  < 0.01, Supplementary Fig.  9B ). This suggests that flow cytometry is a useful screening approach to identify constitutive promoters. Nonetheless, following up on flow cytometry screening results in mycelia may be needed for establishing the precise promoter strength for submerged fermentations.

To further benchmark the SES system we used proteomics to compare the strength of the SES promoter Ao_0583, identified as >4-fold stronger than pTEF1 in both conidia and mycelia, with the starch-inducible pAmyB promoter, one of the strongest known endogenous A. oryzae promoters that is frequently used for high protein expression and secretion from submerged cultures 21 , 50 . Strikingly, Ao_0583 was approximately 6-fold stronger than that of the pAmyB promoter when the strain harboring it was grown under inducing conditions. mCherry levels were estimated to comprise approximately 13% intracellular protein under the Ao_0583 system, and only 1–2% in the pAmyB expression strain (Fig.  3E and Supplementary Fig.  10 ). While mCherry levels did not differ between glucose and dextrin when using the SES, pAmyB expression increased on dextrin, the predicted inducer of pAmyB (Fig.  3E and Supplementary Fig.  10 ). Thus, the SES permits high protein expression independently of the carbon source and avoids the complex multi-step regulation and global metabolic changes involved in pAmyB-driven expression in A. oryzae 60 . To our knowledge, the expression levels afforded by the SES far outperform any characterized promoter in A. oryzae .

In addition to the limited set of available mono-directional promoters, there is a lack of bidirectional promoters for filamentous fungi. Bidirectional promoters, which are available in yeast, could accelerate genetic engineering in edible filamentous fungi by enabling assembly of multi-step metabolic pathways or multi-protein complexes through fewer transformations 61 , 62 . We addressed this challenge in two ways. First, we created a synthetic bidirectional promoter (Syn-BD), as the modular nature of the SES enables different parts to be combined to create highly programmable modes of gene expression 63 . By combining two core promoters ( gpdA and hhfA ) with 2× UAS binding sites, we created a 485-bp bidirectional promoter was sufficient to drive bidirectional gene expression using the SES (Fig.  3F ). Second, we computationally identified candidate endogenous bidirectional promoters using publicly available RNAseq data collected across diverse growth conditions (Supplementary Data file  2 ). Out of five computationally identified bidirectional promoter candidates (Supplementary Table  7 ), two (p2-1 and p4-2) could drive mCherry and GFP expression in a bidirectional fashion (Supplementary Fig.  11 ). The p4-2 promoter displayed similar levels of expression as the rationally designed Syn-BD and a control bidirectional promoter composed of concatenated pAmyB-pTEF1 sequences pointing in separate directions (Fig.  3G ). Interestingly, the sequence of the identified p4-2 endogenous promoter in A. oryzae has the same length and genomic context as the H3/H4 histone promoter which was previously identified and evaluated in a range of Aspergilli , but not in A. oryzae 64 . This suggests that our computational pipeline might be broadly useful to identify bidirectional promoters in filamentous fungi. Overall, these results expand the set of gene regulation tools and promoters available for engineering the edible A. oryzae .

Edible mycelium bioengineered for enhanced nutritional value and sensory appeal

Having established a synthetic biology toolkit for A. oryzae , we next sought to deploy our tools to bioengineer its edible mycelium, as a first step toward enhancing its value as mycoprotein. We were inspired by the recent commercial success of bioengineered Saccharomyces used for brewing, which have been modified for improved cost savings, sustainability, and sensory profiles 17 . To explore whether genetic modification could similarly enhance foods made with filamentous fungi, we set out to modify endogenous biosynthetic pathways that could potentially improve the nutritional value and sensory appeal of the edible A. oryzae mycelium for alternative meat applications.

We initially focused our engineering efforts on ergothioneine, a bioactive amino acid and powerful antioxidant. Low plasma levels of ergothioneine are correlated with cardiovascular disease and neurological decline, and humans encode a specific ergothioneine transporter that uptakes ergothioneine from the diet, underscoring the potential importance of this molecule in human health 65 . While many foods contain low levels of ergothioneine, fungi are the major dietary source 66 . Work in the model ascomycete mold Neurospora crassa has revealed that fungal ergothioneine biosynthesis involves two enzymes, Egt1 and Egt2, that convert cysteine, S-adenosylmethionine, and histidine, to ergothioneine 67 , 68 , 69 (Fig.  4A ). N. crassa Egt1 and Egt2 were recently co-expressed in A. oryzae using plasmid-based random integration 70 . Rice cultured with transformants in solid-state fermentation had elevated ergothioneine levels, but the levels in the biomass alone were not investigated. Untransformed A. oryzae produced low levels of ergothioneine, suggesting that this fungus may harbor endogenous pathways for production 70 .

A Fungal biosynthesis of ergothioneine, a powerful antioxidant associated with several health benefits in humans. The characterized biosynthetic pathway from the fungus Neurospora crassa involves the enzymes Egt1 and Egt2. B A. oryzae homologs of N. crassa Egt1 and Egt2 were identified bioinformatically (see Supplementary Table  7 and Supplementary Fig.  12 for details) and expressed from neutral loci using a bidirectional promoter (strain VMR-Eg1-2) or as two separate genes at two different genomic locations, with each gene under the control of its own promoter (strain VMR-Eg1_2). The strategy is described in Supplementary Fig.  12 . Oyster mushroom, the dietary mushroom with the highest ergothioneine content, was included for comparison. Biomass was analyzed by LC–MS. Results are average and SEM of three biological replicates. C Engineering of heme biosynthesis in A. oryzae biomass. The strategy is described in Supplementary Fig.  15 . Heme was quantified using LC–MS in the biomass. The intracellular heme levels in the engineered strain were 4-fold higher than in the background strain, RIB40, and 40% of those found in IMPOSSIBLE™ burger made from plants, a leading plant-based meat product incorporating heme for flavor and color, was included for comparison. Results are average and SEM of three biological replicates. D Color of harvested background and engineered heme strain after culturing. The engineered strain overproducing heme (VMR-HEM_v1) was distinctly red in color, while RIB40 was off-white. The harvested fungal biomass could be readily formulated into an imitation meat patty with minimal processing. The color difference remained upon cooking, further enhancing the meat-like appearance of the naturally textured fibrous biomass.

Instead of introducing foreign genes, we hypothesized that by changing the expression of potential endogenous A. oryzae genes involved in ergothioneine biosynthesis, we could elevate production in the edible biomass to levels found in dietary mushrooms. To identify candidates, we searched the A. oryzae genome for homologs of N. crassa Egt1 and Egt2. We found two A. oryzae ortholog candidates sharing 49.2 and 45.1% amino acid sequence (Supplementary Table  8 ). Sequence alignment indicated conservation of key residues or functional groups bioinformatically predicted to be involved in substrate binding in Egt1, as well as residues structurally confirmed to participate in catalysis in Egt2 68 (Supplementary Fig.  12 ). We then integrated the A. oryzae homologs (named AO_Egt1 and AO_Egt2) at neutral loci and drove expression of both genes from either a bidirectional promoter (strain VMR-Eg1-2), or as two separate genes in two separate genomic locations (strain VMR-Eg1_2) (Supplementary Fig.  12 ). High-resolution Liquid Chromatography-Mass Spectrometry (LC–MS) was used to detect ergothioneine in samples (Supplementary Fig.  13 ). Consistent with previous observations 70 , we detected low levels of ergothioneine in the mycelium in the background strain RIB40 (Fig.  4B and Supplementary Fig.  13 ). However, the bidirectional promoter and separate promoter strains elevated ergothioneine 11-fold and 21-fold, respectively, over RIB40 (Fig.  4B ). While the ergothioneine levels in VMR-Eg1-2 was similar to those found in oyster mushroom, the highest known dietary ergothioneine source 66 , the mean levels in strain VMR-Eg1_2 were 1.5-fold higher. We observed no major difference in protein content between the wild-type and engineered strains; however, ergothioneine overproduction was associated with a slight growth defect, suggesting a metabolic burden of ergothioneine production under the growth conditions (Supplementary Fig.  14 ). Overall, these results implicate the endogenous genes AO_Egt1 and AO_Egt2 in ergothioneine biosynthesis in A. oryzae and validate the metabolic engineering approach to alter the molecular composition of mycoprotein.

Having validated our tools to increase levels of bioactive molecules for enhanced nutritional value, we asked whether a similar approach could be applied to sensory properties of the edible biomass to more closely mimic animal meat. For example, even though the A. oryzae biomass has a meat-like fibrous texture owing to its microscopic morphology, the biomass, which is off-white, would necessitate color addition for many meat applications. As a first step toward improving the meat-like flavor composition and appearance of the edible biomass using bioengineering, we initially targeted the biosynthesis of heme, an essential cofactor that catalyzes a wide range of reactions across all domains of life and gives red meat its color and contributes to flavor upon cooking 71 . IMPOSSIBLE Foods, a leading plant-based meat producer, has taken advantage of these properties of heme and adds a purified soy Leghemoglobin (LegH) produced with the yeast Pichia pastoris to its products based on plant protein isolates to create realistic alternatives that look like red meat 72 , 73 . Other plant-based meat producers have now followed suit with similar hemoglobin addition strategies 74 .

