Single-cell RNA sequencing and spatial transcriptome reveal potential molecular mechanisms of lung cancer brain metastasis.

BACKGROUND: Lung cancer is a highly aggressive and prevalent disease worldwide. By the time it is first diagnosed, distant metastases have usually already occurred. Among them, the prognosis of patients with brain metastasis from lung cancer is very poor. Therefore, it is particularly important to identify the evolutionary status of tumor cells during lung cancer brain metastases and discover the underlying mechanisms of lung cancer brain metastases. METHODS: In this study, we analysed three types of data: single-cell RNA sequencing, bulk RNA sequencing, and spatial transcriptome. Firstly, we identified early metastatic epithelial cell clusters (EMEC) using CNV and trajectory analysis in scRNA-seq data. Secondly, we integrated scRNA-seq and spatial transcriptome data with the help of MIA (Multimodal intersection analysis) to explore the biological characteristics of EMEC. Finally, we used bulk RNA-seq data to validate the molecular characteristics of EMEC. RESULT: A total of 55,763 single cells were obtained and divided into 9 cell types. In brain metastasis, we found a significantly higher proportion of epithelial cells. In addition, we identified a specific subpopulation of epithelial cells, which was named as "early metastatic epithelial cell clusters (EMEC)". It is enriched in oxidative phosphorylation, coagulation, complement. Moreover, we also found that EMEC underwent cellular communication with other immune cells through ligand-receptor pairs such as MIF-(CD74 + CXCR4) and MIF-(CD74 + CD44). Next, we validated that EMEC were associated with poor clinical prognosis using three independent external datasets. Finally, spatial transcriptome analysis revealed specificity in the spatial distribution of EMEC, which shifted from the peripheral regions to the central regions of the tumour as the depth of tumor invasion progressed. CONCLUSION: This study reveals the potential molecular mechanisms of lung cancer brain metastasis from both single-cell and spatial transcriptomic perspectives, providing biological insights and clinical reference value for detecting patients suffering from lung cancer brain metastasis.

Lumican promotes calcific aortic valve disease through H3 histone lactylation.

BACKGROUND AND AIMS: Valve interstitial cells (VICs) undergo a transition to intermediate state cells before ultimately transforming into the osteogenic cell population, which is a pivotal cellular process in calcific aortic valve disease (CAVD). Herein, this study successfully delineated the stages of VIC osteogenic transformation and elucidated a novel key regulatory role of lumican (LUM) in this process. METHODS: Single-cell RNA-sequencing (scRNA-seq) from nine human aortic valves was used to characterize the pathological switch process and identify key regulatory factors. The in vitro, ex vivo, in vivo, and double knockout mice were constructed to further unravel the calcification-promoting effect of LUM. Moreover, the multi-omic approaches were employed to analyse the molecular mechanism of LUM in CAVD. RESULTS: ScRNA-seq successfully delineated the process of VIC pathological transformation and highlighted the significance of LUM as a novel molecule in this process. The pro-calcification role of LUM is confirmed on the in vitro, ex vivo, in vivo level, and ApoE-/-//LUM-/- double knockout mice. The LUM induces osteogenesis in VICs via activation of inflammatory pathways and augmentation of cellular glycolysis, resulting in the accumulation of lactate. Subsequent investigation has unveiled a novel LUM driving histone modification, lactylation, which plays a role in facilitating valve calcification. More importantly, this study has identified two specific sites of histone lactylation, namely, H3K14la and H3K9la, which have been found to facilitate the process of calcification. The confirmation of these modification sites' association with the expression of calcific genes Runx2 and BMP2 has been achieved through ChIP-PCR analysis. CONCLUSIONS: The study presents novel findings, being the first to establish the involvement of lumican in mediating H3 histone lactylation, thus facilitating the development of aortic valve calcification. Consequently, lumican would be a promising therapeutic target for intervention in the treatment of CAVD.

Macrophage Membrane-Encapsulated Dopamine-Modified Poly Cyclodextrin Multifunctional Biomimetic Nanoparticles for Atherosclerosis Therapy.

