Functional role of TrIL-1ß in Takifugu rubripes defense against Cryptocaryon irritans infection.

The Japanese puffer, Takifugu rubripes, is a commercially important fish species in China that is under serious threat from white spot disease (cyptocaryoniasis), which leads to heavy economic losses. We previously found that interleukin-1ß (IL-1ß), an important cytokine with a potential role in resistance against pathogens, was one of the most significantly differentially up-regulated proteins in the gills and spleen of T. rubripes infected by the protozoan parasite Cryptocaryon irritans. In this study, we assessed the potential function of T. rubripes IL-1ß (TrIL-1ß) in fish infected with C. irritans. Phylogenetic analysis indicated that the TrIL-1ß protein sequence was most closely related to that of Atlantic salmon (Salmo salar) (67.2 %). The incubation experiments revealed that TrIL-1ß may reduce trophont activity by destroying membranes. Immunofluorescence experiments showed that recombinant TrIL-1ß promoted the expression of endogenous IL-1ß, which penetrated and disrupted the cell membranes of trophonts. Transmission electron microscopy showed that the IL-1ß group had less tissue damage compared with control groups of fish. IL-1ß-small interfering RNA and IL-1ß overexpression experiments were performed in head kidney primary cells, and challenge experiments were performed in vitro. Quantitative RT-PCR results showed that TrIL-1ß regulated and activated MyD88/NF-κB and MyD88/MAPK/p38 signaling pathways during C. irritans infection. TrIL-1ß also promoted the differential expression of IgM, showing that it was involved in humoral immunity of T. rubripes. The cumulative mortality experiment show that TrIL-1ß could protect fish against C. irritans infection. These results enrich current knowledge about the molecular structure of TrIL-1ß. They also suggested that recombinant TrIL-1ß could be used as an adjuvant in a subunit vaccine against C. irritans infection, which is of profound importance for the prevention and control of parasitic diseases in T. rubripes.

Efficacy of cardiopulmonary rehabilitation nursing in enhancing pulmonary function, sleep quality, and living ability in COVID-19 patients.

OBJECTIVE: The purpose of this study was to elucidate the impact of cardiopulmonary rehabilitation nursing on the pulmonary function, sleep quality, and living ability of patients afflicted with Coronavirus Disease 2019 (COVID-19). METHODS: A total of 98 patients with COVID-19 treated at The People's Hospital of Guang'an between September 2021 and January 2023 were retrospectively collected as the research subjects. Among them, 48 patients who received standard nursing care from September 2021 to September 2022 were set as the control group, and 50 patients who underwent cardiopulmonary rehabilitation nursing from October 2022 to January 2023 were set as the research group. The pulmonary function indicators [including Forced Expiratory Volume in 1 second (FEV1) and Left Ventricular Ejection Fraction (LVEF)], sleep quality [evaluated using the Pittsburgh Sleep Quality Index (PSQI)], and living ability [assessed by the 36-Item Short Form Survey (SF-36) scale] pre- and post-intervention were compared between the two groups. RESULTS: Pre-intervention, FEV1, LVEF, PSQI scores, inflammatory factor levels [C-reactive protein (CRP), procalcitonin (PCT)], and SF-36 scores showed no significant differences between the two groups (P>0.05). Post-intervention, the research group exhibited notably enhanced FEV1 and LVEF, lower PSQI scores, lower CRP and PCT, and higher SF-36 scores compared with the control group, with statistical significance (P<0.05). Multifactorial logistic regression analysis showed that non-receipt of cardiopulmonary rehabilitation, age ≥60 years, concurrent respiratory failure, coexistent heart failure, and acid-base imbalance were independent risk factors of adverse outcomes in COVID-19 patients (P<0.05). CONCLUSION: Application of cardiopulmonary rehabilitation nursing in COVID-19 patients can significantly improve pulmonary function, sleep quality, and overall quality of life, and relieve the inflammatory state of the patients, thereby enhancing prognosis. This approach has certain value of popularization and application.

Ribosomal DNA and Neurological Disorders.

Ribosomal DNA (rDNA) is important in the nucleolus and nuclear organization of human cells. Defective rDNA repeat maintenance has been reported to be closely associated with neurological disorders, such as Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia, depression, suicide, etc. However, there has not been a comprehensive review on the role of rDNA in these disorders. In this review, we have summarized the role of rDNA in major neurological disorders to sort out the correlation between rDNA and neurological diseases and provided insights for therapy with rDNA as a target.

