Conserved cis-elements enable NODULES WITH ACTIVATED DEFENSE1 regulation by NODULE INCEPTION during nodulation.

Symbiotic nitrogen fixation within nitrogen-fixing clade (NFC) plants is thought to have arisen from a single gain followed by massive losses in the genomes of ancestral non-nodulating plants. However, molecular evidence supporting this model is limited. Here, we confirm through bioinformatic analysis that NODULES WITH ACTIVATED DEFENSE1 (NAD1) is present only in NFC plants and is thus an NFC-specific gene. Moreover, NAD1 was specifically expressed in nodules. We identified three conserved nodulation-associated cis-regulatory elements (NACE1-3) in the promoter of LjNAD1 from Lotus japonicus that are required for its nodule specific expression. A survey of NFC plants revealed that NACE1 and NACE2 are specific to the Fabales and Papilionoideae, respectively, while NACE3 is present in all NFC plants. Moreover, we found that nodule inception (NIN) directly binds to all three NACEs to activate NAD1 expression. Mutation of L. japonicus LjNAD1 resulted in the formation of abnormal symbiosomes with enlarged symbiosome space and frequent breakdown of bacteroids in nodules, resembling phenotypes reported for Medicago truncatula Mtnad1 and Mtnin mutants. These data point to NIN-NAD1 as an important module regulating rhizobial accommodation in nodules. The regulation of NAD1 by NIN in the NFC ancestor represent an important evolutionary adaptation for nodulation.

GmNAC039 and GmNAC018 activate the expression of cysteine protease genes to promote soybean nodule senescence.

Root nodules are major sources of nitrogen for soybean (Glycine max (L.) Merr.) growth, development, production, and seed quality. Symbiotic nitrogen fixation is time-limited, as the root nodule senesces during the reproductive stage of plant development, specifically during seed development. Nodule senescence is characterized by the induction of senescence-related genes, such as papain-like cysteine proteases (CYPs), which ultimately leads to the degradation of both bacteroids and plant cells. However, how nodule senescence-related genes are activated in soybean is unknown. Here, we identified 2 paralogous NAC transcription factors, GmNAC039 and GmNAC018, as master regulators of nodule senescence. Overexpression of either gene induced soybean nodule senescence with increased cell death as detected using a TUNEL assay, whereas their knockout delayed senescence and increased nitrogenase activity. Transcriptome analysis and nCUT&Tag-qPCR assays revealed that GmNAC039 directly binds to the core motif CAC(A)A and activates the expression of 4 GmCYP genes (GmCYP35, GmCYP37, GmCYP39, and GmCYP45). Similar to GmNAC039 and GmNAC018, overexpression or knockout of GmCYP genes in nodules resulted in precocious or delayed senescence, respectively. These data provide essential insights into the regulatory mechanisms of nodule senescence, in which GmNAC039 and GmNAC018 directly activate the expression of GmCYP genes to promote nodule senescence.

scapGNN: A graph neural network-based framework for active pathway and gene module inference from single-cell multi-omics data.

Although advances in single-cell technologies have enabled the characterization of multiple omics profiles in individual cells, extracting functional and mechanistic insights from such information remains a major challenge. Here, we present scapGNN, a graph neural network (GNN)-based framework that creatively transforms sparse single-cell profile data into the stable gene-cell association network for inferring single-cell pathway activity scores and identifying cell phenotype-associated gene modules from single-cell multi-omics data. Systematic benchmarking demonstrated that scapGNN was more accurate, robust, and scalable than state-of-the-art methods in various downstream single-cell analyses such as cell denoising, batch effect removal, cell clustering, cell trajectory inference, and pathway or gene module identification. scapGNN was developed as a systematic R package that can be flexibly extended and enhanced for existing analysis processes. It provides a new analytical platform for studying single cells at the pathway and network levels.

The ortholog of human REEP1-4 is required for autophagosomal enclosure of ER-phagy/nucleophagy cargos in fission yeast.