We reasoned that by modulating the expression of key heme biosynthetic enzymes, we could elevate intracellular heme in the edible fungal biomass to levels found in leading meat alternatives incorporating heme for flavor and color. Fungal heme biosynthesis is carefully regulated at the transcriptional and post-translational levels and involves eight dedicated enzymes, which are split between the mitochondria and the cytosol 75 (Supplementary Fig.  15 ). We identified potential heme biosynthesis proteins in A. oryzae by searching the genome for sequences found in S. cerevisiae 76 (Supplementary Table  9 ). There is limited experimental information about heme biosynthesis in filamentous fungi, but based on successful engineering efforts from yeast 77 , 78 , and studies of individual heme biosynthetic enzymes in different Aspergilli 75 , 79 , 80 , 81 , we initially targeted expression of predicted rate-limiting enzymes, including ALAS (biosynthetic enzyme#1), PBGD (#3), UROD (#4), and CPO (#5). Additionally, we mutated key cysteine residues in the Heme Regulatory Motif (HRM) of ALAS to remove potential feedback inhibition by heme 82 (Supplementary Fig.  15 ). Importantly, high levels of free heme and the porphyrin intermediates can be toxic to the cell, causing oxidative damage and hampering growth 77 , 83 . To address this potential challenge, we expressed two copies of Soy Leghemoglobin, the FDA-approved protein used in IMPOSSIBLE meat 72 , as a potential heme sink, using both the SES and the significantly weaker pTEF1 promoter. Though the regulation of heme biosynthesis has not been characterized in detail in filamentous fungi, simultaneous elevation of biosynthetic enzymes and a heme-binding protein was necessary to increase heme levels without causing excessive toxicity in S. cerevisiae 77 , 78 . The final engineered strain A. oryzae contained a total of separate six modifications (Supplementary Fig.  15 ).

We used high-resolution LC–MS to detect heme across all samples (Supplementary Fig.  16 ). The biomass of the engineered strain contained 4-fold higher levels of heme compared to the non-engineered strain, on a dry weight basis. These levels of heme in the engineered strain were nearly half (40%) of those found in IMPOSSIBLE meat (Fig.  4C ). Increasing levels further may require tuning pathway flux or making additional modifications beyond biosynthetic enzyme expression levels, as was recently shown in S. cerevisae 76 . However, to our knowledge, this is the highest levels of intracellular heme in fungal mycelium and a rare example of heme biosynthesis engineering in filamentous fungi. Given the importance of heme for enzyme production for biofuels and medical applications 78 , 84 , we envision that these strains and approaches could have broad applicability for engineering efforts beyond food.

Upon harvesting the biomass of the heme overproducer, we noticed that it was red in color, compared to the off-white color of the background strain. In contrast to other plant-based meat alternatives, which require extensive processing and ingredient addition to transform off-flavor plant protein isolates (such as soy or pea) to meat alternatives, this bioengineered mycoprotein required minimal post-harvest processing for formulation into an imitation red meat patty following a standard mycoprotein production protocol 11 (Fig.  4D ). The only processing needed was removing excess liquid from the biomass prior to grinding and cooking. The color difference between the background and engineered strains remained after cooking, enhancing the meat-like appearance of the naturally textured, fibrous fungal biomass (Fig.  4D ). There was no decrease in the growth yield or protein content (46%, on a dry weight basis) in the engineered heme strain relative to the background strain (Supplementary Fig.  17 ). The engineered mycoprotein also contained all the essential amino acids, suggesting a promising nutritional profile (Supplementary Fig.  17 ). Taken together, these data suggest that the engineered edible fungal mycelium could have promise in meat alternative applications.

Filamentous fungi are widely used for the industrial production of enzymes and metabolites and recently have found more widespread use in both sustainable materials and foods 12 . However, genetic tools for these organisms have historically been limited in both sophistication and scope, preventing both engineering efforts and fundamental studies. Recent advances in CRISPR-Cas9 technology have dramatically improved the possibilities of modifying diverse mushrooms and molds, including the food-safe, edible fungus A. oryzae 27 , 28 , 29 , 34 . In contrast to industrial strains such as T. reesei , which was recently used to produce milk and egg proteins at lab scale 14 , the historically consumed A. oryzae has potential uses across fermented foods, food protein production, cellular agriculture, and mycoprotein 13 , 20 , 22 , 23 .

To enable bioengineering for these diverse applications, we developed a ready-to-use toolkit that is now available to the research community and includes DNA parts for integration and regulation of genes and pathways. Similar toolkits are available in S. cerevisiae , where they have significantly expanded opportunities for genetic engineering 45 . We hope that our tools will be similarly useful for expanding the engineering possibilities in A. oryzae , alongside other recently developed genome modification methods such as base editing 85 , in vivo DNA assembly in NHEJ-deficient strains 86 , and protein expression screening 87 . Additionally, we envision that the computational and experimental approaches used here – for identification and evaluation of neutral loci and design and identification of promoters – could be broadly useful for constructing genetic toolkits and engineering diverse fungal hosts.

We used our tools to enhance the molecular composition and appearance of the mycelium as a first step toward improving its nutritional and sensory properties. First, we engineered A. oryzae mycoprotein to overproduce the nutraceutical ergothioneine at levels that are higher than those in mushrooms, the highest known natural source from the diet. While ergothioneine has been produced in a range of microbial hosts for the purpose of isolating the nutraceutical 88 , 89 , our work represents a proof of concept of modifying endogenous ergothioneine biosynthesis for mycoprotein applications. Separately, we engineered the A. oryzae mycelium to overproduce heme, a key flavor-and-color molecule in red meat, at levels that are close to half those found in leading plant-based meats. Our engineering of the edible fungal biomass for alternative meat presents an alternative approach to fungal food beyond the production of secreted animal proteins, which is a less efficient fermentation process and has a higher environmental footprint than biomass production 6 , 9 . Future engineering targets for edible fungal biomass could include lipid pathways for flavor, amino acids for nutrition, structural alteration for texture improvement, or enzymes for improved growth on affordable, complex feedstocks. However, it is important to note that our work represents early prototypes, and further assessment of the sensory attributes, consumer acceptability, potential food safety concerns, and the regulatory landscape around genetically modified organisms (GMO), is needed to bring engineered edible fungi from lab bench to the table.

A. oryzae , like many of the strains that form the basis of fungal foods available on the market, has been genetically modified through extensive selection and breeding throughout human history 90 , 91 . Genetic modification using contemporary gene editing tools such as CRISPR-Cas9 represents a natural next step in this long history of microbial gene modification to suit human needs and holds promise to further expand fungal strain diversity and accelerate the adaptation of fungal strains to the demands of current production methods and consumer preferences. Bioengineered edible plants and yeasts have demonstrated reduced environmental impact, improved nutrition, and improved flavor profiles compared to their non-engineered counterparts and are already available on the market 17 , 92 , 93 . We anticipate similar possibilities with genetic modification of edible filamentous fungi, as synthetic biology in these organisms is uniquely positioned to address the pressing environmental, ethical, and public health challenges of industrial animal agriculture.

All primers used for genome modification are shown in Supplementary Table  10 . All strains and plasmids used for strain construction are listed and described in Supplementary Tables  11 - 12 . The sequence files corresponding to each strain and plasmid can be found in the JBEI Public Registry ( https://public-registry.jbei.org/ ) 94 . All plasmids were propagated in Escherichia coli strain DH10B and purified by Miniprep (Qiagen). The plasmids generated in this study were based on the pTWIST_amp backbone (TWIST biosciences) and were constructed by Gibson assembly 95 using Gibson assembly master mix (New England Biolabs). PCR amplification was performed using NEB Q5 polymerase according to the manufacturer’s instructions (New England Biolabs). All genes were codon optimized for A. oryzae and ordered either as G-blocks from IDT or as complete, sequence-verified genes from IDT or TWIST biosciences. The coding sequences of heterologous genes in all plasmids were validated by Sanger sequencing (Azenta) or whole-plasmid sequencing (Primordium).

Growth conditions

A. oryzae strains were always grown at 30 °C. A variety of media were used in the transformation and cultivation of A. oryzae , and these are indicated below. They are referenced in the materials and methods section. Media were supplemented with 5 g/L uridine (Sigma–Aldrich, #U6381) or 2 g/L uracil (Sigma–Aldrich, #U1128) when supplementation to support growth of pyrG mutants was needed. Supplementation is indicated as UU throughout the manuscript.

GP medium (per 1 L of medium)

5 g yeast extract, 10 g polypeptone, 0.5 g MgSO 4 ⋅ 7H 2 O, 5 g KH 2 PO 4 . 20 g glucose was used as the carbon source unless otherwise indicated. Alternatively, 20 g dextrin (Sigma–Aldrich, #31400) was used as the carbon source.

PDA + 5-FOA + Uridine + Uracil (PDA + 5-FOA + UU) medium (per 1 L of medium)

39 g Potato Dextrose Agar (PDA, Sigma–Aldrich, #70139-500 G), 5 g uridine,2 g uracil, and 1 mg/mL 5-fluoroorotic acid (ThermoFisher, #R0812).

Bottom Agar + Methionine (BA + Met, per 0.5 L of medium)

1 g NH 4 Cl, 0.5 g (NH 4 ) 2 SO 4 , 0.25 g KCl, 0.25 g NaCl, 0.5 g KH 2 PO 4 , 0.25 g MgSO 4 •7H 2 O, 0.01 g FeSO 4 , 109.3 g sorbitol, 7.5 g agar, 10 g glucose, 0.75 g methionine. pH was adjusted to 5.5 prior to autoclaving.

Top Agar + Methionine (TA + Met, per 0.5 L of medium)

Same as BA + Met, but 4 g agar instead of 7.5 g agar per 0.5 L.

Minimal Medium Agar + Methionine (MMA + Met, per 0.5 L of medium)

Same as BA + Met, but no sorbitol added as the osmotic stabilizer.

CDA medium (per 1 L of medium)

3 g NaNO 3, 2 g KCl, 1 g KH 2 PO 4 , 0.5 g MgSO 4  × 7 H 2 O, 0.02 g FeSO 4 ·7H 2 O, 15 g agar. 20 g glucose was used as the carbon source unless otherwise indicated. Alternatively, 20 g dextrin (Sigma–Aldrich, #31400) was used as the carbon source.

CDA(Leu) medium

same as CDA medium but containing 10 mM leucine as the sole nitrogen source instead of the 3 g/L NaNO 3 .

