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Cell atlases serve as vital references for automating cell labeling in new samples, yet existing classification algorithms struggle with accuracy. Here we introduce SIMS (scalable, interpretable machine learning for single cell), a low-code data-efficient pipeline for single-cell RNA classification. We benchmark SIMS against datasets from different tissues and species. We demonstrate SIMS's efficacy in classifying cells in the brain, achieving high accuracy even with small training sets (

The presented study demonstrates the capability of the template-based electrokinetic assembly (TEA) and guidance to manipulate and capture individual biological cells within a microfluidic platform. Specifically, dielectrophoretic (DEP) focusing of K-562 cells towards lithographically-defined wells on the microelectrodes and positioning singles cells withing these wells was demonstrated. K-562 lymphoblast cells, are widely used in immunology research. The DEP guidance, particularly involving positive DEP (pDEP), enables the controlled guidance and positioning of conductive and dielectric particles, including biological cells, opening new directions for the accurate and efficient microassembly of biological entities, which is crucial for single cell analysis and other applications in biotechnology. The investigation explores the use of glassy carbon and gold as electrode materials. It was established previously that undiluted physiological buffer is unsuitable for inducing positive DEP (pDEP); therefore, the change of media into a lower ionic concentration is necessary. After pDEP was observed, the cells are resubmerged in the Iscoves modified Dulbeccos medium (IMEM), a cell culturing media, and incubated. A dead/alive staining assay was performed on the cells to determine their survival in the diluted buffer for the period required to capture them. The staining assay confirmed the cells survival after being immersed in the diluted biological buffer necessary for electrokinetic handling. The results indicate the promise of the proposed electrokinetic bio-sorting technology for applications in tissue engineering, lab-on-a-chip devices, and organ-on-a-chip models, as well as contributing to the advancement of single cell analysis.

Single cell–inductively coupled plasma–mass spectrometry (SC-ICP-MS) offers an attractive option for rapidly measuring trace metal heterogeneity at the single cell level. Chemical fixation has been previously applied to mammalian cells prior to sample introduction so that they can be resuspended in a solution suitable for SC-ICP-MS. However, the effect of fixation on the elemental composition of suspended cells is unknown, and robust methodologies are urgently needed so that the community can measure the effects of intracellular pathogens on elemental composition of their host cells. We demonstrate that different fixatives impact measured cell elemental composition. We have compared suspensions treated using different fixatives (methanol 60–100% in H2O and 4% paraformaldehyde in phosphate-buffered saline solution), and the number of distinguishable single cell events, keeping a constant particle number concentration. Significantly more single cell events (n = 3, P ≤ 0.05) were observed for Ca and Mg when cells were fixed in 4% paraformaldehyde than for the methanol-based fixatives, confirming the hypothesis that methanol fixatives cause leaching of these elements from the cells. The impact of fixation on Mn and Zn was less pronounced. Microbial and viral infection of eukaryotic cells can have profound effects on their elemental composition, but chemical fixation is necessary to render infected cells safe before analysis. We have successfully applied our methodology to a macrophage model of tuberculosis demonstrating utility in understanding metal homeostasis during microbial infection of mammalian cells. [ABSTRACT FROM AUTHOR]

Background: Intervertebral disc degeneration (IDD) is a major cause for low back pain. Studies showed the association between senescence and degenerative diseases. Cell senescence can promote the occurrence and development of degenerative diseases through multiple mechanisms including inflammatory stress, oxidative stress and nutritional deprivation. The roles of senescence and senescence‐associated genes (SAGs) remains unknown in IDD. Methods: Four differently expressed SAGs were identified as hub SAGs using "limma" package in R. We then calculated the immune infiltration of IDD patients, and investigated the relation between hub SAGs and immune infiltration. Enrichment analysis was performed to explore the functions of hub SAGs in IDD. Nomogram and LASSO model based on hub SAGs was constructed to predict the risk of severe degeneration (SD) for IDD patients. Subsequently, single cell analysis was conducted to describe the expression pattern of hub SAGs in intervertebral disc tissue. Results: We identified ASPH, CCND1, IGFBP3 and SGK1 as hub SAGs. Further analysis demonstrated that the hub SAGs might mediate the development of IDD by regulating immune infiltration and multiple pathways. The LASSO model based on the four hub SAGs showed good performance in predicting the risk of SD. Single cell analysis revealed that ASPH, CCND1 and SGK1 mainly expressed in nucleus pulposus cells, while IGFBP3 mainly expressed in epithelial cells. Eleven candidate drugs targeting hub SAGS were predicted for IDD patients through Comparative Toxicogenomics Database (CDT). PCR and immunohistochemical analysis showed that the levels of four hub SAGs were higher in SD than MD (mild degeneration) patients. Conclusions: We performed a comprehensive analysis of SAGs in IDD, which revealed their functions and expression pattern in intervertebral disc tissue. Based on hub SAGs, we established a predictive model and explored the potential drugs. These findings provide new understandings of SAG mechanism and promising therapeutic strategies for IDD. [ABSTRACT FROM AUTHOR]

Background: Hepatocellular carcinoma (HCC) poses a significant global health challenge due to its poor prognosis and limited therapeutic modalities. Anoikis and ErbB signaling pathways are pivotal in cancer cell proliferation and metastasis, but their relevance in HCC remains insufficiently explored. Methods: This study evaluates the prognostic significance of anoikis and ErbB signaling pathways in HCC by utilizing data from The Cancer Genome Atlas (TCGA), the International Cancer Genome Consortium (ICGC), three additional independent validation cohorts, and an in-house cohort. Advanced bioinformatics analyses and 167 machine learning models based on leave-oneout cross-validation (LOOCV) were used to predict HCC prognosis and assess outcomes of immune-targeted therapies. Additionally, key biological processes of the anoikis and ErbB signaling pathways in HCC were further investigated. Results: The single sample Gene Set Enrichment Analysis revealed a strong correlation between upregulated ErbB signaling in high anoikis-expressing tumors and poor clinical outcomes. The development of the Anoikis-ErbB Related Signature (AERS) using the LASSO + RSF model demonstrated robust predictive capabilities, as validated across multiple patient cohorts, and proved effective in predicting responses to immune-targeted therapies. Further investigation highlighted activated NOTCH signaling pathways and decreased macrophage infiltration was associated with resistance to sorafenib and immune checkpoint inhibitors, as evidenced by bulk and single-cell RNA sequencing (scRNA-seq). Conclusion: AERS provides a novel tool for clinical prognosis and paves the way for immune-targeted therapeutic approaches, underscoring the potential of integrated molecular profiling in enhancing treatment strategies for HCC. [ABSTRACT FROM AUTHOR]