Atherosclerotic plaques exhibit high cholesterol deposition and oxidative stress resulting from high reactive oxygen species (ROS). These are the major components in plaques and the main pro-inflammatory factor. Therefore, it is crucial to develop an effective therapeutic strategy that can simultaneously address the multiple pro-inflammatory factors via removing cholesterol and inhibiting the overaccumulated ROS. In this study, we constructed macrophage membrane-encapsulated biomimetic nanoparticles (MM@DA-pCD@MTX), which not only alleviate cholesterol deposition at the plaque lesion via reverse cholesterol transport but also scavenge the overaccumulated ROS. ß-Cyclodextrin (ß-CD) and the loaded methotrexate (MTX) act synergistically to induce cholesterol efflux for inhibiting the formation of foam cells. Among them, MTX up-regulated the expression of ABCA1, CYP27A1, and SR-B1. ß-CD increased the solubility of cholesterol crystals. In addition, the ROS scavenging property of dopamine (DA) was perfectly preserved in MM@DA-pCD@MTX, which could scavenge the overaccumulated ROS to alleviate the oxidative stress at the plaque lesion. Last but not least, MM-functionalized "homing" targeting of atherosclerotic plaques not only enables the targeted drug delivery but also prolongs in vivo circulation time and drug half-life. In summary, MM@DA-pCD@MTX emerges as a potent, multifunctional therapeutic platform for AS treatment, offering a high degree of biosafety and efficacy in addressing the complex pathophysiology of atherosclerosis.

Natural product fargesin interferes with H3 histone lactylation via targeting PKM2 to inhibit non-small cell lung cancer tumorigenesis.

Non-small cell lung cancer (NSCLC) is one of the most common malignant tumors. There is an urgent need to find more effective drugs that inhibit NSCLC. Fargesin (FGS) has demonstrated anti-tumor effects; however, its efficacy and the molecular mechanism of inhibiting NSCLC are unclear. Herein, we investigated FGS' inhibitory effects on NSCLC by CCK8 and EdU assays and cell cycle analysis of A549 cells in vitro and in a nude mouse tumor transplantation model in vivo. FGS (10-50 µM) significantly inhibited cell proliferation and down-regulated expression levels of CDK1 and CCND1. Transcriptomic analysis showed that FGS regulated the cell metabolic process pathway. Differential metabolites with FGS treatment were enriched in glycolysis and pyruvate pathways. Cell metabolism assay were used to evaluate the oxygen consumption rate (OCR), Extracellular acidification rate (ECAR) in A549 cells. FGS also inhibited the production of cellular lactate and the expression of LDHA, LDHB, PKM2, and SLC2A1. These genes were identified as important oncogenes in lung cancer, and their binding to FGS was confirmed by molecular docking simulation. Notably, the over-expression and gene silencing experiments signified PKM2 as the molecular target of FGS for anti-tumorigenesis. Moreover, the H3 histone lactylation, were correlated with tumorigenesis, were inhibited with FGS treatment. Conclusively, FGS inhibited the aerobic glycolytic and H3 histone lactylation signaling pathways in A549 NSCLC cells by targeting PKM2. These findings provide evidence of the therapeutic potential of FGS in NSCLC.

Elevated expression of TNFRSF4 impacts immune cell infiltration and gene mutation in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a highly heterogeneous disease, which makes prognostic prediction challenging.We aimed to investigate association of TNFRSF4 expression with the immune infiltration and gene mutation in HCC. METHODS: In this study, the expression profiles and corresponding clinical data of HCC patients were downloaded from the Cancer Genome Atlas (TCGA) database.Kaplan-Meier and Cox regression were used to evaluate the clinical value of TNFRSF4. ESTIMATE and CIBERSORT algorithms were applied to investigate the infiltration ratio of 22 immune cells. The WGCNA and LASSO COX algorithms were performed, establishing a prognostic risk model that was then validated by HCC samples from GEO. Finally, the effects on gene mutation occurring in HCC patients of TNFRSF4 expression and risk score were appraised. RESULTS: In HCC tissues, it was found the TNFRSF4 expression profile was significantly different with age, gender, tumor grade, disease stage, prominently affecting the survival outcome and prognosis of patients. Univariate and multivariate COX regression analysis suggested that TNFRSF4 was an independent prognostic marker. Samples of high/low expression of TNFRSF4 were screened for differential genes, and then the WGCNA and LASSO COX constructed a 13-gene signature, excellently dividing samples into hign/low risk groups. Compared with the low-risk group, the overall survival (OS) of high-risk group was markedly lower, with P< 0.0001. By ROC curve analysis, the predictive ability of the 13-gene signature was further confirmed. Both the high/low TNFRSF4 expression and the high/low risk score were demonstrated to exert effects on the frequency of gene mutation in HCC. CONCLUSIONS: As an independent prognostic marker of HCC, TNFRSF4 was found simultaneously to affect the immune infiltration of cells and the frequency of gene mutations.