Strain construction

A. oryzae strains were genetically modified using protoplast transformation (see standard transformation protocol below). All A. oryzae strains are described in Supplementary Table  12 . Regenerated protoplasts were restreaked onto MMA + Met plates to obtain single colonies and purify the potentially heterokaryotic conidia. Following 48 h of growth at 30 °C, the conidia of individual, single colonies were transferred to MMA + Met slants for growth for 48–72 h at 30 °C. These purified strains represented the strains used in all assays and characterizations. To confirm the insertion of genes at the correct locus, colony PCR was performed on conidia on slants using PHIRE direct plant PCR kit (ThermoFisher, #F130WH) by boiling conidia in 20 µL of dilution buffer for 10 min at 95 °C and using 1 µL of the conidial spore suspension as the template for PCR, which was set up according to the manufacturer’s instructions. Strains harboring the correct insertions were saved as glycerol stocks by suspending conidia in 30% glycerol (v/v). For the simultaneous targeting of wA and niaD loci in a single transformation, DNA templates of plasmids were prepared as described below and 10 µg of each plasmid, along 5 µL of each RNP complex (four total, two per locus) were added at the DNA-RNP incubation step. To check for spore coloration ( wA and yA mutants), strains were grown on PDA medium for 5 days at 30 °C. To check for nitrate assimilation ( niaD mutant), strains were grown on CDA and CDA-Leu for 5 days at 30 °C. To check for pyrG mutation and the associated uridine/uracil auxotrophy, strains were grown on CDA and CDA supplemented with 2 g/L uracil and 5 g/L uridine for 5 days at 30 °C. To assess the targeting efficiency at individual loci, and to evaluate the effect of homology arm length on integration at the wA locus, colony PCR of 10 individual strains was performed, and those displaying the correct band by PCR were deemed successful integrations. Varying homology arm lengths of the wA fixing template were obtained by linearizing the full-length template with different primers (see primer table, Supplementary Table  10 ). Flow cytometry or microscopy assays were used to assess the expression of fluorescent proteins (see below for details). To create the engineered strain VMR-Eg1-2, A. oryzae RIB40 pyrG mutant was transformed with the linear DNA template originating from JBx_250940 and the two 5′ and 3′ RNP complexes targeting the chro1-3 neutral locus. To create the engineered strain VMR-Eg1_2, A. oryzae RIB40 pyrG mutant was transformed with the linear DNA template originating from JBx_250936 and the two 5′ and 3′ RNP complexes targeting the chro1-3 neutral locus, and subsequently with the linear DNA template originating from JBx_250938 and the two 5′ and 3′ RNP complexes targeting the chro2-2 neutral locus. For the engineered strain overproducing heme (VMR-HEM_v1), A. oryzae RIB40 pyrG was sequentially transformed with linearized DNA originating from plasmids JBx_250942, JBx_250944, JBx_250946, JBx_250948, JBx_250950, JBx_236225, as well as the specific 5′ and 3′ RNP complexes associated with the target locus for integration. For the cultivation of RIB40, and the engineered strains overproducing ergothioneine and heme, 5 × 10 5 conidia were inoculated into 50 mL of GP-glucose medium (ergothioneine strains and corresponding RIB40 control) or 50 mL GP-dextrin medium (heme strain and corresponding RIB40 control) in 250 mL Erlenmeyer flasks. The strains were grown for 96 h at 30 °C, shaking at 160 rpm. Biomass was harvested by vacuum filtration over Miracloth. Biomass was lyophilized for extraction of metabolites and was dried for 7 days at 50 °C prior to recording of the dry mass.

Transformation of A. oryzae

Preparation of linearized dna fixing templates for transformation.

To generate linear DNA to be transformed into A. oryzae as fixing templates alongside CRISPR-Cas9 RNP complexes, the DNA was linearized using PCR from the corresponding plasmids harboring the DNA fixing template of interest. Briefly, 1 ng of plasmid DNA was used as the template for a 60 µL PCR reaction using the Q5 high-fidelity polymerase master mix (New England Biolabs, #M0492S) and following the manufacturer’s instructions for the PCR protocol. For each DNA template, five 60 µL reactions were set up in parallel to obtain sufficient DNA for transformation. The PCR reactions were then combined and purified using the QIAquick PCR purification kit (Qiagen) and were eluted at the final step in 25 µL of sterile water. This typically gave sufficient quantities of the large amount of DNA needed for protoplast transformation (>10 µg in 20 µL).

Preparation of RNP complexes for transformation

All CRISPR-Cas9 reagents were obtained from IDT, including the Alt-R S.p. HiFi Cas9 Nuclease V3 (IDT, #1081061), Alt-R CRISPR-Cas9 crRNA XT 2 nmol (customized sequence), and Alt-R CRISPR-Cas9 tracrRNA 100 nmol (IDT, #1072534). RNA duplex buffer was included as part of the tracrRNA. The crRNA, which is the sequence-specific RNA that targets the region of interest, was resuspended in 20 µL water for 100 µM final concentration. To hybridize the sequence-specific crRNA to the universal tracrRNA to generate the final sgRNA, 0.5 µL of crRNA (5 µM final concentration) was mixed with 0.5 µL tracrRNA (5 µM final concentration) in 9 µL RNA duplex buffer and the mixture was then heated for 5 min at 95 °C and was then left to cool. This hybridized mixture represents the final sgRNA which is ready to bind to the Cas9 protein to form the RNP complex. To create the final RNP complexes for transformation, 2.16 µL of the hybridized crRNA-tracrRNA (the sgRNA, final concentration 540 nM) was mixed with 0.18 µL of Alt-R S.p. HiFi Cas9 Nuclease V3 (final concentration 540 nM) and 17.66 µL buffer (9 mM HEPES, 67 mM KCl, pH 7.5). The mixture was incubated at room temperature for 20 min to form the RNP complex and was then transferred to ice. As specified below in the transformation protocol, 5 µL of each RNP complex was used per 200 µL protoplast transformation. The RNP complexes were always prepared fresh on the day of the transformation and were never subjected to freeze-thaw.

Standard transformation protocol

Protoplast-mediated transformation was used to transform Aspergillus oryzae . We followed the protocol from 28 , with minor modifications. To generate mycelial biomass for protoplast generation, pyrG mutant strains were grown in duplicate in 50 mL GP medium supplemented with uracil and uridine in 250 mL flasks at 30 °C, shaking at 160 rpm. Following 72 h of growth, mycelia were harvested by pressing the liquid from the mycelia in a 20 mL syringe harboring a sterile cotton ball.

Dry mycelia from one flask were then put in 10 mL of TF1 solution (Per 500 mL of water: 2.9 g maleic acid, 39.5 g (NH 4 ) 2 SO 4 , pH adjusted to 5.5 and filter sterilized) harboring 0.1 grams of YATALASE enzyme (Takara Bio, #T017) which was used to digest the cell wall. This was incubated shaking for two and a half hours at 30 °C and 160 RPM, and the tissue was pressed within a sterile syringe with cotton to collect the protoplasts within the flow-through. The flow-through was checked for cloudiness, which indicated the generation of protoplasts. The resulting protoplast solution (7–8 mL usually, as sometimes not all protoplast solution could be pushed through the cotton ball due to clogging) was centrifuged at 475 g for 10 min, and the supernatant was then discarded. The protoplasts were then gently resuspended in 10 mL of TF2 solution pre-warmed at 30 °C (Per 1 L of water: 218.5 g sorbitol, 10.95 g CaCl 2 •6H 2 O, 2.05 g NaCl, 1.21 g Tris buffer, pH adjusted to 7.5 and filter sterilized). The protoplast solution resuspended in TF2 was centrifuged at 475 g for 10 min to discard the supernatant. The protoplasts were resuspended in 1–2 mL of TF2 solution and 200 μL of protoplast solution was placed into 15 mL volume centrifuge tubes. At least 1.2 × 10 7 protoplasts/mL was needed, so it is useful to consider this concentration when resuspending in the 1–2 mL TF2 solution. Concentrations of ~10 8 protoplasts per mL were typically obtained in the standard digestion protocol.

To transform the protoplasts, a total of 10 µg of PCR-linearized DNA fixing template (in 20 µL sterile water) was added to the 200 μL protoplast solution. Then 5 µL of each of the two pre-formed sgRNA-Cas9 RNP complexes were added and the protoplast-DNA-RNP mixture was incubated on ice for 30 min. After 30 min, sequentially and slowly, 250 μL, 250 μL, and 850 μL aliquots of TF3 solution were added (TF3 solution: per 1 L of water: 600 g Polyethylene glycol (4000), 10.95 g CaCl 2 •6H 2 O, 1.21 g Tris buffer, pH adjusted to 7.5 followed by autoclaving) and left to incubate at room temperature for 30 min. A total of 5 mL of TF2 solution was then added to each protoplast solution then centrifuged for 10 min at 475 g to discard the supernatant. The protoplast pellet was resuspended in 500 μL of TF2. At this point, a bottom agar plate pre-warmed at 30 °C already brought out from its heating location (see above for bottom agar recipe). The 500 μL of protoplast suspension was then mixed with 5 mL of a liquid layer of top agar (see above for recipe) pre-warmed to 50 °C, then quickly spread uniformly across the bottom agar and left to solidify at room temperature. Transformants were left for 72 h to regenerate the protoplasts. Transformants were then restreaked according to the procedure described in “strain construction” above.

Miniaturized transformation protocol

To speed up and miniaturize the transformation protocol to make it compatible with a 96-well plate format, it was modified according to the process below. Protoplasts were generated by digestion according to the standard protocol. However, only 50 µL protoplasts were used for the transformation, and 1.75 µL of each RNP complex was added alongside 2.5 µg DNA to these protoplasts and the mixture was incubated on ice for 30 min as in the standard protocol. Then, only 212.5 µL TF3 solution was added to the DNA-RNP-protoplast solution, and the entire mixture was spread on a bottom agar plate following the standard washing, using an L-shaped spreader. No top agar was used in the regeneration of the protoplasts. All steps following the plating of protoplasts were the same as described above in the standard protocol. All results reported in the paper followed the standard transformation protocol unless otherwise indicated.