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and causes significant morbidity, ultimately leading to kidney failure. PKD pathogenesis is characterized by complex and dynamic alterations in multiple cell types during disease progression, hampering a deeper understanding of disease mechanism and the development of therapeutic approaches. Here, we generate a single-nucleus multimodal atlas of an orthologous mouse PKD model at early, mid, and late timepoints, consisting of 125,434 single-nucleus transcriptomic and epigenetic multiomes. We catalog differentially expressed genes and activated epigenetic regions in each cell type during PKD progression, characterizing cell-type-specific responses to Pkd1 deletion. We describe heterogeneous, atypical collecting duct cells as well as proximal tubular cells that constitute cyst epithelia in PKD. The transcriptional regulation of the cyst lining cell marker GPRC5A is conserved between mouse and human PKD cystic epithelia, suggesting shared gene regulatory pathways. Our single-nucleus multiomic analysis of mouse PKD provides a foundation to understand the earliest changes molecular deregulation in a mouse model of PKD at a single-cell resolution. [ABSTRACT FROM AUTHOR]

Background: Intervertebral disc degeneration (IVDD) is one of the important causes of lower back pain, seriously affecting people's health and quality of life. This research employs single-cell analysis to identify the specific cellular subtypes and key regulatory genes associated with IVDD. Methods: We analyzed the single-cell data and screened cells that closely associated with the development of IVDD. The differential expression of feature genes between IVDD and control groups was analyzed. Additionally, drugs and regulatory transcription factors that interact with feature genes were predicted and clinically validated by reverse transcription quantitative real-time PCR (RT-qPCR), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA). Results: Our study identified the Chond2 cell subtype associated with IVDD and selected four feature genes influencing the development of IVDD, namely IGFBP3, ACAN, VAPA and TMEM45A, through the high-dimensional weighted gene co-expression network analysis (hdWGCNA) analysis, least absolute shrinkage and selection operator (LASSO), and random forest (RF). Besides, compared to the MDD group, IGFBP3 and TMEM45A were significantly upregulated in the SDD group, while ACAN and VAPA showed no significant difference between the two groups. ELISA testing revealed a positive correlation between IGFBP3 concentration and the grading of IVDD. Furthermore, Celecoxib may be used to treat IVDD by inhibiting IGFBP3. Conclusion: Our study identified the Chond2 cell subtype associated with IVDD and selected four feature genes influencing the development of IVDD, namely IGFBP3, ACAN, VAPA and TMEM45A. Our findings establish a robust theoretical foundation for the clinical diagnosis and treatment of IVDD patients.

In recent years, rapid advances in research methods have made single cell analysis possible. Systemic sclerosis (SSc), a disease characterized by the triad of immune abnormalities, fibrosis, and vasculopathy, has also been the subject of various analyses. To summarize the results of single cell analysis in SSc accumulated to date and to deepen our understanding of SSc. Four databases were used to perform a database search on 23rd June 2023. Assessed Grading of Recommendations Assessment, Development and Evaluation certainty of evidence were performed according to PRISMA guidelines. The analysis was completed on July 2023. 17 studies with 358 SSc patients were included. Three studies used PBMCs, six used skin, nine used lung with SSc-interstitial lung diseases (ILDs), and one used lung with SSc-pulmonary arterial hypertension (PAH). The cells studied included immune cells such as T cells, natural killer cells, monocytes, macrophages, and dendritic cells, as well as endothelial cells, fibroblasts, keratinocytes, alveolar type I cells, basal epithelial cells, smooth muscle cells, mesothelial cells, etc. This systematic review revealed the results of single cell analysis, suggesting that PBMCs, skin, SSc-ILD, and SSc-PAH show activation and dysfunction of cells associated with immune-abnormalities, fibrosis, and vasculopathy, respectively.

Background: Cancer stem cells (CSCs) are a subset of cells within tumors that possess the unique ability to self-renew and give rise to diverse tumor cells. These cells are crucial in driving tumormetastasis, recurrence, and resistance to treatment. The objective of this study was to pinpoint the essential regulatory genes associated with CSCs in prostate adenocarcinoma (PRAD) and assess their potential significance in the diagnosis, prognosis, and immunotherapy of patients with PRAD. Method: The study utilized single-cell analysis techniques to identify stem cellrelated genes and evaluate their significance in relation to patient prognosis and immunotherapy in PRAD through cluster analysis. By utilizing diverse datasets and employing various machine learning methods for clustering, diagnostic models for PRAD were developed and validated. The random forest algorithm pinpointed HSPE1 as the most crucial prognostic gene among the stem cellrelated genes. Furthermore, the study delved into the association between HSPE1 and immune infiltration, and employed molecular docking to investigate the relationship between HSPE1 and its associated compounds. Immunofluorescence staining analysis of 60 PRAD tissue samples confirmed the expression of HSPE1 and its correlation with patient prognosis in PRAD. Result: This study identified 15 crucial stem cell-related genes through singlecell analysis, highlighting their importance in diagnosing, prognosticating, and potentially treating PRAD patients. HSPE1 was specifically linked to PRAD prognosis and response to immunotherapy, with experimental data supporting its upregulation in PRAD and association with poorer prognosis. Conclusion: Overall, our findings underscore the significant role of stem cellrelated genes in PRAD and unveil HSPE1 as a novel target related to stem cell. [ABSTRACT FROM AUTHOR]

Intracellular protein abundance is routinely measured in mammalian cells using population-based techniques such as western blotting which fail to capture single cell protein levels or using fluorescence microscopy which is although suitable for single cell protein detection but not for rapid analysis of large no. of cells. Flow cytometry offers rapid, high-throughput, multiparameter-based analysis of intracellular protein expression in statistically significant no. of cells at single cell resolution. In past few decades, customized assays have been developed for flow cytometric detection of specific intracellular proteins. This review discusses the scope of flow cytometry for intracellular protein detection in mammalian cells along with specific applications. Technological advancements to overcome the limitations of traditional flow cytometry for the same are also discussed. [ABSTRACT FROM AUTHOR]

Drug responses heterogeneity is often highlighted to justify the need for precision medicine. However, due to the highly complex nature of cell phenotypes in many diseases, one of key challenges is how to obtain the high content features in a cellular population. Here we present a single-cell vibrational phenomics approach, integrating synchrotron infrared microspectroscopy and multivariate calculation, for quantitatively evaluating the cellular responses to drug perturbation with single cell resolution. In a human hepatocellular carcinoma HepG2 cell model, the phenotypic changes induced by two types of drugs, taxol (TAX) and protopanaxadiol (PPD), were analyzed and revealed the response heterogeneity in drug concentration and chemical components. These findings not only provide a label-free strategy for determining the drug response at the single cell level, but also demonstrate the great potential of vibrational phenomics as a drug discovery platform. [ABSTRACT FROM AUTHOR]

In recent years, rapid advances in research methods have made single cell analysis possible. Systemic sclerosis (SSc), a disease characterized by the triad of immune abnormalities, fibrosis, and vasculopathy, has also been the subject of various analyses. To summarize the results of single cell analysis in SSc accumulated to date and to deepen our understanding of SSc. Four databases were used to perform a database search on 23rd June 2023. Assessed Grading of Recommendations Assessment, Development and Evaluation certainty of evidence were performed according to PRISMA guidelines. The analysis was completed on July 2023. 17 studies with 358 SSc patients were included. Three studies used PBMCs, six used skin, nine used lung with SSc-interstitial lung diseases (ILDs), and one used lung with SSc-pulmonary arterial hypertension (PAH). The cells studied included immune cells such as T cells, natural killer cells, monocytes, macrophages, and dendritic cells, as well as endothelial cells, fibroblasts, keratinocytes, alveolar type I cells, basal epithelial cells, smooth muscle cells, mesothelial cells, etc. This systematic review revealed the results of single cell analysis, suggesting that PBMCs, skin, SSc-ILD, and SSc-PAH show activation and dysfunction of cells associated with immune-abnormalities, fibrosis, and vasculopathy, respectively. [ABSTRACT FROM AUTHOR]

