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Three-step plan to cut overlooked methane emissions could help us stop global warming faster

Methane, the second most important greenhouse gas, has been neglected—but now scientists lay out a new plan and a new tool to cut emissions down to size

Safeguarding peer review to ensure quality at scale

Making scientific research open has never been more important. But for research to be trusted, it must be of the highest quality. Facing an industry-wide rise in fraudulent science, Frontiers has increased its focus on safeguarding quality.

Ecopreneur Prof Thomas Crowther to showcase the power of nature-based solutions at Frontiers Forum virtual event

Visionary ecologist and pioneer in reforestation and ecosystem restoration Prof Thomas Crowther will explore the vital role that nature plays in our fight against climate change, at a unique Frontiers Forum virtual event on 11 September.

Pompeii skeleton discovery shows another natural disaster may have made Vesuvius eruption even more deadly

The death of inhabitants of Pompeii during the eruption of Vesuvius in 79CE is mostly attributed to volcanic causes, such as falling ashes and hot gas. This, however, may not be the full picture.

Dune-inspired upgrade for spacesuits allow astronauts to recycle urine into water

Inspired by the sci-fi series, Dune, Cornell researchers created a prototype novel urine collection and filtration system for spacesuits.

What happens when the ocean’s biggest predators start hunting each other? Here are five Frontiers articles you won’t want to miss

At Frontiers, we bring some of the world’s best research to a global audience. But with tens of thousands of articles published each year, it’s impossible to cover all of them. Here are just five amazing papers you may have missed.

Five Research Topics on the human impact of population growth

We highlight five Research Topics tackling some of the most urgent issues emerging from our planet's growing population.

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Scopus is the largest abstract and citation database of peer-reviewed literature: scientific journals, books and conference proceedings. Delivering a comprehensive overview of the world's research output in the fields of science, technology, medicine, social sciences, and arts and humanities, Scopus features smart tools to track, analyse and visualise research.

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Psychology of emotion

Protocol of the study for predicting empathy during VR sessions using sensor data and machine learning

Kizhevska and colleagues propose a study that uses virtual reality technology to investigate the potential relationship between empathy and corresponding changes in physiological attributes.

Image credit: Fig 2 by Kizhevska et al., CC BY 4.0

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Teaching methods for critical thinking in health education of children up to high school: A scoping review

Prokop-Dorner and colleagues reviewed educational interventions used in 115 studies on the development of critical thinking regarding health. The interventions focused mainly on lifestyle-related health issues. The authors recommend more detailed reporting and advocate for a reporting checklist to describe health education interventions.

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Arnould and colleagues investigate emotionality in chickens using changes in facial expressions as an indicator of bird welfare.

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Analysis of changes in the occurrence of ice phenomena in upland and mountain rivers of Poland

Kochanek and colleagues present a study on the number of days with ice for rivers in southern Poland, using existing data from years between 1951-2021. They find decreases of number of days with ice for most measuring stations, with indications that increasing air temperatures of 1°C can result in up to 20 fewer days with ice.

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Todisco and colleagues bring new information about this enigmatic group of butterflies

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Choi and Jiao explore transit deserts

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Philosophy leading the way: An interdisciplinary approach to study communication of severe diagnoses

Consolandi combines philosophy and medicine in studying the communication of severe diagnoses in clinical care settings

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Targeted vibratory therapy as a treatment for proprioceptive dysfunction: Clinical trial in older patients with chronic low back pain

Sakai and colleagues report efficacy of vibratory stimulation in treating lower back pain in older patients.

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The SpringerOpen portfolio has grown tremendously since its launch in 2010, so that we now offer researchers from all areas of science, technology, medicine, the humanities and social sciences a place to publish open access in journals. Publishing with SpringerOpen makes your work freely available online for everyone, immediately upon publication, and our high-level peer-review and production processes guarantee the quality and reliability of the work. Open access books are published by our Springer imprint.

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Peer Review: An Introduction: Where to Find Peer Reviewed Sources

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Why is it so hard to find Peer-Reviewed Sources?

It isn't hard to find peer-reviewed sources: you just need to know where to look!  If you start in the right place, you can usually find a relevant, peer-reviewed source for your research in as few clicks as a Google search, and you can even use many of the search techniques you use in Google and Wikipedia.

The easiest way to find a peer-reviewed article is by using one of the Library's numerous databases. All of the Library's databases are listed in the Online Journals and Databases index. The databases are divided by name and discipline.

Departmental libraries and library subject guides have created subject-focused lists of electronic and print research resources that are useful for their disciplines. You can search the library directory  for links to the departmental libraries at the University of Illinois Library, or search library websites by college  if you're not sure which departmental library serves your subject.

Peer-Reviewed Resources for Disciplinary Topics

There are numerous print and digital resources for specific disciplines, areas of study, and specialist fields.  To find research resources and databases for your area, consult the comprehensive directory of LibGuides , the websites of specialist libraries, and above all, contact a librarian for help !

Here are a few major databases for finding peer-reviewed research sources in the humanities, social sciences, and sciences:

• MLA International Bibliography This link opens in a new window Indexes critical materials on literature, languages, linguistics, and folklore. Proved access to citations from worldwide publications, including periodicals, books, essay collections, working papers, proceedings, dissertations and bibliographies. Use MLA International Bibliography in the NEW EBSCO user interface . more... less... Alternate Access Link
• Web of Science (Core Collection) This link opens in a new window Web of Science indexes core journal articles, conference proceedings, data sets, and other resources in the sciences, social sciences, arts, and humanities.

A scholarly, multidisciplinary database providing indexing and abstracts for over 10,000 publications, including monographs, reports, conference proceedings, and others. Also includes full-text access to over 5,000 journals. Offers coverage of many areas of academic study including: archaeology, area studies, astronomy, biology, chemistry, civil engineering, electrical engineering, ethnic & multicultural studies, food science & technology, general science, geography, geology, law, mathematics, mechanical engineering, music, physics, psychology, religion & theology, women's studies, and other fields. 

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• IEEE Xplore This link opens in a new window Provides full-text access to IEEE transactions, IEEE and IEE journals, magazines, and conference proceedings published since 1988, and all current IEEE standards; brings additional search and access features to IEEE/IEE digital library users. Browsable by books & e-books, conference publications, education and learning, journals and magazines, standards and by topic. Also provides links to IEEE standards, IEEE spectrum and other sites.
• Scopus This link opens in a new window Scopus is the largest abstract and citation database including peer-reviewed titles from international publishers, Open Access journals, conference proceedings, trade publications and quality web sources. Subject coverage includes: Chemistry, Physics, Mathematics and Engineering; Life and Health Sciences; Social Sciences, Psychology and Economics; Biological, Agricultural and Environmental Sciences.
• Business Source Ultimate This link opens in a new window Provides bibliographic and full text content, including indexing and abstracts for scholarly business journals back as far as 1886 and full text journal articles in all disciplines of business, including marketing, management, MIS, POM, accounting, finance and economics. The database full text content includes financial data, books, monographs, major reference works, book digests, conference proceedings, case studies, investment research reports, industry reports, market research reports, country reports, company profiles, SWOT analyses and more. Use Business Source Ultimate in the NEW EBSCO user interface . more... less... Alternate Access Link
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Reviews of Peer-Reviewed Journals in the Humanities and Social Sciences

We give you the scuttlebutt on academic journals—aiding you in selecting the right journal for publication—in reviews that are sometimes snarky, sometimes lengthy, always helpful. Written by Princeton University graduate students and Wendy Laura Belcher.

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Identifying the best journal to publish in is not easy. However, it is essential to select correctly, since you can only send your article to one journal at a time, never simultaneously. Based on the advice in Writing Your Journal Article in Twelve Weeks: A Guide to Academic Publishing Success ,  below are some of the ways to find the information you need to make your decision. Here’s how you can find out about:

All journals in your field or discipline.  For brief, online, up-to-date information about journals,  Ulrich’s Periodicals Directory  is the most comprehensive. Do not use the search on the home page, but go to advanced search, select the limiters “Active,” “Journal,” “Academic/Scholarly,” and “ Refereed / Peer-reviewed”   and then enter your keyword (e.g., film, gender, African history). A list of peer-reviewed journals will appear. Under each journal, you can find such information as the journal’s website, publisher, frequency, and a brief review from Magazines for Libraries . Unfortunately, this directory is not free; your library needs to own a subscription to access it.

Journal efficiency (or, gossip about journals) . For detailed information about individuals’ experiences with submitting articles to various journals, including comments on length of backlog and turnaround time, editorial promptness, and peer reviewer helpfulness, see the excellent Humanities Journal Wiki . Not all journals are there, but many are. The entries have to be read with a grain of salt (like any comment system, it is biased toward the negative), but trends in the behavior of the journal can become visible if enough scholars comment.

Journal prestige . Statistical information about which humanities journals are considered better than other journals is not easy to find and is not especially trustworthy. However, two places you can check rankings are the European Reference Index for the Humanities (ERIH)  and the ranking of journals in  JStor alone. You can also check out the meta-list of rankings, Harzing’s  Journal Quality List  (the Harzing 2015 list specifically). which includes many of the websites that provide rankings of journals: outside the United States, many universities compile their own rankings. Academics generally hate humanities lists, see the Guardian article on the topic. The ranking of social science journals can be found in a variety of places, including SCImago , ISI  Journal Citation Reports , and so on.

Journal impact . For information on journals that are cited frequently, use Harzing’s Publish or Perish software. It’s outstanding and free. To find a specific journal’s most cited articles, find its ISSN and insert it in “the Phrase” field.

Acceptance rates . Information about acceptance rates and circulation for peer-reviewed journals in the humanities is often tough to find at their websites. The best source of this information on literary scholarship journals is the MLA Directory of Periodicals , which provides a wealth of other information as well. However, since the information there about acceptance rates and circulation are reported by the journal, these are often slightly inflated. That is, they say their circulation rate is higher than it is and their acceptance rate is lower than it is. MLA doesn’t provide an acceptance rate directly; you have to calculate it by dividing the number of accepted submissions by the number of total submissions (e.g., 40 articles accepted for publication/ 100 articles submitted, equals 40 percent acceptance rate).

Topic trends . The following article analyzed 21,367 scholarly articles in literary journals from 1889–2013 to depict trends in topics over time. “ The Quiet Transformations of Literary Studies: What Thirteen Thousand Scholars Could Tell Us ” by Andrew Goldstone and Ted Underwood in New Literary History (summer 2014).

Our rankings. Using Harzing’s Publish or Perish software, we found the most highly cited articles in specific journals and list those articles on the journal page. We would like to post something here about which journals, therefore, are the most cited, but we haven’t done that yet. It is interesting to note that perhaps the most cited article in the humanities published since 2010 is Homi Bhabha’s two-page (!) introduction to the Simone de Beauvoir special issue in French Politics, Culture and Society , which has been cited 1,182 times.

Questions? Email the webmaster Wendy Laura Belcher.

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The home of cutting-edge research on the development of alternative sustainable technologies. Editorial Board Chair: Javier Pérez-Ramírez Impact factor: 9.3 Time to first decision (peer reviewed only): 35 days

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JAMA , published continuously since 1883, is an international peer-reviewed general medical journal. JAMA is a member of the JAMA Network, a consortium of peer-reviewed, general medical and specialty publications.

Journal Reach. JAMA is the most widely circulated general medical journal in the world, with more than 125,000 recipients of the print journal, more than 2.6 million recipients of electronic tables of contents and alerts, and over 30 million annual visits to the journal's website. JAMA 's reach includes a growing social media presence (more than 1.3 million social media followers [Facebook, X, Instagram, LinkedIn]) and vast international news media exposure.

Frequency of Publication and Access. JAMA is published 48 times per year in print/online issues, and new articles are published every weekday online. Without any author fees, all research articles are made free access online 6 months after publication on the JAMA website. In addition, the online version is freely available or nearly so to institutions in low- and middle-income countries through the World Health Organization's HINARI program.

Editorial Information. JAMA 's acceptance rate is 13% of the more than 10,000 annual submissions and 6% of the more than 5,000 research manuscripts received. JAMA 's Journal Impact Factor is 63.1, among the top 5 journals in medicine and science. For information about JAMA ’s reach and impact, decision and publication timelines, and author services, see below and the JAMA Editorial . For more information on the types of articles published and editorial policies, see the journal's Instructions for Authors .

Editorial Team. The Editor in Chief of JAMA is Kirsten Bibbins-Domingo, PhD, MD, MAS. See the Editors & Publishers for additional information on the journal's Editors and Editorial Board.