Neuropeptides affecting social behavior in mammals: Oxytocin.

Oxytocin (OXT), a neuropeptide consisting of only nine amino acids, is synthesized in the paraventricular and supraoptic nuclei of the hypothalamus. Although OXT is best known for its role in lactation and parturition, recent research has shown that it also has a significant impact on social behaviors in mammals. However, a comprehensive review of this topic is still lacking. In this paper, we systematically reviewed the effects of OXT on social behavior in mammals. These effects of OXT from the perspective of five key behavioral dimensions were summarized: parental behavior, anxiety, aggression, attachment, and empathy. To date, researchers have agreed that OXT plays a positive regulatory role in a wide range of social behaviors, but there have been controversially reported results. In this review, we have provided a detailed panorama of the role of OXT in social behavior and, for the first time, delved into the underlying regulatory mechanisms, which may help better understand the multifaceted role of OXT. Levels of OXT in previous human studies were also summarized to provide insights for diagnosis of mental disorders.

DNA Methylation Analysis Reveals Potential Mechanism in Takifugu rubripes Against Cryptocaryon irritans Infection.

Takifugu rubripes (T. rubripes) is a valuable commercial fish, and Cryptocaryon irritans (C. irritans) has a significant impact on its aquaculture productivity. DNA methylation is one of the earliest discovered ways of gene epigenetic modification and also an important form of modification, as well as an essential type of alteration that regulates gene expression, including immune response. To further explore the anti-infection mechanism of T. rubripes in inhibiting this disease, we determined genome-wide DNA methylation profiles in the gill of T. rubripes using whole-genome bisulfite sequencing (WGBS) and combined with RNA sequence (RNA-seq). A total of 4659 differentially methylated genes (DMGs) in the gene body and 1546 DMGs in the promoter between the infection and control group were identified. And we identified 2501 differentially expressed genes (DEGs), including 1100 upregulated and 1401 downregulated genes. After enrichment analysis, we identified DMGs and DEGs of immune-related pathways including MAPK, Wnt, ErbB, and VEGF signaling pathways, as well as node genes prkcb, myca, tp53, and map2k2a. Based on the RNA-Seq results, we plotted a network graph to demonstrate the relationship between immune pathways and functional related genes, in addition to gene methylation and expression levels. At the same time, we predicted the CpG island and transcription factor of four immune-related key genes prkcb and mapped the gene structure. These unique discoveries could be helpful in the understanding of C. irritans pathogenesis, and the candidate genes screened may serve as optimum methylation-based biomarkers that can be utilized for the correct diagnosis and therapy T. rubripes in the development of the ability to resist C. irritans infection.

Chemotherapy combined with bevacizumab for small cell lung cancer with brain metastases: A case report.

BACKGROUND: Small cell lung cancer (SCLC) is a common and aggressive subtype of lung cancer. It is characterized by rapid growth and a high mortality rate. Approximately 10% of patients with SCLC present with brain metastases at the time of diagnosis, which is associated with a median survival of 5 mo. This study aimed to summarize the effect of bevacizumab on the progression-free survival (PFS) and overall survival of patients with brain metastasis of SCLC. CASE SUMMARY: A 62-year-old man was referred to our hospital in February 2023 because of dizziness and numbness of the right lower extremity without headache or fever for more than four weeks. The patient was diagnosed with limited-stage SCLC. He received 8 cycles of chemotherapy combined with maintenance bevacizumab therapy and achieved a PFS of over 7 mo. CONCLUSION: The combination of bevacizumab and irinotecan effectively alleviated brain metastasis in SCLC and prolonged PFS.

Copper exposure causes alteration in the intestinal microbiota and metabolites in Takifugu rubripes.