Single-cell SERS imaging of dual membrane receptors on cancer cells cultured on substrates with different stiffness. [Display omitted] Membrane receptors perform a diverse range of cellular functions, accounting for more than half of all drug targets. The mechanical microenvironment regulates cell behaviors and phenotype. However, conventional analysis methods of membrane receptors often ignore the effects of the extracellular matrix stiffness, failing to reveal the heterogeneity of cell membrane receptors expression. Herein, we developed an in-situ surface-enhanced Raman scattering (SERS) imaging method to visualize single-cell membrane receptors on substrates with different stiffness. Two SERS substrates, Au@4-mercaptobenzonitrile@Ag@Sgc8c and Au@4-pethynylaniline@Ag@SYL3c, were employed to specifically target protein tyrosine kinase-7 (PTK7) and epithelial cell adhesion molecule (EpCAM), respectively. The polyacrylamide (PA) gels with tunable stiffness (2.5–25 kPa) were constructed to mimic extracellular matrix. The simultaneous SERS imaging of dual membrane receptors on single cancer cells on substrates with different stiffness was achieved. Our findings reveal decreased expression of PTK7 and EpCAM on cells cultured on stiffer substrates and higher migration ability of the cells. The results elucidate the heterogeneity of membrane receptors expression of cells cultured on the substrates with different stiffness. This single-cell analysis method offers an in-situ platform for investigating the impacts of extracellular matrix stiffness on the expression of membrane receptors, providing insights into the role of cell membrane receptors in cancer metastasis. [ABSTRACT FROM AUTHOR]

Previous research has unveiled a stem cell-like transcriptome enrichment in the aldehyde dehydrogenase-expressing (ALDHhigh) mesenchymal stromal cell (MStroC) fraction. However, considering the heterogeneity of MStroCs, with only a fraction of them presenting bona fide stem cells (MSCs), the actual potency of ALDH as an MSC-specific selection marker remains an issue. To address this, the proliferative and differentiation potential of individual ALDHhigh and ALDHlow MStroCs incubated at low oxygen concentrations, estimated to mimic stem cell niches (0.1% O 2), were assayed using single-cell clonal analysis, compared to standard conditions (20% O 2). We confirm that a high proliferative capacity and multi-potent MSCs are enriched in the ALDHhigh MStroC population, especially when cells are cultured at 0.1% O 2. Measurements of reduced/oxidized glutathione and mitochondrial superoxide anions with MitoSoX (MSX) indicate that this advantage induced by low oxygen is related to a decrease in the oxidative and reactive oxygen species (ROS) levels in the stem cell metabolic setup. However, ALDH expression is neither specific nor exclusive to MSCs, as high proliferative capacity and multi-potent cells were also found in the ALDHlow fraction. Furthermore, single-cell assays performed after combined cell sorting based on ALDH and MSX showed that the MSXlow MStroC population is enriched in stem/progenitor cells in all conditions, irrespective of ALDH expression or culture oxygen concentration. Importantly, the ALDHhighMSXlow MStroC fraction exposed to 0.1% O 2 was almost exclusively composed of genuine MSCs. In contrast, neither progenitors nor stem cells (with a complete absence of colony-forming ability) were detected in the MSXhigh fraction, which exclusively resides in the ALDHlow MStroC population. Our study reveals that ALDH expression is not exclusively associated with MSCs. However, cell sorting using combined ALDH expression and ROS content can be utilized to exclude MStroCs lacking stem/progenitor cell properties. [Display omitted] [ABSTRACT FROM AUTHOR]

The aetiology of bone metastasis in prostate cancer (PCa) remains unclear. This study aims to identify hub genes involved in this process. We utilized machine learning, GO, KEGG, GSEA, Single‐cell analysis, ROC methods to identify hub genes for bone metastasis in PCa using the TCGA and GEO databases. Potential drugs targeting these genes were identified. We validated these results using 16 specimens from patients with PCa and analysed the relationship between the hub genes and clinical features. The impact of APOC1 on PCa was assessed through in vitro experiments. Seven hub genes with AUC values of 0.727–0.926 were identified. APOC1, CFH, NUSAP1 and LGALS1 were highly expressed in bone metastasis tissues, while NR4A2, ADRB2 and ZNF331 exhibited an opposite trend. Immunohistochemistry further confirmed these results. The oxidative phosphorylation pathway was significantly enriched by the identified genes. Aflatoxin B1, benzo(a)pyrene, cyclosporine were identified as potential drugs. APOC1 expression was correlated with clinical features of PCa metastasis. Silencing APOC1 significantly inhibited PCa cell proliferation, clonality, and migration in vitro. This study identified 7 hub genes that potentially facilitate bone metastasis in PCa through mitochondrial metabolic reprogramming. APOC1 emerged as a promising therapeutic target and prognostic marker for PCa with bone metastasis. [ABSTRACT FROM AUTHOR]

Background: Osteoarthritis (OA) is a disabling and highly prevalent condition affecting millions worldwide. Recently discovered, disulfidptosis represents a novel form of cell death induced by the excessive accumulation of cystine. Despite its significance, a systematic exploration of disulfidptosis-related genes (DRGs) in OA is lacking. Methods: This study utilized three OA-related datasets and DRGs. Differentially expressed (DE)-DRGs were derived by intersecting the differentially expressed genes (DEGs) from GSE114007 with DRGs. Feature genes underwent screening through three machine learning algorithms. High diagnostic value genes were identified using the receiver operating characteristic curve. Hub genes were confirmed through expression validation. These hub genes were then employed to construct a nomogram and conduct enrichment, immune, and correlation analyses. An additional validation of hub genes was performed through in vitro cell experiments. Results: SLC3A2 and PDLIM1 were designated as hub genes, displaying excellent diagnostic performance. PDLIM1 exhibited low expression in early chondrocyte differentiation, rising significantly in the late stage, while SLC3A2 showed high overall expression, declining in the late differentiation stage. Cellular experiments corroborated the correlation of SLC3A2 and PDLIM1 with chondrocyte inflammation. Conclusions: Two hub genes, SLC3A2 and PDLIM1, were identified in relation to disulfidptosis, providing potential directions for diagnosing and treating OA. [ABSTRACT FROM AUTHOR]

Many different cell types and complex molecular pathways are involved in intervertebral disc degeneration (IDD). We used a multi-omics approach combining single-cell RNA sequencing (scRNA-seq), differential gene expression analysis, and Mendelian randomization (MR) to clarify the underlying genetic architecture of IDD. We identified 1,164 differentially expressed genes (DEGs) across four important cell types associated with IDD using publicly available single-cell datasets. A thorough gene network analysis identified 122 genes that may be connected to programmed cell death (PCD), a crucial route in the etiology of IDD. SLC40A1, PTGS2, and GABARAPL1 have been identified as noteworthy regulatory genes that may impede the advancement of IDD. Furthermore, distinct cellular subpopulations and dynamic gene expression patterns were revealed by functional enrichment analysis and pseudo-temporal ordering of chondrocytes. Our results highlight the therapeutic potential of GABARAPL1, PTGS2, and SLC40A1 targeting in the treatment of IDD.