Generation of pyrG mutants

The two transformation protocols above describe how to transform pyrG strains using a linear DNA fixing template. To generate pyrG mutants in the first place, as we demonstrated with five different A. oryzae strains obtained from NRRL, the standard protocol was changed slightly according to the following modifications. No linear DNA was transformed. Only the two RNP complexes targeting the pyrG gene were added to incubate on ice with the protoplasts. At the final step, strains were plated onto top and bottom agar supplemented with 1 mg/mL 5-FOA 2 g/L uracil, and 5 g/L uridine. Following the regeneration of protoplasts, instead of being restreaked on MMA + Met plates for single colonies and then transferred to MMA + Met slants, strains were restreaked on MMA + Met supplemented with 1 mg/mL 5-FOA and 2 g/L uracil and 5 g/L uridine. Surviving colonies were transferred to slants with the same medium. Between two and three colonies of each strain were analyzed for auxotrophy by plating on CDA, CDA + Uracil + Uridine. One of these strains was subjected to further analysis by amplification of the pyrG gene and flanking regions using primers pyrG-2F and pyrG-2R. The 2640-bp amplicon was purified and subjected to sequencing. The sequences were aligned using Snapgene.

Excision of pyrG marker

To set up the excision to remove the pyrG marker via locus-specific recycling, we followed the protocol described in ref. 96 with minor modifications: spore suspensions were generated by adding 0.5–1 mL sterile water to MMA + Met slants harboring single colonies subjected to colony PCR. Slants were vortexed to resuspend conidia, and then 500 µL of suspension harboring between 10 5 and 10 6 conidia/mL was spread onto PDA + 5-FOA + Uridine + Uracil plates. The conidia were spread using an L-shaped spreader and plates were left to dry for 1–2 h. The plates were then incubated at 30 °C for 5–7 days, at which point healthy, robustly growing colonies appeared on the plates. Conidia from individual colonies that appeared healthy were transferred to PDA + 5-FOA + uridine + uracil slants, whereby they were subject to an additional 4–5 days of growth. These strains had the pyrG marker excised. To verify the marker excision, conidia from slants were subjected to colony PCR (as described in standard transformation protocol), or PCR on extracted genomic DNA. To extract genomic DNA, a small amount of conidia was transferred to 300 µL lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 1 mM EDTA, 10 mM Tris pH 8) in a bead-beating tube (Lysing Matrix Z, MP Biomedicals, catalog#: 116961050-CF). Bead beating was performed for 1 min. Then, samples were incubated at 65 °C for 30 min, vortexing every 10 min. 300 µL of phenol:chloroform:isoamyl alcohol 25:24:1 reagent was then added (Sigma–Aldrich, #P3803) and tubes were vortexed for 5 min and were then centrifuged at max speed for 10 min to separate the layers. 120 µL of the top aqueous layer was transferred to a new tube, and 210 µL of ice-cold 100% ethanol was added to precipitate the DNA. The DNA pellet was washed twice with 70% and was resuspended in 30 µL sterile water. 1 µL was used as the template for PCR reactions using the PHIRE direct plant PCR kit (ThermoFisher, #F130WH).

Whole-genome sequencing, assembly, annotation, and phylogenetic analysis of diverse A. oryzae strains obtained from NRRL

A. oryzae strains subjected to sequencing were obtained from NRRL (NRRL numbers: #2215, #5592, #32614, #1911, #6574). They were grown in GP-glucose medium (50 mL medium in 250 mL flasks) for 72 h prior to harvesting by vacuum filtration and flash-freezing in liquid nitrogen. gDNA extraction, sample quality assessment, DNA library preparation, sequencing, and bioinformatics analysis were conducted at Azenta Life Sciences. Genomic DNA was extracted using DNeasy Plant Mini Kit following manufacturer’s instructions (Qiagen). Genomic DNA was quantified using the Qubit 2.0 Fluorometer (ThermoFisher Scientific). NEBNext® Ultra™ II DNA Library Prep Kit for Illumina, clustering, and sequencing reagents was used throughout the process following the manufacturer’s recommendations. Briefly, the genomic DNA was fragmented by acoustic shearing with a Covaris S220 instrument. Fragmented DNA was cleaned up and end repaired. Adapters were ligated after adenylation of the 3′ends followed by enrichment by limited cycle PCR. DNA libraries were validated using a High Sensitivity D1000 ScreenTape on the Agilent TapeStation (Agilent Technologies) and were quantified using Qubit 2.0 Fluorometer. The DNA libraries were also quantified by real-time PCR (Applied Biosystems). The sequencing library was clustered onto lanes of an Illumina HiSeq 4000 (or equivalent) flow cell. After clustering, the flow cell was loaded onto the Illumina HiSeq instrument according to the manufacturer’s instructions. The samples were sequenced using a 2 × 150 bp Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into FastQ files and de-multiplexed using Illumina bcl2FastQ 2.17 software. One mismatch was allowed for index sequence identification.

Assembly, annotation, and prediction of biosynthetic gene clusters

The reads were filtered with TrimmomaticPE version 0.39 97 with the following parameters: LEADING:30 TRAILING:30 MINLEN:120. The filtered reads were used for de novo assembly using the SPAdes 98 genome assembler v3.13.1-1 with the following parameters --careful --cov-cutoff 100. The resulting assemblies were then processed with AUGUSTUS 99 v3.4.0, to obtain coding sequences and protein predictions. Augustus was executed using a gene model for Aspergillus oryzae to identify start and stop codons, introns, and exons. For the prediction of natural product production repertoire of the strains, the assembled genomes and their gene calling files were used for functional annotation and mining for natural products biosynthetic gene clusters using antiSMASH version 7 100 .

Phylogenetic analysis

The taxonomic affiliation of the A. oryzae strains used in this study (tree in Supplementary Fig.  2 ) was defined using a multilocus phylogenetic tree constructed with the genomes of 59 Aspergillus spp. strains which were obtained from the GenBank database. These genomes were processed with AUGUSTUS 99 v3.4.0, to obtain coding sequences and protein predictions. The predicted proteomes of the Aspergillus dataset. Given the closeness of the strains, a genome from a distantly related taxonomic group ( Trichoderma atroviridae ) was added to the dataset to reduce the number of shared orthologs. The core genome was then calculated using BPGA 101 this analysis led to a set 237 conserved proteins that were sorted, aligned 102 , and trimmed 103 , after this process 167 protein sequences remained. They were then concatenated, and an evolutionary model was calculated for each of the 167 protein partitions. Then a phylogenetic tree was calculated with IQtree2 v2.0.7 104 using maximum likelihood with 10,000 bootstrap replicates. The entire process was executed automatically using a script available at https://github.com/WeMakeMolecules/Core-to-Tree .

Computational identification of candidate neutral, highly transcribed integration sites for protein expression

These sites were identified from a dataset deposited under BioProject accession: “ PRJDB8293 ”. This set of Illumina RNAseq data included 18 libraries which were obtained from A. oryzae RIB40 growing in 50 mL cultures in Czapek–Dox liquid medium supplemented with 1% (w/v) Triton X-100 at 30 °C. This dataset was deposited previously by Wong et al. 105 . The specific datasets that were used are shown in Supplementary Table  13 . The reads were downloaded from the GenBank FTP using fastq-dump v2.113, the reads were then aligned to the A. oryzae reference genome (NCBI RefSeq assembly GCF_000184455.2) using subread package v 2.0.3 106 . To select highly expressed genes, we counted the number of reads that were mapped to each gene in the A. oryzae RIB40 genome using featureCounts v2.0.3 107 . The read counts were calculated independently for each run. As the read counts depend on the depth and processing of each sample, a single cutoff cannot be established. Instead, we used this value to rank genes from most expressed to not expressed using the numbers of reads mapped per library (Supplementary Table  13 ). Then we reasoned that gene that ranked top in all libraries, could be safely considered highly expressed. For selection of neutral, highly transcribed integration sites, we first identified all the intergenic regions in the genome and calculated the average read count of the genes flaking them. Then, we sorted these regions from highest to lowest by their average flanking gene read count. For each of the 18 libraries we selected the top 500 intergenic regions and filtered out those that were not found in all conditions; therefore, we selected intergenic regions that are flanked by constitutive, highly expressed genes. This led to a set of 334 regions that were filtered by length (>4.8 kb). Finally, we selected 10 promising intergenic regions spread across A. oryza e (Fig.  2 , Supplementary Table  4 ).

Computational identification of candidate endogenous bidirectional promoters

For the development of new bidirectional promoters for A. oryzae , we used the annotated genome of strain RIB40 to mine for all the coding sequences whose start codons are in opposite directions. Then we selected the gene pairs that were highly expressed in most conditions using the Wong et al dataset 105 described above. We then identified the promoter region. We used the same dataset or read counts used for the identification of neutral, highly transcribed integration sites.

Identification of core promoters for expression in A. oryzae using the synthetic expression system

To identify candidate core promoters from the A. oryzae genome, we searched for highly expressed genes from publicly available transcriptome data 108 . The gene list of A. oryzae RIB40 grown in CD-glucose in liquid cultures without ER stress was sorted by RPKM to generate a list of the top most highly expressed genes. The top eight most highly expressed genes (unique genes, no duplicates) were selected as candidate strong promoters. Additionally, pdcA , which appeared at #15 in the rank of this list was selected as other studies suggest this is one of the most highly expressed genes in A. oryzae 109 . thiA , which was ranked #20 in this list, was also selected, because it has successfully been used for overexpression in A. oryzae previously 49 . Finally, hhfA , which ranked #55 in the list, was selected, as it was part of the p4-2 bidirectional promoter used in this study. For each of these genes, the genetic DNA 200 bp upstream of the start codon was used as core promoter sequence and ordered as synthetic dsDNA for downstream cloning and transformation. Two additional core promoters that did not come from the transcriptome rank analysis and were not native sequences to A. oryzae were also included. These were An_201205 from A. niger , which was used previously in the development of a Synthetic expression system in A. niger and T. reesei 18 , as well as the core promoter for Afl_ecm33 from gene AFL2G_04718 in Aspergillus flavus . Afl_ecm33 has been successfully used before to express a secondary metabolite in A. oryzae (promoter P4 in this study 48 ). For the An_201205 core promoter, the sequence was identical to the one used previously 18 , but for Afl_ecm33, the 200 bp upstream of the start codon were selected as the core promoter.