JAMA is a member of the International Committee of Medical Journal Editors , cosponsors the International Congress on Peer Review and Biomedical Publication , and is a member of the African Journal Partnership Project and is partnered with the Malawi Medical Journal .

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To promote the science and art of medicine and the betterment of the public health

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The top 10 journal articles from 2023 examined the effects of social media, CBT for substance use, and the psychology of gig work

APA’s 89 journals published more than 5,500 articles in 2023. Here are the top 10 most read

Vol. 55 No. 1 Print version: page 22

1. Looking through a filtered lens: Negative social comparison on social media and suicidal ideation among young adults.

Spitzer, E. G., et al.

Young adults who engage in comparisons to others on social media and thus feel bad about themselves are more likely to think about suicide, this research in Psychology of Popular Media (Vol. 12, No. 1) suggests. Researchers surveyed 456 college students about their frequency of social media use and used scales to assess participants’ tendency to engage in negative social comparison on Instagram and Facebook, suicidal ideation, and thwarted belongingness (i.e., feeling as if lacking connections or meaningful relationships with others). Results indicated that participants who engaged in negative social comparisons were more likely to report suicidal ideation than those who did not. Specifically, on Instagram, those who negatively compared themselves to others the most also showed the highest levels of association between thwarted belongingness and suicidal ideation. These findings suggest the need for limits on social media use and education around its mental health effects. DOI: 10.1037/ppm0000380

2. Self-compassion and women’s experience of social media content portraying body positivity and appearance ideals.

Rutter, H., et al.

The type of social media content women view can affect their self-compassion—how kind to themselves and accepting of their flaws they are—suggests this study in Psychology of Popular Media (advance online publication). In two experiments, a total of 247 women viewed content consistent with appearance ideals (fitspiration body photos; faces with makeup), appearance-neutral content (landscapes), or body-positive content (body-positive body photos, body-positive quotes, faces without makeup). In both experiments, women who viewed content consistent with appearance ideals reported a state of worse self-compassion and worse thoughts about themselves than those who viewed body-positive or appearance-neutral content. Women who already had daily low self-compassion or high disordered-eating symptoms were the most affected by viewing content consistent with appearance ideals. On the contrary, viewing body-positive content increased the state of self-compassion relative to viewing appearance-neutral content. DOI: 10.1037/ppm0000453

3. Reducing social media use improves appearance and weight esteem in youth with emotional distress.

Thai, H., et al.

Reducing smartphone social media use to 1 hour per day might improve body image and weight esteem in youth with emotional distress who are heavy social media users, this study in Psychology of Popular Media (advance online publication) suggests. The researchers randomly assigned 220 participants (ages 17 to 25 who used social media at least 2 hours per day) to either a 4-week intervention in which they limited their social media use to 1 hour per day or to a control condition with unrestricted access to social media. After the 4-week intervention, the group with restricted social media use felt better about their appearance and weight relative to before the intervention, whereas the other group showed no changes. Thus, reducing smartphone social media use appears to be a good method to improve how youths feel about their appearance and weight and could become a component in the prevention and treatment of body image-related disturbances. DOI: 10.1037/ppm0000460

4. Interventions to reduce the negative impact of online highly visual social networking site use on mental health outcomes: A scoping review.

Herriman, Z., et al.

In this review, published in Psychology of Popular Media (advance online publication), researchers identified 39 studies published between 2011 and 2022 that examined how interventions designed to reduce the negative impact of online highly visual social networking site (e.g., Facebook, Instagram) use impact mental health. Most of the studies were conducted on Western adults younger than age 35 and varied widely in terms of the variables assessed, making it difficult to highlight overall conclusions. Nevertheless, results indicate that interventions focused on reducing the exposure to highly visual social media platforms benefited well-being but may also reduce social connectedness. Interventions focused on social media literacy programs may reduce addiction and improve body image. Other interventions that adopted varied psychological approaches did not appear to lead to significant results. The researchers also highlighted the gaps in research that should be addressed to improve the efficacy of such interventions, including a need for interventions that are more guided by psychological theories and assessments of these interventions that are rigorous and include diverse populations. DOI: 10.1037/ppm0000455

5. On the outside looking in: Social media intensity, social connection, and user well-being: The moderating role of passive social media use.

Roberts, J. A., & David, M. E.

According to this study in the Canadian Journal of Behavioural Science (Vol. 55, No. 3) , heavy passive social media use may be linked with a weaker sense of social connection and well-being. In two survey-based studies with 226 participants in the United States, researchers found that passive engagement with social media (viewing social media but not regularly posting or interacting through the platform) was associated with less social connection, lower well-being, and higher stress. In a third, experimental study, with 160 participants, the researchers asked participants to use social media heavily (10 minutes) or lightly (5 minutes) and engage with it actively or passively. The results indicated that heavy social media use had a negative impact on feelings of social connection when used passively but a positive effect when used actively. DOI: 10.1037/cbs0000323

6. Social media usage is associated with lower knowledge about anxiety and indiscriminate use of anxiety coping strategies.

Wolenski, R., & Pettit, J. W.

Social media might not be the best source to learn about anxiety and how to reduce it, this study in Psychology of Popular Media (advance online publication) suggests. Young adults (N=250) responded to an online survey in which they reported their sources of information about anxiety, the strategies they use to cope with anxiety, and their anxiety symptoms and severity. The researchers also tested participants’ knowledge about anxiety. Participants rated the internet (e.g., Wikipedia, medical websites) as their most used information source, followed by friends and family, therapy, and social media. Participants with an anxiety diagnosis or severe symptoms sought information on social media more frequently than the other participants. Across all participants, those who sought information on social media more frequently showed a lower knowledge about anxiety and were more likely to report using both adaptive and maladaptive strategies to reduce anxiety. On the contrary, using the internet was associated with more knowledge about anxiety. These findings suggest the need to promote the dissemination of accurate information about anxiety on social media. DOI: 10.1037/ppm0000456

7. The psychological scaffolding of arithmetic

Grice, M., et al.

In this article in Psychological Review (advance online publication), the authors propose that arithmetic has a biological origin, rather than philosophical, logical, or cognitive basis. This assertion rests on four principles of perceptual organization—monotonicity, convexity, continuity, and isomorphism—that shape how humans and other animals experience the world. According to the authors, these principles exclude all possibilities except the existence of arithmetic. Monotonicity is the idea that things change in the same direction, so that approaching objects appear to expand, while retreating objects appear to shrink. Convexity deals with betweenness, such that the four corners of a soccer pitch define the playing field even without boundary lines connecting them. Continuity describes the smoothness with which objects appear to move in time and space. Isomorphism is the idea of analogy, allowing people to recognize that cats are more similar to dogs than rocks. The authors’ analysis suggests that arithmetic is not necessarily an immutable truth of the universe but rather follows as a natural consequence of our perceptual system. DOI: 10.1037/rev0000431

8. An evaluation of cognitive behavioral therapy for substance use disorders: A systematic review and application of the society of clinical psychology criteria for empirically supported treatments.

Boness C. L., et al.

Cognitive behavioral therapy (CBT) is an empirically supported treatment for substance use disorder (SUD), is the conclusion of this review in Clinical Psychology: Science and Practice (Vol. 30, No. 2) . The researchers reviewed five meta-analyses of the effect of CBT on SUD, but only one had sufficient quality for inclusion to evaluate the size of the effects of CBT. This meta-analysis found that CBT produced small to moderate effects on SUD when compared with minimal treatment (e.g., waitlist, brief psychoeducation) and nonspecific treatment (e.g., treatment as usual, drug counseling). These effects were smaller in magnitude when compared with other active treatments (e.g., motivational interviewing, contingency management). The effects of CBT on SUD tended to diminish over time (i.e., CBT was most effective at early follow-up of 1 to 6 months posttreatment compared with late follow-up of at least 8 months posttreatment). The researchers recommend CBT to be used as an evidence-based approach to SUD but highlight the need for more research to identify patient characteristics that might moderate response to CBT and the best deployment of CBT (e.g., as a standalone or an adjunct intervention). DOI: 10.1037/cps0000131

9. A network approach to understanding parenting: Linking coparenting, parenting styles, and parental involvement in rearing adolescents in different age groups.

Liu, S., et al.

Mothers’ and fathers’ behaviors that promote a sense of family integrity (i.e., coparenting integrity), warmth, and emotional involvement are central components of the parenting network in two-parent families, according to this study in Developmental Psychology (Vol. 59, No. 4) . Researchers used network analysis to explore different facets of maternal and paternal coparenting (e.g., integrity, conflict), parenting styles (e.g., rejection, warmth), and parental involvement (e.g., emotional support, discipline) in two-parent families in China with a total of 4,852 adolescents at different stages of adolescence. They found that maternal and paternal coparenting integrity, warm parenting style, and emotional involvement were key to the parenting network, as indicated by the central spot they occupied in the network analysis. They also found that the expected influence of these characteristics varied for adolescents in different developmental stages—maternal integrity, warmth, and emotional involvement were important throughout adolescence, but paternal integrity, warmth, and emotional involvement were particularly important in early adolescence. The results suggest that supportive parenting might be a prime target for enhancing parenting systems. DOI: 10.1037/dev0001470

10. Seeking connection, autonomy, and emotional feedback: A self-determination theory of self-regulation in attention-deficit hyperactivity disorder.

Champ, R.E., et al.

In this article in Psychological Review (Vol. 130, No. 3) , the authors propose a new framework on the basis of self-determination theory (SDT) for understanding attention-deficit hyperactivity disorder (ADHD) and developing treatment approaches. The researchers suggest that using SDT, which proposes that humans have a natural tendency toward growth and self-actualization, supporting intrinsic motivation and self-organization, can offer a new positive understanding of ADHD and its symptoms. This approach counters the negative characterizations of ADHD; moves beyond symptom reduction and the focus on how ADHD presents motivation, engagement, and self-regulation issues; and instead focuses on potential positive aspects of ADHD and well-being. In addition, the framework highlights the need to help individuals with ADHD better understand how they function, tell the difference between biological and individual needs, and develop self-autonomy and self-regulation skills. According to this SDT approach, treatments that are autonomy supportive and increase self-determination could improve the functioning of individuals with ADHD. DOI: 10.1037/rev0000398

PsycArticles is available by subscription to institutions throughout the world. These are the articles published this year that were downloaded most often between January and September 2023.

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• Open access
• Published: 19 August 2024

Immune checkpoint gene signature assesses immune infiltration profiles in bladder cancer and identifies KRT23 as an immunotherapeutic target

• Dongshan Chen 1 , 2   na1 ,
• Haoyuan Cao 1   na1 ,
• Xiang Zheng 1   na1 ,
• Haojun Wang 1 ,
• Zengchi Han   ORCID: orcid.org/0009-0004-1544-5229 3 &
• Wei Wang   ORCID: orcid.org/0000-0003-2642-3338 1  

BMC Cancer volume  24 , Article number:  1024 ( 2024 ) Cite this article

259 Accesses

Metrics details

In the past few decades, researchers have made promising progress, including the development of immune checkpoint inhibitors (ICIs) in the therapy of bladder cancer (BLCA). Existing studies mainly focus on single immune checkpoint inhibitors but lack relevant studies on the gene expression profiles of multiple immune checkpoints.

RNA-sequencing profiling data and clinical information of BLCA patients and normal human bladder samples were acquired from the Cancer Genome Atlas and Gene Expression Omnibus databases and analyzed to identify different expression profiles of immune checkpoint genes (ICGs) after consensus clustering analysis. Based on the 526 intersecting differentially expressed genes, the LASSO Cox regression analysis was utilized to construct the ICG signature.

According to the expression of ICGs, BLCA patients were divided into three subtypes with different phenotypic and mechanistic characteristics. Furthermore, the developed ICG signature were independent predictors of outcome in BLCA patients, and was correlated with the immune infiltration, the expression of ICGs and chemotherapeutic effect.

Conclusions

This study systematically and comprehensively analyzed the expression profile of immune checkpoint genes, and established the ICG signature to investigate the differences in ICGs expression and tumor immune microenvironment, which will help risk stratification and accelerate precision medicine. Finally, we identified KRT23 as the most critical model gene, and highlighted KRT23 as a potential target to enhance immunotherapy against BLCA.