Isolation of tumor-specific T cells and their antigen receptors (TCRs) from malignant pleural effusions (MPE) may facilitate the development of TCR-transduced adoptive cellular immunotherapy products for advanced lung cancer patients. However, the characteristics and markers of tumor-specific T-cells in MPE are largely undefined. To this end, to establish the phenotypes and antigen specificities of CD8+ T cells, we performed single-cell RNA and TCR sequencing of samples from three advanced lung cancer patients. Dimensionality reduction on a total of 4,983 CD8+ T cells revealed 10 clusters including naïve, memory, and exhausted phenotypes. We focused particularly on exhausted T cell clusters and tested their TCR reactivity against neoantigens predicted from autologous cancer cell lines. Four different TCRs specific for the same neoantigen and one orphan TCR specific for the autologous cell line were identified from one of the patients. Differential gene expression analysis in tumor-specific T cells relative to the other T cells identified CXCL13, as a candidate gene expressed by tumor-specific T cells. In addition to expressing CXCL13, tumor-specific T cells were present in a higher proportion of T cells co-expressing PDCD1(PD-1)/TNFRSF9(4-1BB). Furthermore, flow cytometric analyses in advanced lung cancer patients with MPE documented that those with high PD-1/4-1BB expression have a better prognosis in the subset of 57 adenocarcinoma patients (p = .039). These data suggest that PD-1/4-1BB co-expression might identify tumor-specific CD8+ T cells in MPE, which are associated with patients’ prognosis. (233 words)

ABSTRACT Background Intervertebral disc degeneration (IDD) is a major cause for low back pain. Studies showed the association between senescence and degenerative diseases. Cell senescence can promote the occurrence and development of degenerative diseases through multiple mechanisms including inflammatory stress, oxidative stress and nutritional deprivation. The roles of senescence and senescence‐associated genes (SAGs) remains unknown in IDD. Methods Four differently expressed SAGs were identified as hub SAGs using “limma“ package in R. We then calculated the immune infiltration of IDD patients, and investigated the relation between hub SAGs and immune infiltration. Enrichment analysis was performed to explore the functions of hub SAGs in IDD. Nomogram and LASSO model based on hub SAGs was constructed to predict the risk of severe degeneration (SD) for IDD patients. Subsequently, single cell analysis was conducted to describe the expression pattern of hub SAGs in intervertebral disc tissue. Results We identified ASPH, CCND1, IGFBP3 and SGK1 as hub SAGs. Further analysis demonstrated that the hub SAGs might mediate the development of IDD by regulating immune infiltration and multiple pathways. The LASSO model based on the four hub SAGs showed good performance in predicting the risk of SD. Single cell analysis revealed that ASPH, CCND1 and SGK1 mainly expressed in nucleus pulposus cells, while IGFBP3 mainly expressed in epithelial cells. Eleven candidate drugs targeting hub SAGS were predicted for IDD patients through Comparative Toxicogenomics Database (CDT). PCR and immunohistochemical analysis showed that the levels of four hub SAGs were higher in SD than MD (mild degeneration) patients. Conclusions We performed a comprehensive analysis of SAGs in IDD, which revealed their functions and expression pattern in intervertebral disc tissue. Based on hub SAGs, we established a predictive model and explored the potential drugs. These findings provide new understandings of SAG mechanism and promising therapeutic strategies for IDD.

The formation of new and functional cardiomyocytes requires a 3-step process: dedifferentiation, proliferation, and redifferentiation, but the critical genes required for efficient dedifferentiation, proliferation, and redifferentiation remain unknown. In our study, a circular trajectory using single-nucleus RNA sequencing of the pericentriolar material 1 positive (PCM1+) cardiomyocyte nuclei from hearts 1 and 3 days after surgery-induced myocardial infarction (MI) on postnatal Day 1 was reconstructed and demonstrated that actin remodeling contributed to the dedifferentiation, proliferation, and redifferentiation of cardiomyocytes after injury. We identified four top actin-remodeling regulators, namely Tmsb4x, Tmsb10, Dmd, and Ctnna3, which we collectively referred to as 2D2P. Transiently expressed changes of 2D2P, using a polycistronic non-integrating lentivirus driven by Tnnt2 (cardiac-specific troponin T) promoters (Tnnt2-2D2P-NIL), efficiently induced transiently proliferative activation and actin remodeling in postnatal Day 7 cardiomyocytes and adult hearts. Furthermore, the intramyocardial delivery of Tnnt2-2D2P-NIL resulted in a sustained improvement in cardiac function without ventricular dilatation, thickened septum, or fatal arrhythmia for at least 4 months. In conclusion, this study highlights the importance of actin remodeling in cardiac regeneration and provides a foundation for new gene-cocktail-therapy approaches to improve cardiac repair and treat heart failure using a novel transient and cardiomyocyte-specific viral construct.

Abstract Background Periodontitis is strongly associated with type 2 diabetes (T2D) that results in serious complications and mortality. However, the pathogenic role of periodontitis in the development of T2D and the underlain mechanism have not been fully elucidated. Methods A Mendelian randomization (MR) was performed to estimate the causality between two diseases. Bioinformatics tools, including gene ontology and pathway enrichment analyses, were employed to analyze the common differentially expressed genes (DEGs) in periodontitis and T2D. MR and colocalization analyses were then utilized to investigate the causal associations between potential pathogenic gene expression and the risk of T2D. Single cell-type expression analysis was further performed to detect the cellular localization of these genes. Results Genetically predicted periodontitis was associated with a higher risk of T2D (OR, 1.469; 95% CI, 1.117–1.930; P = 0.006) and insulin resistance (OR 1.034; 95%CI 1.001–1.068; P = 0.041). 79 common DEGs associated with periodontitis and T2D were then identified and demonstrated enrichment mainly in CXC receptor chemokine receptor binding and interleutin-17 signaling pathway. The integration of GWAS with the expression quantitative trait locis of these genes from the peripheral blood genetically prioritized 6 candidate genes, including 2 risk genes (RAP2A, MCUR1) and 4 protective genes (WNK1, NFIX, FOS, PANX1) in periodontitis-related T2D. Enriched in natural killer cells, RAP2A (OR 4.909; 95% CI 1.849–13.039; P = 0.001) demonstrated high risk influence on T2D, and exhibited strong genetic evidence of colocalization (coloc.abf-PPH4 = 0.632). Conclusions This study used a multi-omics integration method to explore causality between periodontitis and T2D, and revealed molecular mechanisms using bioinformatics tools. Periodontitis was associated with a higher risk of T2D. MCUR1, RAP2A, FOS, PANX1, NFIX and WNK1 may play important roles in the pathogenesis of periodontitis-related T2D, shedding light on the development of potential drug targets.

