Bioinformatic analysis and experimental validation of cuproptosis-related LncRNA as a novel biomarker for prognosis and immunotherapy of oral squamous cell carcinoma.

BACKGROUND: The novel form of regulatory cell death, cuproptosis, is characterized by proteotoxicity, which ultimately leads to cell death. Its targeting has emerged as a promising therapeutic approach for oral squamous cell carcinoma (OSCC). Long noncoding RNAs (lncRNAs) participate in epigenetic regulation and have been linked to the progression, prognosis, and treatment of OSCC. Thus, this study aimed to identify new cuproptosis-related lncRNAs (CRLs), establish predictive models for clinical prognosis, immune response, and drug sensitivity, and provide novel insights into immune escape and tumor drug resistance. METHODS: The present study screened eight CRLs (THAP9-AS1, STARD4-AS1, WDFY3-AS2, LINC00847, CDKN2A-DT, AL132800.1, GCC2-AS1, AC005746.1) using Lasso Cox regression analysis to develop an eight-CRL prognostic model. Patients were categorized into high- and low-risk groups using risk scores. To evaluate the predictive ability of the model, Kaplan-Meier analysis, ROC curves, and nomograms were employed. Furthermore, the study investigated the differences in immune function and anticancer drug sensitivity between the high- and low-risk groups. To validate the expression of CRLs in the model, OSCC cell lines were subjected to quantitative real-time fluorescence PCR (qRT-PCR). RESULTS: The results of the study showed that the high-risk group had a shorter overall survival (OS) time in OSCC patients. Cox regression analysis demonstrated that the high-risk score was an independent risk factor for a poor prognosis. The validity of the model was confirmed using ROC curve analysis, and a nomogram was developed to predict the prognosis of OSCC patients. Furthermore, patients in the high-risk group with high TMB had a poorer prognosis. Patients in the low-risk group responded better to immunotherapy than those in the high-risk group. Additionally, the risk scores were significantly associated with drug sensitivity in OSCC patients. Finally, the findings of qRT-PCR supported the reliability of the proposed risk model. CONCLUSION: The study identified and established the 8-CRL model, which represents a novel pathway of lncRNA regulation of cuproptosis in OSCC. This model provides guidance for the prognosis and treatment of OSCC and offers a new insight into immune escape and tumor drug resistance.

Network pharmacological analysis and experimental study of cucurbitacin B in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a malignant tumor with a high incidence and poor prognosis. Cucurbitacin B (CuB) is a tetracyclic triterpenoid small-molecule compound extracted from plants, such as Cucurbitaceae and Brassicaceae, which has powerful anticancer effects. However, the effect and mechanism of CuB on OSCC remain unclear. Within the framework of the current study, network pharmacology was used to analyze the relationship between CuB and OSCC. The network pharmacology analysis showed that CuB and OSCC share 134 common targets; among them, PIK3R1, SRC, STAT3, AKT1, and MAPK1 are the key targets. The molecular docking analysis showed that CuB binds five target proteins. The results of the enrichment analysis showed that CuB exerted effects on OSCC through various pathways; of these pathways, PI3K-AKT was the most important pathway. The results of the in vitro cell experiments showed that CuB could inhibit the proliferation and migration of SCC25 and CAL27 cells, block the cell cycle in the G2 phase, induce cell apoptosis, and regulate the protein expression of the PI3K-AKT signaling pathway. The results of the in vivo animal experiments showed that CuB could inhibit 4NQO-induced oral cancer in mice. Therefore, network pharmacology, molecular docking, cell experiments, and animal experiments showed that CuB could play a role in OSCC by regulating multiple targets and pathways.

Differential Soft Sensor-Based Measurement of Interactive Force and Assistive Torque for a Robotic Hip Exoskeleton.

With the emerging of wearable robots, the safety and effectiveness of human-robot physical interaction have attracted extensive attention. Recent studies suggest that online measurement of the interaction force between the robot and the human body is essential to the aspects above in wearable exoskeletons. However, a large proportion of existing wearable exoskeletons monitor and sense the delivered force and torque through an indirect-measure method, in which the torque is estimated by the motor current. Direct force/torque measuring through low-cost and compact wearable sensors remains an open problem. This paper presents a compact soft sensor system for wearable gait assistance exoskeletons. The contact force is converted into a voltage signal by measuring the air pressure within a soft pneumatic chamber. The developed soft force sensor system was implemented on a robotic hip exoskeleton, and the real-time interaction force between the human thigh and the exoskeleton was measured through two differential soft chambers. The delivered torque of the hip exoskeleton was calculated based on a characterization model. Experimental results suggested that the sensor system achieved direct force measurement with an error of 10.3 ± 6.58%, and torque monitoring for a hip exoskeleton which provided an understanding for the importance of direct force/torque measurement for assistive performance. Compared with traditional rigid force sensors, the proposed system has several merits, as it is compact, low-cost, and has good adaptability to the human body due to the soft structure.