Copper is an environmental pollutant, and copper in aquatic environments mainly comes from soil and water. It enters the environment through atmospheric deposition, sewage discharge, and industrial production, and enters aquatic organisms, causing toxicity. Takifugu rubripes (T. rubripes) is a marine fish with high economic value. Due to the toxic effects of heavy metals on aquatic organisms such as fish, it can affect the gut community and metabolites of fish. The gut is an important channel for fish to communicate with the outside world and a necessary pathway for the metabolism of nutrients and toxic substances in the fish body. Studies have shown that due to changes in global water emissions and the high sensitivity of aquatic organisms to the environment, copper may pose greater potential hazards to aquatic organisms. Copper poses a greater risk to aquatic species than other heavy metals and metal/metal like pollutants (such as cadmium, lead, mercury, arsenic, etc.) . In order to elucidate the effects of copper exposure on the gut of T. rubripes. In this study, we exposed T. rubripes to 0, 50, 100, or 500 µg/L of copper for three days, the effects of copper exposure on the gut microbiota structure and metabolites of the T. rubripes were investigated using 16 S rRNA gene and metabolomics techniques. The research results indicate that with the increase copper concentration, the intestinal tissue of T. rubripes undergoes significant damage. 16 S rRNA sequencing results show that copper exposure alters the structure and metabolites of intestinal microbiota. Copper exposure of 100 and 500 µg/L inhibited the colonization of the bacterial gut, disrupted the intestinal barrier, and made the fish susceptible to the pathogens. Liquid chromatography-mass spectrometry analysis showed that copper regulated the production of metabolites such as L-histidine, arachidonic acid, and L-glutamic acid, which are related to energy and immunity. Microbiome-metabolome correlation analysis showed that Subdoligranulum, Family_XIII_AD3011_group, and Clostridium_sensu_stricto_1 were the key bacteria for copper ion intervention, and they might up-regulate the levels of metabolites such as indole-3-acetic acid, 3-indoleacrylic acid, and 5-hydroxyindole in the tryptophan metabolism pathway. In summary, our research has demonstrated that copper exposure can cause pathological changes in the intestinal tissue of the T. rubripes. High concentrations of copper ions can affect the colonization of the T. rubripes microbiota in the intestine, damage the fish's immune system, and alter the structure and metabolites of the intestinal microbiota, this can lead to intestinal metabolic dysfunction. providing a reference for the evaluation of the biological toxicity effects of heavy metal elements in the marine environment. This study provides a reference for evaluating the biological toxicity effects of heavy metal elements in marine environments.

Dual roles of CCDC102A in governing centrosome duplication and cohesion.

In animal cells, the dysregulation of centrosome duplication and cohesion maintenance leads to abnormal spindle assembly and chromosomal instability, contributing to developmental disorders and tumorigenesis. However, the molecular mechanisms involved in maintaining accurate centrosome number control and tethering are not fully understood. Here, we identified coiled-coil domain-containing 102A (CCDC102A) as a centrosomal protein exhibiting a barrel-like structure in the proximal regions of parent centrioles, where it prevents centrosome overduplication by restricting interactions between Cep192 and Cep152 on centrosomes, thereby ensuring bipolar spindle formation. Additionally, CCDC102A regulates the centrosome linker by recruiting and binding C-Nap1; it is removed from the centrosome after Nek2A-mediated phosphorylation at the onset of mitosis. Overall, our results indicate that CCDC102A participates in controlling centrosome number and maintaining centrosome cohesion, suggesting that a well-tuned system regulates centrosome structure and function throughout the cell cycle.

Carboxylesterase-activated near-infrared fluorescence probe for highly sensitive imaging of liver tumors.

Carboxylesterases (CESs) are critical for metabolizing ester-containing biomolecules and are specifically important in liver metabolic disorders. The modulation of CESs is also an important issue in pharmacology and clinical applications. Herein, we present a near-infrared (NIR) CES fluorescent probe (NCES) based on the protection-deprotection of the hydroxyl group for monitoring CES levels in living systems. The NCES probe has good selectivity and sensitivity for CESs with a limit of detection (LOD) of 5.24 mU mL-1, which allows for tracing the fluctuation of cellular CES after treatment with anticancer drugs and under inflammation and apoptosis states. Furthermore, NCES can be successfully applied for guiding liver cancer surgery with high-contrast in vivo imaging and detecting clinical serum samples from liver cancer patients. This work showed that the NCES probe has great potential in drug development, imaging applications for medical diagnosis, and early-stage detection for clinical liver diseases.

One-Base-Gap Circular Probe-Mediated Dual Amplification for Isothermal Detection of N6-Methyladenosine Modifications.

