Advanced Platelet-Rich Fibrin Extract Treatment Promotes the Proliferation and Differentiation of Human Adipose-Derived Mesenchymal Stem Cells through Activation of Tryptophan Metabolism.

BACKGROUND: Advanced platelet-rich fibrin extract (APRFE) contains a high concentration of various cytokines that are helpful for improving stem cells repair function. OBJECTIVE: However, the underlying mechanism of APRFE improving stem cell repairing is not clear. METHODS: We produced APRFE by centrifuging fresh peripheral blood samples and isolated and identified human adipose-derived mesenchymal stem cells (ADMSCs). The abundance of cytokines contained in APRFE was detected by the Enzyme-linked immunosorbent assay (ELISA). The ADMSCs treated with or without APRFE were collected for transcriptome sequencing. RESULTS: Based on the sequencing data, the expression profiles were contracted. The differentially expressed genes and lncRNA (DEGs and DElncRNAs) were obtained using for the differential expression analysis. The lncRNA-miRNA-mRNA network was constructed based on the miRNet database. The further enrichment analysis results showed that the biological functions were mainly related to proliferation, differentiation, and cell-cell function. To explore the role of APRFE, the protein-protein interaction network was constructed among the cytokines included in APRFE and DEGs. Furthermore, we constructed the global regulatory network based on the RNAInter and TRRUST database. The pathways in the global regulatory network were considered as the core pathways. We found that the DEGs in the core pathways were associated with stemness scores. CONCLUSION: In summary, we predicted that APRFE activated three pathways (tryptophan metabolism, mTOR signaling pathway, and adipocytokine signaling) to promote the proliferation and differentiation of ADMSCs. The finding may be helpful for guiding the application of ADMSCs in the clinic.

Exploration the global single-cell ecological landscape of adenomyosis-related cell clusters by single-cell RNA sequencing.

Background: Adenomyosis (AM) is a common benign uterine disease that threatens the normal life of patients. Cells associated with microenvironmental immune ecology are crucial in AM, although they are not as well understood at the cellular level. Methods: Single-cell sequencing (scRNA-seq) data were used to construct an AM global single-cell map, to further identify relevant cell clusters and infer chromosomal copy number variation (CNV) in AM samples. The biological functions of cell clusters were explored and cellular evolutionary processes were inferred by enrichment analysis and pseudotime analysis. In addition, a gene regulatory network (GRN) analysis was constructed to explore the regulatory role of transcription factors in AM progression. Results: We obtained the expression profiles of 42260 cells and identified 10 cell clusters. By comparing the differences in cell components between AM patients and controls, we found that significant abundance of endometrial cells (EC), epithelial cells (Ep), endothelial cells (En), and smooth muscle cells (SMC) in AM patients. Cell clusters with high CNV levels possessing tumour-like features existed in the ectopic endometrium samples. Moreover, the Ep clusters were significantly involved in leukocyte transendothelial cell migration and apoptosis, suggesting an association with cell apoptosis and migration. En clusters were mainly involved in pathways in cancer and apoptosis, indicating that En has certain malignant features. Conclusion: This study identified cell clusters with immune-related features, investigated the changes in the immune ecology of the microenvironment of these cells during AM, and provided a new strategy for the treatment of AM.

Rg1 Promotes the Proliferation and Adipogenic Differentiation of Human Adipose-Derived Stem Cells via FXR1/Lnc-GAS5-AS1 Pathway.