Single-Cell Transcriptomic Map of the Human and Mouse Bladders.

BACKGROUND: Having a comprehensive map of the cellular anatomy of the normal human bladder is vital to understanding the cellular origins of benign bladder disease and bladder cancer. METHODS: We used single-cell RNA sequencing (scRNA-seq) of 12,423 cells from healthy human bladder tissue samples taken from patients with bladder cancer and 12,884 cells from mouse bladders to classify bladder cell types and their underlying functions. RESULTS: We created a single-cell transcriptomic map of human and mouse bladders, including 16 clusters of human bladder cells and 15 clusters of mouse bladder cells. The homology and heterogeneity of human and mouse bladder cell types were compared and both conservative and heterogeneous aspects of human and mouse bladder evolution were identified. We also discovered two novel types of human bladder cells. One type is ADRA2A+ and HRH2+ interstitial cells which may be associated with nerve conduction and allergic reactions. The other type is TNNT1+ epithelial cells that may be involved with bladder emptying. We verify these TNNT1+ epithelial cells also occur in rat and mouse bladders. CONCLUSIONS: This transcriptomic map provides a resource for studying bladder cell types, specific cell markers, signaling receptors, and genes that will help us to learn more about the relationship between bladder cell types and diseases.

Transcriptomic analysis reveals the functions of H2S as a gasotransmitter independently of Cys in Arabidopsis.

Numerous studies have revealed the gasotransmitter functions of hydrogen sulfide (H2S) in various biological processes. However, the involvement of H2S in sulfur metabolism and/or Cys synthesis makes its role as a signaling molecule ambiguous. The generation of endogenous H2S in plants is closely related to the metabolism of Cys, which play roles in a variety of signaling pathway occurring in various cellular processes. Here, we found that exogenous H2S fumigation and Cys treatment modulated the production rate and content of endogenous H2S and Cys to various degrees. Furthermore, we provided comprehensive transcriptomic analysis to support the gasotransmitter role of H2S besides as a substrate for Cys synthesis. Comparison of the differentially expressed genes (DEGs) between H2S and Cys treated seedlings indicated that H2S fumigation and Cys treatment caused different influences on gene profiles during seedlings development. A total of 261 genes were identified to respond to H2S fumigation, among which 72 genes were co-regulated by Cys treatment. GO and KEGG enrichment analysis of the 189 genes, H2S but not Cys regulated DEGs, indicated that these genes mainly involved in plant hormone signal transduction, plant-pathogen interaction, phenylpropanoid biosynthesis, and MAPK signaling pathway. Most of these genes encoded proteins having DNA binding and transcription factor activities that play roles in a variety of plant developmental and environmental responses. Many stress-responsive genes and some Ca2+ signal associated genes were also included. Consequently, H2S regulated gene expression through its role as a gasotransmitter, rather than just as a substrate for Cys biogenesis, and these 189 genes were far more likely to function in H2S signal transduction independently of Cys. Our data will provide insights for revealing and enriching H2S signaling networks.

Integrative Single-Cell Analysis Reveals Transcriptional and Epigenetic Regulatory Features of Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) frequently features a high level of tumor heterogeneity. Elucidating the chromatin landscape of ccRCC at the single-cell level could provide a deeper understanding of the functional states and regulatory dynamics underlying the disease. Here, we performed single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) on 19 ccRCC samples, and whole-exome sequencing was used to understand the heterogeneity between individuals. Single-cell transcriptome and chromatin accessibility maps of ccRCC were constructed to reveal the regulatory characteristics of different tumor cell subtypes in ccRCC. Two long noncoding RNAs (RP11-661C8.2 and CTB-164N12.1) were identified that promoted the invasion and migration of ccRCC, which was validated with in vitro experiments. Taken together, this study comprehensively characterized the gene expression and DNA regulation landscape of ccRCC, which could provide new insights into the biology and treatment of ccRCC. SIGNIFICANCE: A comprehensive analysis of gene expression and DNA regulation in ccRCC using scATAC-seq and scRNA-seq reveals the DNA regulatory programs of ccRCC at the single-cell level.