The formation of new and functional cardiomyocytes requires a 3-step process: dedifferentiation, proliferation, and redifferentiation, but the critical genes required for efficient dedifferentiation, proliferation, and redifferentiation remain unknown. In our study, a circular trajectory using single-nucleus RNA sequencing of the pericentriolar material 1 positive (PCM1+) cardiomyocyte nuclei from hearts 1 and 3 days after surgery-induced myocardial infarction (MI) on postnatal Day 1 was reconstructed and demonstrated that actin remodeling contributed to the dedifferentiation, proliferation, and redifferentiation of cardiomyocytes after injury. We identified four top actin-remodeling regulators, namely Tmsb4x , Tmsb10 , Dmd , and Ctnna3 , which we collectively referred to as 2D2P. Transiently expressed changes of 2D2P, using a polycistronic non-integrating lentivirus driven by Tnnt2 (cardiac-specific troponin T) promoters (Tnnt2-2D2P-NIL), efficiently induced transiently proliferative activation and actin remodeling in postnatal Day 7 cardiomyocytes and adult hearts. Furthermore, the intramyocardial delivery of Tnnt2-2D2P-NIL resulted in a sustained improvement in cardiac function without ventricular dilatation, thickened septum, or fatal arrhythmia for at least 4 months. In conclusion, this study highlights the importance of actin remodeling in cardiac regeneration and provides a foundation for new gene-cocktail-therapy approaches to improve cardiac repair and treat heart failure using a novel transient and cardiomyocyte-specific viral construct. Transient transfection of the 2D2P genes (Tmsb4x , Tmsb10 , Dmd , and Ctnna3) promotes actin cytoskeletal remodeling and cardiac regeneration, which is associated with the dedifferentiation, proliferation, and redifferentiation of cardiomyocytes. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: Periodontitis is strongly associated with type 2 diabetes (T2D) that results in serious complications and mortality. However, the pathogenic role of periodontitis in the development of T2D and the underlain mechanism have not been fully elucidated. Methods: A Mendelian randomization (MR) was performed to estimate the causality between two diseases. Bioinformatics tools, including gene ontology and pathway enrichment analyses, were employed to analyze the common differentially expressed genes (DEGs) in periodontitis and T2D. MR and colocalization analyses were then utilized to investigate the causal associations between potential pathogenic gene expression and the risk of T2D. Single cell-type expression analysis was further performed to detect the cellular localization of these genes. Results: Genetically predicted periodontitis was associated with a higher risk of T2D (OR, 1.469; 95% CI, 1.117–1.930; P = 0.006) and insulin resistance (OR 1.034; 95%CI 1.001–1.068; P = 0.041). 79 common DEGs associated with periodontitis and T2D were then identified and demonstrated enrichment mainly in CXC receptor chemokine receptor binding and interleutin-17 signaling pathway. The integration of GWAS with the expression quantitative trait locis of these genes from the peripheral blood genetically prioritized 6 candidate genes, including 2 risk genes (RAP2A, MCUR1) and 4 protective genes (WNK1, NFIX, FOS, PANX1) in periodontitis-related T2D. Enriched in natural killer cells, RAP2A (OR 4.909; 95% CI 1.849–13.039; P = 0.001) demonstrated high risk influence on T2D, and exhibited strong genetic evidence of colocalization (coloc.abf-PPH4 = 0.632). Conclusions: This study used a multi-omics integration method to explore causality between periodontitis and T2D, and revealed molecular mechanisms using bioinformatics tools. Periodontitis was associated with a higher risk of T2D. MCUR1, RAP2A, FOS, PANX1, NFIX and WNK1 may play important roles in the pathogenesis of periodontitis-related T2D, shedding light on the development of potential drug targets. [ABSTRACT FROM AUTHOR]

Traditional Chinese medicine (TCM) formulae have been studied extensively in various human diseases and have proven to be effective due to their multicomponent, multi-target advantage. However, its active metabolites are not clear and the specific mechanisms are not well established, which limits its scientific application. Recently, combination therapies are attracting increasing attention from the scientific community in the past few years and are considered as the next paradigm in drug discovery. Here, we tried to define a new concept of "active metabolites combination therapies (AMCT)" rules to elucidate how the bioactive metabolites from TCMs to produce their synergistic effects in this review. The AMCT rules integrate multidisciplinary technologies like molecular biology, biochemistry, pharmacology, analytical chemistry and pharmacodynamics, etc. Meanwhile, emerging technologies such as multi-omics combined analysis, network analysis, artificial intelligence conduce to better elucidate the mechanisms of these combination therapies in disease treatment, which provides new insights for the development of novel active metabolites combination drugs. AMCT rules will hopefully further guide the development of novel combination drugs that will promote the modernization and international needs of TCM. [ABSTRACT FROM AUTHOR]

Copyright of Advances in Biochemistry / Postepy Biochemii is the property of Polish Biochemical Society / Acta Biochimica Polonica and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Raman scattering can provide information on molecular fingerprints, which have been widely applied in various fields of material science and nanobiotechnology. Notably, low interference with water molecules in obtaining the Raman spectra between 500 and 2000 cm−1 made it a powerful spectroscopic tool in biology, such as imaging and signaling for a living cell. To be a robust tool for cell biology, the performance of obtaining molecular-specific information with high sensitivity, high resolution in real time, and without inducing cell damage is strongly required. The conventional fluorescence-based method has been suffered from the rapid photobleaching of organic fluorophores and the lack of molecular information. In contrast, Raman scattering is a promising spectroscopic tool to acquire cellular information, and the extremely low signal intensity of Raman scattering could be amplified by incorporating the plasmonic nanomaterials. Along with the fundamental research focus on surface-enhanced Raman scattering (SERS), the practical approaches of SERS for cellular imaging as a new tool for drug screening and monitoring cellular signals have been extensively explored based on new optical setups and new designing strategies for the nanostructures. Diverse nanostructure and surface chemistry for targeting or sensing have been played pivotal roles in acquiring cellular information and high resolution cell imaging. In this regard, this review focused on the recent advances of SERS-based technologies for a live cell imaging investigated such as potential drug screening, signaling for chemicals or biomolecules in cell, in situ sensing, and high spatiotemporal resolution. [ABSTRACT FROM AUTHOR]

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SERS is a rapidly advancing and non-destructive technique that has been proven to be more reliable and convenient than other traditional analytical methods. Due to its sensitivity and specificity, this technique is earning its place as a routine and powerful tool in biological and medical studies, especially for the analysis of living cells and subcellular components. This paper reviewed the research progress of single-cell SERS that has been made in the last few years and discussed challenges and future perspectives of this technique. The reviewed SERS platforms have been categorized according to their nature into the following types: (1) colloid-based, substrate-based, or hybrid; (2) ligand-based or ligand-free, and (3) label-based or label-free. The advantages and disadvantages of each type and their potential applications in various fields are thoroughly discussed.

