Circular RNAs: Characterization, cellular roles, and applications.

Most circular RNAs are produced from the back-splicing of exons of precursor mRNAs. Recent technological advances have in part overcome problems with their circular conformation and sequence overlap with linear cognate mRNAs, allowing a better understanding of their cellular roles. Depending on their localization and specific interactions with DNA, RNA, and proteins, circular RNAs can modulate transcription and splicing, regulate stability and translation of cytoplasmic mRNAs, interfere with signaling pathways, and serve as templates for translation in different biological and pathophysiological contexts. Emerging applications of RNA circles to interfere with cellular processes, modulate immune responses, and direct translation into proteins shed new light on biomedical research. In this review, we discuss approaches used in circular RNA studies and the current understanding of their regulatory roles and potential applications.

Elements of successful NIH grant applications.

Is there a formula for a competitive NIH grant application? The Serenity Prayer may provide one: "Grant me the serenity to accept the things I cannot change, the ability to change the things I can, and the wisdom to know the difference." But how to tell the difference? In this Perspective, we provide an inclusive roadmap-elements of NIH funding. Collectively, we have over 30 y of peer review experience as NIH Scientific Review Officers in addition to over 30 y of program experience as NIH Program Officers. This article distills our NIH experience. We use Euclid's 13-book landmark, The Elements, as our template to humbly share what we learned. We have three specific aims: inform, guide, and motivate prospective applicants. We also address ways that support diversity and inclusion among applicants and young investigators in biomedical research. The elements we describe come from a wide range of sources. Some themes will be general. Some will be specific. All will be candid. The ultimate goal is a competitive application, serenity, and hopefully both.

Web-accessible application for identifying pathogenic transcripts with RNA-seq: Increased sensitivity in diagnosis of neurodevelopmental disorders.

For neurodevelopmental disorders (NDDs), a molecular diagnosis is key for management, predicting outcome, and counseling. Often, routine DNA-based tests fail to establish a genetic diagnosis in NDDs. Transcriptome analysis (RNA sequencing [RNA-seq]) promises to improve the diagnostic yield but has not been applied to NDDs in routine diagnostics. Here, we explored the diagnostic potential of RNA-seq in 96 individuals including 67 undiagnosed subjects with NDDs. We performed RNA-seq on single individuals' cultured skin fibroblasts, with and without cycloheximide treatment, and used modified OUTRIDER Z scores to detect gene expression outliers and mis-splicing by exonic and intronic outliers. Analysis was performed by a user-friendly web application, and candidate pathogenic transcriptional events were confirmed by secondary assays. We identified intragenic deletions, monoallelic expression, and pseudoexonic insertions but also synonymous and non-synonymous variants with deleterious effects on transcription, increasing the diagnostic yield for NDDs by 13%. We found that cycloheximide treatment and exonic/intronic Z score analysis increased detection and resolution of aberrant splicing. Importantly, in one individual mis-splicing was found in a candidate gene nearly matching the individual's specific phenotype. However, pathogenic splicing occurred in another neuronal-expressed gene and provided a molecular diagnosis, stressing the need to customize RNA-seq. Lastly, our web browser application allowed custom analysis settings that facilitate diagnostic application and ranked pathogenic transcripts as top candidates. Our results demonstrate that RNA-seq is a complementary method in the genomic diagnosis of NDDs and, by providing accessible analysis with improved sensitivity, our transcriptome analysis approach facilitates wider implementation of RNA-seq in routine genome diagnostics.

epidecodeR: a functional exploration tool for epigenetic and epitranscriptomic regulation.

Recent technological advances in sequencing DNA and RNA modifications using high-throughput platforms have generated vast epigenomic and epitranscriptomic datasets whose power in transforming life science is yet fully unleashed. Currently available in silico methods have facilitated the identification, positioning and quantitative comparisons of individual modification sites. However, the essential challenge to link specific 'epi-marks' to gene expression in the particular context of cellular and biological processes is unmet. To fast-track exploration, we generated epidecodeR implemented in R, which allows biologists to quickly survey whether an epigenomic or epitranscriptomic status of their interest potentially influences gene expression responses. The evaluation is based on the cumulative distribution function and the statistical significance in differential expression of genes grouped by the number of 'epi-marks'. This tool proves useful in predicting the role of H3K9ac and H3K27ac in associated gene expression after knocking down deacetylases FAM60A and SDS3 and N6-methyl-adenosine-associated gene expression after knocking out the reader proteins. We further used epidecodeR to explore the effectiveness of demethylase FTO inhibitors and histone-associated modifications in drug abuse in animals. epidecodeR is available for downloading as an R package at https://bioconductor.riken.jp/packages/3.13/bioc/html/epidecodeR.html.