Flow cytometry assays of conidia for fluorescence quantification

The overall approach followed the method for promoter evaluation described in ref. 47 but with minor modifications. For all flow cytometry assays, strains were grown on PDA + 5-FOA + UU slants (excised neutral loci strains) or PDA slants (all others) for 5–6 days at 30 °C to allow robust development of conidia. Conidia were harvested by the addition of 1 mL of sterile water, followed by vortexing. 250 µL conidial suspension was then transferred to a 96-well plate (Corning, Falcon Tissue Culture Plate, #353072). Flow cytometry assays were performed on the BD Accuri C6 instrument (BD Biosciences) using the following settings: Run limit = 50,000 events, FSC-H threshold <80,000, agitation = 1 cycle every 1 wells. Raw fluorescence data were converted into MEFL (mean equivalents of Fluorescein, for GFP) or METR (mean equivalents of Texas Red, for mCherry), using a fluorescence beads standard (Spherotech, #RCP-30-5A). At least three biological replicates were run for each sample. FlowJo software (version 10) was used to analyze the data.

Fluorescence microscopy

Strains were grown on either CDA, CDA-Leu, or CDA-dextrin for 4–5 days at 30 °C. Fluorescent protein expression was then imaged in mycelia (edge of the colony) using Leica Microscope DM6B (Leica) and the associated Leica LAS X software (v.5.1.0).

Proteomic comparison of mCherry expression across media and promoters

to compare the expression of mCherry under endogenous promoters and the core promoters in the synthetic expression system, conidia from three different transformants per construct were inoculated at 5 × 10 5 conidia in 50 mL of either CD-dextrin or CD-glucose medium in 250 mL Erlenmeyer flasks. Strains were grown for 96 h at 30 °C, 160 rpm shaking. Biomass was harvested by vacuum filtration over Miracloth and was then lyophilized prior to proteomics.

Proteomics analysis

Protein was extracted and tryptic peptides were prepared by following established proteomic sample preparation protocol 110 . Briefly, cell pellets were resuspended in Qiagen P2 Lysis Buffer (Qiagen, Hilden, Germany, Cat.#19052) to promote cell lysis. Proteins were precipitated with addition of 1 mM NaCl and 4× vol acetone, followed by two additional wash with 80% acetone in water. The recovered protein pellet was homogenized by pipetting mixing with 100 mM Ammonium bicarbonate in 20% Methanol. Protein concentration was determined by the DC protein assay (BioRad, Hercules, CA). Protein reduction was accomplished using 5 mM tris 2-(carboxyethyl)phosphine (TCEP) for 30 min at room temperature, and alkylation was performed with 10 mM iodoacetamide (IAM; final concentration) for 30 min at room temperature in the dark. Overnight digestion with trypsin was accomplished with a 1:50 trypsin:total protein ratio. The resulting peptide samples were analyzed on an Agilent 1290 UHPLC system coupled to a Thermo Scientific Orbitrap Exploris 480 mass spectrometer for discovery proteomics 111 . Briefly, peptide samples were loaded onto an Ascentis® ES-C18 Column (Sigma–Aldrich, St. Louis, MO) and separated with a 10 min LC gradient from 98% solvent A (0.1% FA in H2O) and 2% solvent B (0.1% FA in ACN) to 65% solvent A and 35% solvent B. Eluting peptides were introduced to the mass spectrometer operating in positive-ion mode and were measured in data-independent acquisition (DIA) mode with a duty cycle of 3 survey scans from m/z 380 to m/z 985 and 45 MS2 scans with precursor isolation width of 13.5  m/z to cover the mass range. DIA raw data files were analyzed by an integrated software suite DIA-NN 112 . The database used in the DIA-NN search (library-free mode) is the latest A. oryzae UniProt proteome FASTA sequences plus the protein sequences of the heterologous proteins and common proteomic contaminants. DIA-NN determines mass tolerances automatically based on first-pass analysis of the samples with automated determination of optimal mass accuracies. The retention time extraction window was determined individually for all MS runs analyzed via the automated optimization procedure implemented in DIA-NN. Protein inference was enabled, and the quantification strategy was set to Robust LC = High Accuracy. Output main DIA-NN reports were filtered with a global FDR = 0.01 on both the precursor level and protein group level. The total peak area of tryptic peptides of identified proteins was used to plot the quantity of the targeted proteins in the samples.

Extraction and LC–MS analysis of ergothioneine and heme in fungal mycelium and reference samples

Extraction and analysis of heme.

Extraction was performed according to the protocol specified in ref. 113 , with minor modifications. Lyophilized fungal biomass was ground into a homogeneous powder using a mortar and pestle. Approximately 30 mg of the powder was then transferred to a bead-beating tube (Lysing Matrix Z, MP Biomedicals, catalog#: 116961050-CF) and 1 mL of TE buffer (10 mM Tris, 1 mM EDTA, pH 8) was added. The tube was vortexed to suspend the powder and was then subjected to bead beating for 2 × 1 min using the Biospec Mini Beadbeater. 750 µL of the bead-beaten solution was then transferred to 15 mL conical tubes containing 4 mL of 8:2 acetonitrile:1.7 M HCl. The tubes were then vortexed for 20 min. Then, 1 mL of saturated 0.25 g MgSO 4 •7H 2 O was added to each tube, followed by the addition of 0.1 g NaCl. This created a separation of the aqueous and organic layers. The tubes were then vortexed for 10 min, followed by spinning down at 5000 rcf for 10 min to separate the layers. 100 µL of the top layer was transferred to an LC–MS vial for analysis. In addition to the wild-type and engineered biomass, we extracted heme from lyophilized plant-based ground beef (IMPOSSIBLE Foods Inc, 12 oz IMPOSSIBLE™ burger made from plants).

LC–MS analysis of heme

For the LC–MS analysis, analytes were chromatographically separated with a Kinetex XB-C18 column (50-mm length, 2.1-mm internal diameter, 2.6-µm particle size; Phenomenex, Torrance, CA) at 50 °C using a 1260 Infinity HPLC system (Agilent Technologies). The injection volume for each measurement was 5 µL. The mobile phase was composed of 0.1% formic acid in water (as mobile phase A) and 0.1% formic acid in acetonitrile (as mobile phase B). Analytes were separated via the following gradient: linearly increased from 20%B to 45.5%B in 1.7 min, linearly increased from 45.5%B to 95%B in 0.2 min, held at 95%B for 1.6 min, linearly decreased from 95%B to 20%B in 0.2 min, held at 20%B for 1.3 min. A flow rate of 1 mL/min was used throughout. The total run time was 5 min. The HPLC system was coupled to an Agilent Technologies 6520 Quadrupole Time-of-Flight Mass Spectrometer (QTOF-MS) with a 1:4 post-column split. Nitrogen gas was used as both the nebulizing and drying gas to facilitate the production of gas-phase ions. Drying and nebulizing gases were set to 10 L/min and 30 psi, respectively, and a drying gas temperature of 330 °C was used throughout. Fragmentor, skimmer, and OCT1 RF voltages were set to 250 V, 65 V, and 400 V, respectively. Electrospray ionization (ESI) was conducted in the positive-ion mode with a capillary voltage of 3.5 kV. MS experiments were carried out in the full-scan mode ( m/z 60–1100) at 0.86 spectra per second for the detection of [M + H] + ions. The instrument was tuned for a range of m/z 50–1700. Prior to LC-ESI-TOF MS analysis, the TOF MS was calibrated with the Agilent ESI-Low TOF tuning mix. Mass accuracy was maintained via reference ion mass correction, which was performed with purine and HP-0921 (Agilent Technologies). Data acquisition was carried out by MassHunter Workstation Software version B.08.00 (Agilent Technologies). Data processing was carried out by MassHunter Workstation Qualitative Analysis version B.06.00 and MassHunter Quantitative Analysis version 10.00. External calibration curves were used to quantify the analytes. Hemin chloride (Sigma–Aldrich, #3741) was used as the standard. The mass spectrum in the standards and samples corresponded to that from other experimentally validated studies of intracellular heme 114 . Calculated concentrations obtained from LC–MS analysis were normalized to the initial dry sample weight used for extraction.

Extraction and analysis of ergothioneine

All samples were lyophilized prior to analysis. For extraction from solid, samples were ground into a fine powder using a mortar and pestle and then approximately 30 mg was transferred to tubes for homogenization (Lysing Matrix Z, MP Biomedicals, catalog#: 116961050-CF). 1 mL of 20% methanol with 0.1% formic acid was added and samples were subjected to bead beating for 2 × 1 min. Following bead-beating, samples were spun down at 12,000 RCF for 10 min to separate the solids. 500 µL supernatant was transferred to a centrifugal spin filter to remove any particulates larger molecules (3 kDa cutoff) (Amicon Ultra, Sigma–Aldrich, Catalog # UFC500324). The flow-through was collected and subjected to analysis by LC–MS. In addition to wild-type and engineered fungal biomass, we extracted ergothioneine from the fruiting body of the oyster mushroom ( Pleurotus ostreatus ). The mushroom was purchased fresh (from Berkeley Bowl in Berkeley, CA) and subjected to lyophilization prior to extraction using the procedure above.