Selective macroautophagy of the endoplasmic reticulum (ER) and the nucleus, known as ER-phagy and nucleophagy, respectively, are processes whose mechanisms remain inadequately understood. Through an imaging-based screen, we find that in the fission yeast Schizosaccharomyces pombe, Yep1 (also known as Hva22 or Rop1), the ortholog of human REEP1-4, is essential for ER-phagy and nucleophagy but not for bulk autophagy. In the absence of Yep1, the initial phase of ER-phagy and nucleophagy proceeds normally, with the ER-phagy/nucleophagy receptor Epr1 coassembling with Atg8. However, ER-phagy/nucleophagy cargos fail to reach the vacuole. Instead, nucleus- and cortical-ER-derived membrane structures not enclosed within autophagosomes accumulate in the cytoplasm. Intriguingly, the outer membranes of nucleus-derived structures remain continuous with the nuclear envelope-ER network, suggesting a possible outer membrane fission defect during cargo separation from source compartments. We find that the ER-phagy role of Yep1 relies on its abilities to self-interact and shape membranes and requires its C-terminal amphipathic helices. Moreover, we show that human REEP1-4 and budding yeast Atg40 can functionally substitute for Yep1 in ER-phagy, and Atg40 is a divergent ortholog of Yep1 and REEP1-4. Our findings uncover an unexpected mechanism governing the autophagosomal enclosure of ER-phagy/nucleophagy cargos and shed new light on the functions and evolution of REEP family proteins.

Measuring transcription factor function with cell type-specific somatic transgenesis in chicken embryos.

Retroviral-mediated misexpression in chicken embryos has been a powerful research tool for developmental biologists in the last two decades. In the RCASBP retroviral vectors that are widely used for in vivo somatic transgenesis, a coding sequence of interest is under the transcriptional control of a strong viral promoter in the long terminal repeat. While this has proven to be effective for studying secreted signalling proteins, interpretation of the mechanisms of action of nuclear factors is more difficult using this system since it is not clear whether phenotypic effects are cell-autonomous or not, and therefore whether they represent a function of the endogenous protein. Here, we report the consequences of retroviral expression using the RCANBP backbone, in which the transcription factor Dlx5 is expressed under the control of chondrocyte-specific regulatory sequences from the Col2a1 gene. To our knowledge, this is the first demonstration of a tissue-specific phenotype in the chicken embryo.

Soybean ethylene response factors GmENS1 and GmENS2 promote nodule senescence.

The final phase in root nodule development is nodule senescence. The mechanism underlying the initiation of nodule senescence requires further elucidation. In this study, we investigate the intrinsic signals governing soybean (Glycine max L. Merr.) nodule senescence, uncovering ethylene as a key signal in this intricate mechanism. Two AP2/ethylene response factor (ERF) transcription factor (TF) genes, GmENS1 and GmENS2 (Ethylene-responsive transcription factors required for Nodule Senescence), exhibit heightened expression levels in both aged nodules and nodules treated with ethylene. An overexpression of either GmENS1 or GmENS2 accelerates senescence in soybean nodules, whereas the knockout or knockdown of both genes delays senescence and enhances nitrogenase activity. Furthermore, our findings indicate that GmENS1 and GmENS2 directly bind to the promoters of GmNAC039, GmNAC018, and GmNAC030, encoding 3 NAC (NAM, ATAF1/2, and CUC2) TFs essential for activating soybean nodule senescence. Notably, the nodule senescence process mediated by GmENS1 or GmENS2 overexpression is suppressed in the soybean nac039/018/030 triple mutant compared with the wild-type control. These data indicate GmENS1 and GmENS2 as pivotal TFs mediating ethylene-induced nodule senescence through the direct activation of GmNAC039/GmNAC018/GmNAC030 expression in soybean.

FAM122A functions as a tumor suppressor in oral squamous cell carcinoma.