Die Analyse von Einzelzellen birgt großes Potenzial für die Diagnostik und die Therapie verschiedenster Erkrankungen. In der Onkologie werden bereits im Rahmen der Liquid Biopsy einzelne zirkulierende Tumorzellen im Blut charakterisiert, um Krankheitsverläufe zu überwachen oder die Therapie optimal anzupassen. Die Weiterentwicklung der Einzelzellanalyse beispielsweise durch Spatial-Omics-Technologien sowie die bioinformatische Auswertung verschiedener Informationsebenen lässt hoffen, dass in Zukunft bei komplexen physiologischen Prozessen pathogene Abweichungen in nur wenigen Zellen frühzeitig identifiziert werden können. [ABSTRACT FROM AUTHOR]

In‐depth profiling of embryogenesis‐associated endogenous and exogenous metabolic changes can reveal potential bio‐effects resulting from human‐made chemicals and underlying mechanisms. Due to the lack of potent tools for monitoring spatiotemporal distribution and bio‐transformation behavior of dynamic metabolites at single‐cell resolution, however, how and to what extent environmental chemicals may influence or interfere embryogenesis largely remain unclear. Herein, a zero‐sample‐loss micro‐biopsy‐based mass spectrometric platform is presented for quantitative, chemo‐selective, high‐coverage, and minimal‐destructive profiling of development‐associated cis‐diol metabolites, which are critical for signal transduction and epigenome regulation, at both cellular level and tissue level of Xenopus laevis. Using this platform, three extraordinary findings that are otherwise hard to achieve are revealed: 1) there are characteristically different cis‐diol metabolic signatures among oocytes, anterior and posterior part of tailbud‐stage embryos; 2) halogenated cis‐diols heavily accumulate at the posterior part of tailbud‐stage embryos of Xenopus laevis; 3) dimethachlon, a kind of exogenous fungicide that is widely used as pesticide, may be bio‐transformed and accumulated in vertebrate animals in environment. Thus, this study opens a new avenue to simultaneously monitoring intercellular and intraembryonic heterogeneity of endogenous and exogenous metabolites, providing new insights into metabolic remolding during embryogenesis and putting a warning on potential environmental risk. [ABSTRACT FROM AUTHOR]

Diabetic foot ulcers (DFUs) are a serious chronic complication of diabetes mellitus and a leading cause of disability and death in diabetic patients. However, current treatments remain unsatisfactory. Although macrophages are associated with DFU, their exact role in this disease remains uncertain. This study sought to detect macrophage‐related genes in DFU and identify possible therapeutic targets. Single‐cell datasets (GSE223964) and RNA‐seq datasets (GSM68183, GSE80178, GSE134431 and GSE147890) associated with DFU were retrieved from the gene expression omnibus (GEO) database for this study. Analysis of the provided single‐cell data revealed the distribution of macrophage subpopulations in the DFU. Four independent RNA‐seq datasets were merged into a single DFU cohort and further analysed using bioinformatics. This included differential expression (DEG) analysis, multiple machine learning algorithms to identify biomarkers and enrichment analysis. Finally, key results were validated using reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) and Western bolt. Finally, the findings were validated using RT‐qPCR and western blot. We obtained 802 macrophage‐related genes in single‐cell analysis. Differential expression analysis yielded 743 DEGs. Thirty‐seven macrophage‐associated DEGs were identified by cross‐analysis of marker genes with macrophage‐associated DEGs. Thirty‐seven intersections were screened and cross‐analysed using four machine learning algorithms. Finally, HMOX1 was identified as a potentially valuable biomarker. HMOX1 was significantly associated with biological pathways such as the insulin signalling pathway. The results showed that HMOX1 was significantly overexpressed in DFU samples. In conclusion, the analytical results of this study identified HMOX1 as a potentially valuable biomarker associated with macrophages in DFU. The results of our analysis improve our understanding of the mechanism of macrophage action in this disease and may be useful in developing targeted therapies for DFU. [ABSTRACT FROM AUTHOR]

Evidence from numerous studies has revealed the synchronous progression of aging in bone and muscle; however, little is known about the underlying mechanisms. To this end, human muscles and bones are harvested and the aging‐associated transcriptional dynamics of two tissues in parallel using single‐cell RNA sequencing are surveyed. A subset of lipid‐associated macrophages (triggering receptor expressed on myeloid cells 2, TREM2+ Macs) is identified in both aged muscle and bone. Genes responsible for muscle dystrophy and bone loss, such as secreted phosphoprotein 1 (SPP1), are also highly expressed in TREM2+ Macs, suggesting its conserved role in aging‐related features. A common transition toward pro‐inflammatory phenotypes in aged CD4+ T cells across tissues is also observed, activated by the nuclear factor kappa B subunit 1 (NFKB1). CD4+ T cells in aged muscle experience Th1‐like differentiation, whereas, in bone, a skewing toward Th17 cells is observed. Furthermore, these results highlight that degenerated myocytes produce BAG6‐containing exosomes that can communicate with Th17 cells in the bone through its receptor natural cytotoxicity triggering receptor 3 (NCR3). This communication upregulates CD6 expression in Th17 cells, which then interact with TREM2+ Macs through CD6‐ALCAM signaling, ultimately stimulating the transcription of SPP1 in TREM2+ Macs. The negative correlation between serum exosomal BCL2‐associated athanogene 6 (BAG6) levels and bone mineral density further supports its role in mediating muscle and bone synchronization with aging. [ABSTRACT FROM AUTHOR]

Background/Aim: Tyrosine kinase inhibitor (TKI) therapy, a principal treatment for advanced non-small cell lung cancer (NSCLC), frequently encounters the development of drug resistance. The tumor microenvironment (TME) plays a critical role in the progression of NSCLC, yet the relationship between endothelial cells (ECs) and cancerassociated fibroblasts (CAFs) subpopulations in TKI treatment resistance remains largely unexplored. Materials and Methods: The BioProject database PRJNA591860 project was used to analyze scRNA-seq data including 49 advancedstage NSCLC samples across three different time points: pretargeted therapy (naïve), post-partial response (PR) to targeted therapy, and post-progressive disease (PD) stage. The data involved clustering stromal cells into multiple CAFs and ECs subpopulations. The abundance changes and functions of each cluster during TKI treatment were investigated by KEGG and GO analysis. Additionally, we identified specific transcription factors and metabolic pathways via DoRothEA and scMetabolism. Moreover, cellcell communications between PD and PR stages were compared by CellChat. Results: ECs and CAFs were clustered and annotated using 49 scRNA-seq samples. We identified seven ECs subpopulations, with OIT3 ECs showing enrichment in the PR phase with a drug-resistance phenotype, and ACKR1 ECs being prevalent in the PD phase with enhanced cell adhesion. Similarly, CAFs were clustered into 7 subpopulations. PLA2G2A CAFs were predominant in PR, whereas POSTN CAFs were prevalent in PD, characterized by an immunomodulatory phenotype and increased collagen secretion. CellChat analysis showed that ACKR1 ECs strongly interacted with macrophage through the CD39 pathway and POSTN CAFs secreted Tenascin-C (TNC) to promote the progression of epithelial cells, primarily malignant ones, in PD. Conclusion: This study reveals that POSTN CAFs and ACKR1 ECs are associated with resistance to TKI treatment, based on single-cell sequencing. [ABSTRACT FROM AUTHOR]