Machine learning algorithm and deep neural networks identified a novel subtype in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. Due to the lack of specific characteristics in the early stage of the disease, patients are usually diagnosed in the advanced stage of disease progression. OBJECTIVE: This study used machine learning algorithms to identify key genes in the progression of hepatocellular carcinoma and constructed a prediction model to predict the survival risk of HCC patients. METHODS: The transcriptome data and clinical information were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). The differential expression analysis and COX proportional-hazards model participated in the identification of survival-related genes. K-Means, Random forests, and LASSO regression are involved in identifying novel subtypes of HCC and screening key genes. The prediction model was constructed by deep neural networks (DNN), and Gene Set Enrichment Analysis (GSEA) reveals the metabolic pathways where key genes are located. RESULTS: Two subtypes were identified with significantly different survival rates (p< 0.0001, AUC = 0.720) and 17 key genes associated with the subtypes. The accuracy rate of the deep neural network prediction model is greater than 93.3%. The GSEA analysis found that the survival-related genes were significantly enriched in hallmark gene sets in the MSigDB database. CONCLUSIONS: In this study, we used machine learning algorithms to screen out 17 genes related to the survival risk of HCC patients, and trained a DNN model based on them to predict the survival risk of HCC patients. The genes that make up the model are all key genes that affect the formation and development of cancer.

ROS responsive polydopamine nanoparticles to relieve oxidative stress and inflammation for ameliorating acute inflammatory bowel.

Oxidative stress is a key factor in the development of inflammatory diseases. Elimination of reactive oxygen species (ROS) in the inflamed colon has been confirmed as an effective strategy to alleviate inflammatory bowel disease (IBD). The conventional approaches will cause systemic absorption and potential side effects. To address these issues, we develop a nanomedicine (LS@PDA NPs) that is capable of delivering to target inflammatory lesions by electrostatic adsorption, subsequently effectively scavenging the excess ROS and alleviating inflammation to ameliorate ulcerative colitis (UC). In the DSS induced acute colitis mice model, LS@PDA NPs can significantly reduce the production of pro-inflammatory cytokines, alleviate oxidative stress, and promote the favorable recovery of the damaged colonic tissue. These results indicate that LS@PDA NPs are able to effectively alleviate intestinal inflammation and provide strong theoretical support for the treatment of other inflammatory diseases.

Nonsense-Mediated Decay Targeted RNA (ntRNA): Proposal of a ntRNA-miRNA-lncRNA Triple Regulatory Network Usable as Biomarker of Prognostic Risk in Patients with Kidney Cancer.

The most prevalent subtype of renal cell carcinoma (RCC), kidney renal clear cell carcinoma (KIRC) may be associated with a poor prognosis in a high number of cases, with a stage-specific prognostic stratification currently in use. No reliable biomarkers have been utilized so far in clinical practice despite the efforts in biomarker research in the last years. Nonsense-mediated mRNA decay (NMD) is a critical safeguard against erroneous transcripts, particularly mRNA transcripts containing premature termination codons (called nonsense-mediated decay targeted RNA, ntRNA). In this study, we first characterized 296 differentially expressed ntRNAs that were independent of the corresponding gene, 261 differentially expressed miRNAs, and 4653 differentially expressed lncRNAs. Then, we constructed a hub ntRNA-miRNA-lncRNA triple regulatory network associated with the prognosis of KIRC. Moreover, the results of immune infiltration analysis indicated that this network may influence the changes of the tumor immune microenvironment. A prognostic model derived from the genes and immune cells associated with the network was developed to distinguish between high- and low-risk patients, which was a better prognostic than other models, constructed using different biomarkers. Additionally, correlation of methylation and ntRNAs in the network suggested that some ntRNAs were regulated by methylation, which is helpful to further study the causes of abnormal expression of ntRNAs. In conclusion, this study highlighted the possible clinical implications of ntRNA functions in KIRC, proposing potential significant biomarkers that could be utilized to define the prognosis and design personalized treatment plans in kidney cancer management in the next future.