Peer Review reports

Introduction

Bladder cancer (BLCA) is the seventh most common type of malignant tumor worldwide [ 1 ]. Moreover, approximately 549,393 people were newly diagnosed with bladder cancer, and 199,922 patients died of the disease in 2018 [ 2 ]. Besides basic diagnosis based on symptoms and physical exams, exfoliated urothelial cells and cystoscopy are the primary methods of diagnosing bladder cancer, making BLCA one of the most expensive cancers. Approximately 80% of newly diagnosed patients present with non-muscle-invasive bladder cancer (NMIBC), and 10–20% of them will progress and invade the bladder muscle [ 3 ]. Muscular invasive bladder cancer (MIBC) is more prone to lymph node and organ metastasis, and the MIBC survival rate of five-years remains low [ 4 ]. Traditional therapy for bladder cancer mainly includes surgical resection and local or systemic chemotherapy, but the postoperative recurrence and the distant metastasis are difficult to control [ 5 ]. Therefore, it is urgent to develop new detection and treatment strategies to reduce the risk of recurrence and progression.

Although cisplatin-based combination chemotherapy realized the ideal reduction of death risk induced by bladder cancer, chemoresistance is developed and remains one of the chief reasons for cancer recurrence [ 6 ]. Additionally, the age-related and/or disease-related comorbidities may delay or impede the receipt of cisplatin-based perioperative chemotherapy [ 7 ]. As our understanding of the interaction between tumor cells and the tumor immune microenvironment (TIME) deepens, the increasing use of immune checkpoint inhibitors (ICIs) has revealed encouraging clinical efficacy in treatment. Interestingly, compared with other cancers, MIBC is one of the tumors with the highest mutation profiles, while cancers with high mutation burden, such as melanoma and non-small cell lung cancer, respond very well to ICIs [ 8 , 9 , 10 , 11 ]. In the past few decades, researchers had made promising progresses in the treatment of bladder cancer, including ICIs [ 12 ]. For example, a press in 2023 indicated that the combination of cisplatin/gemcitabine with nivolumab demonstrated statistically significant improvements in progression-free survival (PFS) and overall survival (OS) compared to using cisplatin/gemcitabine alone [ 13 ]. At present, the US Food and Drug Administration (FDA) has approved several ICIs for the treatment of cisplatin-ineligible patients and patients with advanced BLCA who progress despite cisplatin-based chemotherapy [ 14 ]. Despite it showed great prospects relative to past therapies, it must be recognized that only a subset of patients achieves long-term durable responses [ 15 ]. A major challenge in the ICIs therapy area is to investigate the mechanism of ICGs in BLCA and to identify patients that may respond to specific immunotherapies.

In the present study, we divided BLCA patients into three ICG clusters and built the ICG signature on the basis of the differentially expressed genes among different clusters to predict progression and outcome of BLCA. Besides, we discussed the correlation of ICG signature with the tumor immune microenvironment, and further investigated the potential mechanisms between ICGs and basic TIME characterizations. Here, our study identifies a new ICG signature to clarified the progress mechanism in bladder cancer and provides potential biomarkers and therapeutic targets.

Overview of genetic changes and expression variations of immune checkpoint genes in BLCA

Among the 412 samples in TCGA-BLCA dataset, 92 samples (about 22.33%) showed ICGs mutations, and KDR showed the highest frequency of mutations (Fig.  1 A). Additionally, waterfall diagram showed that missense mutation and nonsense mutation were the most common mutation types. Through the analysis of the frequency of the copy number variation (CNV) changes, we also found that most of CNV changes of ICGs were concentrated in the gain of copy number, and the gain of CD244, CD48, SLAMF1 and CD160 were the most obvious (Fig.  1 B). The CNV changes of the ICGs on the chromosome were shown in Fig.  1 C. In order to study the tumorigenic mechanism at the expression level, we further analyzed the expression levels of ICGs between normal and tumor samples. 30 ICGs were identified as genes with statistically differential expression finally (Fig.  1 D). Those findings revealed the pathogenesis of BLCA more comprehensively, and may provide guidance for medication guide of ICIs.

Survey of genetic changes and expression variations of immune checkpoint genes in BLCA. ( A ) The landscape of mutation profiles of 68 ICGs in 412 bladder cancer patients from TCGA-BLCA cohort. ( B ) The CNV variation frequency of ICGs. red circle: amplified frequency; green circle: missing frequency. ( C ) The location of CNV alteration of ICGs on chromosomes. CNV, copy number variations. ( D ) The expression difference of 68 ICGs between normal and BLCA samples. *** p  < 0.001, ** p  < 0.01, * p  < 0.05

Tumor classification based on the ICGs

After merging the GEO and TCGA databases, we performed the survival analysis to clarify the prognostic implication of these ICGs in BLCA samples. The K-M analysis of all the prognosis related ICGs in the merged cohort was shown in Supplementary Table S1 . In addition, we analyzed the correlation network containing all ICGs of prognostic functions (Fig.  2 A). Based on the expression levels of 53 ICGs, we performed the consensus clustering analysis and divided all samples into three clusters to investigate the relationship between these ICGs and BLCA subtypes (Fig.  2 B). The OS of three subtypes was significantly different ( p  < 0.05). And the survival advantage of cluster C was higher than that of cluster B and A successively (Fig.  2 C). The correlation heatmap demonstrated the expression of ICGs and the clinical manifestations including age, gender, grade and TMN stage of these three different clusters (Fig.  2 D). The result showed that most ICGs expressed highly in cluster C and lowly in cluster B. Then we analyzed immune atlas of ICG clusters and found that activated immune cell infiltration, including the presence of dendritic cells (DCs), macrophages, natural killer cells (NKs), neutrophil, along with activated B, CD4 and CD8 T cells, was abundant in cluster C, which typically indicates significant survival benefits (Fig.  2 E). Finally, the principal component analysis indicated that patients were well divided into three clusters (Fig.  2 F).

Tumor classification based on the ICGs. ( A ) The correlation network of all the prognosis related ICGs with correlations of absolute values greater than 0.2. Red indicates positive correlation and blue indicates negative correlation. ( B ) The consensus score matrix of all samples when k = 3 in merged cohorts (TCGA-BLCA, GSE13507 and GSE31684) based on the expression of ICGs. ( C ) OS curves for the three ICG clusters based on 670 patients with BLCA. ( D ) The heatmap of gene expression and clinical correlation among different clusters of BLCA. ( E ) Differential expression of immune cells among the BLCA ICG clusters. *** p  < 0.001, ** p  < 0.01, * p  < 0.05. ( F ) PCA analysis of the different ICG clusters based on the prognosis related ICGs

GSVA and functional enrichment analysis between ICG clusters

We performed GSVA to detect differential pathway activity of these three clusters and provides an intuitive and stable context for assessing biological activity (Fig.  3 A and C). The result showed cluster C was significantly enriched in enrichment pathways related to complete immune activation, including CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION, T_CELL_RECEPTOR_SIGNALING_PATHWAY, B_CELL_RECEPTOR_SIGNALING_PATHWAY, CHEMOKINE_SIGNALING_PATHWAY, NOD_LIKE_RECEPTOR_SIGNALING_PATHWAY, NATURAL_KILLER_CELL_MEDIATED_CYTOTOXICITY and TOLL_LIKE_RECEPTOR_SIGNALING_PATHWAY (Fig.  3 B and C). Surprisingly, compared with cluster B, cluster A was significantly enriched in innate immune cell infiltration including CHEMOKINE_SIGNALING_PATHWAY, T_CELL_RECEPTOR_SIGNALING_PATHWAY, B_CELL_RECEPTOR_SIGNALING_PATHWAY and NATURAL_KILLER_CELL_MEDIATED_CYTOTOXICITY, while the prognosis of the patients in cluster A was significantly worse than that of patients in cluster B (Fig.  3 A). Thus, we suspected that this may be due to the aberrant activity of REGULATION_OF_ACTIN_CYTOSKELETON and ECM_RECEPTOR_INTERACTION signaling pathway.

GSVA and Functional Enrichment Analysis Between ICG clusters. ( A - C ) The heatmap of GSVA enrichment analysis among different ICG clusters. Red indicates the active pathway, and blue indicates inhibition pathway. ( A : cluster A vs. cluster B ; B : cluster A vs. cluster C ; C : cluster B vs. cluster C ). ( D ) The Venn plot of intersection DEGs. ( E ) Bubble of GO enrichment analysis. ( F ) Bubble of the top 30 KEGG signaling pathways. BP: biological process; CC: cellular component; MF: molecular function; KEGG: Kyoto encyclopedia of genes and genomes

Subsequently, we determined DEGs among different subtypes and extracted 526 intersecting DEGs for subsequent analysis (Fig.  3 D). GO enrichment analysis and KEGG pathway analysis were then performed to further explore the differences in the functions and pathways of these intersecting DEGs. BPs were mainly enriched in T cell activation, negative regulation of immune system process, leukocyte cell-cell adhesion and regulation of T cell activation. CCs were mainly enriched in external side of plasma membrane, secretory granule membrane, endocytic vesicle and MHC protein complex. MFs were mainly enriched in receptor ligand activity, signaling receptor activator activity, chemokine activity, immune receptor activity and cytokine receptor binding (Fig.  3 E). KEGG were mainly enriched in cytokine − cytokine receptor interaction, phagosome, antigen processing and presentation, Th1 and Th2 cell differentiation and TNF signaling pathway (Fig.  3 F). The results indicated that the intersecting DEGs were clearly correlated with the immunomodulation functions, inflammatory cell chemotaxis and epithelial mesenchymal transition (EMT) related factors.

Tumor classification based on the intersecting DEGs

The Univariate Cox regression analysis was used to analyze the prognostic values of intersecting DEGs in the merged cohort, and a total of 127 genes were considered as genes associated with prognosis (supplementary Table S2 ). We then performed the unsupervised clustering analysis to verify the correlation between these prognosis related intersection DEGs and BLCA subtypes (Fig.  4 A). Based on the highest intragroup correlations and lowest intergroup correlations when k = 2, we classified all BLCA patients into two clusters, and we found that the prognosis of patients from cluster A was significantly better than that of the patients from cluster B (Fig.  4 B). The expression of prognosis related intersection DEGs and the clinical information including age, gender, grade, TNM stage were showed in a heatmap, and we found there were significant expression differences between two clusters and little differences in clinical features (Fig.  4 C). The analysis of ICGs different expression showed that the expression levels of most ICGs were significantly reduced in the cluster A, while expression of CD24 , CD96 , CEACAM1 , TNFRSF14 , TNFRSF25 and VTCN1 were increased significantly in cluster A (Fig.  4 D).

Tumor classification based on the intersecting DEGs. ( A ) The consensus score matrix of all samples when k = 2 based on the expression of intersecting DEGs. ( B ) Survival curve for the two clusters. ( C ) Heatmap and the clinicopathologic characters of the two clusters classified by these intersecting DEGs. ( D ) Differential expression of ICGs among the intersecting DEGs clusters. *** p  < 0.001, ** p  < 0.01, * p  < 0.05

Establishment and validation of a ICG signature

The samples in the merged dataset were randomly assigned into training cohort and testing cohort. We performed the least absolute shrinkage and LASSO Cox regression analysis using 127 prognosis related genes in the training cohort to build the 9-gene risk signature (Fig.  5 A). The regression coefficients of the 9 signature genes were shown in Table  1 . The alluvial map was used to visualize the model construction and testing (Fig.  5 B). Furthermore, the risk score of the patients in ICG cluster A was higher than that in ICG cluster B and ICG cluster C in succession (Fig.  5 C), while the patients in genecluster B favored high-risk scores compared to those with in genecluster A (Fig.  5 D). It was obvious to note that increased risk scores were shown to correlate with poor prognosis. Wilcoxon test was applied to investigate the relationship between the expression of these ICGs and BLCA risk subtypes (Fig.  5 E).

Establishment of a ICG signature. ( A ) LASSO Cox regression analysis. ( B ) Alluvial diagram showing the changes of ICG clusters, intersecting DEGs clusters, the signature and fustat. ( C ) The ICG signature risk scores in different ICG clusters. ( D ) The ICG signature risk scores in different intersecting DEGs clusters. ( E ) Differential expression of ICGs between low- and high-risk subgroups. *** p  < 0.001, ** p  < 0.01, * p  < 0.05

We divided patients in total, training and testing cohorts into high and low risk groups with median risk score of training cohort. We found that the low group had a clear survival advantage over the high group in total (Fig.  6 A), training (Fig.  6 B) and testing cohorts (Fig.  6 C). The subsequent ROC analysis demonstrated that this ICG signature could reliably evaluate the efficiency of prognosis prediction, and we found that AUC in training cohort was 0.777 for 1-year, 0.709 for 2-year, and 0.706 for 3-year survival (Fig.  6 E). In addition, the signature was respectively validated in the total and testing cohorts (Fig.  6 D and F). The risk scatterplot and heatmap revealed the difference of survival status distribution and model gene expression between low-risk and high‐risk groups in total, training, and testing cohorts (Fig.  6 G and I). Moreover, we observed consistent results in the GSE13507 and GSE31684 dataset (Supplementary Fig S1 ). The 9 ICGs‐related genes forming the signature exhibited distinct expression diverse expression patterns, including five relatively highly expressed genes ( TNFAIP6 , CPVL , SLFN11 , KRT23 and SPRR3 ) and four relatively lowly expressed genes ( CD3D , GBP4 , PLEKHA6 and PPP1R3C ), consistent with the risk coefficient.