Intrahepatic cholangiocarcinoma (iCCA) is an aggressive liver malignancy with limited treatment options and a dismal prognosis. The tumor immune microenvironment (TIME) is crucial for iCCA progression, yet its comprehensive characterization remains incomplete. This study utilized mass cytometry by time of flight (CyTOF) to comprehensively analyze immune cell populations in fresh iCCA tumor samples and adjacent peritumor liver tissues. Notably, NK cell percentages significantly decreased in iCCA lesions compared to peritumor liver tissues. Conversely, an enrichment of immunosuppressive CD39+Foxp3+CD4+ regulatory T cells (CD39+T-regs) and exhausted-like CD8+ T cells (with pronounced CD39 and PD-1 expression) within TIME was identified and confirmed by multiplex immunofluorescence staining in an independent patient cohort (n = 140). Crucially, tumor-infiltrating CD39+T-regs and CD39+PD-1+CD8+ T cells emerged as independent prognostic indicators associated with an unfavorable prognosis in iCCA. These findings unveil the intricate immune landscape within iCCA, offering valuable insights for disease management and novel cancer immunotherapies.

The dissociation of tissue and cell aggregates into single cells is of high interest for single cell analysis studies, primary cultures, tissue engineering, and regenerative medicine. However, current methods are slow, poorly controlled, variable, and can introduce artifacts. We previously developed a microfluidic device that contains two separate dissociation modules, a branching channel array and nylon mesh filters, which was used as a polishing step after tissue processing with a microfluidic digestion device. Here, we employed the integrated disaggregation and filtration (IDF) device as a standalone method with both cell aggregates and traditionally digested tissue to perform a well-controlled and detailed study into the effect of mechanical forces on dissociation, including modulation of flow rate, device pass number, and even the mechanism. Using a strongly cohesive cell aggregate model, we found that single cell recovery was highest using flow rates exceeding 40 ml/min and multiple passes through the filter module, either with or without the channel module. For minced and digested kidney tissue, recovery of diverse cell types was maximal using multiple passes through the channel module and only a single pass through the filter module. Notably, we found that epithelial cell recovery from the optimized IDF device alone exceeded our previous efforts, and this result was maintained after reducing digestion time to 20 min. However, endothelial cells and leukocytes still required extended digestion time for maximal recover. These findings highlight the significance of parameter optimization to achieve the highest cell yield and viability based on tissue sample size, extracellular matrix content, and strength of cell-cell interactions.

Background: Osteoarthritis (OA) is a disabling and highly prevalent condition affecting millions worldwide. Recently discovered, disulfidptosis represents a novel form of cell death induced by the excessive accumulation of cystine. Despite its significance, a systematic exploration of disulfidptosis-related genes (DRGs) in OA is lacking. Methods: This study utilized three OA-related datasets and DRGs. Differentially expressed (DE)-DRGs were derived by intersecting the differentially expressed genes (DEGs) from GSE114007 with DRGs. Feature genes underwent screening through three machine learning algorithms. High diagnostic value genes were identified using the receiver operating characteristic curve. Hub genes were confirmed through expression validation. These hub genes were then employed to construct a nomogram and conduct enrichment, immune, and correlation analyses. An additional validation of hub genes was performed through in vitro cell experiments. Results: SLC3A2 and PDLIM1 were designated as hub genes, displaying excellent diagnostic performance. PDLIM1 exhibited low expression in early chondrocyte differentiation, rising significantly in the late stage, while SLC3A2 showed high overall expression, declining in the late differentiation stage. Cellular experiments corroborated the correlation of SLC3A2 and PDLIM1 with chondrocyte inflammation. Conclusions: Two hub genes, SLC3A2 and PDLIM1, were identified in relation to disulfidptosis, providing potential directions for diagnosing and treating OA.

The lipidome is currently understudied but fundamental to life. Within the brain, little is known about cell-type lipid heterogeneity, and even less is known about cell-to-cell lipid diversity because it is difficult to study the lipids within individual cells. Here, we used single-cell mass spectrometry-based protocols to profile the lipidomes of 154 910 single cells across ten individuals consisting of five developmental ages and five brain regions, resulting in a unique lipid atlas available via a web browser of the developing human brain. From these data, we identify differentially expressed lipids across brain structures, cortical areas, and developmental ages. We inferred lipid profiles of several major cell types from this data set and additionally detected putative cell-type specific lipids. This data set will enable further interrogation of the developing human brain lipidome.

Abstract In‐depth profiling of embryogenesis‐associated endogenous and exogenous metabolic changes can reveal potential bio‐effects resulting from human‐made chemicals and underlying mechanisms. Due to the lack of potent tools for monitoring spatiotemporal distribution and bio‐transformation behavior of dynamic metabolites at single‐cell resolution, however, how and to what extent environmental chemicals may influence or interfere embryogenesis largely remain unclear. Herein, a zero‐sample‐loss micro‐biopsy‐based mass spectrometric platform is presented for quantitative, chemo‐selective, high‐coverage, and minimal‐destructive profiling of development‐associated cis‐diol metabolites, which are critical for signal transduction and epigenome regulation, at both cellular level and tissue level of Xenopus laevis. Using this platform, three extraordinary findings that are otherwise hard to achieve are revealed: 1) there are characteristically different cis‐diol metabolic signatures among oocytes, anterior and posterior part of tailbud‐stage embryos; 2) halogenated cis‐diols heavily accumulate at the posterior part of tailbud‐stage embryos of Xenopus laevis; 3) dimethachlon, a kind of exogenous fungicide that is widely used as pesticide, may be bio‐transformed and accumulated in vertebrate animals in environment. Thus, this study opens a new avenue to simultaneously monitoring intercellular and intraembryonic heterogeneity of endogenous and exogenous metabolites, providing new insights into metabolic remolding during embryogenesis and putting a warning on potential environmental risk.

Abstract Evidence from numerous studies has revealed the synchronous progression of aging in bone and muscle; however, little is known about the underlying mechanisms. To this end, human muscles and bones are harvested and the aging‐associated transcriptional dynamics of two tissues in parallel using single‐cell RNA sequencing are surveyed. A subset of lipid‐associated macrophages (triggering receptor expressed on myeloid cells 2, TREM2+ Macs) is identified in both aged muscle and bone. Genes responsible for muscle dystrophy and bone loss, such as secreted phosphoprotein 1 (SPP1), are also highly expressed in TREM2+ Macs, suggesting its conserved role in aging‐related features. A common transition toward pro‐inflammatory phenotypes in aged CD4+ T cells across tissues is also observed, activated by the nuclear factor kappa B subunit 1 (NFKB1). CD4+ T cells in aged muscle experience Th1‐like differentiation, whereas, in bone, a skewing toward Th17 cells is observed. Furthermore, these results highlight that degenerated myocytes produce BAG6‐containing exosomes that can communicate with Th17 cells in the bone through its receptor natural cytotoxicity triggering receptor 3 (NCR3). This communication upregulates CD6 expression in Th17 cells, which then interact with TREM2+ Macs through CD6‐ALCAM signaling, ultimately stimulating the transcription of SPP1 in TREM2+ Macs. The negative correlation between serum exosomal BCL2‐associated athanogene 6 (BAG6) levels and bone mineral density further supports its role in mediating muscle and bone synchronization with aging.