Compromised glycolysis contributes to foot process fusion of podocytes in diabetic kidney disease: Role of ornithine catabolism.

INTRODUCTION: Compromised glycolysis in podocytes contributes to the initiation of diabetic kidney disease (DKD). Podocyte injury is characterized by cytoskeletal remodeling and foot process fusion. Compromised glycolysis in diabetes likely leads to switch of energy supply in podocyte. However, the underlying mechanism by which disturbed energy supply in podocytes affects the cytoskeletal structure of podocytes remains unclear. METHODS: Metabolomic and transcriptomic analyses were performed on the glomeruli of db/db mice to examine the catabolism of glucose, fatty, and amino acids. Ornithine catabolism was targeted in db/db and podocyte-specific pyruvate kinase M2 knockout (PKM2-podoKO) mice. In vitro, expression of ornithine decarboxylase (ODC1) was modulated to investigate the effect of ornithine catabolism on mammalian target of rapamycin (mTOR) signaling and cytoskeletal remodeling in cultured podocytes. RESULTS: Multi-omic analyses of the glomeruli revealed that ornithine metabolism was enhanced in db/db mice compared with that in db/m mice under compromised glycolytic conditions. Additionally, ornithine catabolism was exaggerated in podocytes of diabetic PKM2-podoKO mice compared with that in diabetic PKM2flox/flox mice. In vivo, difluoromethylornithine (DFMO, inhibitor of ODC1) administration reduced urinary albumin excretion and alleviated podocyte foot process fusion in db/db mice. In vitro, 2-deoxy-d-glucose (2-DG) exposure induced mTOR signaling activation and cytoskeletal remodeling in podocytes, which was alleviated by ODC1-knockdown. Mechanistically, a small GTPase Ras homolog enriched in the brain (Rheb), a sensor of mTOR signaling, was activated by exposure to putrescine, a metabolic product of ornithine catabolism. CONCLUSION: These findings demonstrate that compromised glycolysis in podocytes under diabetic conditions enhances ornithine catabolism. The metabolites of ornithine catabolism contribute to mTOR signaling activation via Rheb and cytoskeletal remodeling in podocytes in DKD.

A Transendothelial Leukocyte Transmigration Model Based on Computational Fluid Dynamics and BP Neural Network.

The mechanism of immune infiltration involving immune cells is closely related to various diseases. A key issue in immune infiltration is the transendothelial transmigration of leukocytes. Previous studies have primarily interpreted the leukocyte infiltration of from biomedical perspective. The physical mechanism of leukocyte infiltration remains to be explored. By integrating the immune cell transmigration computational fluid dynamics (CFD) data, the paper builds a time-dependent leukocyte transmigration prediction model based on the bio-inspired methods, namely back propagation neural networks (BPNN) model. The model can efficiently predict the immune cell transmigration in a special microvascular environment, and obtain good prediction accuracy. The model accurately predicted the cell movement and flow field changes during the transmigration. In the test data set, it has high prediction accuracy for cell deformation, motion velocity and flow lift forces during downstream motion, and maintains a good prediction accuracy for drag force. The two prediction models achieved the prediction of leukocyte transmigration in a specific microvascular environment and maintained a high prediction accuracy, indicating the feasibility and robustness of the BPNN model applied to the prediction of immune cell infiltration. Compared with traditional CFD simulations, BPNN models avoid complex and time-dependent physical modeling and computational processes.

Mechano Growth Factor Accelerates ACL Repair and Improves Cell Mobility of Mechanically Injured Human ACL Fibroblasts by Targeting Rac1-PAK1/2 and RhoA-ROCK1 Pathways.