Validation of the ICG signature. ( A - C ) The KM analysis of the overall survival of the high-risk and low-risk groups in total ( A ), training ( B ) and testing cohorts ( C ). ( D - F ) The ROC analysis to evaluate the predictive efficiency of the ICG signature in total ( D ), training ( E ) and testing cohorts ( F ). ( G - I ) Distribution of the risk scores, survival status, and expression of the nine risk genes in total ( G ), training ( H ) and testing cohorts ( I )

Subsequently, we conducted an in-depth analysis of differentially expressed genes between high-risk and low-risk patient groups, uncovering significant biological disparities between the two groups (Supplementary Fig S2 A and S2 B). Our GO analysis identified several biological processes and cellular compartments where the DEGs are enriched, including collagen fibril organization, extracellular matrix organization, collagen-containing extracellular matrix (Supplementary Fig S2C). The KEGG enrichment analysis identified significant enrichment of pathways related to cytoskeleton organization in muscle cells, ECM-receptor interaction, IL-17 signaling, and protein digestion and absorption in the DEGs (Supplementary Fig S2 D). These findings suggest that the dysregulation of these pathways may contribute to the aggressive behavior of bladder cancer, including increased cell motility, altered ECM-cell interactions, immune evasion, and altered nutrient utilization. Subsequently, we constructed a PPI network of the identified DEGs to identify potential hubs and interactors (Supplementary Fig S2 E). These proteins may play crucial roles in bladder cancer development and could serve as potential therapeutic targets or drug discovery leads.

Evaluation of the ICG signature As shown in Fig.  7 A, the risk scores and all significant factors for prognosis (including age, gender, grade, TNM stage) were integrated into a novel prognostic nomogram to provide a reliable quantitative tool for predicting the prognosis of BLCA patients. The nomogram could effectively predict the 1, 3 and 5 year survival rates of BLCA patients.

Molecular characteristics and clinical utility of the ICG signature. ( A ) Nomograph for predicting the overall survival of BLCA patients at 1, 3, and 5 years. ( B ) Correlation heatmap of the levels of infiltration of the immune cells with nine risk genes. ( C ) Distribution of immune scores, stromal scores and ESTIMATE scores in low- and high-risk subgroups. ( D - E ) Differences in the mutational status within the different subgroups. ( F ) Expression difference of the three hot immune checkpoint genes (PD-L1, PD-1, and CTLA4) in different subgroups. ( G ) Box plots of differential chemosensitivity between two subgroups

A comparative analysis between the high- and low-risk groups stratified by our ICG signature revealed notable differences in tumor immune dysfunction and exclusion (TIDE), microsatellite instability (MSI), dysfunction, and exclusion scores. Specifically, the low-risk group exhibited higher TIDE and T-cell dysfunction scores, as well as a lower T-cell exclusion score, indicating a significant variation in the responsiveness to immunotherapy between the two risk groups (Supplementary Fig.  3 A- 3  C). However, there was no discernible difference in the MSI score between the two subgroups (Supplementary Fig. 3D). Subsequently, ROC curve analysis was employed to assess the prognostic superiority of our ICG signature model over the existing TIDE and 18-gene T-cell inflammation signature (TIS) models. The TIDE model evaluates the degree of immune cell dysfunction and exclusion within the tumor microenvironment, while the 18-gene TIS model delves into the expression of a panel of immune-related genes to predict patient prognosis [ 16 ]. Our model demonstrated an AUC value that exceeded those of both the TIDE and 18-gene TIS models (Supplementary Fig. 3E), suggesting that the ICG signature holds the potential to enhance the accuracy of risk stratification and prognosis prediction for bladder cancer patients when compared to the existing methods.

Identification of the immune characteristics and tumor mutational burden (TMB)

Correlation analysis of 9 model genes and invasive immune cell types was used to study the potential impact of ICG signature on BLCA immune microenvironment (Fig.  7 B). Further investigation on the correlation between infiltrating immunocytes and risk scores revealed that risk scores had positive correlations with macrophages M0, macrophages M2, neutrophils, dendritic cells activated, T cells CD4 memory resting and mast cells activated, and risk scores had negative correlations with T cells CD8, T cells regulatory (Tregs), T cells gamma delta, Macrophages M1 and activated T cells CD4 memory (supplementary Fig S4 ). To quantify differences of tumor microenvironment (TME) between high and low-risk groups, ESTIMATE scores were calculated and demonstrated that high-risk group had significantly higher ESTIMATE, stromal, and immune scores compared to low-risk group (Fig.  7 C).

Afterwards, we compared the differences of TMB between different risk subgroups. We found that the frequency of tumor mutation in high-risk group (95.07%) was higher than that in low-risk group (92.57%), and the most common types of mutation were missense mutation and nonsense mutation (Fig.  7 D and E). Next, we determined the top 20 genes with the highest mutation rate in the risk subgroups and found that TP53 was the common mutation gene, with the mutation rates of over 40% in both risk groups.

The role of ICG signature in immunotherapy and chemotherapy

Previous studies have shown that anti PD1/PD-L1 immunotherapy is long-term effective for patients with high TMB status [ 17 ], and only a handful of patients benefit from ICI therapies. Therefore, we further explored the differential expression of most common ICIs, and found that three hot immune checkpoint genes (PD-L1, PD-1, and CTLA4) in the TCGA-BLCA cohort were up regulated in low-risk group, indicating that a better response to immunotherapy (Fig.  7 F). As previously mentioned, combination chemotherapy based on cisplatin is still the first-line treatment standard for locally advanced or metastatic urothelial carcinoma [ 15 ], and methotrexate, docetaxel and paclitaxel were also shown to have significant and curative effect in bladder cancer [ 18 , 19 , 20 ]. Thus, we investigated the role of the risk model in predicting the efficacy of chemotherapy in BLCA patients. We found that high risk patients were inclined to possess lower half inhibition concentration (IC50) of chemotherapy drugs (such as cisplatin docetaxel and paclitaxel), while the sensitivity of the low-risk group to methotrexate was higher than that of the high-risk group (all p  < 0.05, Fig.  7 G). These results suggested that this ICG signature may help in selecting chemotherapeutic drugs and judging the therapeutic effects.

Furthermore, we profiled T cells using single-cell RNA to assess the T cell composition of the tumor environment. Series GSE149652 was downloaded from GEO database and composed of human bladder tumors and non-malignant tissue of 2 standard-of-care-untreated patients (“untreated”), 1 chemotherapy-treated patient (gemcitabine + carboplatin, “chemo”), and 4 anti-PD-L1-treated patients (“anti-PD-L1”). Unsupervised clustering of immune cell infiltration data revealed the presence of T cell components, including CD4 T, CD8 T, Tregs, and NK cells (Fig.  8 A). Next, we assessed the T cell-inflamed and checkpoint gene differences in different treatment groups and found that the T cell-inflamed and checkpoint gene expressions in anti-PD-L1 group were generally higher than those in the other two groups (Fig.  8 B).

T cell infiltration pattern. ( A ) SNE plot of total sample cells and T cell infiltration profiles of all samples. ( B ) T cell-inflamed and checkpoint gene differences in different treatment groups. ( C and D ) To further evaluate the importance of these genes for the prognostic contribution of BLCA patients, we performed a random forest analysis of these genes based on risk scores and patient survival status. ( E - F ) The expression difference of KRT23 in different T cell types

To further evaluate the importance of these model genes for the prognostic contribution of BLCA patients, we performed a random forest analysis of these genes based on risk scores and patient survival status and found that the mean decrease Gini of KRT23 was higher in both analyses (Fig.  8 C and D). Immune infiltration analysis revealed that KRT23 expression was negatively correlated with the infiltration of CD8 + T and CD4 + cells in bladder cancer, while positively correlated with the infiltration of dendritic cells and neutrophils, indicating a complex role for KRT23 in shaping the immune microenvironment of bladder cancer (Supplementary Fig S5). Additionally, we mainly analyzed the KRT23 expression in different T cell types and found KRT23 was generally low expressed in T cells, which was similar to the results of data merged by TCGA and GEO database (Fig.  8 E and F).

Knockdown of KRT23 inhibited BLCA Cell proliferation, migration and invasion, and promoted apoptosis

The expression of KRT23 increased in the normal, low grade and high grade samples in the HPA database progressively (Fig.  9 A). Next, we performed RT-qPCR in 4 cell lines, consisting of 3 BLCA cell lines and 1 normal bladder epithelium cell line, and found that KRT23 has a strong expression in the bladder cancer cells (Fig.  9 B). We selected T24 and J82 cells to further verify the importance of KRT23 .

KRT23 Knockdown. ( A ) Immunohistochemical analysis of KRT23 in normal, low grade and high grade samples. ( B ) The gene expression levels of KRT23 were evaluated by real-time qPCR. ( C ) The knockdown of KRT23 by siRNA was detected by RT-PCR. ( D ) Cell colony formation assay. ( E ) CCK-8 assay. ( F ) Wound scratch assay. ( G ) Transwell Assay. ( H ) Flow cytometric dot plots. *** p  < 0.01, ** p  < 0.01, * p  < 0.05

To further explore the biological function of KRT23 in BLCA cells, the expression of KRT23 was knocked down in T24 and J82 cells using two different siRNAs. The efficiency of transfection of KRT23 siRNA was verified in mRNA levels (Fig.  9 C). First, we examined the colony formation and proliferative capacity of BLCA cells after KRT23 knockdown. Inhibition of KRT23 expression markedly suppressed the colony formation and cell proliferation (Fig.  9 D and E). Subsequently, we performed the wound healing assay scratch assay and transwell assay, and the results showed that KRT23 knockdown impaired the migration and invasion ability of the T24 and J82 cells (Fig.  9 F and G). The apoptosis rate in the KRT23 knockdown groups of T24 and J82 cells was significantly higher than those in the si-NC groups (Fig.  9 H). To evaluate the correlation between KRT23 and clinical outcome, we detected KRT23 expression in tumor and adjacent tissues of 6 BLCA patients using western blotting, and the results showed that KRT23 was highly expressed in tumor tissues (Fig.  10 A). Those results indicated that KRT23 knockdown inhibited proliferation and promotes apoptosis, and KRT23 plays an important role in the process of promoting tumorigenesis.

Knockdown of KRT23 inhibited tumor proliferation in vivo. ( A ) Western blotting analysis was conducted to detect the expression of KRT23 in BLCA tissues and adjacent normal tissues ( n  = 6). ( B ) The tumor volumes were measured at the indicated time points. ( C ) The representative images of tumor. Tumor weight was measured after isolation from the mice. ( D ) IHC of PCNA and Ki67 in the tumors. *** p  < 0.01, ** p  < 0.01, * p  < 0.05. Original blots are presented in supplementary Fig S6

Knockdown of KRT23 inhibited tumor proliferation in vivo

Subsequently, we explored the impact of OGT on tumor formation in mice via a xenograft model. Control (shCtrl) or KRT23 knockdown (shKRT23) T24 cells were injected into the flanks of male nude mice. Compared to the control group, the tumors in KRT23 knockdown were significantly small and light in weight (Fig.  10 B and C). At the end of this experiment, the mice were killed and their tumors were isolated. Immunohistochemistry of tumors showed that PCNA and ki67 were decreased due to the decreased expression of KRT23 (Fig.  10 D), which indicated the inhibitory effect of KRT23 knockdown on tumor proliferation and was consistent with the findings in vitro.