Commonly used sample introduction systems for inductively coupled plasma mass spectrometry (ICP-MS) are generally not well-suited for single particle ICP-MS (spICP-MS) applications due to their high sample requirements and low efficiency. In this study, the first completely 3D-printed, polymer SIS was developed to facilitate spICP-MS analysis. The system is based on a microconcentric pneumatic nebulizer and a single-pass spray chamber with an additional sheath gas flow to further facilitate the transport of larger droplets or particles. The geometry of the system was optimized using numerical simulations. Its aerosol characteristics and operational conditions were studied via optical particle counting and a course of spICP-MS measurements, involving nanodispersions and cell suspensions. In a comparison of the performance of the new and the standard (quartz microconcentric nebulizer plus a double-pass spray chamber) systems, it was found that the new sample introduction system has four times higher particle detection efficiency, significantly better signal-to-noise ratio, provides ca. 20% lower size detection limit, and allows an extension of the upper limit of transportable particle diameters to about 25 µm. [ABSTRACT FROM AUTHOR]

Introduction: Kidney renal clear cell carcinoma (KIRC), as a main type of malignant kidney cancers, has a poor prognosis. Epithelial-mesenchymal transformation (EMT) exerts indispensable role in tumor progression and metastasis, including in KIRC. This study aimed to mine more EMT related details and build prognostic signature for KIRC. Methods: The KIRC scRNA-seq data and bulk data were downloaded from GEO and TCGA databases, respectively. The cell composition in KIRC was calculated using CIBERSORT. Univariate Cox regression analysis and LASSO Cox regression analysis were combined to determine the prognostic genes. Gene set variation analysis and cell-cell communication analysis were conducted to obtain more functional information. Additionally, functional analyses were conducted to determine the biological roles of si-LGALS1 in vitro. Results: We totally identified 2,249 significant differentially expressed genes (DEGs) in KIRC samples, meanwhile a significant distinct expression pattern was found in KIRC, involving Epithelial Mesenchymal Transition pathway. Among all cell types, significantly higher proportion of epithelial cells were observed in KIRC, and 289 DEGs were identified in epithelial cells. After cross analysis of all DEGs and 970 EMT related genes, SPARC, TMSB10, LGALS1, and VEGFA were optimal to build prognostic model. Our EMT related showed good predictive performance in KIRC. Remarkably, si-LGALS1 could inhibit migration and invasion ability of KIRC cells, which might be involved in suppressing EMT process. Conclusion: A novel powerful EMT related prognostic signature was built for KIRC patients, based on SPARC, TMSB10, LGALS1, and VEGFA. Of which, si-LGALS1 could inhibit migration and invasion ability of KIRC cells, which might be involved in suppressing EMT process. [ABSTRACT FROM AUTHOR]

Background: Although lactate metabolism-related genes (LMRGs) have attracted attention for their effects on cancer immunity, little is known about their function in clear cell renal cell carcinoma (ccRCC). The aim of this study was to examine the cellular specificity of lactate metabolism and how it affected the first-line treatment outcomes in ccRCC. Methods: GSE159115 was used to examine the features of lactatemetabolism at the single-cell level. Utilizing the transcriptome, methylation profile, and genomic data from TCGA-KIRC, a multi-omics study of LMRG expression characteristics was performed. A prognostic index based on a gene-pair algorithm was created to assess how LMRGs affected patients' clinical outcomes. To simulate the relationship between the prognostic index and the frontline treatment, pRRophetic and Subclass Mapping were used. E-MTAB-1980, E-MTAB-3267, Checkmate, and Javelin-101 were used for external validation. Results: The variable expression of some LMRGs in ccRCC can be linked to variations in DNA copy number or promoter methylation levels. Lactate metabolism was active in tumor cells and vSMCs, and LDHA, MCT1, and MCT4 were substantially expressed in tumor cells, according to single-cell analysis. The high-risk patients would benefit from immune checkpoint blockade monotherapy (ICB) and ICB plus tyrosine kinase inhibitors (TKI) therapy, whereas the low-risk individuals responded to mTOR-targeted therapy. Conclusions: At the single-cell level, our investigation demonstrated the cellular specificity of lactate metabolism in ccRCC. We proposed that the lactate-related gene pair index might be utilized to identify frontline therapy responders in ccRCC patients as well as predict prognosis. [ABSTRACT FROM AUTHOR]

[This corrects the article DOI: 10.3389/fmed.2020.00499.].

ABSTRACTIn its canonical interpretation, quorum sensing (QS) allows single cells in a bacterial population to synchronize gene expression and hence perform specific tasks collectively once the quorum cell density is reached. However, growing evidence in different bacterial species indicates that considerable cell-to-cell variation in the QS activation state occurs during growth, often resulting in coexisting subpopulations of cells in which QS is active (quorate cells) or inactive (non-quorate cells). Heterogeneity has been observed in the las QS system of the opportunistic pathogen Pseudomonas aeruginosa. However, the molecular mechanisms underlying this phenomenon have not yet been defined. The las QS system consists of an incoherent feedforward loop in which the LasR transcriptional regulator activates the expression of the lasI synthase gene and rsaL, coding for the lasI transcriptional repressor RsaL. Here, single-cell-level gene expression analyses performed in ad hoc engineered biosensor strains and deletion mutants revealed that direct binding of RsaL to the lasI promoter region increases heterogeneous activation of the las QS system. Experiments performed with a dual-fluorescence reporter system showed that the LasR-dependent expression of lasI and rsaL does not correlate in single cells, indicating that RsaL acts as a brake that stochastically limits the transition of non-quorate cells to the quorate state in a subpopulation of cells expressing high levels of this negative regulator. Interestingly, the rhl QS system that is not controlled by an analogous RsaL protein showed higher homogeneity with respect to the las system.IMPORTANCESingle-cell analyses can reveal that despite experiencing identical physico-chemical conditions, individual bacterial cells within a monoclonal population may exhibit variations in gene expression. Such phenotypic heterogeneity has been described for several aspects of bacterial physiology, including QS activation. This study demonstrates that the transition of non-quorate cells to the quorate state is a graded process that does not occur at a specific cell density and that subpopulations of non-quorate cells also persist at high cell density. Here, we provide a mechanistic explanation for this phenomenon, showing that a negative feedback regulatory loop integrated into the las system has a pivotal role in promoting cell-to-cell variation in the QS activation state and in limiting the transition of non-quorate cells to the quorate state in P. aeruginosa.