BACKGROUND: Human adipose-derived stem cells (hASCs) play an important role in regenerative medicine. OBJECTIVE: Exploring the mechanism of Rg1 in the promotion of the proliferation and adipogenic differentiation of hASCs is important in regenerative medicine research. METHODS: To observe ginsenoside Rg1 in promoting the proliferation and adipogenic differentiation of hASCs, Rg1 medium at different concentrations was established and tested using the cell counting kit-8 (CCK-8) assay, oil red O staining, alizarin red, and alcian blue. Compared to the control, differentially expressed genes (DEGs) were screened via DEG analysis, which was carried out in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. To explore the relationship among mRNA, long non-coding RNA (lncRNA) and microRNA (miRNA), we constructed a competing endogenous RNA (ceRNA) network. RESULTS: In this study, Rg1 was observed to promote the proliferation and adipogenic differentiation of hASCs. Additionally, enriched BPs and KEGG pathways may be involved in the promotion process, where FXR1 and Lnc-GAS5-AS1 were found to be regulatory factors. The regulatory network suggested that Rg1 could regulate the adipocytokine signaling pathway and IL-17 signaling pathway via FXR1 and Lnc-GAS5-AS1, which served as the mechanism encompassing the promotion of Rg1 on the proliferation and adipogenic differentiation of hASCs. CONCLUSION: A comprehensive transcriptional regulatory network related to the promotion ability of Rg1 was constructed, revealing mechanisms regarding Rg1's promotion of the proliferation and adipogenic differentiation of hASCs. The present study provides a theoretical basis for optimizing the function of hASCs.

Corrigendum to "Altered Genes and Biological Functions in Response to Severe Burns".

[This corrects the article DOI: 10.1155/2021/8836243.].

An N6-Methyladenosine-Related Gene Set Variation Score as a Prognostic Tool for Lung Adenocarcinoma.

N6-methyladenosine (m6A) is the most prevalent type of RNA modification, and we hypothesized that patterns of m6A-related genes may be useful for estimating risk of lung adenocarcinoma (LUAD). An m6A-related gene set variation score (m6A-GSVS) was generated using RNA-sequencing data from LUAD patients in The Cancer Genome Atlas (TCGA). We investigated the association of m6A-GSVS with stemness, tumor mutational burden (TMB), expression of three immune checkpoints, levels of tumor-infiltrating lymphocytes (TILs), and patient prognosis. We found that m6A-GSVS was higher in LUAD than in healthy lung tissue, and it strongly correlated with stemness and TMB. Activated CD4 + T cells were more numerous in LUAD samples that had higher m6A-GSVS than in those with lower scores. Biological processes and pathways, including "Cell cycle," "DNA replication," and "RNA degradation," were significantly enriched in samples with high scores. Furthermore, m6A-GSVS was an independent prognostic indicator in LUAD. In conclusion, we proposed an m6A-GSVS in LUAD. It is a putative indicator for evaluating the ability to RNA m6A, an independent prognostic indicator and associated with tumor stemness.

Altered Genes and Biological Functions in Response to Severe Burns.

Severe burns are acute wounds caused by local heat exposure, resulting in life-threatening systemic effects and poor survival. However, the specific molecular mechanisms remain unclear. First, we downloaded gene expression data related to severe burns from the GEO database (GSE19743, GSE37069, and GSE77791). Then, a gene expression analysis was performed to identify differentially expressed genes (DEGs) and construct protein-protein interaction (PPI) network. The molecular mechanism was identified by enrichment analysis and Gene Set Enrichment Analysis. In addition, STEM software was used to screen for genes persistently expressed during response to severe burns, and receiver operating characteristic (ROC) curve was used to identify key DEGs. A total of 2631 upregulated and 3451 downregulated DEGs were identified. PPI network analysis clustered these DEGs into 13 modules. Importantly, module genes mostly related with immune responses and metabolism. In addition, we identified genes persistently altered during the response to severe burns corresponding to survival and death status. Among the genes with high area under the ROC curve in the PPI network gene, CCL5 and LCK were identified as key DEGs, which may affect the prognosis of burn patients. Gene set variation analysis showed that the immune response was inhibited and several types of immune cells were decreased, while the metabolic response was enhanced. The results showed that persistent gene expression changes occur in response to severe burns, which may underlie chronic alterations in physiological pathways. Identifying the key altered genes may reveal potential therapeutic targets for mitigating the effects of severe burns.

Long Non-coding RNA Expression Patterns in Stomach Adenocarcinoma Serve as an Indicator of Tumor Mutation Burden and Are Associated With Tumor-Infiltrating Lymphocytes and Microsatellite Instability.