Robust ring-opening reaction via asymmetrically coordinated Fe single atoms scaffolded by spoke-like mesoporous carbon nanospheres.

The ability to construct metal single-atom catalysts (SACs) asymmetrically coordinated with organic heteroatoms represents an important endeavor toward developing high-performance catalysts over symmetrically coordinated counterparts. Moreover, it is of key importance in creating supporting matrix with porous architecture for situating SACs as it greatly impacts the mass diffusion and transport of electrolyte. Herein, we report the crafting of Fe single atoms with asymmetrically coordinated nitrogen (N) and phosphorus (P) atoms scaffolded by rationally designed mesoporous carbon nanospheres (MCNs) with spoke-like nanochannels for boosting ring-opening reaction of epoxide to produce an array of pharmacologically important ß-amino alcohols. Notably, interfacial defects in MCN derived from the use of sacrificial template create abundant unpaired electrons, thereby stably anchoring N and P atoms and in turn Fe atoms on MCN. Importantly, the introduction of P atom promotes the symmetry-breaking of common four N-coordinated Fe sites, resulting in the Fe-N3P sites on MCN (denoted Fe-N3P-MCN) with an asymmetric electronic configuration and thus superior catalytic capability. As such, the Fe-N3P-MCN catalysts manifest a high catalytic activity for ring-opening reaction of epoxide (97% yield) over the Fe-N3P docked on nonporous carbon surface (91%) as well as the sole Fe-N4 SACs grounded on the same MCN support (89%). Density functional theory calculations reveal that Fe-N3P SAC lowers the activation barrier for the C-O bond cleavage and the C-N bond formation, thus accelerating the ring-opening of epoxide. Our study provides fundamental and practical insights into developing advanced catalysts in a simple and controllable manner for multistep organic reactions.

The discovery of a catalytic RNA within RNase P and its legacy.

Sidney Altman's discovery of the processing of one RNA by another RNA that acts like an enzyme was revolutionary in biology and the basis for his sharing the 1989 Nobel Prize in Chemistry with Thomas Cech. These breakthrough findings support the key role of RNA in molecular evolution, where replicating RNAs (and similar chemical derivatives) either with or without peptides functioned in protocells during the early stages of life on Earth, an era referred to as the RNA world. Here, we cover the historical background highlighting the work of Altman and his colleagues and the subsequent efforts of other researchers to understand the biological function of RNase P and its catalytic RNA subunit and to employ it as a tool to downregulate gene expression. We primarily discuss bacterial RNase P-related studies but acknowledge that many groups have significantly contributed to our understanding of archaeal and eukaryotic RNase P, as reviewed in this special issue and elsewhere.

Emerging concepts of migrasome: An up-and-coming organelle from biology to the clinic.

Since the migrasome concept was first proposed in 2015, extensive research has been conducted on these novel organelles, which grow on retracted fibers at the posterior end of migrating cells. Recently, molecular markers, biological functions, and clinical values based on the initial formation mechanism of migrasomes have emerged. Additionally, researchers are recognizing the significant role that migrasomes play in the pathological and diagnostic processes of clinical diseases. In this review, we summarize recent advances in the biology and clinical application of migrasomes and provide a comprehensive view of the prospective challenges surrounding their clinical application.

Lactate and Lactylation: Clinical Applications of Routine Carbon Source and Novel Modification in Human Diseases.

Cell metabolism generates numerous intermediate metabolites that could serve as feedback and feed-forward regulation substances for posttranslational modification. Lactate, a metabolic product of glycolysis, has recently been conceptualized to play a pleiotropic role in shaping cell identities through metabolic rewiring and epigenetic modifications. Lactate-derived carbons, sourced from glucose, mediate the crosstalk among glycolysis, lactate, and lactylation. Furthermore, the multiple metabolic fates of lactate make it an ideal substrate for metabolic imaging in clinical application. Several studies have identified the crucial role of protein lactylation in human diseases associated with cell fate determination, embryonic development, inflammation, neoplasm, and neuropsychiatric disorders. Herein, this review will focus on the metabolic fate of lactate-derived carbon to provide useful information for further research and therapeutic approaches in human diseases. We comprehensively discuss its role in reprogramming and modification during the regulation of glycolysis, the clinical translation prospects of the hyperpolarized lactate signal, lactyl modification in human diseases, and its application with other techniques and omics.