Exceeded mechanical stress leads to a sublethal injury to anterior cruciate ligament (ACL) fibroblasts, and it will hinder cell mobility and ACL regeneration, and even induce osteoarthritis. The mechano growth factor (MGF) could be responsible for mechanical stress and weakening its negative effects on cell physiological behaviors. In this study, effects of MGF on cell mobility and relevant molecules expression in injured ACL fibroblasts were detected. After an injurious mechanical stretch, the analysis carried out, at 0 and 24 h, respectively, showed that the cell area, roundness, migration, and adhesion of ACL fibroblasts were reduced. MGF (10, 100 ng/mL) treatment could improve cell area, roundness and promote cell migration and adhesion capacity compared with the injured group without MGF. Further study indicated that cell mobility-relevant molecules (PAK1/2, Cdc42, Rac1, RhoA, and ROCK1) expression in ACL fibroblasts was down-regulated at 0 or 24 h after injurious stretch (except Rac1 and RhoA at 0 h). Similarly, MGF improved cell mobility-relevant molecule expression, especially the ROCK1 expression level in ACL fibroblasts at 0 or 24 h after injurious stretch. Protein expression of ROCK1 in injured ACL fibroblasts was also reduced and could be recovered by MGF treatment. In a rabbit partial ACL transection (ACLT) model, ACL exhibited poor regenerative capacity in collagen and extracellular matrix (ECM) synthesis after partial ACLT for 2 or 4 weeks, and MGF remarkably accelerated ACL regeneration and restored its mechanical loading capacity after partial ACLT for four weeks. Our findings suggest that MGF weakens the effects of pathological stress on cell mobility of ACL fibroblasts and accelerates ACL repair, and might be applied as a future treatment approach to ACL rupture in the clinic.

Integrated bioinformatical and in vitro study on drug targets for liver cirrhosis based on unsupervised consensus clustering and immune cell infiltration.

Liver cirrhosis is one of the most common cause of death in the world. The progress of liver cirrhosis involves health, liver cirrhosis and liver cancer, leading to great challenges in the diagnosis of the disease. Drug targets, which could be obtained conveniently, can help clinicians improve prognosis and treatment. Liver cirrhosis is associated with serum calcium levels. And studies reported Tanshinone IIA plays a therapeutic role in liver injury through activating calcium-dependent apoptosis. In this study, we explored the diagnostic key targets of Tanshinone IIA in liver cirrhosis through exploration of comprehensive dataset including health, liver cirrhosis and liver cancer patients. The unsupervised consensus clustering algorithm identified 3 novel subtypes in which differentially expressed genes (DEGs) between both subtypes were found by pairwise comparison. Then, 4 key drug targets of Tanshinone IIA were determined through the intersection of these DEGs. The diagnostic performance of target genes was assessed and further verified in the external dataset. We found that the 4 key drug targets could be used as effective diagnostic biomarkers. Then the immune scores in the high and low expression groups of target genes were estimated to identify significantly expressed immune cells. In addition, the immune infiltration of high and low target gene expression groups in several immune cells were significantly different. The findings suggest that 4 key drug targets may be a simple and useful diagnostic tool for predicting patients with cirrhosis. We further studied the carcinogenesis role of AKR1C3 and TPX2 in vitro. Both mRNA and protein expression in hepatoma carcinoma cells was detected using qRT-PCR and Western blot. And the knockdown of AKR1C3 and TPX2 significantly suppressed cell proliferation, migration and invasion.

Biomechanics Assist Measurement, Modeling, Engineering Applications, and Clinical Decision Making in Medicine.

Biomechanical studies of surgeries and medical devices are usually performed with human or animal models [...].

Hyperphosphorylation of Tau Due to the Interference of Protein Phosphatase Methylesterase-1 Overexpression by MiR-125b-5p in Melatonin Receptor Knockout Mice.