Despite significant advances in monitoring opportunities and unique therapeutic options, the survival of BLCA patients has not significantly changed in the past 30 years [ 21 ]. In view of the high frequency mutations identified by next-generation sequencing [ 22 , 23 ], different biomarkers and therapies will be required to improve patients’ outcome in different clinical application. The effect of perioperative cisplatin-based combination chemotherapy on the survival rate of BLCA patients is still limited [ 24 ]. Currently, with the deepening of the research on immune checkpoint mechanism, immune-checkpoint inhibitors (CPIs) had shown great potential in the treatment of multiple types of malignancies and approved by FDA for first-line metastatic therapy in patients with cis-ineligible and PD-L1positive tumors (i.e., atezolizumab, pembrolizumab) and second-line therapies following failure of platinum-based chemotherapy(i.e., atezolizumab, pembrolizumab, nivolumab, avelumab and durvalumab) [ 24 , 25 , 26 , 27 , 28 ].

Based on reliable evidence from the background of metastatic urothelial carcinoma, CPIs are well tolerated and effective, and are expected to change the foundations of MIBC treatment in the near future [ 29 , 30 ]. Therefore, to discover the ICGs molecular mechanisms underlying the development and progression of BLCA and to improve the prognosis and survival rate of BLCA patients, developing highly accurate, cost-effective, definitive test for diagnosis and surveillance is an urgent need.

This study systematically compared the altered expressions of ICGs between normal and BLCA samples, and analyzed the prognostic value of ICGs in BLCA. A total of 37 of 53 ICGs in merged cohort were associated with the OS of BLCA as shown by K-M survival analysis ( p  < 0.05). Based on the expression of ICGs, the consensus clustering analysis was performed to classify BLCA patients into three clusters. The prognosis of patients in cluster C was significantly better than that in clusters A and B. Stratified analysis of BLCA infiltrated immunocytes revealed innate immune cell infiltration, including the presence of DCs, macrophages, NKs, neutrophil, along with activated B, CD 4 and CD 8 T cells, was abundant in cluster C. It is known that resident innate immune cells provide anti-tumor activity by detecting tumor associated antigens (TAAs) and damage associated molecular patterns (DAMP), which is of vital importance in cancer immune editing [ 31 ]. DCs are the most important antigen-presenting cells, and can process TAA and DAMP and present them to cytotoxic T lymphocytes (CTLs), causing activation and subsequent tumor infiltration [ 32 ]. NK cells can regulate the immune surveillance and clearance of malignant cells, and their presence in the tumor microenvironment is related to better survival and lower progression rates of several solid tumors [ 31 , 33 ]. Besides, an increasing body of evidence suggests that the density of infiltrating T cells and B cells have a positive association with longer OS or disease-free survival (DFS) [ 34 , 35 , 36 ]. Consistently, cluster C was significantly enriched in immune mechanism related pathways. Accordingly, we suspected that the infiltration of immune cells and the activation of immune pathways are important reasons for cluster C to gain survival advantage. To investigate the molecular differences among different clusters, we further identified the DEGs between the different clusters and identified 526 overlapping DEGs. Based on the results of GO and KEGG analyses, we’re convinced that ICGs could increase the activity of the immune system to promote immune-mediated elimination of tumor cells.

Existing literature mainly focuses on the study of single immune checkpoint inhibitors, but there is a lack of research on the gene expression profiles of multiple immune checkpoints. To comprehensively and systematically quantify the pattern of genetic modification of ICGs in individual tumors, we performed the LASSO and Cox analysis of the ICGs genes to establish an independent prognostic model which was well validated in the total and testing cohorts. The ICG signature was further identified as a stable and universally applicable prognostic assessment instrument for BLCA by the ROC curve analysis. Base on the ICG signature and other significant factors for prognosis, we structured nomogram to predict the 1, 3, and 5 year survival rates of BLCA patients.

In our signature, we found 5 of the 9 genes were inflammation related gene or antigen presenting related genes, functioning as significant components in inflammatory process of tumor initiation and progression. Because of the significant and strong association with the infiltrating immune cells, two genes ( CD3D and GBP4 ) were paid special attention. Stratified analysis of BLCA immune microenvironment revealed a significant positive correlation between CD3D and GBP4 and antitumor immune cells (CD4 T cells, CD8 cells and macrophages M1) [ 36 , 37 ], which was consistent with the risk coefficient of these two genes. The delta subunit of the CD3 complex encoded by CD3D gene is involved in T-cell development and signal transduction, which is essential for the initiation of antigen-specific T cell responses to pathogens, autoantigens and tumors [ 38 ]. CD3D defects could result in severe combined immunodeficiency autosomal recessive T-cell-negative/B-cell-positive/NK-cell-positive [ 39 ]. Besides, the expression of CD3D was consistently associated with both RFS and OS of breast cancer (BC), and was also found to be related to the response to neoadjuvant chemotherapy especially in estrogen receptor negative breast cancer, thus potentially identifying individuals responding to chemotherapy [ 40 , 41 ]. Five members ( GBP1 , GBP2 , GBP3 , GBP4 and GBP5 ) of the Guanylate-binding proteins (GBPs) family are the most abundant cellular proteins in response to IFN-γ, suggesting that GBPs may be important components of the innate immunity [ 42 , 43 ]. Increasing evidence suggests that TMB serves as a clinically valuable biomarker for identifying patients who may benefit from immune treatment [ 8 ]. In this study, we found that the frequency of tumor mutation in high-risk group was higher than that in low-risk group, and TP53 was the most common mutation gene. The wild-type TP53 protein plays a crucial role in cell apoptosis and cell cycle regulation following DNA damage. Cells with TP53 mutations can evade apoptosis after DNA damage and transform into cancer cells. Additionally, p53 loss or mutation in cancers can disrupt myeloid and T cell function, enabling immune evasion and advancing cancer progression [ 44 ]. The accumulation of mutant TP53 protein is considered a highly specific marker for malignant tumors [ 45 ]. Therefore, we speculate that the shorter OS and “cold” tumor microenvironment in high-risk BLCA patients may partly be attributed to the heightened TP53 mutations, which exert a specific inhibitory effect on the cancer-associated immune system. However, the mechanisms by which TP53 mutations impact the immune phenotype and prognosis of BLCA require further investigation.

Further investigation on the correlation between infiltrating immunocytes and risk scores revealed that risk scores had positive correlations with macrophages M2, and risk scores had negative correlations with Macrophages M1 and T cells CD8. The infiltrating macrophages in tumors are referred to as tumor-associated macrophages, with their activated forms primarily including two types: M1-type and M2-type macrophages. M1-type macrophages could enhance T cell effector functions through releasing pro-inflammatory signals and presenting antigens. Conversely, M2 macrophages possess immunosuppressive traits, dampening effector T cell proliferation. Consequently, the polarization of M1 macrophages towards the M2 phenotype within the tumor microenvironment plays a critical role in fostering tumor immunosuppression and enabling evasion from immune surveillance, thereby indicating a bleak outlook for cancer therapy [ 46 ]. Together, the above results indicate that patients at high risk often exhibit an immunosuppressive “cold” tumor microenvironment, elucidating their resistance to immune checkpoint therapy.

Given that high tumor mutation burden has been recognized as a predictive biomarker of immune checkpoint blocking response [ 47 ], we explored whether the developed ICG signature could stratify patients according to the sensitivity of patients to ICIs. Our findings showed that three hot immune checkpoint genes (PD-L1, PD-1, and CTLA4) were significantly downregulated in high-risk group, indicating that the signature could provide new ideas for drug design of immune checkpoints. Up to now, cisplatin-based combination chemotherapy regimens, such as methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC), as well as gemcitabine and cisplatin/carboplatin (GC), remain the first-line treatment standard for locally advanced or metastatic urothelial carcinoma, but the optimal chemotherapy regimen has not been definitively established. In this scenario, immunotherapy utilizing checkpoint inhibitors is the preferred second-line option, but paclitaxel or docetaxel can be utilized as second- or later-line chemotherapy and have demonstrated significant and potentially curative effects in bladder cancer [ 19 , 20 ]. In cases where patients are ineligible for immunotherapy or have received checkpoint inhibitors, docetaxel may still be considered as salvage therapy. Several Phase II trials have confirmed the effectiveness of docetaxel in treating metastatic bladder cancer [ 48 ]. Additionally, paclitaxel has shown activity as a standalone agent in urothelial cancer, achieving a 42% response rate in advanced and/or metastatic bladder cancer according to a Phase II trial [ 49 ]. Our study revealed that the ICG signature we developed could directly predict the immunotherapy and chemotherapy response, and facilitate the selection of therapy medications and assessment of treatment efficacy, which may guide more precision therapy for BLCA.

KRT23 was identified as the most critical gene by random forest analysis in this signature. As a newly discovered member of the keratin family, KRT23 belongs to the acidic type I keratins [ 50 ]. Many studies showed that KRT23 expression in tumors has been associated with increased aggressiveness and decreased patient survival in many kinds of cancer, including pancreatic cancer, colorectal carcinoma, breast cancer and hepatocellular carcinoma [ 51 , 52 ]. In ovarian cancer, KRT23 upregulation promoted migration and invasion of cancer cells via EMT by regulating the TGF-β/Smad signaling pathway. Our results demonstrated that KRT23 knockdown impaired proliferation, migration and invasion, and induced the apoptosis of BLCA cells. Previous research has found that KRT23 knockdown increased the secretion of CD8 + T cell-related chemokines (CCL5, CXCL9, and CXCL10), and subsequently promoting the recruitment of CD8 + T cells [ 53 ]. Consistent with this, we observed an inverse correlation between the expression level of KRT23 and the abundance of T cell infiltration. Tumors are typically classified into two categories based on T cell infiltration within the TME: non-inflamed tumors (lacking T cell infiltration) and inflamed tumors (with T cell infiltration) [ 54 ]. Immunotherapy, including immune checkpoint inhibitors (ICIs) such as anti-PD-1, anti-PD-L1 and anti-CTLA-4 therapies, is generally ineffective in non-inflamed tumors due to the absence of T cell immunity. Consequently, inflamed tumors generally respond better to immunotherapy, including ICIs [ 55 , 56 ]. In light of these discoveries, KRT23 emerges as a promising biomarker for predicting immune cell infiltration in tumors and developing novel immunotherapy strategies. However, further biological experiments are needed to clarify the regulatory mechanism and to promote the signature as new anticancer drug targets.

Taken together, this study systematically and comprehensively analyzed the expression profile of immune checkpoint genes in BLCA and their correlation with tumor mutation. In addition, we established the ICG signature to investigate the differences in immune checkpoint expression and tumor immune microenvironment, which will help risk stratification and accelerate precision medicine. Finally, we identified KRT23 as the most critical model gene and highlighted it as a potential target to enhance immunotherapy against BLCA.

Materials and methods

Data source and preprocessing.

Overall RNA sequencing data and clinical information of BLCA patients and normal human bladder samples were obtained from the TCGA data portal ( https://tcga-data.nci.nih.gov/tcga/ ) and GEO database ( https://www.ncbi.nlm.nih.gov/geo/ , ID: GSE13507 and GSE31684). The patient characteristics of the collected patients in TCGA and GEO database were shown in Supplementary Table S3 . Processed single-cell RNA sequencing data came from tumor immune single-cell hub 2 (TISCH2) database ( http://tisch.comp-genomics.org ) based on GSE149652 data from the GEO database [ 57 ]. Moreover, somatic mutation data are downloaded from TCGA database and presented with the mutation landscape by “maftools” package. The Wilcoxon test was applied to identify differentially expressed ICGs. Then, we used univariate Cox regression analysis and Kaplan-Meier survival estimates to extract ICGs associated with prognosis using the R packages ‘survival’ and ‘survminer’ (p-value < 0.05). The correlation of ICGs were visualized by the “igraph” package in the R software ( https://www.r-project.org/ ).

ICG clusters

We screened and identified 68 immune checkpoint genes with the key words of immune checkpoint, immune checkpoint inhibitors, immune checkpoint blockade, and checkpoint blockade in the Pubmed database ( https://pubmed.ncbi.nlm.nih.gov/ ). All of the ICGs were shown in Supplementary Table S4 . We merged BLCA samples of TCGA, GSE13507 and GSE31684 datasets into one train ( n  = 664) to analyze the prognostic value of the ICGs for BLCA. After merging the GEO and TCGA databases, we found that the remaining 53 ICGs were expressed in different datasets. According to 53 ICGs expression level, we performed unsupervised cluster analysis by R package “Consensus Cluster Plus” to explore the underlying molecular subtype between the BLCA patients [ 58 ]. The maximum cluster count (k) was set as 9, and the optimal cluster count value was chosen for further study. Kaplan Meier analysis was used for survival analysis and the Log rank analysis for comparison to evaluate overall survival (OS) differences with ‘survival’ package in R. Principal component analysis (PCA) was displayed to visualize the similarities and differences among grouped clusters. We followed the methods of Ninghong Song1 et al. 2022 [ 59 ].