LC–MS analysis of ergothioneine

For LC–MS analysis, analytes were chromatographically separated with a Kinetex HILIC column (100-mm length, 4.6-mm internal diameter, 2.6-µm particle size; Phenomenex, Torrance, CA) at 20 °C using a 1260 Infinity HPLC system (Agilent Technologies, Santa Clara, CA, USA). The injection volume for each measurement was 2 µL. The mobile phase was composed of 10 mM ammonium formate (prepared from a pre-made solution from Sigma–Aldrich, St. Louis, MO, USA) and 0.2% formic acid (from an original stock at ≥98% chemical purity from Sigma–Aldrich) in water (as mobile phase A) and 10 mM ammonium formate and 0.2% formic acid in 90% acetonitrile with the remaining solvent being water (as mobile phase B). The solvents used were of LC–MS grade and purchased from Honeywell Burdick & Jackson, CA, USA. Analytes were separated via the following gradient: linearly decreased from 90%B to 70%B in 4 min, held at 70%B for 1.5 min, linearly decreased from 70%B to 40%B in 0.5 min, held at 40%B for 2.5 min, linearly increased from 40%B to 90%B in 0.5 min, held at 90%B for 2 min. The flow rate was changed as follows: 0.6 mL/min for 6.5 min, linearly increased from 0.6 mL/min to 1 mL/min for 0.5 min, held at 1 mL/min for 4 min. The total run time was 11 min. The HPLC system was coupled to an Agilent Technologies 6520 Quadrupole Time-of-Flight Mass Spectrometer (QTOF-MS) with a 1:4 post-column split. Nitrogen gas was used as both the nebulizing and drying gas to facilitate the production of gas-phase ions. Drying and nebulizing gases were set to 12 L/min and 25 psi, respectively, and a drying gas temperature of 350 °C was used throughout. Fragmentor, skimmer, and OCT1 RF voltages were set to 100 V, 50 V, and 250 V, respectively. Electrospray ionization (ESI) was conducted in the positive-ion mode with a capillary voltage of 3.5 kV. MS experiments were carried out in the full-scan mode ( m/z 70–1100) at 0.86 spectra per second for the detection of [M + H] + ions. The instrument was tuned for a range of m/z 50–1700. Prior to LC-ESI-TOF MS analysis, the TOF MS was calibrated with the Agilent ESI-Low TOF tuning mix. Mass accuracy was maintained via reference ion mass correction, which was performed with purine and HP-0921 (Agilent Technologies). Data acquisition was carried out by MassHunter Workstation Software version B.08.00 (Agilent Technologies). Data processing was carried out by MassHunter Workstation Qualitative Analysis version B.06.00 and MassHunter Quantitative Analysis version 10.00. External calibration curves were used to quantify the analytes. Ergothioneine (Sigma–Aldrich, #7521-25MG) was used as the standard. The mass spectrum in the standards and samples corresponded to that from other experimentally validated studies of intracellular ergothioneine 115 . Calculated concentrations obtained from LC–MS analysis were normalized to the initial dry sample weight used for extraction.

Protein and amino acid analysis in fungal mycelium

Protein content was analyzed by combustion method, directly following the Method 990.03 described in ref. 116 . The combustion was performed using Leco FP-528 Nitrogen Combustion Analyzer (Leco). Crude Protein was calculated as Nitrogen × 6.25. Amino acid composition was analyzed using acid hydrolysis of lyophilized fungal biomass, directly following the protocols of Method 994.12 described in ref. 116 , Method 982.30 in ref. 117 , as well as the methods described in ref. 118 .

Statistics and Reproducibility

No statistical method was used to predetermine sample size. n  = 3 was chosen as the minimal number of replicates for experimental characterization. We determined this to be sufficient based on internal controls (using previously characterized promoters and fluorescent genes) to capture biological variability between transformants/strains. All microscopy images and PCR results for confirming insertion/excision displayed are representative of at least three biological replicates. No data were excluded from the analyses. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.

Reporting summary

Further information on research design is available in the  Nature Portfolio Reporting Summary linked to this article.

Data availability

The authors declare that all data supporting the findings of this study are available within the paper, supplementary information, the supplementary data files, and in the source data file. Source data are provided as a Source Data file. Strains and plasmids (and their associated sequences) generated in this study have been deposited in the JBEI Public Registry ( https://public-registry.jbei.org/ ). See Supplementary Tables  11 – 12 for plasmids and strain information. Outputs of computational analysis for identification of candidate endogenous neutral loci and bidirectional promoters are available in Supplementary data files 1 and 2. Output of mass spectrometry data are available as source data. The generated mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE 94 partner repository with the dataset identifier “ PXD043152 ”. The genome sequences of the A. oryzae strains obtained from NRRL and sequenced as part of this study have been deposited to the Sequence Read Archive under Bioproject “ PRJNA987873 ”. The previously published transcriptome data used to identify endogenous neutral loci and bidirectional promoters in Aspergillus oryzae can be found in GenBank under BioProject accession: “ PRJDB8293 ”. CAoGD (Comprehensive Aspergillus oryzae Genome Database) v.2.4 was used to identify the genomic location and sequence identity of endogenous promoters and genes targeted in transformations ( https://nribf21.nrib.go.jp/CAoGD/ ).  Source data are provided with this paper.

Code availability

No custom code was used in the analysis of data, and all the previously published software used as well as the relevant commands has been cited in the methods. The automatic phylogenomic analysis from the core genomes of Aspergillus oryzae strains was executed using the script available at https://github.com/WeMakeMolecules/Core-to-Tree . DIA-NN is freely available for download from https://github.com/vdemichev/DiaNN .

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Acknowledgements

V.M.R. was supported by the Miller Institute at the University of California, Berkeley. P.C.M. and J.D.K. were supported by Novo Nordisk Foundation grant no. NNF20CC0035580. C.V.D.L. was supported by Novo Nordisk Foundation grant NNF21OC0065495. This work was part of the DOE Joint BioEnergy Institute ( https://www.jbei.org ) supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U.S. Department of Energy.

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VMR and JDK conceptualized the study. VMR developed and optimized transformation methods, established the neutral loci, endogenous promoters, and vectors for transformation, and engineered the final strains. CVDL evaluated parts for the Synthetic expression system and conducted transformation of A. oryzae NRRL strains. PCM conducted bioinformatics analysis, including genome annotation and assembly, phylogenetic analysis, and identification of endogenous neutral loci and bidirectional promoters. YC and CJP conducted proteomics experiments and analysis. RK and EEKB conducted targeted LC–MS experiments and analysis. All authors approved the final manuscript.

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Data mining model for scientific research classification: the case of digital workplace accessibility

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• Radka Nacheva   ORCID: orcid.org/0000-0003-3946-2416 1 ,
• Maciej Czaplewski   ORCID: orcid.org/0000-0003-1888-8776 2 &
• Pavel Petrov   ORCID: orcid.org/0000-0002-1284-2606 1  

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Research classification is an important aspect of conducting research projects because it allows researchers to efficiently identify papers that are in line with the latest research in each field and relevant to projects. There are different approaches to the classification of research papers, such as subject-based, methodology-based, text-based, and machine learning-based. Each approach has its advantages and disadvantages, and the choice of classification method depends on the specific research question and available data. The classification of scientific literature helps to better organize and structure the vast amount of information and knowledge generated in scientific research. It enables researchers and other interested parties to access relevant information in a fast and efficient manner. Classification methods allow easier and more accurate extraction of scientific knowledge to be used as a basis for scientific research in each subject area. In this regard, this paper aims to propose a research classification model using data mining methods and techniques. To test the model, we selected scientific articles on digital workplace accessibility for the disabled retrieved from Scopus and Web of Science repositories. We believe that the classification model is universal and can be applied in other scientific fields.

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The project "Impact of digitalization on innovative approaches in human resources management" is implemented by the University of Economics—Varna, in the period 2022–2025. The authors express their gratitude to the Bulgarian Scientific Research Fund, Ministry of Education and Science of Bulgaria for the support provided in the implementation of the project "Impact of digitalization on innovative approaches in human resources management," Grant No. BG-175467353-2022-04/12-12-2022, contract No. KP-06-H-65/4 – 2022.

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An Entity Extraction Pipeline for Medical Text Records Using Large Language Models: Analytical Study

Authors of this article:

Original Paper

• Lei Wang 1, 2 * , MS   ; 
• Yinyao Ma 3 * , MS, MD   ; 
• Wenshuai Bi 1 , MS   ; 
• Hanlin Lv 1 , MD, PhD   ; 
• Yuxiang Li 1, 2 , PhD  

1 BGI Research, Wuhan, China

2 Guangdong Bigdata Engineering Technology Research Center for Life Sciences, BGI Research, Shenzhen, China

3 Department of Obstetrics, People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning, China

*these authors contributed equally

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Hanlin Lv, MD, PhD

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Background: The study of disease progression relies on clinical data, including text data, and extracting valuable features from text data has been a research hot spot. With the rise of large language models (LLMs), semantic-based extraction pipelines are gaining acceptance in clinical research. However, the security and feature hallucination issues of LLMs require further attention.

Objective: This study aimed to introduce a novel modular LLM pipeline, which could semantically extract features from textual patient admission records.

Methods: The pipeline was designed to process a systematic succession of concept extraction, aggregation, question generation, corpus extraction, and question-and-answer scale extraction, which was tested via 2 low-parameter LLMs: Qwen-14B-Chat (QWEN) and Baichuan2-13B-Chat (BAICHUAN). A data set of 25,709 pregnancy cases from the People’s Hospital of Guangxi Zhuang Autonomous Region, China, was used for evaluation with the help of a local expert’s annotation. The pipeline was evaluated with the metrics of accuracy and precision, null ratio, and time consumption. Additionally, we evaluated its performance via a quantified version of Qwen-14B-Chat on a consumer-grade GPU.

Results: The pipeline demonstrates a high level of precision in feature extraction, as evidenced by the accuracy and precision results of Qwen-14B-Chat (95.52% and 92.93%, respectively) and Baichuan2-13B-Chat (95.86% and 90.08%, respectively). Furthermore, the pipeline exhibited low null ratios and variable time consumption. The INT4-quantified version of QWEN delivered an enhanced performance with 97.28% accuracy and a 0% null ratio.

Conclusions: The pipeline exhibited consistent performance across different LLMs and efficiently extracted clinical features from textual data. It also showed reliable performance on consumer-grade hardware. This approach offers a viable and effective solution for mining clinical research data from textual records.