Deciphering the pharmacological mechanisms of Scutellaria baicalensis Georgi on oral leukoplakia by combining network pharmacology, molecular docking and experimental evaluations.

BACKGROUND: Oral leukoplakia (OLK), an uncharacterized pathological condition that occurs as a white patch in the oral mucosa, is the most common precancerous condition. Scutellaria baicalensis Georgi (SBG) is a medicinal plant with a wide range of pharmacological effects. Increased evidence shows that SBG has potential therapeutic effects on OLK. However, the therapeutic mechanisms of SBG against OLK have not yet been completely elucidated. PURPOSE: This study aimed to clarify the active components and multi-target mechanisms of SBG against OLK via network pharmacology, molecular docking and experimental evaluations. STUDY DESIGN AND METHODS: The active components and related targets of SBG were screened by the TCMSP database and Swiss Target Prediction database. Potential therapeutic targets of OLK were collected using the GeneCards and OMIM databases. Then, we established protein-protein interaction (PPI), compound-target-disease (C-T-D), and compound-target-pathway (C-T-P) networks by Cytoscape to identify the main components, core targets, and pharmacological pathways of SBG against OLK via applying data mining techniques and topological parameters. Metascape database was utilized for GO and KEGG pathway analysis. Molecular docking techniques were used to estimate the binding force between the components and the hub genes. Subsequently, a series of in vitro experiments, specifically CCK-8 assay, clone formation assay, wound healing assay, flow cytometry, RT-qPCR and western blotting were conducted for further verification. RESULTS: There were 25 active components and 31 related target genes in SBG against OLK. PPI analysis showed that Akt1, VEGFA, EGFR, HIF1A and PTGS2 shared the highest centrality among all target genes. KEGG pathway analysis found that PI3K-Akt signaling pathway may occupy core status in the anti-OLK system. Molecular docking results showed that the main active components of SBG had a strong binding affinity to the hub genes. In vitro experiments showed that the leading component baicalein may inhibit proliferation, block cells in the S phase, induce DOK cell apoptosis, and downregulate the mRNA expression of 5 hub genes by inhibiting PI3K/Akt signaling pathway activation. CONCLUSION: The most predominant component of SBG against OLK was baicalein and the key pathway was PI3K/Akt. The main components and hub genes had robust binding abilities. In vitro experiments showed that baicalein could inhibit the proliferation of DOK cells, induce apoptosis, block the cell cycle, and inhibit the mRNA expression level of the hub genes by inhibiting the PI3K/Akt pathway.

Myelin and lymphocyte protein serves as a prognostic biomarker and is closely associated with the tumor microenvironment in the nephroblastoma.

Nephroblastoma, also known as Wilms' tumor (WT), is the most common renal tumor that occurs in children. Although the efficacy of treatment has been significantly improved by a series of comprehensive treatments, some patients still have poor prognosis. Myelin and lymphocyte (MAL) protein, a highly hydrophobic integrated membrane-bound protein, has been implicated in many tumors and is also closely linked to kidney development. However, the relationship between MAL and WT has not yet been elucidated. Therefore, we attempted to evaluate the feasibility of MAL as a promising prognosis factor for WT. The differential expression of MAL was investigated using TARGET database and was verified using the Gene Expression Omnibus database and real-time quantitative PCR. The prognostic ability of MAL was determined using Kaplan-Meier and Cox regression analyses. Pearson correlation analysis was applied to explore the relationship between MAL expression and methylation sites. The ESTIMATE and CIBERSORT algorithms showed that MAL expression was associated with the WT tumor microenvironment. Gene Set Enrichment Analysis (GSEA) indicated that multiple signaling pathways closely associated with tumorigenesis were differentially enriched between the high- and low-MAL groups. In conclusion, our study comprehensively explored the potential of MAL as a prognosis factor for WT. Meanwhile, we also demonstrated that MAL, as a prognostic factor for WT, may be closely related to the tumor microenvironment.

Gasotransmitter H2S accelerates seed germination via activating AOX mediated cyanide-resistant respiration pathway.