Gene set variation analysis (GSVA)

GSVA was used to assess changes in pathways and biological process movement among different clusters. We downloaded the “c2.cp.kegg.v7.4.symbols” gene set from the molecular signatures database for GSVA. The adjusted p value less than 0.05 is considered statistically significant.

Distinguish the differentially expressed genes (DEGs) related to ICGs

The differentially expressed genes among different clusters were analyzed with “limma” package, and the venn diagram of the overlapped genes between each cluster was draw by “VennDiagram” package. The significance criteria for selecting differentially expressed genes related to ICGs was set as an adjusted p  < 0.05 and the value of |logFC| > 1. To further explore the biological processes and signal pathways of the intersection genes, Gene Ontology (GO) functions of the overlapping genes and enhanced the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were analyzed with the “clusterProfiler” package.

Establishment and validation of the prognostic signature

The relationship between the intersection DEGs and survival was explored by the univariate Cox analysis. The cut-off p -value was set to 0.05, and 127 intersection genes related to survival were screened for further study. All BLCA patients ( n  = 664) were randomly divided to a training cohort ( n  = 332) (50% for identifying the prognostic model) and a testing cohort ( n  = 332) (50% for validating the prognostic model). The LASSO Cox regression model was utilized to minimize overfitting between survival-related genes and to obtain a more concise prognostic genes combination by “glmnet” package. Finally, we construct the 9-gene risk signature based on multivariate Cox regression, and calculated the risk score by multiplying the expression level of each gene by the corresponding regression coefficient. The BLCA samples were assigned into high- and low-risk groups according to the median risk score. The area under the ROC curve (AUC value) obtained from the R package “timeROC” was used to evaluate the diagnostic efficacy of prognostic model. The waterfall map of high- and low-risk group mutation landscape was presented by maftools function. The immunohistochemistry (IHC) staining images of risk genes in normal and BLCA tissues using the same antibodies were analyzed and obtained from the Human Protein Atlas (HPA) database ( http://www.proteinatlas.org/ ).

Assessment of tumor immune microenvironment

CIBERSORT was applied to evaluate the immunocyte infiltration levels of BLCA, and the ESTIMATE algorithm was used to evaluate the immune scores and the stromal scores. We performed correlation analysis on the ICG signature to further reveal the connection with immune cells. In addition, the most common immune checkpoint genes, such as PD-1, PD-L1 and CTLA4, were extracted and evaluated the relevance with ICG signature in TCGA-BLCA dataset.

Drug sensitivity

Using “pRRophetic” package in R, we estimated the chemotherapeutic response determined of the common anticancer drugs by the half-maximal inhibitory concentration (IC50) and compared the difference of chemosensitivity between the high- and the low-risk group.

Cell culture, RT-PCR and siRNA transfection

The immortalized cells of human bladder epithelium (SV-HUC-1) and human BLCA cell line (T24, UMUC3 and J82) were cultured in Ham’s F-12 K (Gibco/BRL, Grand Island, NY), RPMI-1640 medium (HyClone, Logan, UT, USA) and MEM (Gibco/BRL, Grand Island, NY) supplemented with complete medium (10% fetal bovine serum) at 37 °C and 5% CO2. Total RNA was isolated using TRIzol reagent (Invitrogen, Frederick, MD, USA), and cDNA was synthesized using standard techniques. Quantitative RT-PCR analyses were performed using SYBR Green Fast qPCR Mix (ABclonal, RK21203, Wuhan, China) on Prism ® 7500 (Applied Biosystems, California, USA). The KRT23 primers were 5′- CCATGCAGAATCTCAACGAC.

-3′ (sense) and 5′- GGTGTGTGATGTTTTCCTCA-3′ (antisense). The GAPDH primers were 5′- GATGGAGGAGGCTCAGCA − 3′ (sense) and 5′- CTCAGCCAATGGGACCTG − 3′ (antisense).

The sequences of KRT23-target-specifc-siRNA were as follows: siRNA1: 5′-AUGAGAAUGAGCUCUUUCCUC-3′(sense), 5′-GGAAAGAGCUCAUUCUCAUGA-3′(antisense); siRNA2:5′-GGAGGAUAUGAGACAAGAAUA-3′(sense), 5′-UUCUUGUCUCAUAUCCUCCAG-3′(antisense). The shRNA expression vector targeting KRT23 was synthesized based on the sequence of siRNA2. The INTERFERin (Polyplus, Illkirch-Graffenstaden, France) was used to transfect siRNA and vector into cells according to the manufacturer’s instructions.

Proliferation studies

2000 cells per well of T24 and J82 cell lines were seeded in 96-well plates after KRT23 siRNA transfection. The cell viability was determined at 12 h, 24 h, 48 h, 72 h and 96 h. T24 and J82 cell suspension with logarithmic growth phase was inoculated with 1000 cells/well in a 6-well plate. After 2 weeks, the cells were fixed with 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet for 20 min.

Wound scratch assay and transwell assay

After transfected with KRT23 siRNA, the 200 µL eppen-dorf tip was used to scratch the cell plate. The complete medium was then replaced with serum-free medium, and cell migration was observed at 0 h and 48 h with an inverted microscope.

Migration and invasion of T24 and J82 cells was analyzed through the Transwell assay. 3 × 10 4 cells in medium without serum were seeded in the upper chamber of the Transwell apparatus without/with Matrigel (ABW, 0827045, Xiamen, China). Then 500 µL medium with 10% fetal bovine serum (FBS) was added to the lower chamber. The membrane of chamber was fixed for 30 min and stained with 0.1% crystal violet after 24 h.

Apoptosis assay

Apoptosis of T24 and J82 cells were detected using the Annexin V-FITC apoptosis detection kits (Elabscience, E-CK-A211, Wuhan, China). 1 × 10 5 cells in 500 µL1× binding buffer were labeled with Annexin V-FITC (5 µL) and propidium iodide (PI) (5 µL) for 15 min at room temperature in the dark. Specimens were analyzed with a Accuri C6 Plus flow cytometer within 1 h of labeling.

Western blotting

Western blotting was performed as previously studies described [ 60 ]. The following antibodies were used: anti-KRT23 (Abcam, ab156569, 1:1000 dilution) and anti-GAPDH (ABclonal, a19056, 1:50000 dilution).

Tumor tissue preparation

we collected six fresh bladder cancer samples and their paired adjacent normal samples from BLCA patients who underwent radical cystectomy at Beijing Chaoyang Hospital Affiliated Capital Medical University, without any preoperative treatment. The study received approval from the Medical Ethical Committees of the Beijing Chaoyang Hospital Affiliated Capital Medical University. All patients were informed about this study.

Tumor xenograft assay and immunohistochemistry staining

Six-week-old female BALB/c nude mice (Vital River Laboratories, China) were randomly divided into two groups ( n  = 4 per group). Next, 1 × 10 7 KRT23 knockdown (shKRT23 group) and negative control (shCtrl group) T24 cells were subcutaneously injected into the right flank of two groups of mice. All animals were deeply anesthetized before euthanasia to ensure they were unconscious and did not experience pain. All animals were deeply anesthetized before euthanasia to ensure they were unconscious and did not experience pain. Isoflurane was used as the anesthetic, administered through inhalation by placing the animals in an induction chamber and exposing them to 4–5% isoflurane in oxygen for 2–3 min until deep anesthesia was achieved. After confirming deep anesthesia, euthanasia was performed using cervical dislocation, a method that is quick and ensures a humane death. All experimental procedures were approved by the Animal Ethics Committee of Beijing Chaoyang Hospital, Capital Medical University.

IHC staining for PCNA (Boster, BM0104, 1:200 dilution) and ki67 (Abcam, ab16667, 1:500 dilution) was conducted following the manufacturer’s instructions as described in previous study [ 61 ].

Statistical analysis

The Student’s t test was utilized for statistical comparison of paired data, while the ANOVA test for comparison of more than two groups and Pearson’s chi-square tests for comparison of categorical variables. Logarithmic rank test was used to determine the significance of differences in survival curves. Statistical significance was set at p  < 0.05.

Data availability

The datasets analyzed during the current study are available in the TCGA data portal ( https://tcga-data.nci.nih.gov/tcga/ ), GEO database ( http://www.ncbi.nlm.nih.gov/geo , ID: GSE13507 and GSE31684), HPA database ( http://www.proteinatlas.org/ ) and TISCH2 database ( http://tisch.comp-genomics.org/home/ ).

Abbreviations

Bladder cancer

• Immune checkpoint inhibitors

Immune checkpoint genes

Non-muscle-invasive bladder cancer

Muscular invasive bladder cancer

Food and Drug Administration

Tumor immune microenvironment

Principal component analysis

Gene set variation analysis

Differentially expressed genes

Kyoto Encyclopedia of Genes and Genomes

Gene Ontology

Half-maximal inhibitory concentration

Copy number variation

Guanylate-binding proteins

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Dongshan Chen, Haoyuan Cao and Xiang Zheng have contributed equally to this work and share first authorship

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Department of Urology, Beijing Chaoyang Hospital Affiliated Capital Medical University, 8 Gong Ti Nan Road, Chaoyang District, Beijing, 100020, China

Dongshan Chen, Haoyuan Cao, Xiang Zheng, Haojun Wang & Wei Wang

Department of Urology, Qilu Hospital of Shandong University, Wenhuaxi Road #107, Jinan, 250012, China

Dongshan Chen

Department of Urology, The Second Affiliated Hospital of Shandong University of Traditional Chinese Medicine, NO.1 Jingba Road, Shizhong District, Jinan, 250001, China

Zengchi Han

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WW and HZC conceived of and directed the project. CDS, ZX and CHY designed the study. CDS and CHY performed the experiments. WHJ, ZX and CDS collected and analyzed the data. All the authors listed have approved the manuscript that is enclosed for publication.

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Chen, D., Cao, H., Zheng, X. et al. Immune checkpoint gene signature assesses immune infiltration profiles in bladder cancer and identifies KRT23 as an immunotherapeutic target. BMC Cancer 24 , 1024 (2024). https://doi.org/10.1186/s12885-024-12790-w

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• Systematic Map Protocol
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What evidence exists relating the impact of different grassland management practices to soil carbon in livestock systems? A systematic map protocol

• Camille Rousset   ORCID: orcid.org/0000-0002-9251-3959 1 ,
• Carmen Segura 2 , 9 ,
• Anina Gilgen 1 ,
• Marta Alfaro 3 ,
• Luís André Mendes 4 ,
• Mike Dodd 5 ,
• Batnyambuu Dashpurev 6 ,
• Mike Bastidas 7 ,
• Julian Rivera 8 ,
• Lutz Merbold 1 &
• Eduardo Vázquez 4  

Environmental Evidence volume  13 , Article number:  22 ( 2024 ) Cite this article

Metrics details

Grasslands are essential for providing vital resources in the livestock sector and delivering invaluable ecosystem services such as biodiversity and soil carbon (C) sequestration. Despite their critical importance, these ecosystems face escalating threats from human disturbances, human degradation, and climate change, compromising their ability to effectively stock C. Restoring degraded grasslands emerges as a pragmatic and cost-effective approach to tackling climate change. However, the successful implementation of grassland management toward this goal, faces significant challenges. A systematic mapping approach will help to compile a comprehensive global inventory of studies investigating the impact of differing grassland management practices on soil carbon. In addition, the potential for trade-offs with other greenhouse gas emissions further underlines the value of a systematic assessment. This approach aims to identify knowledge clusters (i.e., well-represented subtopics that are amenable to full synthesis) for potential systematic reviews and pinpoint knowledge gaps requiring further primary research efforts, all contributing to a better understanding of the evidence surrounding this topic.

Following systematic evidence synthesis standards, we developed the question to address in the systematic map protocol using the PICO framework. We established a preliminary search string by combining search terms for the Population (Grasslands), Intervention (management) and Outcome (soil carbon) categories, as well as with one additional group (Study types—to focus on farm and field experiments). We will conduct a comprehensive literature search of relevant peer-reviewed and grey literature using Web of Science, Scopus, CABI platforms, Google Scholar, and specialised websites (e.g., Agrotrop). Searches will be conducted in the English, Spanish, Portuguese, French, German, and Mongolian languages, as per the linguistic capabilities of the research team. The comprehensiveness of the search will be assessed by comparing the literature collected to a test-list of forty relevant articles. The repeatability of the literature screening process will be ensured by a list of inclusion/exclusion criteria and inter-reviewer consistency statistical tests. Data extraction will be organised into four complementary sections (article information, PICO categories, study characteristics, measurable parameters), on which we will perform queries to produce the tables, figures and evidence maps that will compose the systematic map. The results will identify and describe knowledge gaps and clusters.