Long non-coding RNAs (lncRNAs) are crucial in controlling important aspects of tumor immunity. However, whether the expression pattern of lncRNAs in stomach adenocarcinoma (STAD) reflects tumor immunity is not fully understood. We screened differentially expressed lncRNAs (DElncRNAs) between high and low tumor mutation burden (TMB) STAD samples. Using the least absolute shrinkage and selection operator method, 33 DElncRNAs were chosen to establish a lncRNA-based signature classifier for predicting TMB levels. The accuracy of the 33-lncRNA-based signature classifier was 0.970 in the training set and 0.950 in the test set, suggesting the expression patterns of the 33 lncRNAs may be an indicator of TMB in STAD. Survival analysis showed that a lower classifier index reflected better prognosis for STAD patients, and the index showed correlation with expression of immune checkpoint molecules (PD1, PDL1, and CTLA4), tumor-infiltrating lymphocytes, and microsatellite instability. In conclusion, STAD samples with different tumor mutation burdens have different lncRNA expression patterns. The 33-lncRNA-based signature classifier index may be an indicator of TMB and is associated expression of immune checkpoints, tumor-infiltrating lymphocytes, and microsatellite instability.

Exploring the mechanism of resistance to sorafenib in two hepatocellular carcinoma cell lines.

Sorafenib has long been the only approved systemic therapy for advanced hepatocellular carcinoma (HCC), but most patients show primary or acquired drug resistance. In the present study, RNA was extracted from sorafenib-resistant and -sensitive clones of the HCC cell lines HepG2 and Huh7. Protein-protein interaction networks of the up- and down-regulated genes common to the two sorafenib-resistant cell lines were extracted and subjected to modular analysis in order to identify functional modules. Functional enrichment analysis showed the modules were involved in different biological processes and pathways. These results indicate that sorafenib resistance in HCC is complicated and heterogeneous. The potential regulators of each functional module, including transcription factors, microRNAs and long non-coding RNAs, were explored to construct a comprehensive transcriptional regulatory network related to sorafenib resistance in HCC. Our results provide new insights into sorafenib resistance of HCC at the level of transcriptional regulation.

Landscape of transcription and expression regulated by DNA methylation related to age of donor and cell passage in adipose-derived mesenchymal stem cells.

Adipose-derived mesenchymal stem cells (ADSCs) are pluripotent stromal cells that can differentiate into a variety of cell types, including skin cells. High-throughput sequencing was performed on cells of different ages and cell passage, obtaining their methylation, mRNA expression, and protein profile data. The stemness of each sample was then calculated using the TCGAbiolinks package in R. Co-expression modules were identified using WGCNA, and a crosstalk analysis was performed on the corresponding modules. The ClusterProfile package was used for the functional annotation of module genes. Finally, the regulatory network diagram was visualized using the Cytoscape software. First, a total of 16 modules were identified, where 3 modules were screened that were most relevant to the phenotype. 29 genes were screened in combination of the RNA seq, DNA methylation seq and protein iTRAQ. Finally, a comprehensive landscape comprised of RNA expression, DNA methylation and protein profiles of age relevant ADSCs was constructed. Overall, the different omics of ADSCs were comprehensively analyzed in order to reveal mechanisms pertaining to their growth and development. The effects of age, cell passage, and stemness on the therapeutic effect of ADSCs were explored. Additionally, a theoretical basis for selecting appropriate ADSC donors for regenerative medicine was provided.

Single-cell RNA-seq reveals the immune escape and drug resistance mechanisms of mantle cell lymphoma.

Objective: Mantle cell lymphoma (MCL) is a rare subtype of non-Hodgkin lymphoma (NHL) with high heterogeneity and a high recurrence rate. How heterogenous cell populations contribute to relapse remains to be elucidated. Methods: We performed single cell RNA sequencing (scRNA-seq) on approximately 4,000 bone marrow cells sampled from one patient with multidrug resistant MCL. We identified 10 subpopulations comprising 4 malignant B cell subtypes, 3 T cell subtypes, 2 dendritic cell subtypes and 1 natural killer (NK) cell subtype. Subsequently, we identified cell markers, including a series of genes associated with immune escape and drug resistance. In addition, we explored the roles of these genes in the mechanism of immune escape and drug resistance, and we verified the expression imbalance and clinical prognostic potential by using GEO datasets including 211 MCL samples. Results: The major immune escape mechanisms of MCL included anti-perforin activity, decreased immunogenicity and direct inhibition of apoptosis and cell killing, as mediated by type I and II B cells. The drug resistance mechanisms of different cell clusters included drug metabolism, DNA damage repair, apoptosis and survival promotion. Type III B cells closely communicate with other cells. The key genes involved in the resistance mechanisms showed dysregulated expression and may have significant clinical prognostic value. Conclusion: This study investigated potential immune escape and drug resistance mechanisms in MCL. The results may guide individualized treatment and promote the development of therapeutic drugs.