Family with sequence similarity 122a (FAM122A), identified as an endogenous inhibitor of protein phosphatase 2A (PP2A) previously, is involved in multiple important physiological processes, and essential for the growth of acute myeloid leukemia and hepatocellular carcinoma cells. However, the function of FAM122A in oral squamous cell carcinoma (OSCC) is undetermined. In this study, by analyzing TCGA and GEO databases, we found that the expression of FAM122A was significantly down-regulated in head and neck squamous cell carcinoma and OSCC patients, meanwhile this low expression was tightly associated with the poor prognosis and advanced clinical stage during OSCC development. The similar low expression pattern of FAM122A could also been seen in OSCC cell lines compared with normal human oral keratinocytes. Further, we demonstrated that FAM122A knockdown significantly promoted the growth, clonogenic potential as well as migration capabilities of OSCC cells, while these alterations could be rescued by the re-expression of FAM122A. Over-expression of FAM122A suppressed OSCC cell proliferation and migration. FAM122A also inhibited the epithelial-mesenchymal transition (EMT) in OSCC cells by the up-regulation of epithelial marker E-cadherin and down-regulation of mesenchymal markers Fibronectin and Vimentin, which is presumably mediated by transforming growth factor ß receptor 3 (TGFBR3), a novel tumor suppressor. In addition, FAM122A could induce T cell infiltration in OSCC, indicating that FAM122A might influence the immune cell activity of tumor environment and further interfere the tumor development. Collectively, our results suggest that FAM122A functions as a tumor suppressor in OSCC and possibly acts as a predictive biomarker for the diagnosis and/or treatment of OSCC.

Role of HCN channels in the functions of basal ganglia and Parkinson's disease.

Parkinson's disease (PD) is a motor disorder resulting from dopaminergic neuron degeneration in the substantia nigra caused by age, genetics, and environment. The disease severely impacts a patient's quality of life and can even be life-threatening. The hyperpolarization-activated cyclic nucleotide-gated (HCN) channel is a member of the HCN1-4 gene family and is widely expressed in basal ganglia nuclei. The hyperpolarization-activated current mediated by the HCN channel has a distinct impact on neuronal excitability and rhythmic activity associated with PD pathogenesis, as it affects the firing activity, including both firing rate and firing pattern, of neurons in the basal ganglia nuclei. This review aims to comprehensively understand the characteristics of HCN channels by summarizing their regulatory role in neuronal firing activity of the basal ganglia nuclei. Furthermore, the distribution and characteristics of HCN channels in each nucleus of the basal ganglia group and their effect on PD symptoms through modulating neuronal electrical activity are discussed. Since the roles of the substantia nigra pars compacta and reticulata, as well as globus pallidus externus and internus, are distinct in the basal ganglia circuit, they are individually described. Lastly, this investigation briefly highlights that the HCN channel expressed on microglia plays a role in the pathological process of PD by affecting the neuroinflammatory response.

Lectin-Based SP3 Technology Enables N-Glycoproteomic Analysis of Mouse Oocytes.

N-glycosylation is one of the most universal and complex protein post-translational modifications (PTMs), and it is involved in many physiological and pathological activities. Owing to the low abundance of N-glycoproteins, enrichment of N-glycopeptides for mass spectrometry analysis usually requires a large amount of peptides. Additionally, oocyte protein N-glycosylation has not been systemically characterized due to the limited sample amount. Here, we developed a glycosylation enrichment method based on lectin and a single-pot, solid-phase-enhanced sample preparation (SP3) technology, termed lectin-based SP3 technology (LectinSP3). LectinSP3 immobilized lectin on the SP3 beads for N-glycopeptide enrichment. It could identify over 1100 N-glycosylation sites and 600 N-glycoproteins from 10 µg of mouse testis peptides. Furthermore, using the LectinSP3 method, we characterized the N-glycoproteome of 1000 mouse oocytes in three replicates and identified a total of 363 N-glycosylation sites from 215 N-glycoproteins. Bioinformatics analysis revealed that these oocyte N-glycoproteins were mainly enriched in cell adhesion, fertilization, and sperm-egg recognition. Overall, the LectinSP3 method has all procedures performed in one tube, using magnetic beads. It is suitable for analysis of a low amount of samples and is expected to be easily adaptable for automation. In addition, our mouse oocyte protein N-glycosylation profiling could help further characterize the regulation of oocyte functions.

Utilizing human cerebral organoids to model breast cancer brain metastasis in culture.