Globally, grasslands cover about 40% of the Earth’s ice-free surface, and their importance for human livelihood is primarily related to fodder and forage production for the livestock sector [ 1 , 2 , 3 ]. Although livestock production is viewed with increasingly criticism given its greenhouse gas (GHG) emissions, grasslands also contribute to global food and nutrition security [ 4 ], rural livelihood, and supply valuable ecosystem services across a wide range of geological and climatic conditions [ 5 , 6 ]. Grasslands are known to help maintain above and belowground biodiversity, filter water and combat erosion [ 5 , 6 ]. Given that up to 30% of the Earth’s terrestrial C is stored in grasslands [ 7 ], they play a key role in climate regulation; therefore, grassland management strategies have received increasing national and global interest as potential pathways for sequestering C [ 8 , 9 , 10 , 11 ].

Soil C stocks (referring to the mass of carbon in a sample of known bulk density for a nominated depth and commonly restricted to the fraction < 2 mm in size [ 12 ]) under grasslands are very vulnerable to disturbance and degradation from human management [ 13 , 14 ]. These ecosystems can become sinks or sources of CO 2 , depending on multiple variables [ 13 , 15 , 16 ]. Soil organic carbon (SOC) exhibits temporal and spatial variability, being constantly accumulated, decomposed, and mineralized. Although both SOC and soil inorganic carbon (SIC) are present in soils, this paper emphasizes SOC, as it is the primary focus in both scientific and political discussions. Over the past decades, the total area of grassland has decreased while that of arable land has increased, suggesting ongoing conversion of grasslands to croplands [ 17 ]. Concurrently, close to 50% of the world's natural grassland has experienced various degrees of degradation [ 18 ]. Meanwhile, the demand for livestock products, continues to increase, especially in emerging-economy countries [ 19 ]. Less grazing area for greater forage demand has led to an increase in grazing intensity [ 1 , 16 ], ensuing that a noticeable fraction of the world’s grasslands is hosting a livestock population that exceeds local carrying capacity [ 20 ]. This overgrazing as well as other unsustainable grassland management practices (e.g., excessive fertilisation of meadows or biomass removal), can have serious negative impacts for the environment, including negative effects on soil C storage [ 1 , 10 , 13 , 15 ]. A better understanding of the effect of different grassland management practices on soil C stocks can help guide efforts to reverse the grassland degradation trend and preserve the important soil C stocks of the world’s grasslands [ 1 , 13 , 15 , 16 ].

Considering the large area covered by grasslands globally and that approximately 50% of the area is degraded [ 14 ], the potential for C sequestration (referring to the process of capturing and storing atmospheric carbon dioxide in soils) by restoring grasslands offers a very cost-efficient nature-based solution to mitigate climate change [ 12 , 21 ]. Several reviews and studies have shown the potential for changes in grassland management practices—such as rotational grazing, fertilisation, sowing legumes and improved forages or the establishment of silvopastoral systems—to restore degraded grasslands and increase their soil C stocks [ 10 , 11 , 15 ]. In the context of this study, grassland management refers to a range of human-directed practices implemented with the aim of restoring grasslands while simultaneously enhancing forage quantity and quality, as well as soil C stocks. These may be considered as alternatives to current or locally conventional management practice recognised as leading to degradation. Implementing these strategies on a regional and global scale remains problematic because of four main challenges (Fig.  1 ): (i) a lack of robust evidence regarding the effect of grassland management practices on soil C stocks, (ii) uncertainties caused by variation in SOC measurements and the complexity of interactions between edaphoclimatic conditions and management practices, (iii) understudied potential trade-offs with other greenhouse gas (GHG) emissions and other environmental consequences resulting from these practice changes, and (iv) the technical and economic constraints faced by farmers, limiting their adoption [ 10 , 13 , 22 ].

Challenges in implementing grassland management strategies at a global scale: Unveiling critical questions for systematic inquiry. The questions to be addressed in the systematic map are underlined in red.

Despite a growing number of reviews and articles on the subject, there is still limited evidence that implementing certain promising practices, such as silvopastoral systems or sowing improved forages or legumes, is positive for increasing soil C stocks in grasslands. For example, the meta-analysis on the topic performed by Conant et al. [ 10 ] included only one, two and seven studies focused on these practices, respectively. In addition, the geographical distribution of evidence supporting the benefit of most of these practices is not evenly distributed. Africa and Asia are under-represented compared with Europe, North America, South America and Oceania [ 10 ]. This is particularly problematic given that Africa and Asia have the highest estimates for potential C storage through grassland restoration [ 13 ]. In the context of a research system where modelling studies are increasingly used, the field studies evidence becomes more critical to address substantial uncertainties in the models [ 8 ].

The second challenge is the large uncertainty in the predicted soil C sequestration potential of various grassland management practices on a global scale, as their effects are highly context dependent [ 10 , 13 , 23 ]. The C sequestration potential of specific practices will depend on climate, soil characteristics, vegetation (i.e. species composition, presence/absence of C3 or C4 grasses, biological nitrogen fixers etc.), intensity of biomass removal, as well as animal stocking densities and the ingested amount of biomass produced [ 7 , 8 , 24 , 25 , 26 ]. This explains why the study scale is often limited to local or subregional scales [ 23 , 27 , 28 , 29 , 30 ], and thus, upscaling to regional and global scales remains challenging [ 31 ]. In addition, the characteristics of management practices are highly site-specific, since what is considered conventional in one situation may be considered alternative in another. Environment and discourse situations can lead to variations in terminology. As an example, the term “silvopastoral systems” encompasses the dehesa or montado ecosystems of Spain and Portugal, as well as the high-density silvopastoral systems planted with rows of Leucaena trees in tropical regions, although their functionality and C sequestration potential are very different [ 29 , 32 , 33 ]. The name used to designate certain practices may also vary from one region or author to another, as there is no scientific standardisation for the name or definition of the practices. This can be the case for rotational grazing strategies which are referred to as “rotational grazing”, “regenerative rotational grazing”, “intensive short-duration rotational grazing”, “cell grazing”, “technograzing”, “mob grazing” or “multi-paddock grazing”, even though the intent of these practices is similar [ 27 , 34 , 35 , 36 ]. This lack of category uniformity needs to be remedied. It is also essential to note that despite the recent efforts made by international organisations to standardise the methods used to assess changes in soil C stocks [ 31 , 37 ] there are still significant divergences in measuring, reporting and verifying these changes, which is also a source of uncertainty.

The third challenge is a better understanding of the potential trade-off with other GHG emissions of soil C sequestration and the proposed changes in management practices. While increased soil C stock resulting from changes in grassland management is generally associated with positive environmental outcomes (C sequestration, improved soil health, climate resilience, among others), it is essential to acknowledge potential challenges and negative consequences. For instance, management strategies affecting SOC cycle are likely to affect soil nitrous oxide (N 2 O) emissions as C and N biogeochemical cycles are strongly interlinked [ 38 , 39 ]. This potential trade-off could offset the climatic benefits of increased soil C stocks. Although many studies and meta-analyses have evaluated the impact of increased soil C on GHG emissions for croplands [ 38 , 39 , 40 , 41 ], studies evaluating the impact of grassland managements on GHG emissions are rare, with the exception of those evaluating the impact of N fertilisation on N 2 O emissions [ 42 , 43 ]. A compilation of the evidence could help to identify clusters of knowledge for certain practices and gaps of knowledge that require further empirical research.

The final challenge to the overall implementation of different grassland management practices lies in the technical and economical constraints that may limit adoption by many farmers [ 10 , 13 , 22 ]. The development of policies and extension services to promote those practices as well as to address the adoption barriers are thus necessary [ 10 ]. Policymakers and stakeholders usually require a comprehensive body of robust evidence to support the development of policies focused on the promotion of grassland management practices that will achieve restoration goals [ 44 ].

Addressing these four challenges (Fig.  1 ), encourages a detailed collation of the available evidence as well as the development of a comprehensive and systematic database considering the types of management practice being studied, and the contexts in which each study has been carried out. This may help to reduce uncertainties about the potential for soil C enhancement in grasslands in certain regions and potential trade-offs, but also highlight other regions or types of practices where evidence is scarce and further studies are needed. To the best of our knowledge, there is currently no comprehensive, systematic, and updated assessment of the existing evidence on the impact of differing grassland management practices on soil C stocks. The review by Conant et al. [ 10 ] is the most comprehensive analysis of existing evidence on the topic to date. However, a sufficient number of articles have been published since this 2017 review, justifying the need for a systematic re-evaluation of the evidence up to the present time. Our objective in this work is not to ascertain the directional impact (positive or negative or neutral) of a practice on soil C. Instead, our focus is on cataloguing the various practices that have been subject to study. Therefore, our approach is a qualitative examination rather than a quantitative assessment of the effects of these practices on SOC.

Objectives of the review

The impacts of grassland management on soil C have been subject to several conventional literature reviews [ 11 , 13 , 15 , 16 , 45 ]. To our knowledge, no study has evaluated these impacts systematically. Therefore, the objective of the forthcoming systematic map is to fill this gap of knowledge, answering the following question:

“What evidence exists relating the impact of different grassland management practices to soil carbon in livestock systems?”

This systematic map aims to (i) identify a comprehensive list of studies around the globe, on the impact of different grassland management practices on soil C, (ii) supply a robust database of studies with their main descriptive information (meta-data) and to ensure open access availability of this resource, (iii) generate an evidence map indicating the location of the existing studies, (iv) create a list of knowledge clusters [ 46 ] of management practices and regions well studied that would be suitable for a full systematic review, and (v) create a list of knowledge gaps of practices and regions underrepresented in studies that warrant further primary research effort. All these outputs will help to understand the existing evidence relating the impact of grassland management practices on soil C.

The PICO (Population, Intervention, Comparison, Outcome) method, a structured approach used in literature reviews to formulate clear research questions, will be used with the following key elements:

Managed permanent grasslands and pastures (including silvo-pastoral systems) distributed worldwide. Unmanaged grasslands not subject to animal husbandry are not included.

Intervention

Any grassland management practices used as alternative to a locally standard/conventional/nominal/common practice adopted in each study for the purpose of restoring grasslands while maintaining or improving forage quantity and quality and/or soil C stocks.

The standard/nominal/common local management practices used for comparison in each study.

Soil C stocks, concentration and related terms (Table  1 and Table S3). While not every article specifically addresses soil C stocks, many studies measure related measurements such as SOC concentration or total C. Similarly, some studies refer to soil health or soil quality, which include different soil properties such as carbon storage. Consequently, we have opted for a broader terminology in our search string to comprehensively cover all aspects of C change related to management practices documented in the literature.

This systematic map has been designed based on the Collaboration for Environmental Evidence Guidelines and Standards for Evidence Synthesis in Environmental Management [ 47 ]. The preparation of this systematic map protocol has been made following the ROSES reporting standards [ 48 ].

Searching for articles

While the primary focus will be on peer-reviewed articles, other forms of literature such as book chapters or conference proceedings will also be considered. The year of publication will not be restricted.

Academic bibliographic databases

The academic bibliographic databases used for this study are listed in Table  2 . The literature search will be mainly done using the subscriptions of the Swiss Consortium. The search string was built up in agreement by all authors of the study. The search string was designed to include three substrings representing the population , intervention and outcome of the PICO question and connected by the Boolean operator “AND” (Table  1 ). The search strings will be adapted to the syntax requirements of each database (Table  2 ). The searches in bibliography databases will be performed in English using the refinement criterion ‘Language’ in the academic databases. Additionally, an initial screening process will be performed on the academic bibliographic platforms to eliminate articles that are clearly irrelevant based on their subject categories. For example, certain articles may have been categorised under art, sociology, or veterinary topics subsequent to the primary search.

To ensure that the systematic map remains up-to-date, several strategies for updating searches will be implemented. First, regular searches will be conducted in relevant databases and repositories to identify newly published literature related to the impact of grassland management practices on soil C. These searches will be conducted at predefined intervals throughout the systematic mapping process. Additionally, automated alerts will be set up in databases and search engines to notify the research team of any newly published articles that meet the inclusion criteria. These alerts will be tailored to specific search terms and filters used in the systematic map protocol.