Hydrogen sulfide (H2S) has been witnessed as a crucial gasotransmitter involving in various physiological processes in plants. H2S signaling has been reported to involve in regulating seed germination, but the underlying mechanism remains poorly understood. Here, we found that endogenous H2S production was activated in germinating Arabidopsis seeds, correlating with upregulated both the transcription and the activity of L-cysteine desulfhydrase (EC 4.4.1.28, LCD and DES1) responsible for H2S production. Moreover, seed germination could be significantly accelerated by exogenous NaHS (the H2S donor) fumigation and over-expressing DES1, while H2S-generation defective (lcd/des1) seeds exhibited decreased germination speed. We also confirmed that the alternative oxidase (AOX), a cyanide-insensitive terminal oxidase, can be stimulated by imbibition. Furthermore, exogenous H2S fumigation and over-expressing DES1 could significantly reinforced imbibition induced increase of both the AOX1A expression and AOX protein abundance, while this increase could be obviously weakened in lcd/des1. Additionally, exogenous H2S fumigation mediated post-translational modification to keep AOX in its reduced and active state, which might involve H2S induced improvement of the reduced GSH content and the cell reducing power. The promotive effect of H2S on germination was clearly impaired by inducing aox1a mutation, indicating that AOX acts downstream of H2S signaling to accelerate seed germination. Consequently, H2S signaling was activated during germination then acted as a trigger to induce AOX mediated cyanide-resistant respiration to accelerate seed germination. Our study correlates H2S signaling to cyanide-resistant respiration, providing evidence for more extensive studies of H2S signaling.

Single-Cell RNA Sequencing of Metastatic Testicular Seminoma Reveals the Cellular and Molecular Characteristics of Metastatic Cell Lineage.

Background: Testicular cancer is the most common solid malignancy in young men. Given the many histological classifications of testicular tumors, seminoma is one of the most treatable cancers. The survival rate in early-stage disease was more than 90%. Thus, seminoma at the cellular and molecular levels, especially at the single-cell level, is worth studying. Methods: We performed a single-cell RNA sequencing (scRNA-seq) study on a patient who was diagnosed with testicular seminoma with lymph node metastasis. This study presented tumor tissue, PBMC, pelvic and renal hilus lymph node in a total of 18,206 high-quality single-cell transcriptome information. The characteristics of metastatic cell lineage were revealed by the comparison between different tumor cell subtypes at the scRNA level. Results: A single-cell map of testicular seminoma with lymph node metastasis was constructed by scRNA-seq. We discovered the gene expression characteristics of the tumor cells in testicular seminoma, especially metastatic tumor cells. KRT8 and KRT18 were commonly expressed in the three tumor cell subtypes. However, TCF7L1, SCG3 and SV2C were the specifically expressed genes of tumor cell subtypes in primary tumor sites. Some molecular markers specifically expressed by the metastatic cell lineage, such as POU5F1, were identified. Conclusions: We revealed the molecular characteristics of testicular seminoma at the single-cell level, especially the metastatic tumor cells. This study could provide new insights into the diagnosis and treatment of testicular seminoma.

Corrigendum: Single-cell transcriptomics reveals the complexity of the tumor microenvironment of treatment-naive osteosarcoma.

[This corrects the article DOI: 10.3389/fonc.2021.709210.].

Single-Cell RNA Sequencing in Multiple Pathologic Types of Renal Cell Carcinoma Revealed Novel Potential Tumor-Specific Markers.

BACKGROUND: Renal cell carcinoma (RCC) is the most common type of kidney cancer. Studying the pathogenesis of RCC is particularly important, because it could provide a direct guide for clinical treatment. Given that tumor heterogeneity is probably reflected at the mRNA level, the study of mRNA in RCC may reveal some potential tumor-specific markers, especially single-cell RNA sequencing (scRNA-seq). METHODS: We performed an exploratory study on three pathological types of RCC with a small sample size. This study presented clear-cell RCC (ccRCC), type 2 pRCC, and chRCC in a total of 30,263 high-quality single-cell transcriptome information from three pathological types of RCC. In addition, scRNA-seq was performed on normal kidneys. Tumor characteristics were well identified by the comparison between different pathological types of RCC and normal kidneys at the scRNA level. RESULTS: Some new tumor-specific markers for different pathologic types of RCC, such as SPOCK1, PTGIS, REG1A, CP and SPAG4 were identified and validated. We also discovered that NDUFA4L2 both highly expressed in tumor cells of ccRCC and type 2 pRCC. The presence of two different types of endothelial cells in ccRCC and type 2 pRCC was also identified and verified. An endothelial cell in ccRCC may be associated with fibroblasts and significantly expressed fibroblast markers, such as POSTN and COL3A1. At last, by applying scRNA-seq results, the activation of drug target pathways and sensitivity to drug responses was predicted in different pathological types of RCC. CONCLUSIONS: Taken together, these findings considerably enriched the single-cell transcriptomic information for RCC, thereby providing new insights into the diagnosis and treatment of RCC.