PtPd Atomic Layer Shelled PdCu Hollow Nanoparticles on Partially Unzipped Carbon Nanotubes for Breaking the Activity-Stability Trade-Off toward the Hydrogen Oxidation Reaction in Alkaline Media.

Developing highly active yet stable catalysts for the hydrogen oxidation reaction (HOR) in alkaline media remains a significant challenge. Herein, we designed a novel catalyst of atomic PtPd-layer shelled ultrasmall PdCu hollow nanoparticles (HPdCu NPs) on partially unzipped carbon nanotubes (PtPd@HPdCu/W-CNTs), which can achieve a high mass activity, 5 times that of the benchmark Pt/C, and show exceptional stability with negligible decay after 20,000 cycles of accelerated degradation test. The atomically thin PtPd shell serves as the primary active site for the HOR and a protective layer that prevents Cu leaching. Additionally, the HPdCu substrate not only tunes the adsorption properties of the PtPd layer but also prevents corrosive Pt from reaching the interface between NPs and the carbon support, thereby mitigating carbon corrosion. This work introduces a new strategy that leverages the distinct advantages of multiple components to address the challenges associated with slow kinetics and poor durability toward the HOR.

PyChelator: a Python-based Colab and web application for metal chelator calculations.

BACKGROUND: Metal ions play vital roles in regulating various biological systems, making it essential to control the concentration of free metal ions in solutions during experimental procedures. Several software applications exist for estimating the concentration of free metals in the presence of chelators, with MaxChelator being the easily accessible choice in this domain. This work aimed at developing a Python version of the software with arbitrary precision calculations, extensive new features, and a user-friendly interface to calculate the free metal ions. RESULTS: We introduce the open-source PyChelator web application and the Python-based Google Colaboratory notebook, PyChelator Colab. Key features aim to improve the user experience of metal chelator calculations including input in smaller units, selection among stability constants, input of user-defined constants, and convenient download of all results in Excel format. These features were implemented in Python language by employing Google Colab, facilitating the incorporation of the calculator into other Python-based pipelines and inviting the contributions from the community of Python-using scientists for further enhancements. Arbitrary-precision arithmetic was employed by using the built-in Decimal module to obtain the most accurate results and to avoid rounding errors. No notable differences were observed compared to the results obtained from the PyChelator web application. However, comparison of different sources of stability constants showed substantial differences among them. CONCLUSIONS: PyChelator is a user-friendly metal and chelator calculator that provides a platform for further development. It is provided as an interactive web application, freely available for use at https://amrutelab.github.io/PyChelator , and as a Python-based Google Colaboratory notebook at https://colab. RESEARCH: google.com/github/AmruteLab/PyChelator/blob/main/PyChelator_Colab.ipynb .

Dashing Growth Curves: a web application for rapid and interactive analysis of microbial growth curves.

BACKGROUND: Recording and analyzing microbial growth is a routine task in the life sciences. Microplate readers that record dozens to hundreds of growth curves simultaneously are increasingly used for this task raising the demand for their rapid and reliable analysis. RESULTS: Here, we present Dashing Growth Curves, an interactive web application ( http://dashing-growth-curves.ethz.ch/ ) that enables researchers to quickly visualize and analyze growth curves without the requirement for coding knowledge and independent of operating system. Growth curves can be fitted with parametric and non-parametric models or manually. The application extracts maximum growth rates as well as other features such as lag time, length of exponential growth phase and maximum population size among others. Furthermore, Dashing Growth Curves automatically groups replicate samples and generates downloadable summary plots for of all growth parameters. CONCLUSIONS: Dashing Growth Curves is an open-source web application that reduces the time required to analyze microbial growth curves from hours to minutes.

Holomics - a user-friendly R shiny application for multi-omics data integration and analysis.