BACKGROUND: Metastasis, the spread, and growth of malignant cells at secondary sites within a patient's body, accounts for over 90% of cancer-related mortality. Breast cancer is the most common tumor type diagnosed and the leading cause of cancer lethality in women in the United States. It is estimated that 10-16% breast cancer patients will have brain metastasis. Current therapies to treat patients with breast cancer brain metastasis (BCBM) remain palliative. This is largely due to our limited understanding of the fundamental molecular and cellular mechanisms through which BCBM progresses, which represents a critical barrier for the development of efficient therapies for affected breast cancer patients. METHODS: Previous research in BCBM relied on co-culture assays of tumor cells with rodent neural cells or rodent brain slice ex vivo. Given the need to overcome the obstacle for human-relevant host to study cell-cell communication in BCBM, we generated human embryonic stem cell-derived cerebral organoids to co-culture with human breast cancer cell lines. We used MDA-MB-231 and its brain metastatic derivate MDA-MB-231 Br-EGFP, other cell lines of MCF-7, HCC-1806, and SUM159PT. We leveraged this novel 3D co-culture platform to investigate the crosstalk of human breast cancer cells with neural cells in cerebral organoid. RESULTS: We found that MDA-MB-231 and SUM159PT breast cancer cells formed tumor colonies in human cerebral organoids. Moreover, MDA-MB-231 Br-EGFP cells showed increased capacity to invade and expand in human cerebral organoids. CONCLUSIONS: Our co-culture model has demonstrated a remarkable capacity to discern the brain metastatic ability of human breast cancer cells in cerebral organoids. The generation of BCBM-like structures in organoid will facilitate the study of human tumor microenvironment in culture.

Antiprogrammed death ligand 1 therapy failed to reduce the risk of developing brain metastases in patients with extensive-stage small cell lung cancer: A retrospective analysis.

BACKGROUND: Immunotherapy (IO) has demonstrated promising results in treating extensive-stage small cell lung cancer (ES-SCLC), and the management of ES-SCLC brain metastases (BMs) is now receiving significant clinical attention. The objective of this study was to evaluate the role of IO in the clinical management of BMs. METHODS: Between January 2020 and December 2021, the study included the records of 250 patients who were diagnosed with ES-SCLC. Overall survival (OS), progression-free survival, intracranial progression-free survival, and the cumulative incidence of BMs were calculated using the Kaplan-Meier method and were compared using the log-rank test. In addition, the Cox regression model was used to analyze prognostic factors. RESULTS: In the entire group, 85 patients had baseline BMs (IO plus chemotherapy [IO + ChT], n = 38; ChT alone, n = 47), and 165 patients (IO + ChT, n = 86; ChT alone, n = 79) did not have BMs at the time of initial diagnosis. The median follow-up was 22.4 months. The OS benefit with first-line antiprogrammed death ligand 1 therapy was maintained regardless of whether patients had BMs (with BMs, 17.97 vs. 13.14 months [p = .03]; without BMs, 18.46 vs. 15.05 months [p = .047]). However, in patients without BMs, IO did not delay the median time to developing brain progression (10.84 vs. 10.74 months; p = .84), and it did not significantly reduce the risk of developing intracranial metastases (the 2-year actuarial risk of developing BMs was 57.0% vs. 50.6%, respectively). CONCLUSIONS: Antiprogrammed death ligand 1 therapy improved OS regardless of the presence of BMs. However, IO did not delay the median time to brain progression or reduce the risk of intracranial metastasis in patients without baseline BMs. The findings of this study have important clinical implications for the future management of BMs from ES-SCLC.

Celastrol inhibits platelet function and thrombus formation.

INTRODUCTION: Celastrol is an active pentacyclic triterpenoid extracted from Tripterygium wilfordii and has anti-inflammatory and anti-tumor properties. Whether Celastrol modulates platelet function remains unknown. Our study investigated its role in platelet function and thrombosis. METHODS: Human platelets were isolated and incubated with Celastrol (0, 1, 3 and 5 µM) at 37 °C for 1 h to measure platelet aggregation, granules release, spreading, thrombin-induced clot retraction and intracellular calcium mobilization. Additionally, Celastrol (2 mg/kg) was intraperitoneally administrated into mice to evaluate hemostasis and thrombosis in vivo. RESULTS: Celastrol treatment significantly decreased platelet aggregation and secretion of dense or alpha granules induced by collagen-related peptide (CRP) or thrombin in a dose-dependent manner. Additionally, Celastrol-treated platelets showed a dramatically reduced spreading activity and decreased clot retraction. Moreover, Celastrol administration prolonged tail bleeding time and inhibited formation of arterial/venous thrombosis. Furthermore, Celastrol significantly reduced calcium mobilization. CONCLUSION: Celastrol inhibits platelet function and venous/arterial thrombosis, implying that it might be utilized for treating thrombotic diseases.