Introduction

Clinical text data have been widely recognized in data research due to their inclusion of multisource information [ 1 , 2 ] (eg, patient subjective statements, past objective facts, doctors’ diagnostic processes, and summary records). Extracting useful information from text data could serve as a crucial supplement to the study of disease progression; it could complement objective indicators dependent on laboratory tests and examinations [ 3 ], which has consistently been a hot research topic [ 4 , 5 ].

Historically, methods for text data extraction mainly include the following:

• Manual annotation: scales are designed based on clinical and research experience, followed by manual field extraction [ 6 - 8 ].
• Rule extraction: concepts from established knowledge base, such as International Classification of Diseases, Tenth Revision [ 9 ], are used for concept term extraction. This process is typically based on similarity algorithms and manual assistance to extract terms and their attributes (eg, negations and dependency relationships) [ 10 ].
• Named entity recognition or natural language processing algorithms: supervised learning methods, such as pretrained models like T5 [ 11 ], Bidirectional Encoder Representations from Transformers (BERT) [ 12 ], and BERT’s variants [ 13 - 15 ], with manual annotation to enhance semantic comprehension capabilities [ 16 , 17 ].

The task of extracting features from vast unstructured text presents itself as a daunting, labor-intensive, and time-consuming endeavor [ 18 ], for the following reasons:

• It is challenging to determine the dimension of extracted features initially, and from another perspective, confining the feature dimension means constraining the research scope from the outset [ 19 ].
• Given the inherent subjectivity and potential biases of recording subjects, solely relying on algorithms without annotation typically results in low accuracy and recall [ 20 ].
• Achieving higher accuracy with a broader feature scope, and the required human effort involved, is typically nonlinear [ 4 ], and the difficulty becomes apparent when confronted with massive real-world data.

The advent of large language models (LLMs) has paved a new path for the dilemma in clinical text extraction [ 21 - 23 ]. In the realm of natural language understanding research, generative large models, represented notably by ChatGPT [ 24 ] since 2022, have achieved unimaginable capabilities in semantic dimensions, leveraging the emergent intelligence from vast parameter scales. However, there are numerous considerations and limitations in their application, as follows:

• High-performing LLMs, such as OpenAI ChatGPT and Google Bard [ 25 , 26 ], are currently not open source, and patient data need to be submitted to their platform for analysis, presenting security challenges [ 27 , 28 ].
• Open-source LLMs with high intelligence generally require a large number of parameters (10-100 billion), which are hard to support on consumer-level graphics processing units (GPUs) [ 29 ].
• Low-parameter (around 10 billion) LLMs, typically require multistrategy support when dealing with tasks in certain vertical segments [ 30 ] (eg, fine-tuning, knowledge base or knowledge graph support, complex Chain of Thought (CoT) [ 31 ] along with its derivatives, and even global training) and are accompanied by various anomaly issues, including feature hallucination.

Although the application of LLM faces various potential limitations and challenges, as mentioned above, the foundational entity extraction and understanding capabilities of LLMs can still be used for low-cost extraction of clinical text data through meticulous prompt design, guidance combining CoT, and standardized examples [ 30 , 32 ].

In this study, we aimed to extract valuable features from a series of given patient admission records, which include the chief complaint and the medical histories. In light of this task, we introduced a modular LLM approach, which divides the entire extraction path into several smaller steps, with each modular LLM handling these basic steps automatically. We adopted the core idea of LLM agents [ 30 ] and self-consistency with CoT [ 33 ].

To experiment with this approach, we implemented 2 low-parameter LLMs in a local environment and compared their performances within a retrospective cohort of pregnancy to provide a reference that future researchers might draw upon.

Study Preparation

Data sources.

In this study, the text corpus was compiled from two primary sources:

• Chief complaints and medical histories, exemplified in Multimedia Appendix 1 , were extracted from inpatient admission records of an established cohort at the People’s Hospital of Guangxi Zhuang Autonomous Region in China. The established cohort for the preeclampsia risk study consisted of 25,709 pregnancies that received prenatal care between the 11th and 13th weeks of gestation from April 2012 to September 2021.
• Clinical practice guidelines consisted of the 2018 guidelines from the American College of Obstetricians and Gynecologists [ 34 ] and the 2019 guidelines from the National Institute for Health and Care Excellence [ 35 ].

To ensure linguistic consistency, the entire corpus was maintained in Chinese.

Model Deployment

We deployed 2 most exemplary LLMs in the Chinese domain until September 2023 in an intranet security environment independently: Qwen-14B-Chat (QWEN) [ 36 ] and Baichuan2-13B-Chat (BAICHUAN) [ 37 ]. In the environment, the server cluster used NVIDIA DGX-A100 (2×40 G) GPU nodes. The QWEN used 29 GB of storage and 27 GB of GPU memory, while the BAICHUAN used 26 GB of storage and 28.9 GB of GPU memory. Both models operated solely on physically isolated GPUs, and access was facilitated through the OpenAI [ 38 ] format and FastChat [ 39 ]. The LLMs were built upon PyTorch 2.0, with the temperature set to 0 and max_token adjusted task by task.

Experimental Path

In this study, we have introduced an approach that autonomously extracts valuable textual features. Diverging from traditional LLM applications, we used an “external-COT” strategy, dividing the process into several controllable steps, as illustrated in Figure 1 .

The extraction approach could be divided into four parts: (1) concept preparation, that is, extracting existing concepts from the corpus and selecting concerning concepts; (2) corpus preparation, that is, deidentifying raw data and preparing the corpus in accordance with the selected concept; (3) prompt design for different LLM tasks; (4) question-and-answer (Q&A) scales, that is, transforming concepts into question templates and extracting corresponding scales by LLMs.

Prompt Design

The design of prompt templates is fundamental to efficient and accurate extraction. Prior to processing the entire data set, an initial evaluation was conducted on 100 observations to assess the effectiveness of the templates, allowing for continuous refinement of prompt strategies and orientations. An appropriate template was defined based on the following criteria: (1) absence of redundant content generation, (2) consistent and uniform efficiency, and (3) infrequent occurrence of feature hallucination.

We adopted a 4-paragraph structure, referring to the prompt engineering suggestions of QWEN and BAICHUAN, as follows:

• Context section: defines the role and task, provides a basic understanding, and establishes a behavioral baseline for the model.
• Instruction section: outlines the execution steps, uses the CoT methodology, and provides examples to ensure guided model operation.
• Input data section: manages various inputs to meet diverse information needs.
• Output indicator section: specifies the output format and standards, setting clear expectations for the output.

To avoid input bias, the prompt templates for QWEN and BAICHUAN were maintained without any modifications. In addition, we conducted experiments using 100 observations at different levels of concurrency to select the most optimal configuration.

Concept Extraction and Aggregation

We initially extracted all discernible concepts from chief complaints and medical histories using LLMs with a designed prompt 1, and concepts were retained only with a manifestation frequency exceeding 5% occurrences. To reduce potential attention bias and expand the range of identified concepts, we also included concepts from clinical practice guidelines related to preeclampsia, particularly the American College of Obstetricians and Gynecologists 2018 guidelines and the National Institute for Health and Care Excellence 2019 guidelines.

As we defined in prompt 1, the extracted concepts were formatted using the Systematised Nomenclature of Medicine Clinical Terms (SNOMED CT) vocabulary within the Clinical Findings and Observations domain [ 40 ].

To mitigate potential output errors from LLMs, such as concepts not belonging to the Clinical Findings and Observations domain, or even errors outside of the SNOMED CT vocabulary, we implemented a rule-based matching approach to filter extraction inaccuracies.

Furthermore, in this research, we aimed to extract concepts with diverse semantic expressions (including diagnoses, various medical histories, symptoms, observations, interventions, and types of examination). To accomplish this, local experts manually filtered out concepts embedded in structured text, such as dates or numbers.

Question Generation

After the extraction and aggregation of concepts, they were transformed into specific questions by LLMs as question templates for subsequent data extraction. In this section, we leveraged ChatGPT4.0 as a question generator to produce a basic set of questions, which were then refined by local experts for specificity based on its performance across 100 observations.

Q&A Scale Extraction

To avoid contextual and temporal event confusion leading to incorrect responses (eg, confusing current medical history with a past medical history or confusing the patient’s medical history with that of family members), we preextracted the corpus using two strategies: (1) based on the position of the question templates and (2) based on the sentence containing the concepts. The extracted corpus was then labeled with the corresponding question templates for the subsequent extraction of Q&A scales.

The refined corpus, combined with corresponding question templates, guided a systematic extraction process with 2 LLMs, forming Q&A scales for further application.

Each question probed the LLMs, and the extracted sentences formed the basis of the generated responses. This approach enabled a logical mapping of questions to relevant text, ultimately improving the accuracy and efficiency of feature extraction.

Given the practical constraints and the objective of minimizing manual intervention, it was unfeasible to validate all answer scales individually across a Q&A space containing 68 questions and 25,709 observations. Therefore, a 3-fold assessment strategy was developed, as explained in the sections that follow.

Accuracy and Precision

A subset of 1500 observations chosen at random was manually annotated in collaboration with local experts, serving as the gold standard. The precision of positive identifications by both LLMs was assessed against a specified benchmark.

The null ratio of both LLMs was independently measured across all 25,709 observations. Empty or meaningless outputs (symbols and gibberish) were identified as null outputs, and the null ratio was then calculated as the proportion of such responses to the total.

Time Consumption

The efficiency of the extraction process was evaluated by measuring the time taken by the 2 LLMs to respond to the questions across all 25,709 observations.

Ethical Considerations

The study was approved by the People’s Hospital of the Guangxi Zhuang Autonomous Region in China (KT-KJT-2021-67). The requirement for informed consent was waived, due to the retrospective nature of the study, and all clinical data were deidentified and anonymized.

Path Decomposition Overview

Prompt template.

Through trials with the prompt template on 100 observations, we selected the template that demonstrated optimal consistency, as shown in Figure 2 .

Merged Concepts

After merging all concepts, we filtered out those that appeared less than 5% of the time. A total of 117 concepts and terms were listed in Table S1 in Multimedia Appendix 2 .