Melatonin has been indicated to ameliorate tau hyperphosphorylation in the pathogenesis of tau diseases, but the role of melatonin-receptor signal transduction has not been clearly discovered. In this study, we found intensive tau hyperphosphorylation in melatonin receptor knockout mice. Bielschowsky silver staining showed ghostlike neurofibrillary tangles in melatonin receptor-2 knockout (MT2KO) as well as melatonin receptors-1 and -2 knockout (DKO) mice, and an argyrophilic substance was deposited in melatonin receptor-1 knockout (MT1KO) mice. Furthermore, we found significantly decreased activity of protein phosphatase 2A (PP2A) by Western blot and enzyme-linked immunosorbent assay (ELISA), which was partly due to the overexpression of protein phosphatase methylesterase-1 (PME-1), but not glycogen synthase kinase-3ß (GSK-3ß), cyclin-dependent kinase 5 (CDK5) or protein kinase B (Akt). Finally, we observed a significant increase in cyclic adenosine monophosphate (cAMP) and a decrease in miR-125b-5p levels in MT1KO, MT2KO and DKO mice. Using a luciferase reporter assay, we discovered that miR-125b-5p largely decreased the expression of firefly luciferase by interfering with the 3'UTR of PME-1. Furthermore, miR-125b-5p mimics significantly decreased the expression of PME-1, while miR-125b-5p inhibitor induced tau hyperphosphorylation. These results show that melatonin-receptor signal transduction plays an important role in tau hyperphosphorylation and tangle formation.

Pretreatment with mechano growth factor E peptide attenuates osteoarthritis through improving cell proliferation and extracellular matrix synthesis in chondrocytes under severe hypoxia.

Osteoarthritis (OA) is characterized by pain and declining gait function associated with degeneration of cartilage. A severe hypoxic environment occurs due to tissue injury in the joint cavity and may aggravate the development of OA. In this study, the effects of severe hypoxia and treatment with mechano growth factor (MGF) E peptide on metabolism of the extracellular matrix (ECM) during the progression of OA were determined. The results showed that cell viability, cell proliferation, and type II collagen expression in chondrocytes were significantly inhibited by cobalt chloride (CoCl2)-simulated severe hypoxia, whereas cell apoptosis and expression levels of hypoxia inducible factor 1 alpha, type I collagen, and matrix metalloproteinases 1/13 were clearly induced. Pretreatment with MGF E peptide reduced the abovementioned adverse effects induced by CoCl2-simulated severe hypoxia in chondrocytes. Pretreatment also upregulated the proliferation of chondrocytes under severe hypoxia through the PI3K-Akt and MEK-ERK1/2 signaling pathways. In a rat model of monosodium iodoacetate (MIA)-induced OA. MIA treatment induced tissue necrosis and cartilage degeneration, and histological score was significantly decreased. The levels of type II collagen and aggrecan were reduced after MIA treatment for 4 or 6 weeks, and abnormal distribution of ECM occurred in the inner epicondyle after 6 weeks. MGF E peptide also reduced the progression of MIA-induced OA by retarding cartilage degeneration, upregulating type II collagen synthesis, and improving ECM distribution after 4 or 6 weeks. Our findings suggest that MGF attenuates the progression of OA, and thus may be applied for the treatment of OA in the clinic.

Development of a macrophages-related 4-gene signature and nomogram for the overall survival prediction of hepatocellular carcinoma based on WGCNA and LASSO algorithm.

BACKGROUND: Immune system instability and poor prognosis are the two major clinical performance of hepatocellular carcinoma (HCC). Abnormal expression of MiR-424-5p has been reported to accelerate the progression of liver cancer, but it mediated immune cell infiltration imbalance is still unknown. We aim to mine the immune-related genes (IRGs) targeted by miR-424-5p and construct a multi-gene signature to improve the prognostic prediction of HCC. METHODS: The HCC-related data of the cancer genome atlas (TCGA) database and the GSE14520 dataset of the gene expression omnibus (GEO) database were downloaded as the discovery dataset and the validation dataset, respectively. Weighted gene co-expression network analysis (WGCNA), the deconvolution algorithm of CIBERSORT and LASSO algorithm participated in the identification of IRGs and the development of prognostic signature and nomogram. RESULTS: Our study found that the abundance of macrophages M0, M1 and M2 are all drastically changed during the cancerous process. A total of 920 macrophages infiltration-related LRGs were identified and a novel 4-gene signature (CDCA8, CBX2, UCK2 and SOCS2) with superior prognostic independence was established. The prognostic signature based risk score has superior capability to identify high-risk patients and predict overall survival (p < 0.001; AUC = 0.798 for 1 year; AUC = 0.748 for 3 years; AUC = 0.721 for 5 years). And it (C-index = 0.726) has a better prognostic potential than the TNM stage (C-index = 0.619), which is widely adopted in clinical practice. Additionally, the nomogram formed by combining the risk score and TNM stage further improved the accuracy of survival prediction (C-index = 0.733). CONCLUSION: In summary, the immune landscape with abnormal infiltration of macrophages may be one of the prelude to the cancerous process. The novel macrophages-related 4-gene signature is expected to become a potential prognostic marker in liver cancer.