Double-negative T cells with a distinct transcriptomic profile are abundant in the peripheral blood of patients with breast cancer.

BACKGROUND: Double-negative T (DNT) cells comprise a distinct subset of T lymphocytes that have been implicated in immune responses. The aim of this study was to characterize the peripheral DNT population in breast cancer (BC) patients. METHODS: DNT cells were isolated from the peripheral blood samples of BC patients and healthy controls by flow cytometry. The sorted DNT cells were analyzed by the Smart-seq2 for single-cell full-length transcriptome profiling. The differentially expressed genes (DEGs) between the BC and control groups were screened and functionally annotated by Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses using R. The protein-protein interaction (PPI) network of the DEGs was constructed using the CytoHubba and MCODE plug-in of Cytoscape software to identify the core genes. Survival status, DNA methylation level, immune infiltration and immune checkpoint expression were analyzed using Kaplan-Meier Plotter, UALCAN, MethSeuvr, TIMER, and TISIDB respectively. The sequencing results were verified by RT-qPCR. RESULT: The percentage of DNT cells was higher in the BC patients compared to healthy controls. We identified 289 DEGs between the DNT populations of both groups. GO and KEGG pathway analyses revealed that the DEGs were mainly related to immunoglobulin mediated immune response, complement activation, and B cell receptor signaling. The PPI networks of the common DEGs were constructed using Cytoscape, and 10 core genes were identified, including TMEM176B, C1QB, C1QC, RASD2, and IFIT3. The expression levels of these genes correlated with the prognosis and immune infiltration in BC patients, and were validated by RT-qPCR (P < 0.05). CONCLUSIONS: DNT cells are abundant in patients with BC, and might exert anti-tumor immune responses by regulating genes such as TMEM176B and EGR1.

Atomically Dispersed Ce Sites Augmenting Activity and Durability of Fe-Based Oxygen Reduction Catalyst in PEMFC.

In the cathode of proton exchange membrane fuel cells (PEMFCs), Fe and N co-doped carbon (Fe-N-C) materials with atomically dispersed active sites are one of the satisfactory candidates to replace Pt-based catalysts. However, Fe-N-C catalysts are vulnerable to attack from reactive oxygen species, resulting in inferior durability, and current strategies failing to balance the activity and stability. Here, this study reports Fe and Ce single atoms coupled catalysts anchored on ZIF-8-derived nitrogen-doped carbon (Fe/Ce-N-C) as an efficient ORR electrocatalyst for PEMFCs. In PEMFC tests, the maximum power density of Fe/Ce-N-C catalyst reached up to 0.82 W cm-2, which is 41% larger than that of Fe-N-C. More importantly, the activity of Fe/Ce-N-C catalyst only decreased by 21% after 30 000 cycles under H2/air condition. Density functional theory reveals that the strong coupling between the Fe and Ce sites result in the redistribution of electrons in the active sites, which optimizes the adsorption of OH* intermediates on the catalyst and increases the intrinsic activity. Additionally, the admirable radical scavenging ability of the Ce sites ensured that the catalysts gained long-term stability. Therefore, the addition of Ce single atoms provides a new strategy for improving the activity and durability of oxygen reduction catalysts.

Sex differences in stress-induced hyperalgesia and its mechanisms.

Chronic stress induces a variety of physiological and/or psychological abnormalities, including hyperalgesia. Researchers have discovered sex differences in the prevalence of stress-induced hyperalgesia (SIH) in recent years. Sex differences may be one of the reasons for the heterogeneity of susceptibility to stress-related diseases. In this review, the potential mechanisms of sex differences in SIH are discussed, such as hypothalamus-pituitary-adrenal axis responses, regulation of sex hormones, and immune system responses.