Predicting and analyzing the COVID-19 epidemic in China: Based on SEIRD, LSTM and GWR models.

In December 2019, the novel coronavirus pneumonia (COVID-19) occurred in Wuhan, Hubei Province, China. The epidemic quickly broke out and spread throughout the country. Now it becomes a pandemic that affects the whole world. In this study, three models were used to fit and predict the epidemic situation in China: a modified SEIRD (Susceptible-Exposed-Infected-Recovered-Dead) dynamic model, a neural network method LSTM (Long Short-Term Memory), and a GWR (Geographically Weighted Regression) model reflecting spatial heterogeneity. Overall, all the three models performed well with great accuracy. The dynamic SEIRD prediction APE (absolute percent error) of China had been ≤ 1.0% since Mid-February. The LSTM model showed comparable accuracy. The GWR model took into account the influence of geographical differences, with R2 = 99.98% in fitting and 97.95% in prediction. Wilcoxon test showed that none of the three models outperformed the other two at the significance level of 0.05. The parametric analysis of the infectious rate and recovery rate demonstrated that China's national policies had effectively slowed down the spread of the epidemic. Furthermore, the models in this study provided a wide range of implications for other countries to predict the short-term and long-term trend of COVID-19, and to evaluate the intensity and effect of their interventions.

Multiomics global landscape of stemness-related gene clusters in adipose-derived mesenchymal stem cells.

BACKGROUND: Adipose-derived mesenchymal stem cells (AD-MSCs) are a type of stem cell that is abundant and widely used. The molecular characteristics of AD-MSCs from different passages from donors of different ages have not been well elucidated. METHODS: Six kinds of AD-MSCs ((E1, E2, E3, Y1, Y2, and Y3) with E denoting cells derived from an elderly patient, Y denoting cells derived from a young patient, and 1, 2, and 3 representing passages 3, 6, and 10) were obtained from human abdominal adipose tissue. We obtained the protein expression profile, the mRNA expression profile, the lncRNA expression profile, and the methylation profile of each kind of AD-MSC by sequencing. After calculating the stemness indices, genes related to stemness were extracted. The multiomics correlation analysis was performed in the stemness-related genes. In addition, short time-series expression miner (STEM) analysis was performed for all cell passages and donor ages. To further explore the biological functions of the stemness-related genes, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Finally, the lncRNA-KEGG network and transcription factor (TF)-KEGG network were constructed based on the RNAInter database and TRRUST v2 database. RESULTS: The stemness of the Y1, E1, and Y2 cells was higher than that of the E2, Y3, and E3 cells. The stemness was the highest for Y1 cells and the lowest for E3 cells. STEM analysis showed that five stemness-related gene clusters were associated with the cell passages, and only one gene cluster was associated with age. The enrichment analysis results showed that the biological processes (BPs) and KEGG pathways were mainly involved in the proliferation, differentiation, and migration of cells. The global regulatory landscape of AD-MSCs was constructed: 25 TFs and 16 lncRNAs regulated 21 KEGG pathways through 27 mRNAs. Furthermore, we obtained a core stemness-related gene set consisting of ITGAV, MAD2L1, and PCNA. These genes were expressed at higher levels in Y1 cells than in E3 cells. CONCLUSION: The multiomics global landscape of stemness-related gene clusters was determined for AD-MSCs, which may be helpful for selecting AD-MSCs with increased stemness.

FGF2-induced PI3K/Akt signaling evokes greater proliferation and adipogenic differentiation of human adipose stem cells from breast than from abdomen or thigh.