N6-Methyladenosine (m6A) stands out as the predominant internal modification in mammalian RNA, exerting crucial regulatory functions in the metabolism of mRNA. Currently available methods have been limited by an inability to quantify m6A modification at precise sites. In this work, we screened a Bst 2.0 warm start DNA polymerase with the capability of discriminating m6A from adenosine (A) and developed a robust m6A RNA detection method that enables isothermal and ultrasensitive quantification of m6A RNA at single-base resolution. The detection limit of the assay could reach about 0.02 amol, and the quantitative accuracy of the assay was verified in real cell samples. Furthermore, we applied this assay to single-cell analysis and found that the coefficients of variation of the MALAT1 m6A 2611 site in glioblastoma U251 cells showed over 20% higher than in oligodendrocytes MO3.13 cells. This method provides a highly sensitive analytical tool for site-specific m6A detection and quantification, which is expected to provide a basis for precise disease diagnosis and epigenetic transcriptional regulation.

Visual genetic typing of glioma using proximity-anchored in situ spectral coding amplification.

Gliomas are histologically and genetically heterogeneous tumors. However, classical histopathological typing often ignores the high heterogeneity of tumors and thus cannot meet the requirements of precise pathological diagnosis. Here, proximity-anchored in situ spectral coding amplification (ProxISCA) is proposed for multiplexed imaging of RNA mutations, enabling visual typing of brain gliomas with different pathological grades at the single-cell and tissue levels. The ligation-based padlock probe can discriminate one-nucleotide variations, and the design of proximity primers enables the anchoring of amplicons on target RNA, thus improving localization accuracy. The DNA module-based spectral coding strategy can dramatically improve the multiplexing capacity for imaging RNA mutations through one-time labelling, with low cost and simple operation. One-target-one-amplicon amplification confers ProxISCA the ability to quantify RNA mutation copy number with single-molecule resolution. Based on this approach, it is found that gliomas with higher malignant grades express more genes with high correlation at the cellular and tissue levels and show greater cellular heterogeneity. ProxISCA provides a tool for glioma research and precise diagnosis, which can reveal the relationship between cellular heterogeneity and glioma occurrence or development and assist in pathological prognosis.

Tandem mass tag (TMT) quantitative proteomics and phosphoproteomic of Takifugu rubripes infected with Cryptocaryon irritans.

In this study, we identified the differentially expressed proteins in gills stimulated by infected ciliates and analyzed the immune mechanisms of T. rubripes infected with the ciliate Cryptocaryon irritans. Through liquid chromatography analysis, a total of 144 proteins were identified with significant differences, of which 58 were upregulated and 86 were downregulated. Among phosphorylated proteins, we identified a total of 167 significantly different phosphorylated proteins, of which 44 were upregulated, 123 were downregulated, 60 were upregulated, and 208 were downregulated. We analyzed the data of proteomics and Phosphorylated proteome quantification protein omics to finally identify three phosphorylated proteins (RPS27, eNOS and CaM) and two phosphorylated protein kinases(CaMKII and MAPK1) as potential biomarkers for T. rubripes immune responses. We finally identified three phosphorylated proteins (RPS27, eNOS and CaM) and two phosphorylated protein kinases (CaMKII and MAPK1) as potential biomarkers of immune response of T. rubripes. Our research findings provide new insights into the immune mechanism of T. rubripes, which may serve as an effective indicator of C. irritans infection in T. rubripes.

Combination of RCA and DNAzyme for Dual-Signal Isothermal Amplification of Exosome RNA.

The RNA contained in exosomes plays a crucial role in information transfer between cells in various life activities. The accurate detection of low-abundance exosome RNA (exRNA) is of great significance for cell function studies and the early diagnosis of diseases. However, their intrinsic properties, such as their short length and high sequence homology, represent great challenges for exRNA detection. In this paper, we developed a dual-signal isothermal amplification method based on rolling circle amplification (RCA) coupled with DNAzyme (RCA-DNAzyme). The sensitive detection of low-abundance exRNA, the specific recognition of their targets and the amplification of the detection signal were studied and explored. By designing padlock probes to specifically bind to the target exRNA, while relying on the ligation reaction to enhance recognition, the precise targeting of exosome RNA was realized. The combination of RCA and DNAzyme could achieve a twice-as-large isothermal amplification of the signal compared to RCA alone. This RCA-DNAzyme assay could sensitively detect a target exRNA at a concentration as low as 527 fM and could effectively distinguish the target from other miRNA sequences. In addition, this technology was successfully proven to be effective for the quantitative detection of miR-21 by spike recovery, providing a new research approach for the accurate detection of low-abundance exRNA and the exploration of unknown exRNA functions.

Proteomics and Phosphoproteomic Analysis to Identify Spleen of Takifugu rubripes Infected Cryptocaryon irritans.