An organism's observable traits, or phenotype, result from intricate interactions among genes, proteins, metabolites and the environment. External factors, such as associated microorganisms, along with biotic and abiotic stressors, can significantly impact this complex biological system, influencing processes like growth, development and productivity. A comprehensive analysis of the entire biological system and its interactions is thus crucial to identify key components that support adaptation to stressors and to discover biomarkers applicable in breeding programs or disease diagnostics. Since the genomics era, several other 'omics' disciplines have emerged, and recent advances in high-throughput technologies have facilitated the generation of additional omics datasets. While traditionally analyzed individually, the last decade has seen an increase in multi-omics data integration and analysis strategies aimed at achieving a holistic understanding of interactions across different biological layers. Despite these advances, the analysis of multi-omics data is still challenging due to their scale, complexity, high dimensionality and multimodality. To address these challenges, a number of analytical tools and strategies have been developed, including clustering and differential equations, which require advanced knowledge in bioinformatics and statistics. Therefore, this study recognizes the need for user-friendly tools by introducing Holomics, an accessible and easy-to-use R shiny application with multi-omics functions tailored for scientists with limited bioinformatics knowledge. Holomics provides a well-defined workflow, starting with the upload and pre-filtering of single-omics data, which are then further refined by single-omics analysis focusing on key features. Subsequently, these reduced datasets are subjected to multi-omics analyses to unveil correlations between 2-n datasets. This paper concludes with a real-world case study where microbiomics, transcriptomics and metabolomics data from previous studies that elucidate factors associated with improved sugar beet storability are integrated using Holomics. The results are discussed in the context of the biological background, underscoring the importance of multi-omics insights. This example not only highlights the versatility of Holomics in handling different types of omics data, but also validates its consistency by reproducing findings from preceding single-omics studies.

Counter-intuitive discovery in the formulation of poorly water-soluble drugs: Amorphous small-molecule gels.

Amorphous strategies have been extensively used in improving the dissolution of insoluble drugs for decades due to their high free energy. However, the formation of amorphous small-molecule gels (ASMGs) presents a counter-intuitive discovery that significantly limits their practical application. Recently, ASMGs have garnered attention because of their noncovalent structures, excellent biodegradability, and significant potential in various drug delivery systems in the pharmaceutical field. Hence, a comprehensive review is necessary to contribute to a better understanding of recent advances in ASMGs. This review aimed to introduce the main formation mechanisms, summarize possible influencing factors, generalize unique properties, outline elimination strategies, and discuss clinical application potential with preclinical cases of ASMGs. Moreover, few ASMGs are advanced to clinical stages. Intensive clinical research is needed for further development. We hope that this review can provide more efficient and rational guidance for exploring further clinical applications of ASMGs.

Clinical application of liquid biopsy in colorectal cancer: detection, prediction, and treatment monitoring.

Colorectal cancer (CRC) is one of the most prevalent malignancies affecting the gastrointestinal tract and is ranked third among cancers with the highest incidence and second-highest mortality rate worldwide. CRC exhibits a slow progression providing a wide treatment window. The currently employed CRC screening methods have shown great potential to prevent CRC and reduce CRC-related morbidity and mortality. The diagnosis of CRC is achieved by colonoscopy and tissue biopsy, with studies showing that liquid biopsy is more effective in detecting and diagnosing early CRC patients. Increasing number of studies have shown that the tumor components shed into circulating blood can be detected in liquid form, and can be applied in the clinical management of CRC. Analysis of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), or tumor-associated platelets (TEPs) in the blood can be used for early screening and diagnosis of CRC, aid tumor staging, treatment response monitoring, and prediction of CRC recurrence and metastasis in a minimally invasive manner. This chapter provides an updated review of CTCs, ctDNA, and TEPs as novel biomarkers for CRC, highlighting their strengths and limitations.

Unraveling the cross-talk between N6-methyladenosine modification and non-coding RNAs in breast cancer: Mechanisms and clinical implications.

In recent years, breast cancer (BC) has surpassed lung cancer as the most common malignant tumor worldwide and remains the leading cause of cancer death in women. The etiology of BC usually involves dysregulation of epigenetic mechanisms and aberrant expression of certain non-coding RNAs (ncRNAs). N6-methyladenosine (m6A), the most prevalent RNA modification in eukaryotes, widely exists in ncRNAs to affect its biosynthesis and function, and is an important regulator of tumor-related signaling pathways. Interestingly, ncRNAs can also regulate or target m6A modification, playing a key role in cancer progression. However, the m6A-ncRNAs regulatory network in BC has not been fully elucidated, especially the regulation of m6A modification by ncRNAs. Therefore, in this review, we comprehensively summarize the interaction mechanisms and biological significance of m6A modifications and ncRNAs in BC. Meanwhile, we also focused on the clinical application value of m6A modification in BC diagnosis and prognosis, intending to explore new biomarkers and potential therapeutic targets.