Quality Evaluation of Guidelines for the Diagnosis and Treatment of Liver Failure.

OBJECTIVES: This study aimed to systematically assess the methodological quality and key recommendations of the guidelines for the diagnosis and treatment of liver failure (LF), furnishing constructive insights for guideline developers and equipping clinicians with evidence-based information to facilitate informed decision-making. DATA SOURCES: Electronic databases and manual searches from January 2011 to August 2023. STUDY SELECTION: Two reviewers independently screened titles and abstracts, then full texts for eligibility. Fourteen guidelines were included. DATA EXTRACTION AND SYNTHESIS: Two reviewers extracted data and checked by two others. Methodological quality of the guidelines was appraised using the Appraisal of Guidelines for Research and Evaluation II tool. Of the 14 guidelines, only the guidelines established by the Society of Critical Care Medicine and the American College of Gastroenterology (2023) achieved an aggregate quality score exceeding 60%, thereby meriting clinical recommendations. It emerged that there remains ample room for enhancement in the quality of the guidelines, particularly within the domains of stakeholder engagement, rigor, and applicability. Furthermore, an in-depth scrutiny of common recommendations and supporting evidence drawn from the 10 adult LF guidelines unveiled several key issues: controversy exists in the recommendation, the absence of supporting evidence and confusing use of evidence for recommendations, and a preference in evidence selection. CONCLUSIONS: There are high differences in methodological quality and recommendations among LF guidelines. Improving these existing problems and controversies will benefit existing clinical practice and will be an effective way for developers to upgrade the guidelines.

DeepSCP: utilizing deep learning to boost single-cell proteome coverage.

Multiplexed single-cell proteomes (SCPs) quantification by mass spectrometry greatly improves the SCP coverage. However, it still suffers from a low number of protein identifications and there is much room to boost proteins identification by computational methods. In this study, we present a novel framework DeepSCP, utilizing deep learning to boost SCP coverage. DeepSCP constructs a series of features of peptide-spectrum matches (PSMs) by predicting the retention time based on the multiple SCP sample sets and fragment ion intensities based on deep learning, and predicts PSM labels with an optimized-ensemble learning model. Evaluation of DeepSCP on public and in-house SCP datasets showed superior performances compared with other state-of-the-art methods. DeepSCP identified more confident peptides and proteins by controlling q-value at 0.01 using target-decoy competition method. As a convenient and low-cost computing framework, DeepSCP will help boost single-cell proteome identification and facilitate the future development and application of single-cell proteomics.

Roles of naïve CD4+ T cells and their differentiated subtypes in lung adenocarcinoma and underlying potential regulatory pathways.

BACKGROUND: Naïve CD4+ T cells and their differentiated counterparts play a significant regulatory role in the tumor immune microenvironment, yet their effects on lung adenocarcinoma (LUAD) are not fully understood. METHODS: We utilized Mendelian randomization to assess the causal association between naïve CD4+ T cells and LUAD. Employing a modified single-sample Gene Set Enrichment Analysis (ssGSEA) algorithm with The Cancer Genome Atlas (TCGA) database, we determined the infiltration levels of naïve CD4+ T cells and their differentiation subtypes and investigated their correlation with clinical characteristics. Potential regulatory pathways of T helper cells were identified through Mantel tests and Kyoto Encyclopedia of Genes and Genomes (KEGG) database enrichment analysis. RESULTS: Mendelian randomization analysis revealed an inhibitory effect of naïve CD4+ T cells on LUAD (false discovery rate < 0.05), which was corroborated by observational experiments using TCGA database. Specifically, T helper cell type 2 demonstrated a promotive effect on LUAD in terms of overall, disease-free, and progression-free survival (p < 0.05). Moreover, regulatory T cells exhibited a protective effect on LUAD in terms of disease-specific survival (p < 0.01). Concurrently, we explored the overall impact of naïve CD4+ T cell differentiation subtypes on LUAD, revealing upregulation in pathways such as neutrophil degranulation, MAPK family signaling pathways, and platelet activation, signaling, and aggregation. CONCLUSION: Naïve CD4+ T cells and their differentiated counterparts play essential regulatory roles in the tumor immune microenvironment, demonstrating bidirectionality in their effects.Thus, elucidating the mechanisms and developing novel cell differentiation-inducing agents will benefit anti-cancer therapy.