In this study, human adipose stem cells were isolated from subcutaneous fat in the thigh (htASCs), abdomen (haASCs) and breast (hbASCs). Flow cytometry was used to detect cell surface markers, and an enzyme-linked immunosorbent assay was used to detect paracrine activity. Paracrine gene expression in the three cell types was examined using real-time qPCR, and adipogenic ability was assessed using Oil Red O staining. RNA from third-passage haASCs and hbASCs was sequenced. The results showed that the differentiation potential marker markers CD49d and CD54 were similar across hbASCs from 10 subjects. The hbASCs showed higher colony forming ability and expression of fibroblast growth factor-2, tissue inhibitor of metalloproteinase-1 and stromal cell derived factor-1 than htASCs and haASCs. Stimulating hbASCs with FGF2 promoted adipogenic differentiation, while treating the cells with the PI3K inhibitor LY294002 inhibited differentiation. These results suggest that the PI3K/Akt signaling pathway can promote proliferation and adipogenic differentiation of adipose stem cells, and that activation of this pathway by FGF2 may explain why hbASCs show greater proliferation and adipogenic differentiation than haASCs and htASCs.

USP22 promotes development of lung adenocarcinoma through ubiquitination and immunosuppression.

Ubiquitin-specific protease 22 (USP22) expresses highly in lung adenocarcinoma (LUAD), which are associated with poor overall survival (OS). Microarray processing was performed to determine gene expression profiling, in which it was found that knocking down USP22 resulted in abnormal expression of a large number of genes. Differentially expressed genes (DEGs)-based protein-protein interaction (PPI) network was organized into 9 functional modules. These functional modules participated significantly in protein modification-related biological process and were involved in cancer-related pathways. The network was constructed to describe the global regulation of USP22-TF/pivot-module-pathway. It suggested that knocking down USP22 may up-regulate the expression of UBC to promote the pathways of cell cycle and ubiquitin-mediated proteolysis in the development of LUAD. More than that, knocking down USP22 can up-regulate STAT1 to activate JAK1-STAT1-caspase pathway, and promote apoptosis of tumor cell. Receiver operating characteristic (ROC) curve analysis suggested that E2F3, H2AFX, TFAP2A, PITX1, IRF7, and FOXM1 may be the potential diagnosis biomarkers for LUAD. On the other hand, BRCA1, FOXM1 and TFAP2A may be prognostic biomarkers of LUAD. In conclusion, we constructed a global regulation network to show that USP22 may promote the development of LUAD through ubiquitination and immunosuppression.

Increasing the density of nanomedicines improves their ultrasound-mediated delivery to tumours.

Nanomedicines have provided fresh impetus in the fight against cancer due to their selectivity and power. However, these agents are limited when delivered intravenously due to their rapid clearance from the bloodstream and poor passage from the bloodstream into target tumours. Here we describe a novel stealthing strategy which addresses both these limitations and thereby demonstrate that both the passive and mechanically-mediated tumour accumulation of the model nanomedicine adenovirus (Ad) can be substantially enhanced. In our strategy gold nanoparticles were thoroughly modified with 2kDa polyethyleneglycol (PEG) and then linked to Ad via a single reduction-cleavable 5kDa PEG. The resulting Ad-gold-PEG construct was compared to non-modified Ad or conventionally stealthed Ad-poly[N-(2-hydroxypropyl)methacrylamide] (Ad-PHPMA). Notably, although Ad-gold-PEG was of similar size and surface charge to Ad-PHPMA the increase in density, resulting from the inclusion of the gold nanoparticles, provided a substantial enhancement of ultrasound-mediated transport. In an in vitro tumour mimicking phantom, the level and distance of Ad-gold-PEG transport was shown to be substantially greater than achieved with Ad-PHPMA. In in vivo studies 0.1% of an unmodified Ad dose was shown to accumulate in tumours, whereas over 12% of the injected dose was recovered from the tumours of mice treated with Ad-gold-PEG and ultrasound. Ultimately, a significant increase in anti-tumour efficacy resulted from this strategy. This stealthing and density-increasing technology could ultimately enhance clinical utility of intravenously delivered nanoscale medicines including viruses, liposomes and antibodies.

Mass-encoded synthetic biomarkers for multiplexed urinary monitoring of disease.