PFKP Activation Ameliorates Foot Process Fusion in Podocytes in Diabetic Kidney Disease.

Background: Glycolysis dysfunction is an important pathogenesis of podocyte injury in diabetic kidney disease (DKD). Foot process fusion of podocytes and increased albuminuria are markers of early DKD. Moreover, cytoskeletal remodeling has been found to be involved in the foot process fusion of podocytes. However, the connections between cytoskeletal remodeling and alterations of glycolysis in podocytes in DKD have not been clarified. Methods: mRNA sequencing of glomeruli obtained from db/db and db/m mice with albuminuria was performed to analyze the expression profiling of genes in glucose metabolism. Expressions of phosphofructokinase platelet type (PFKP) in the glomeruli of DKD patients were detected. Clotrimazole (CTZ) was used to explore the renal effects of PFKP inhibition in diabetic mice. Using Pfkp siRNA or recombinant plasmid to manipulate PFKP expression, the effects of PFKP on high glucose (HG) induced podocyte damage were assessed in vitro. The levels of fructose-1,6-bisphosphate (FBP) were measured. Targeted metabolomics was performed to observe the alterations of the metabolites in glucose metabolism after HG stimulation. Furthermore, aldolase type b (Aldob) siRNA or recombinant plasmid were applied to evaluate the influence of FBP level alteration on podocytes. FBP was directly added to podocyte culture media. Db/db mice were treated with FBP to investigate its effects on their kidney. Results: mRNA sequencing showed that glycolysis enzyme genes were altered, characterized by upregulation of upstream genes (Hk1, and Pfkp) and down-regulation of downstream genes of glycolysis (Pkm, and Ldha). Moreover, the expression of PFKP was increased in glomeruli of DKD patients. The CTZ group presented more severe renal damage. In vitro, the Pfkp siRNA group and ALDOB overexpression group showed much more induced cytoskeletal remodeling in podocytes, while overexpression of PFKP and suppression of ALDOB in vitro rescued podocytes from cytoskeletal remodeling through regulation of FBP levels and inhibition of the RhoA/ROCK1 pathway. Furthermore, targeted metabolomics showed FBP level was significantly increased in HG group compared with the control group. Exogenous FBP addition reduced podocyte cytoskeletal remodeling and renal damage of db/db mice. Conclusions: These findings provide evidence that PFKP may be a potential target for podocyte injury in DN and provide a rationale for applying podocyte glycolysis enhancing agents in patients with DKD.

WGCNA, LASSO and SVM Algorithm Revealed RAC1 Correlated M0 Macrophage and the Risk Score to Predict the Survival of Hepatocellular Carcinoma Patients.

Background: RAC1 is involved in the progression of HCC as a regulator, but its prognostic performance and the imbalance of immune cell infiltration mediated by it are still unclear. We aim to explore the prognostic and immune properties of RAC1 in HCC. Methods: We separately downloaded the data related to HCC from the Cancer Genome Atlas (TCGA) and GEO database. CIBERSORT deconvolution algorithm, weighted gene co-expression network analysis (WGCNA) and LASSO algorithm participate in identifying IRGs and the construction of prognostic signatures. Results: The study discovered that RAC1 expression was linked to the severity of HCC lesions, and that its high expression was linked to a poor prognosis. Cox analysis confirmed that RAC1 is a clinically independent prognostic marker. M0, M1 and M2 macrophages' abundance are significantly different in HCC. We found 828 IRGs related to macrophage infiltration, and established a novel 11-gene signature with excellent prognostic performance. RAC1-based risk score and M0 macrophage has a good ability to predict overall survival. Conclusion: The immune state of irregular macrophage infiltration may be one of the precursors to carcinogenesis. The RAC1 correlated with M0 macrophage and the risk score to show a good performance to predict the survival of HCC patients.