Single-Cell RNA-seq Identification of the Cellular Molecular Characteristics of Sporadic Bilateral Clear Cell Renal Cell Carcinoma.

Bilateral renal cell carcinoma (RCC) is a rare disease that can be classified as either familial or sporadic. Studying the cellular molecular characteristics of sporadic bilateral RCC is important to provide guidance for clinical treatment. Cellular molecular characteristics can be expressed at the RNA level, especially at the single-cell degree. Single-cell RNA sequencing (scRNA-seq) was performed on bilateral clear cell RCC (ccRCC). A total of 3,575 and 3,568 high-quality single-cell transcriptome data were captured from the left and right tumour tissues, respectively. Gene characteristics were identified by comparing left and right tumours at the scRNA level. The complex cellular environment of bilateral ccRCC was presented by using scRNA-seq. Single-cell transcriptomic analysis revealed high similarity in gene expression among most of the cell types of bilateral RCCs but significant differences in gene expression among different site tumour cells. Additionally, the potential biological function of different tumour cell types was determined by gene ontology (GO) analysis. The transcriptome characteristics of tumour tissues in different locations at the single-cell transcriptome level were revealed through the scRNA-seq of bilateral sporadic ccRCC. This work provides new insights into the diagnosis and treatment of bilateral RCC.

Single-Cell Transcriptomics Reveals the Complexity of the Tumor Microenvironment of Treatment-Naive Osteosarcoma.

Osteosarcoma (OS), which occurs most commonly in adolescents, is associated with a high degree of malignancy and poor prognosis. In order to develop an accurate treatment for OS, a deeper understanding of its complex tumor microenvironment (TME) is required. In the present study, tissues were isolated from six patients with OS, and then subjected to single-cell RNA sequencing (scRNA-seq) using a 10× Genomics platform. Multiplex immunofluorescence staining was subsequently used to validate the subsets identified by scRNA-seq. ScRNA-seq of six patients with OS was performed prior to neoadjuvant chemotherapy, and data were obtained on 29,278 cells. A total of nine major cell types were identified, and the single-cell transcriptional map of OS was subsequently revealed. Identified osteoblastic OS cells were divided into five subsets, and the subsets of those osteoblastic OS cells with significant prognostic correlation were determined using a deconvolution algorithm. Thereby, different transcription patterns in the cellular subtypes of osteoblastic OS cells were reported, and key transcription factors associated with survival prognosis were identified. Furthermore, the regulation of osteolysis by osteoblastic OS cells via receptor activator of nuclear factor kappa-B ligand was revealed. Furthermore, the role of osteoblastic OS cells in regulating angiogenesis through vascular endothelial growth factor-A was revealed. C3_TXNIP+ macrophages and C5_IFIT1+ macrophages were found to regulate regulatory T cells and participate in CD8+ T cell exhaustion, illustrating the possibility of immunotherapy that could target CD8+ T cells and macrophages. Our findings here show that the role of C1_osteoblastic OS cells in OS is to promote osteolysis and angiogenesis, and this is associated with survival prognosis. In addition, T cell depletion is an important feature of OS. More importantly, the present study provided a valuable resource for the in-depth study of the heterogeneity of the OS TME.

Single-cell RNA sequencing of human kidney.

A comprehensive cellular anatomy of normal human kidney is crucial to address the cellular origins of renal disease and renal cancer. Some kidney diseases may be cell type-specific, especially renal tubular cells. To investigate the classification and transcriptomic information of the human kidney, we rapidly obtained a single-cell suspension of the kidney and conducted single-cell RNA sequencing (scRNA-seq). Here, we present the scRNA-seq data of 23,366 high-quality cells from the kidneys of three human donors. In this dataset, we show 10 clusters of normal human renal cells. Due to the high quality of single-cell transcriptomic information, proximal tubule (PT) cells were classified into three subtypes and collecting ducts cells into two subtypes. Collectively, our data provide a reliable reference for studies on renal cell biology and kidney disease.