Biomarkers are becoming increasingly important in the clinical management of complex diseases, yet our ability to discover new biomarkers remains limited by our dependence on endogenous molecules. Here we describe the development of exogenously administered 'synthetic biomarkers' composed of mass-encoded peptides conjugated to nanoparticles that leverage intrinsic features of human disease and physiology for noninvasive urinary monitoring. These protease-sensitive agents perform three functions in vivo: they target sites of disease, sample dysregulated protease activities and emit mass-encoded reporters into host urine for multiplexed detection by mass spectrometry. Using mouse models of liver fibrosis and cancer, we show that these agents can noninvasively monitor liver fibrosis and resolution without the need for invasive core biopsies and substantially improve early detection of cancer compared with current clinically used blood biomarkers. This approach of engineering synthetic biomarkers for multiplexed urinary monitoring should be broadly amenable to additional pathophysiological processes and point-of-care diagnostics.

Structural analysis of the genome of breast cancer cell line ZR-75-30 identifies twelve expressed fusion genes.

BACKGROUND: It has recently emerged that common epithelial cancers such as breast cancers have fusion genes like those in leukaemias. In a representative breast cancer cell line, ZR-75-30, we searched for fusion genes, by analysing genome rearrangements. RESULTS: We first analysed rearrangements of the ZR-75-30 genome, to around 10kb resolution, by molecular cytogenetic approaches, combining array painting and array CGH. We then compared this map with genomic junctions determined by paired-end sequencing. Most of the breakpoints found by array painting and array CGH were identified in the paired end sequencing-55% of the unamplified breakpoints and 97% of the amplified breakpoints (as these are represented by more sequence reads). From this analysis we identified 9 expressed fusion genes: APPBP2-PHF20L1, BCAS3-HOXB9, COL14A1-SKAP1, TAOK1-PCGF2, TIAM1-NRIP1, TIMM23-ARHGAP32, TRPS1-LASP1, USP32-CCDC49 and ZMYM4-OPRD1. We also determined the genomic junctions of a further three expressed fusion genes that had been described by others, BCAS3-ERBB2, DDX5-DEPDC6/DEPTOR and PLEC1-ENPP2. Of this total of 12 expressed fusion genes, 9 were in the coamplification. Due to the sensitivity of the technologies used, we estimate these 12 fusion genes to be around two-thirds of the true total. Many of the fusions seem likely to be driver mutations. For example, PHF20L1, BCAS3, TAOK1, PCGF2, and TRPS1 are fused in other breast cancers. HOXB9 and PHF20L1 are members of gene families that are fused in other neoplasms. Several of the other genes are relevant to cancer-in addition to ERBB2, SKAP1 is an adaptor for Src, DEPTOR regulates the mTOR pathway and NRIP1 is an estrogen-receptor coregulator. CONCLUSIONS: This is the first structural analysis of a breast cancer genome that combines classical molecular cytogenetic approaches with sequencing. Paired-end sequencing was able to detect almost all breakpoints, where there was adequate read depth. It supports the view that gene breakage and gene fusion are important classes of mutation in breast cancer, with a typical breast cancer expressing many fusion genes.

Ultrasound-enhanced drug delivery for cancer.

INTRODUCTION: Ultrasound, which has traditionally been used as a diagnostic tool, is increasingly being used in non-invasive therapy and drug delivery. AREAS COVERED: Of particular interest to this review is the rapidly accumulating evidence that ultrasound may have a key role to play both in improving the targeting and the efficacy of drug delivery for cancer. Currently available ultrasound-triggerable vehicles are first described, with particular reference to the ultrasonic mechanism that can activate release and the suitability of the size range of the vehicle used for drug delivery. Further mechanical and thermal effects of ultrasound that can enhance extravasation and drug distribution following release are then critically reviewed. EXPERT OPINION: Acoustic cavitation is found to play a potentially key role both in achieving targeted drug release and enhanced extravasation at modest pressure amplitudes and acoustic energies, whilst simultaneously enabling real-time monitoring of the drug delivery process. The next challenge in ultrasound-enhanced drug delivery will thus be to develop a new generation of drug-carrying nanoparticles which are of the right size range for delivery to tumours, yet still capable of achieving initiation of cavitation activity and drug release at modest acoustic pressures and energies that have no safety implications for the patient.