Takifugu rubripes is important commercially fish species in China and it is under serious threat from white spot disease (cyptocaryoniasis), which leads to heavy economic losses. In this study, we used proteomics and phosphoproteomic analysis to identify differentially abundant proteins in the spleen of T. rubripes infected with the Cryptocaryon irritans. We identified 5,307 proteins and 6,644 phosphorylated sites on 2,815 phosphoproteins using high-throughput proteomics analysis of the spleen of T. rubripes based on 26,421 unique peptides and 5,013 modified peptides, respectively. The 5,307 quantified host proteins, 40 were upregulated and 43 were downregulated in the infection group compared to the control group. Among the 2815 phosphoproteins, 44/120 were upregulated/downregulated, and 62/151 were upregulated/downregulated in the 6644 quantified phosphosites. Using the combination of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses, screening for significantly different phosphoproteins, motif analysis and protein-protein interaction analysis, we ultimately identified three phosphorylated proteins (G-protein-signaling modulator 1-like, zinc finger protein 850-like, and histone H1-like) and three phosphorylated protein kinases (serine/threonine-protein kinase homolog isoform X2, mitogen-activated protein kinase 5-like, and protein kinase C theta type) as potential biomarkers for T. rubripes immune responses. We then screened the phosphorylation sites of these biomarker proteins for further verification. Based on our results, we speculate that phosphorylation modification of the phosphorylation sites is involved in the immunity of T. rubripes against C. irritans.

Concomitant analyses of intratumoral microbiota and genomic features reveal distinct racial differences in breast cancer.

Racial disparities are most accentuated among Black women as their lifetime risk of breast cancer incidence is lower than white and Asian women but their breast cancer related mortality is the highest among all races. Black women are more likely to develop triple-negative breast cancer at a younger age and harbor more aggressive tumors. In addition to tumor-centric alterations, tumor growth is also influenced by multiple other tumor microenvironment-related features, including resident immune cells and microbiota. Hence, in this study, we conduct concurrent genomic and metagenomic analyses, and uncover distinctive intratumoral microbial community compositions and tumor immune microenvironment-related traits in breast tumors from Asian, Black and white women. Interestingly, unique racially associated genomic nodes are found in the breast tumors from Asian, Black and white women. Examination of the cellular heterogeneity show differential enrichment of 11 out of 64 immune and stroma cell types in the breast tumors from different racial groups. In terms of microbial diversity, significant differences are revealed in alpha and beta-diversity measures. Intriguingly, potential race-specific microbial biomarkers of breast cancer are identified which significantly correlate with genes involved with tumor aggressiveness, angiogenesis, tumor cell migration and metastasis as well as oncogenic pathways-GLI and Notch. Investigating the metabolic features of intratumoral microbes, we find a significant differential enrichment of environmental information processing pathways, oncogenic pathways, and lipid metabolism pathways. Concomitantly investigating tumor-centric, tumor immune microenvironment-related and microbial alterations, our study provides a comprehensive understanding of racial disparities in breast cancer and warrants further exploration.

Coupled CFD-DEM modeling to predict how EPS affects bacterial biofilm deformation, recovery and detachment under flow conditions.

The deformation and detachment of bacterial biofilm are related to the structural and mechanical properties of the biofilm itself. Extracellular polymeric substances (EPS) play an important role on keeping the mechanical stability of biofilms. The understanding of biofilm mechanics and detachment can help to reveal biofilm survival mechanisms under fluid shear and provide insight about what flows might be needed to remove biofilm in a cleaning cycle or for a ship to remove biofilms. However, how the EPS may affect biofilm mechanics and its deformation in flow conditions remains elusive. To address this, a coupled computational fluid dynamic- discrete element method (CFD-DEM) model was developed. The mechanisms of biofilm detachment, such as erosion and sloughing have been revealed by imposing hydrodynamic fluid flow at different velocities and loading rates. The model, which also allows adjustment of the proportion of different functional groups of microorganisms in the biofilm, enables the study of the contribution of EPS toward biofilm resistance to fluid shear stress. Furthermore, the stress-strain curves during biofilm deformation have been captured by loading and unloading fluid shear stress to study the viscoelastic properties of the biofilm. Our predicted emergent viscoelastic properties of biofilms were consistent with relevant experimental measurements.

The Optimization of Chlorella vulgaris Flocculation Harvesting by Chitosan and Calcium Hydroxide.