Dynamic landscape of m6A modifications and related post-transcriptional events in muscle-invasive bladder cancer.

BACKGROUND: Muscle-invasive bladder carcinoma (MIBC) is a serious and more advanced stage of bladder carcinoma. N6-Methyladenosine (m6A) is a dynamic and reversible modifications that primarily affects RNA stability and alternative splicing. The dysregulation of m6A in MIBC can be potential target for clinical interventions, but there have been limited studies on m6A modifications in MIBC and their associations with post-transcriptional regulatory processes. METHODS: Paired tumor and adjacent-normal tissues were obtained from three patients with MIBC following radical cystectomy. The additional paired tissues for validation were obtained from patients underwent transurethral resection. Utilizing Nanopore direct-RNA sequencing, we characterized the m6A RNA methylation landscape in MIBC, with a focus on identifying post-transcriptional events potentially affected by changes in m6A sites. This included an examination of differential transcript usage, polyadenylation signal sites, and variations in poly(A) tail length, providing insights into the broader impact of m6A alterations on RNA processing in MIBC. RESULTS: The prognostic-related m6A genes and m6A-risk model constructed by machine learning enables the stratification of high and low-risk patients with precision. A novel m6A modification site in the 3' untranslated region (3'UTR) of IGLL5 gene were identified, characterized by a lower m6A methylation ratio, elongated poly(A) tails, and a notable bias in transcript usage. Furthermore, we discovered two particular transcripts, VWA1-203 and CEBPB-201. VWA1-203 displayed diminished m6A methylation levels, a truncated 3'UTR, and an elongated poly(A) tail, whereas CEBPB-201 showed opposite trends, highlighting the complex interplay between m6A modifications and RNA processing. Source code was provided on GitHub ( https://github.com/lelelililele/Nanopore-m6A-analysis ). CONCLUSIONS: The state-of-the-art Nanopore direct-RNA sequencing and machine learning techniques enables comprehensive identification of m6A modification and provided insights into the potential post-transcriptional regulation mechanisms on the development and progression in MIBC.

A pathogenesis-related protein, PRP1, negatively regulates root nodule symbiosis in Lotus japonicus.

The legume-rhizobium symbiosis represents a unique model within the realm of plant-microbe interactions. Unlike typical cases of pathogenic invasion, the infection of rhizobia and their residence within symbiotic cells do not elicit a noticeable immune response in plants. Nevertheless, there is still much to uncover regarding the mechanisms through which plant immunity influences rhizobial symbiosis. In this study, we identify an important player in this intricate interplay: Lotus japonicus PRP1, which serves as a positive regulator of plant immunity but also exhibits the capacity to decrease rhizobial colonization and nitrogen fixation within nodules. The PRP1 gene encodes an uncharacterized protein and is named Pathogenesis-Related Protein1, owing to its orthologue in Arabidopsis thaliana, a pathogenesis-related family protein (At1g78780). The PRP1 gene displays high expression levels in nodules compared to other tissues. We observed an increase in rhizobium infection in the L. japonicus prp1 mutants, whereas PRP1-overexpressing plants exhibited a reduction in rhizobium infection compared to control plants. Intriguingly, L. japonicus prp1 mutants produced nodules with a pinker colour compared to wild-type controls, accompanied by elevated levels of leghaemoglobin and an increased proportion of infected cells within the prp1 nodules. The transcription factor Nodule Inception (NIN) can directly bind to the PRP1 promoter, activating PRP1 gene expression. Furthermore, we found that PRP1 is a positive mediator of innate immunity in plants. In summary, our study provides clear evidence of the intricate relationship between plant immunity and symbiosis. PRP1, acting as a positive regulator of plant immunity, simultaneously exerts suppressive effects on rhizobial infection and colonization within nodules.