HOXA9 transcription factor is a double-edged sword: from development to cancer progression.

The HOXA9 transcription factor serves as a molecular orchestrator in cancer stemness, epithelial-mesenchymal transition (EMT), metastasis, and generation of the tumor microenvironment in hematological and solid malignancies. However, the multiple modes of regulation, multifaceted functions, and context-dependent interactions responsible for the dual role of HOXA9 as an oncogene or tumor suppressor in cancer remain obscure. Hence, unravelling its molecular complexities, binding partners, and interacting signaling molecules enables us to comprehend HOXA9-mediated transcriptional programs and molecular crosstalk. However, it is imperative to understand its central role in fundamental biological processes such as embryogenesis, foetus implantation, hematopoiesis, endothelial cell proliferation, and tissue homeostasis before designing targeted therapies. Indeed, it presents an enormous challenge for clinicians to selectively target its oncogenic functions or restore tumor-suppressive role without altering normal cellular functions. In addition to its implications in cancer, the present review also focuses on the clinical applications of HOXA9 in recurrence and drug resistance, which may provide a broader understanding beyond oncology, open new avenues for clinicians for accurate diagnoses, and develop personalized treatment strategies. Furthermore, we have also discussed the existing therapeutic options and accompanying challenges in HOXA9-targeted therapies in different cancer types.

Topical application of phenformin ameliorates the psoriasis-like inflammatory response via the inhibition of c-Myc expression in keratinocytes.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease characterized by a complex pathogenesis involving various types of cells and cytokines. Among those, the pro-inflammatory cytokine IL-23/IL-17A axis plays a crucial role in the development and rapid progression of psoriasis. Phenformin, a derivative of metformin and a member of the biguanide class of drugs, exhibits superior anti-inflammatory and anti-tumor efficacy compared to metformin. However, the potential role of phenformin in anti-psoriatic skin inflammation has not been explored. METHODS: In this study, we utilized a mouse model of psoriasis and an in vitro model using human keratinocytes to investigate whether phenformin can suppress psoriasis-like inflammatory responses. RESULTS: Our results demonstrate that the topical application of phenformin significantly inhibited acute skin inflammatory responses in the psoriasis mouse model induced by imiquimod (IMQ). Additionally, phenformin suppressed the expression of psoriasis-related cytokines IL-17, IL-23, IL-8, and S100A8/S100A9 in an in vitro psoriatic keratinocyte model induced by IMQ. Furthermore, we found that IMQ-induced psoriatic skin and IMQ-treated keratinocytes exhibited high expression of the c-Myc gene, which was downregulated by phenformin. The c-Myc inhibitor JQ1 similarly inhibited the psoriatic inflammatory response and the expression of psoriasis-related cytokines in both in vitro and in vivo models. CONCLUSION: phenformin ameliorates the psoriasis-like inflammatory response by inhibiting c-Myc expression in keratinocytes, suggesting its potential as a topical drug for the treatment of psoriasis.

Quality assessment of enzymatic methyl-seq library constructed using crude cell lysate.

Enzymatic methyl-seq (EM-seq), an enzyme-based method, identifies genome-wide DNA methylation, which enables us to obtain reliable methylome data from purified genomic DNA by avoiding bisulfite-induced DNA damage. However, the loss of DNA during purification hinders the methylome analysis of limited samples. The crude DNA extraction method is the quickest and minimal sample loss approach for obtaining useable DNA without requiring additional dissolution and purification. However, it remains unclear whether crude DNA can be used directly for EM-seq library construction. In this study, we aimed to assess the quality of EM-seq libraries prepared directly using crude DNA. The crude DNA-derived libraries provided appropriate fragment sizes and concentrations for sequencing similar to those of the purified DNA-derived libraries. However, the sequencing results of crude samples exhibited lower reference sequence mapping efficiencies than those of the purified samples. Additionally, the lower-input crude DNA-derived sample exhibited a marginally lower cytosine-to-thymine conversion efficiency and hypermethylated pattern around gene regulatory elements than the higher-input crude DNA- or purified DNA-derived samples. In contrast, the methylation profiles of the crude and purified samples exhibited a significant correlation. Our findings indicate that crude DNA can be used as a raw material for EM-seq library construction.

Comparative transcriptome analysis reveals major genes, transcription factors and biosynthetic pathways associated with leaf senescence in rice under different nitrogen application.