Grey literature

The search for grey literature will be made using the Google Scholar engine which has been demonstrated to be a useful tool for identifying grey literature [ 49 ]. We will evaluate grey literature in five languages in addition to English, corresponding to the native languages of all contributing authors of this article. This approach aims to include studies that may have been overlooked during the English-language academic database search. Therefore, simplified search strings in English, Spanish, French, German, Mongolian and Portuguese will be used (Table S1). In the case of a very large number of matches, the analysis will be limited to the first 500 hits. We acknowledge the limitation of using only Google Scholar for this grey literature search, as it may not capture all types of grey literature for the different languages tested.

Supplementary searches

The protocol preparation team may contact other relevant researchers and stakeholders to ask for additional literature related to the topic. Furthermore, the bibliographies of some selected papers (particularly reviews and meta-studies) relevant on the topic will be screened aiming to find relevant bibliographies (e.g. snowballing/backward citation searching). Theses and dissertation on the topic will be examined.

Estimating the comprehensiveness of the search

To assess the comprehensiveness of the search, we prepared a list of reference articles that were considered relevant for this systematic map based on the experience of the authors of this article (Table S2). We have checked whether the reference articles were captured by our search strategy both in academic bibliographic databases used and in Google Scholar. Using the mentioned search strings in Table  1 in a preliminary search, articles have been found in both the academic bibliographic databases as well as in Google Scholar, confirming the comprehensiveness of our search.

Article screening, study eligibility criteria and analysis

Both the article screening process and data extraction will be conducted using the Covidence software [ 50 ]. Covidence is a web-based collaboration software platform that streamlines the production of systematic and other literature reviews.

Screening process

Since we anticipate that various search databases may duplicate references, we will first eliminate duplicates using the Covidence software tool which identifies duplicates on the basis of the DOI or title. We will also classify the articles by authors to facilitate the identification of any duplicates that may have been overlooked by the software. Then, the studies selected will be screened to decide their inclusion within the systematic map based on the eligibility criteria described below. The screening will be conducted firstly using to the titles, keywords and abstract of the studies, and secondly, using the full-text. To ensure repeatability of the selection process during the title and abstract screening phases, a subsample of 200 articles will be randomly selected and independently reviewed and tested for consistency of selection by all members of the selection team. If applicable, disagreements in the eligibility criteria will be discussed and clarified to ensure the accurate repeatability of the screening process. This repeatability procedure will be reiterate with another random subset of articles until a repeatability of 95% among all the members is reached. Even when consistency will be sufficient (> 95%), reviewers will discuss and solve the remaining disagreements to ensure a high replicability. Special attention will be given to avoid situations where screening team members, who are authors of articles, review their own work. A similar methodology will be applied during the full-text screening phase, using a subsample adjusted to correspond to 5% of the number of articles validated during the title and abstract screening.

Eligibility criteria

We will screen the studies regarding the population, intervention, comparator, outcome, and study design listed in Table S3.

Eligible population

To be included in the systematic map, studies must evaluate permanent grasslands managed for livestock production (including those that have a woody vegetation component, such as silvopastoral systems). This excludes unmanaged grasslands without human intervention and grasslands managed for biofuel production or recreation. Managed natural grasslands with naturally occurring indigenous grass communities will be included. Grassland-cropping rotations or so called “temporary grasslands” [ 51 ] will be excluded, since the objective of the addition of a grassland phase within the crop rotation schemes is a means to mitigate the environmental impacts of intensive arable cropping systems. Likewise, forests including livestock grazing with the objective of benefitting the forest management as described by Öllerer et al. [ 52 ], will be excluded as the targeted grazing is a means for enhancing forest management rather than an end. Due to the unique carbon dynamics observed in wetlands and peatlands (or mires) in contrast to other grassland ecosystems, they will be excluded from our study [ 53 ]. Grazing systems dominated by woody species will also be omitted from the study (i.e. shrublands, scrublands).

Eligible intervention

As mentioned above, to be included in the systematic map, studies must evaluate any permanent grassland management practices used as alternatives to a locally standard/conventional/nominal/common practice adopted in the studies. These practices should aim to restore grasslands while maintaining or improving forage quantity and/or quality and/or soil C stocks. Whether or not the objective is achieved at the end of the experiment, will have no impact on the selection. This definition combines a variety of practices as the used permanent grassland management practice depends strongly on the context of application. Grassland management practices used with objectives other than livestock production or improving soil health, such as the reclamation of polluted soils or biomass production for energy, will not be included. It is important to emphasise that the management practise should be undertaken with the objective of maintaining or increasing grassland quality and yield, rather than facilitating supplementary planting. In the latter case, the grassland is considered as a service plant, and will not be included in the study. The implementation of grazing exclusion, whether through enhanced management practices such as meadow establishment or through temporary exclusion measures such as rest periods, will be included. However, studies focusing on abandoned grasslands will be excluded.

Eligible comparator

An eligible comparator includes any locally standard/conventional/nominal/common practice grassland used by the authors in the study as a comparator to a specific grassland management practice. We will only consider as a comparator those practices which investigate grasslands, i.e. other comparators such as arable cropping agriculture, forest, abandoned grasslands, park or urban soils will be excluded. In addition, we will consider as a comparator, those grassland management practices performed with a different intensity than the targeted intervention practice. If a study involves a comparison between a grassland system and a crop system (or forest or native ecosystems), along with a comparison to a silvopastoral system or another grassland management, it will be included based on the latter comparison.

Eligible outcome

To be included in the systematic map, the study must report either the soil C stock, C concentration or any other terms mentioned in Table S3, in their abstract, keywords or title. In soil science, agricultural and environmental studies, providing soil C stock, C concentration, or related metrics is common as a description of the soil of study site even when soil C is not the primary outcome objective [ 54 , 55 , 56 ]; however, for this study, we included only studies where soil C is one of the primary focuses, and should therefore be indicated in the abstract, keywords, or title, to keep the review manageable considering our available resources.

Eligible study design and other characteristics

We will include field studies under realistic conditions that provide experimental data. Mesocosm experiments (e.g., lab incubations or pot experiments) will be excluded unless they are part of a combined field study. In this study, modelling-only articles will be excluded from the selected list of publications. Models rely on various data inputs, and the quality and reliability of these inputs can vary widely among studies [ 57 ]. This variability can introduce uncertainty and affect the overall reliability of the systematic map's conclusions.

We will include both the control-impact studies (intervention vs comparator) and the before-after intervention studies (comparing soil carbon over time) if a control is provided to evaluate the change in the soil C. We will accept both studies performed on experimental plots as well as the comparison of real farms. We will not restrict the number of replicates and the study duration. In cases where two or more studies report the same data from the same experiment, we will choose one of the studies and exclude others, except if they provide complementary supporting environmental or management information about the site.

Study validity assessment

An assessment of study validity (in terms of the methods, assumptions or peer-review processes used) will not be performed because the objective of the systematic map is to compile all the existing evidence rather than to assess the quality or validity of the existing data [ 47 ]. However, during the extraction phase, data on the quality of the studies (e.g., study characteristics outlined in Table  3 ) will be gathered and subsequently reported in the systematic map. We will remain open to the possibility of incorporating a validity assessment, especially if deemed beneficial based on the quantity and nature of the studies included.

Data coding and extraction strategy

The meta-data will be extracted, organised and compiled in Excel files for all the relevant selected studies. The data extracted from the articles will be organised into four complementary sections (article information, PICO categories, study characteristics, measurable parameters, Table  3 ), on which we will perform queries to produce the tables, figures and evidence maps that will compose the systematic map. Some variables will be represented as multiple-choice options. The extraction, organisation and compilation will be performed by the co-authors in a subset of the relevant studies included (15 articles), to check the consistency between the co-authors in this process. We will report in the systematic map the repeatability of each of the extracted variables.

Study mapping, presentation and analysis

In the systematic map we will provide answers to the primary question developed in the present protocol and the secondary questions presented in Fig.  1 , in the form of descriptive texts supported by tables, figures and maps. As the table described in the data organisation strategy (Table  3 ) might be further developed during full-text assessment, we will provide all final table structures and contents in the upcoming publication, as well as the final database produced in the frame of this review. Descriptive statistics in R or other open-source software will be used to analyse the results. Among the results, we will include “heat maps” that cross-tabulate two variables and illustrate the quantity of evidence (number of studies) within each cell of the table. These heat maps will explore various combinations of variables, such as types of grassland management practices in relation to the study country or study variables (Table  3 ). Furthermore, an evidence atlas will be generated using study latitude and longitude meta-data and presenting studies on an interactive cartographic map. Detailed information about the articles included/excluded at the different stages of the screening process will be reported, in addition to any eventual modifications to the present protocol. Finally, the detection of knowledge gaps will involve the identification of subtopics that are either unexplored or underrepresented in the literature. For that objective, we will use heat maps or bar plots to highlight these gaps. Subtopics devoid of studies or a small number of studies will be documented. Similarly, the evidence clusters will be identified using heat maps and bar plots, and subtopics with more evidence available will be highlighted.

Availability of data and materials

Data sharing is not applicable to this protocol article as no datasets were generated or analysed during the current study.

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Agroscope co-authors would like to thank the Swiss National Science Foundation for its support of the CarboGrass project (Grant No. 31SL30_214523). Rothamsted Research co-author receives strategic funding from the Biotechnology and Biological Sciences Research Council of the United Kingdom (BBSRC). CS acknowledges support from the Growing Health Institute Strategic Programme [BB/X010953/1; BBS/E/RH/230003B; BBS/E/RH/230003C]. AgResearch co-authors are funded by the Ministry for Primary Industries (MPI), project number 2023-EJPSOILS-CarboGrass-AGR. UPM co-authors are funded by the Spanish Ministry of Science and Innovation through the project number PCI2023-143386. CIAT co-author is founded by The CGIAR Research Initiative on Livestock and Climate. CIPAV co-author is funded by the New Zealand Government to support the objectives of the Global Research Alliance on Agricultural Greenhouse Gases (GRA). KIT co-author is funded by the German Ministry for Education and Research (BMBF) (funding number 031B1396).

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Integrative Agroecology Group, Research Division Agroecology & Environment, Reckenholzstr. 191, 8046, AgroscopeZurich, Switzerland

Camille Rousset, Anina Gilgen & Lutz Merbold

Net Zero and Resilient Farming. Rothamsted Research - North Wyke. Okehampton, Okehampton, UK

Carmen Segura

AgResearch Ltd, Ruakura Research Centre, 10 Bisley Road, Hamilton, 3214, New Zealand

Marta Alfaro

Departamento de Producción Agraria, ETSIAAB, Universidad Politécnica de Madrid (UPM), Madrid, Spain

Luís André Mendes & Eduardo Vázquez

AgResearch Ltd, Grasslands Research Centre, Private Bag 11008, Palmerston North, 4442, New Zealand

Institute for Meteorology and Climate Research, Atmospheric Environmental Research (IMK-IFU), Karlsruhe Institute of Technology, Garmisch-Partenkirchen, Germany

Batnyambuu Dashpurev

International Center for Tropical Agriculture (CIAT), Km 17 Recta Cali-Palmira, Palmira, Colombia

Mike Bastidas

Centre for Research On Sustainable Agriculture (CIPAV), Cali, Colombia

Julian Rivera

Department of Soil Science and Agricultural Chemistry, University of Granada, Granada, Spain

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CR and EV jointly contributed to the conceptualization of the study and writing the manuscript. CR created the tables and figures presented. All authors collectively contributed to the development of the eligibility criteria section, with specific focus on refining Table S3. All authors provided feedback, which was reviewed and incorporated into the manuscript. All authors approved the original draft and reviewed manuscripts.

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The topic of this study was discussed and designed in response to a funding call proposed by the Second External Call of the European Joint Projects: soil, entitled “Managing and mapping agricultural soils for enhancing soil functions and services”. A consortium formed by scientific and researchers from ten institutions worldwide distributed applied to the Topic A “Soil restoration practices and management for alleviating land degradation, supporting agro-ecosystem function and maintaining soil organic carbon stocks” with a project proposal entitled “CarboGrass: Impact of grassland management on soil carbon storage.”

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Additional file 1. roses checklist for the systematic map., rights and permissions.

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Rousset, C., Segura, C., Gilgen, A. et al. What evidence exists relating the impact of different grassland management practices to soil carbon in livestock systems? A systematic map protocol. Environ Evid 13 , 22 (2024). https://doi.org/10.1186/s13750-024-00345-2

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Received : 25 March 2024

Accepted : 24 July 2024

Published : 24 August 2024

DOI : https://doi.org/10.1186/s13750-024-00345-2

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