Effect of mechanical injury and IL-1ß on the expression of LOXs and MMP-1, 2, 3 in PCL fibroblasts after co-culture with synoviocytes.

BACKGROUND: Crosstalk between posterior cruciate ligament fibroblasts (PCLfs) and synoviocytes (SCs) significantly modifies the homeostatic balance of the extracellular matrix (ECM) and appears to post a prominent affection for wound healing of PCL. Interleukin-1ß (IL-1ß) is regarded as a critical factor in acute inflammatory events during ligament injury. METHODS: In order to confirm the capability of SCs the response of lysyl oxidases (LOXs) and matrix metalloproteinases (MMPs) to IL-1ß, the complex cues of the joint cavity following PCL injury were simulated and the effect of IL-1ß on the expression of LOXs and MMPs in PCLfs were investigated. PCLfs in both the mono- and co-culture conditions were treated with IL-1ß. Cell lysates were collected from the PCLfs and LOXs and MMP-1, 2, 3 expression quantified using quantitative real-time PCR and western bolting. RESULTS: The results indicated that injury alone elevated the expression of LOXs and MMP-1, 2 and 3. But IL-1ß significantly decreased the LOX, LOXL1, and LOXL3 expression, and simultaneously increased MMP-1, 2 and 3 expressions in injured PCLfs. Furthermore, co-culture further suppressed LOXs, but stimulated MMP-1, 2 and 3 expressions when subjected to both mechanical injury and IL-1ß treatment. This possibly suggests that a number of soluble factors are secreted that act as mediators that amplify the response of SCs. CONCLUSION: The results indicated that the SCs could affect the IL-1ß-induction of LOXs inhibition and MMPs accumulation, which may be the underlying mechanism of the the poor healing response following PCL injury.

Safflower yellow alleviates osteoarthritis and prevents inflammation by inhibiting PGE2 release and regulating NF-κB/SIRT1/AMPK signaling pathways.

BACKGROUND: Safflower yellow (SY) is the main active ingredient of safflower, with various pharmacological effects such as anticoagulating, antioxidant, and anti-arthritis effects. PURPOSE: To investigate the anti-inflammatory and chondrocyte protecting role of SY, which subsequently leads to the inhibition of cartilage degradation. METHODS: Rat chondrocytes were stimulated with tumor necrosis factor α (TNF-α) with or without SY treatment. Following this, CCK-8 assay was performed to detect cytotoxicity. RT-qPCR, Western blotting, and immunofluorescence staining were used to detect the gene/protein expression of typical cartilage matrix genes and related inflammatory markers. Subsequently, EdU assay was used to evaluate cell proliferation. RNA sequencing, online target prediction, and molecular docking were performed to determine the possible molecular targets and pathways. RESULTS: The results showed that SY restored the TNF-α-induced up-regulation of IL-1ß, PTGS2, and MMP-13 and down-regulation of COL2A1 and ACAN. Furthermore, it recovered cell proliferation by suppressing TNF-α. Gene expression profiles identified 717 differentially expressed genes (DEGs) in the cells cultured with or without SY under TNF-α stimulation. After pathway enrichment, PI3K-Akt, TNF, Cytokine-cytokine receptor interaction, NF-κB, NOD-like receptor, and Chemokine signaling pathways were notably selected to highlight NFKBIA, CCL5, CCL2, IL6, and TNF as potential targets in osteoarthritis (OA). SY inhibited TNF-α-induced activation of NF-κB and endoplasmic reticulum (ER) stress by promoting AMPK phosphorylation along with SIRT1 expression. Further, SY reduced MMP-13 expression and targeted COX-2 for decreasing PGE2 release. In addition, anterior cruciate ligament transection-induced OA was ameliorated by local administration of SY. CONCLUSION: These results demonstrate that SY protects chondrocytes and inhibits inflammation by regulating the NF-κB/SIRT1/AMPK pathways and ER stress, thus preventing cartilage degeneration in OA.