The high cost for microalgae harvesting still is the bottleneck of microalgae commercial production. In the present study, the effect of adjusting pH to alkaline conditions with sodium hydroxide/calcium hydroxide and the addition of chitosan together with pH adjustments on the flocculation of Chlorella vulgaris (C. vulgaris) was studied, respectively. A single-factor experiment showed a maximum flocculation efficiency of 96.7% when adjusting the pH to 12 with calcium hydroxide. There was synergistic action between chitosan and calcium hydroxide. Flocculation conditions of C. vulgaris for the combined use of calcium hydroxide and chitosan was optimized by response surface methodology (RSM) with a Box-Behnken design (BBD). Flocculation efficiency reached 97.08% under optimal flocculation conditions when adjustion of pH to 8.97 with 2 g/L calcium hydroxide, a chitosan dosage of 20 mg/L, and a flocculation time of 60 min. The current study presents one method for efficient flocculation harvesting of C. vulgaris at weak alkaline conditions and low chitosan dosage. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01004-1.

A Comprehensive Evaluation of the Application of the Halcyon(2.0) IMRT Technique in Long-Course Radiotherapy for Rectal Cancer.

Objective: To evaluate if the Halcyon(2.0) Intensity Modulation Radiotherapy (IMRT) technique has an advantage in the long-course rectal cancer radiotherapy. Methods: A total of 20 clinical IMRT plans of Halcyon(2.0) for long-course (2Gy in 25 fractions) rectal cancer radiotherapy were randomly selected. Based on the parameters of these plans, 20 TrueBeam (with the Millennium 120 MLC) plans were redesigned, respectively. The dosimetry indexes, field complexity parameters, the Gamma Passing Rates (GPR), and the delivery time of the 2 groups of plans were obtained as measures of the plan quality, the modulation complexity, the delivery accuracy, and the delivery efficiency. The differences between the 2 groups of parameters were analyzed, with P < .05 means statistically significant. Results: In terms of dosimetry, there was no significant or clinical difference between the 2 groups in critical dosimetry parameters. The Monitor Unit of the Halcyon(2.0) fields is lower than the TrueBeam fields by 26.39, while the modulation complexity score (MCS), the mean aperture area variability (AAV), and the mean leaf sequence variability (LSV) of the Halcyon(2.0) fields were 23.8%, 20%, and 2.3% larger than those of the TrueBeam fields, respectively. Neither the ArcCheck-based GPRs nor the portal-dosimetry-based GPRs in both 3%/3 mm and 2%/2 mm criteria showed the difference between the Halcyon(2.0) fields and the TrueBeam fields. The Pearson correlation coefficient between GPR(2%/2 mm) and MCS of the Halcyon(2.0) fields was 0.335, while that of the TrueBeam fields was 0.502. The mean total delivery time of the TrueBeam plans was 195.55 ± 22.86 s, while that of Halcyon(2.0) was 124.25 ± 10.42 s (P < .001), which was reduced approximatively by 36%. Conclusion: For long-course rectal cancer radiotherapy, the Halcyon(2.0) IMRT plans behave almost the same in dosimetry and delivery accuracy as the TrueBeam plans. However, the lower MU and the field modulation complexity, combined with the higher delivery efficiency, make Halcyon(2.0) a feasible and reliable platform in long-course radiotherapy for the rectal cancer.

Dual-Functional Nanocluster Probe-Based Single-Cell Analysis of RNA Splice Variants.

Differential expression of RNA splice variants among individual cells accounts for cell heterogeneity of gene expression, which plays a key role in the regulation of the immune system. However, currently available techniques face difficulties in achieving single-cell analysis of RNA splice variants with high base resolution, high spatial resolution and accurate quantification. Herein, we constructed DNA-templated dual-functional nanocluster probes to achieve in situ imaging and accurate quantification of RNA splice variants at the single-cell level. By designing ultrasmall nanocluster labeled probes to directly target the splicing junction sequence of RNA splice variants, the base recognition resolution is significantly improved. Benefit from the controllable fluorescence of nanoclusters, in situ imaging and genotyping of RNA splice variants are achieved. Due to the atom-precise nanocluster, RNA splice variants can be accurately quantified by laser ablation inductively coupled plasma mass spectrometry at the single-cell level. We further applied the probes to explore the function of MyD88 splice variants in mononuclear macrophages under immune activation. This strategy provides a novel single-cell analysis tool for studying the functional diversity of the immune system and splicing-related immune diseases.