Question Template

Then we selected and transferred 68 concepts into question formats, for further Q&A scales. The detailed questions and their corresponding concepts are listed in Table S2 in Multimedia Appendix 2 .

Scale Extraction

We identified that the optimal performance in Q&A scale extraction occurs with a concurrency of 3 requests, enhancing speed by 17.9% compared to a single request. Furthermore, we used a max_token restriction strategy, capping it at 20, to optimize inference speed.

Ultimately, within the 2D Q&A space formed by the answer scales, there were a total of 68 question columns and 25,709 observations (listed in Table 1 ).

We used accuracy and precision metrics for assessing the accuracy of LLMs across 1500 observations. Furthermore, we used 2 parameters—null ratio and time consumption—in 25,709 observations to evaluate the consistency and efficiency of the 2 LLMs, respectively.

a Not applicable.

b The mean pregnancy weight gain was 13.73 (SD 24.12) for QWEN and 13.75 (SD 31.28) for BAICHUAN.

c G6PD: glucose-6-phosphate dehydrogenase.

Evaluation Metrics

Figure 3 A and 3B and Figure S1 (parts A and B) in Multimedia Appendix 2 illustrate the Q&A space for a sample chunk extracted by QWEN and BAICHUAN with the comparison of manual annotation. The figures demonstrate the exceptional accuracy and precision of QWEN and BAICHUAN. QWEN attained an average accuracy of 95.52% and an average precision of 92.93%, whereas BAICHUAN displayed an average accuracy of 95.86% and an average precision of 90.08%. These figures clearly indicate that the 2 LLMs have more concentrated errors in specific concepts, and overall, they achieve high levels of precision in most extractions.

LLMs demonstrated consistent performance across most questions and excelled in binary, well-defined medical history questions, often reaching 100% accuracy and precision. However, the accuracy performance varied significantly when dealing with questions that involved semantic ambiguities or definitional uncertainties. This inconsistency might be tied to the LLM’s training and inference alignment. Notable disparities were observed in questions pertaining to menstrual color (QWEN: 1000/1500, 66.7%; BAICHUAN: 1097/1500, 73.1%), early pregnancy symptoms (QWEN: 909/1500, 60.7%; BAICHUAN: 1474/1500, 98.3%), vaginal bleeding (QWEN: 593/1500, 39.5%; BAICHUAN: 1455/1500, 97%), bilateral lower limb edema (QWEN: 786/1500, 52.4%; BAICHUAN: 1486/1500, 99.7%), and menstrual flow (QWEN: 1498/1500, 99.8%; BAICHUAN: 605/1500, 40.3%).

Apart from the above, the precision inconsistency performance of concepts could be attributed to their low true positive rate, like insomnia (QWEN: 3/17, 17.7%; BAICHUAN: 8/17, 47.1%), personal history—antiphospholipid syndrome (QWEN: 0/2, 0%; BAICHUAN: 1/2, 50%), and poor pregnancy history—induced abortion (QWEN: 1/2, 50%; BAICHUAN: 2/2, 100%). The exact precision is listed in Table S3 in Multimedia Appendix 2 .

As depicted in Table 1 , both LLMs demonstrated superior performance with minimal null ratios. Specifically, QWEN (Figure S1A in Multimedia Appendix 2 ) exhibited a mean null ratio of 0.02%, in contrast to BAICHUAN (Figure S1B in Multimedia Appendix 2 ), which recorded a slightly higher null ratio of 0.21%. Failure of QWEN extraction was only in pregnancy weight gain (411/25,709, 1.60%), but failures of BAICHUAN extraction were mainly in symptoms (abdominal pain: 1252/25,707, 4.87%; vaginal infection: 275/25,709, 1.07%).

We conducted a comparative analysis of the time performance between QWEN and BAICHUAN on various Q&A scales, discovering that BAICHUAN consistently exhibits higher time consumption across almost all scales, reaching up to 4 times that of QWEN, as illustrated in Figure 4 B.

Figure 4 A compares the time consumption of LLMs in extracting diverse concepts. Although there were significant differences across different concepts, overall, the LLMs demonstrated a consistent performance across these concepts. For queries with clear definitions and concise corpora, such as drug usage and previous pregnancy history, the time consumed was minimal. In the category of medical history, both models exhibited uniform and stable performances (QWEN and BAICHUAN both revealed a time consumption ratio of 1:3).

Additional Research

In clinical practice, to address scenarios of resource constraints, we used a quantified version of the LLM in our study to validate the applicability of this approach. We used an official-release INT4 version of QWEN, QWEN(INT4). The model was deployed on an NVIDIA RTX 3090 GPU (24 GB).

With the same approach listed above, the performance of QWEN(INT4) achieved even better performance. Figure 3 and Figure S1C in Multimedia Appendix 2 demonstrate that the average accuracy of QWEN(INT4) is 97.28%, accompanied by a null ratio of 0%.

Despite a notable correlation in performance extraction between QWEN(INT4) and QWEN, QWEN(INT4) demonstrated superior efficiency on limited hardware, with an average of 31 seconds per observation, compared to 47 seconds for QWEN and 312 seconds for BAICHUAN.

Principal Findings

In this study, the extracted scales incorporated not only the conventional features of interest but also less frequently mentioned dimensions in previous cohorts or guidelines. These included food and drug allergies (6.6% for food allergy and 25.2% for drug allergy), certain pregnancy symptoms (average positive ratio of 0.9% for insomnia and 2.3% for palpitations), menstrual conditions (22.8% for dysmenorrhea), medical history (1% for asthma family history and 0.27% for mental illness family history), and gestational intervention (13.93% for progestogen and 1.4% for aspirin).

As a naturally recruited cohort of pregnancy, the extracted features show comparable proportions or trends compared with similar studies, such as systemic lupus erythematosus (average positive ratio of 0.20% vs 0.03%-0.23% of similar cohorts [ 41 , 42 ]) and antiphospholipid syndrome (average positive ratio of 0.08% vs 0.02%-0.12% of similar cohorts [ 43 ]), thereby corroborating the accuracy of our approach.

Additionally, certain scale deviations were revealed compared to similar studies, notably in fetal paternal smoking history (average positive ratio of 0.04% vs approximately 28.1%-40% in similar studies [ 44 , 45 ]). Although these deviations were few, we conducted a sample retracing to the original texts and determined that the extraction approach was not at fault and accurately reflected the original data. This discrepancy highlights persistent concerns [ 46 ] regarding the data quality in inpatient documentation, originating from patient self-reports and physician documentation, and vulnerability to recall and inquiry bias. Documentation varies among patients, influenced not only by patient conditions but also by physicians’ writing habits. Thus, we regard our approach as a preexperimental data analysis. Despite the presence of biases or missing dimensions, the approach uncovers several dimensions absent in structured medical texts, and valuable insights could still be extracted from the data with appropriate statistics [ 47 ]. In clinical practice, preliminary interviews with documenting physicians are recommended prior to the selection of concepts to enhance data quality and mitigate potential biases.

In the context of the extraction process, even when deployed solely on a standard consumer-grade GPU (NVIDIA RTX 3090), the QWEN(INT4) completed the extraction of 25,709 observations and 68 features within 15 calendar days, averaging 48.9 seconds per observation. In practical applications, deploying 2 instances of QWEN(INT4) on a single graphics card, coupled with an additional deployment in CPU [ 36 ], is hypothesized to reduce the extraction to approximately 7 days. Furthermore, multi-GPU server clusters, prevalent in clinical environments, could markedly reduce processing times, potentially to the scale of hours.

In our study, we experimented with omitting the corpus extraction step, directly using the long text of each observation’s chief complaints and medical histories as raw data for Q&A scale extraction. However, the experiment yielded poor performance in accuracy, precision, and time consumption, as illustrated in Figure 5 and Figure S2 in Multimedia Appendix 2 .

These limitations appear significantly correlated with the current technological constraints of LLMs [ 32 ], which tend to generate “feature hallucinations” more frequently when processing extensive texts [ 48 ], leading to the loss of critical information. We believe that this issue will be resolved as the technology continues to evolve [ 49 ].

Limitations

In our experimental validation, we selected a limited set of concepts, comprising only 68 items, to balance the consideration of time constraints. Despite our efforts to encompass a broad scope, some dimensions inevitably remain unaddressed, which is a limitation in verifying efficiency and accuracy across all dimensions.

Furthermore, the raw data in this study was sourced exclusively from a single hospital, spanning nearly a decade. This duration, while significant, introduces limitations in the generalizability of our approach.

Additionally, the approach used only 2 LLMs. Although we anticipate that future LLMs will be compatible with the current approach, this assumption necessitates further experimental validation.

Conclusions

Our proposed approach offers a potential methodology for clinical text data analysis. It involves extracting and summarizing concepts from the comprehensive text of a defined population, thus selecting research directions of interest, and eventually generating analyzable features for the cohort. This approach demonstrates notable precision and could provide substantial data support for future research endeavors.

Acknowledgments

We are grateful to Yinyao Ma and the clinical team for their exceptional contributions to this project and we thank the technical support provided by China National GeneBank.

This work was supported by Guangxi Key Research and Development Program (AB22035056).

Data Availability

The data sets generated and analyzed during this study are not publicly available due to privacy or ethical restrictions but are available on request from the corresponding author.

Authors' Contributions

LW and YM contributed equally to this study. LW, HL, and YM participated in the study design and drafted the manuscript. YL and HL participated in data collection and outcome rule review. LW and WB performed the statistical analysis, and established machine learning models. YL helped to draft the manuscript. All authors read and approved the final manuscript.

Conflicts of Interest

None declared.

A sample of chief complaints and medical histories.

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Abbreviations

Edited by Q Jin; submitted 15.11.23; peer-reviewed by X Tannier, M Torii; comments to author 12.01.24; revised version received 23.01.24; accepted 14.02.24; published 29.03.24.

©Lei Wang, Yinyao Ma, Wenshuai Bi, Hanlin Lv, Yuxiang Li. Originally published in the Journal of Medical Internet Research (https://www.jmir.org), 29.03.2024.

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