BACKGROUND: Rice (Oryza sativa L.) is one of the most important food crops in the world and the application of nitrogen fertilizer is an effective means of ensuring stable and high rice yields. However, excessive application of nitrogen fertilizer not only causes a decline in the quality of rice, but also leads to a series of environmental costs. Nitrogen reutilization is closely related to leaf senescence, and nitrogen deficiency will lead to early functional leaf senescence, whereas moderate nitrogen application will help to delay leaf senescence and promote the production of photosynthetic assimilation products in leaves to achieve yield increase. Therefore, it is important to explore the mechanism by which nitrogen affects rice senescence, to search for genes that are tolerant to low nitrogen, and to delay the premature senescence of rice functional leaves. RESULTS: The present study was investigated the transcriptional changes in flag leaves between full heading and mature grain stages of rice (O. sativa) sp. japonica 'NanGeng 5718' under varying nitrogen (N) application: 0 kg/ha (no nitrogen; 0N), 240 kg/ha (moderate nitrogen; MN), and 300 kg/ha (high nitrogen; HN). Compared to MN condition, a total of 10427 and 8177 differentially expressed genes (DEGs) were detected in 0N and HN, respectively. We selected DEGs with opposite expression trends under 0N and HN conditions for GO and KEGG analyses to reveal the molecular mechanisms of nitrogen response involving DEGs. We confirmed that different N applications caused reprogramming of plant hormone signal transduction, glycolysis/gluconeogenesis, ascorbate and aldarate metabolism and photosynthesis pathways in regulating leaf senescence. Most DEGs of the jasmonic acid, ethylene, abscisic acid and salicylic acid metabolic pathways were up-regulated under 0N condition, whereas DEGs related to cytokinin and ascorbate metabolic pathways were induced in HN. Major transcription factors include ERF, WRKY, NAC and bZIP TF families have similar expression patterns which were induced under N starvation condition. CONCLUSION: Our results revealed that different nitrogen levels regulate rice leaf senescence mainly by affecting hormone levels and ascorbic acid biosynthesis. Jasmonic acid, ethylene, abscisic acid and salicylic acid promote early leaf senescence under low nitrogen condition, ethylene and ascorbate delay senescence under high nitrogen condition. In addition, ERF, WRKY, NAC and bZIP TF families promote early leaf senescence. The relevant genes can be used as candidate genes for the regulation of senescence. The results will provide gene reference for further genomic studies and new insights into the gene functions, pathways and transcription factors of N level regulates leaf senescence in rice, thereby improving NUE and reducing the adverse effects of over-application of N.

Nitrogen application in pod zone improves yield and quality of two peanut cultivars by modulating nitrogen accumulation and metabolism.

Cultivated peanut (Arachis hypogaea L.) represents one of the most important oil and cash crops world-widely. Unlike many other legumes, peanuts absorb nitrogen through their underground pods. Despite this unique feature, the relationship between yield and nitrogen uptake within the pod zone remains poorly understood. In our pot experiment, we divided the underground peanut part into two zones-pod and root-and investigated the physiological and agronomic traits of two peanut cultivars, SH11 (large seeds, LS) and HY23 (small seeds, SS), at 10 (S1), 20 (S2), and 30 (S3) days after gynophores penetrated the soil, with nitrogen application in the pod zone. Results indicated that nitrogen application increased pod yield, kernel protein content, and nitrogen accumulation in plants. For both LS and SS peanut cultivars, optimal nitrogen content was 60 kg·hm- 2, leading to maximum yield. LS cultivar exhibited higher yield and nitrogen accumulation increases than SS cultivar. Nitrogen application up-regulated the expression of nitrogen metabolism-related genes in the pod, including nitrate reductase (NR), nitrite reductase (NIR), glutamine synthetase (GS), glutamate synthase (NADH-GOGAT), ATP binding cassette (ABC), and nitrate transporter (NRT2). Additionally, nitrogen application increased enzyme activity in the pod, including NR, GS, and GOGAT, consistent with gene expression levels. These nitrogen metabolism traits exhibited higher up-regulations in the large-seeded cultivar than in the small-seeded one and showed a significant correlation with yield in the large-seeded cultivar at S2 and S3. Our findings offer a scientific basis for the judicious application and efficient utilization of nitrogen fertilization in peanuts, laying the groundwork for further elucidating the molecular mechanisms of peanut nitrogen utilization.