Quercetin Protects Against Hypertensive Renal Injury by Attenuating Apoptosis: An Integrated Approach Using Network Pharmacology and RNA Sequencing.

ABSTRACT: Quercetin is known for its antihypertensive effects. However, its role on hypertensive renal injury has not been fully elucidated. In this study, hematoxylin and eosin staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, and Annexin V staining were used to assess the pathological changes and cell apoptosis in the renal tissues of angiotensin II (Ang II)-infused mice and Ang II-stimulated renal tubular epithelial cell line (NRK-52E). A variety of technologies, including network pharmacology, RNA-sequencing, immunohistochemistry, and Western blotting, were performed to investigate its underlying mechanisms. Network pharmacology analysis identified multiple potential candidate targets (including TP53, Bcl-2, and Bax) and enriched signaling pathways (including apoptosis and p53 signaling pathway). Quercetin treatment significantly alleviated the pathological changes in renal tissues of Ang II-infused mice and reversed 464 differentially expressed transcripts, as well as enriched several signaling pathways, including those related apoptosis and p53 pathway. Furthermore, quercetin treatment significantly inhibited the cell apoptosis in renal tissues of Ang II-infused mice and Ang II-stimulated NRK-52E cells. In addition, quercetin treatment inhibited the upregulation of p53, Bax, cleaved-caspase-9, and cleaved-caspase-3 protein expression and the downregulation of Bcl-2 protein expression in both renal tissue of Ang II-infused mice and Ang II-stimulated NRK-52E cells. Moreover, the molecular docking results indicated a potential binding interaction between quercetin and TP53. Quercetin treatment significantly attenuated hypertensive renal injury and cell apoptosis in renal tissues of Ang II-infused mice and Ang II-stimulated NRK-52E cells and by targeting p53 may be one of the potential underlying mechanisms.

Elucidating Iron Metabolism through Molecular Imaging.

Iron is essential for many physiological processes, and the dysregulation of its metabolism is implicated in the pathogenesis of various diseases. Recent advances in iron metabolism research have revealed multiple complex pathways critical for maintaining iron homeostasis. Molecular imaging, an interdisciplinary imaging technique, has shown considerable promise in advancing research on iron metabolism. Here, we comprehensively review the multifaceted roles of iron at the cellular and systemic levels (along with the complex regulatory mechanisms of iron metabolism), elucidate appropriate imaging methods, and summarize their utility and fundamental principles in diagnosing and treating diseases related to iron metabolism. Utilizing molecular imaging technology to deeply understand the complexities of iron metabolism and its critical role in physiological and pathological processes offers new possibilities for early disease diagnosis, treatment monitoring, and the development of novel therapies. Despite technological limitations and the need to ensure the biological relevance and clinical applicability of imaging results, molecular imaging technology's potential to reveal the iron metabolic process is unparalleled, providing new insights into the link between iron metabolism abnormalities and various diseases.

Identification of an immune-related 6-lncRNA panel with a good performance for prognostic prediction in hepatocellular carcinoma by integrated bioinformatics analysis.

Hepatocellular carcinoma (HCC) is one of the most malignant tumors with a poor prognosis. The long non-coding RNA (lncRNA) has been found to have great potential as a prognostic biomarker or therapeutic target for cancer patients. However, the prognostic value and tumor immune infiltration of lncRNAs in HCC has yet to be fully elucidated. To identify prognostic biomarkers of lncRNA in HCC by integrated bioinformatics analysis and explore their functions and relationship with tumor immune infiltration. The prognostic risk assessment model for HCC was constructed by comprehensively using univariate/multivariate Cox regression analysis, Kaplan-Meier survival analysis, and the least absolute shrinkage and selection operator regression analysis. Subsequently, the accuracy, independence, and sensitivity of our model were evaluated, and a nomogram for individual prediction in the clinic was constructed. Tumor immune microenvironment (TIME), immune checkpoints, and human leukocyte antigen alleles were compared in high- and low-risk patients. Finally, the functions of our lncRNA signature were examined using Gene Ontology, Kyoto Encyclopedia of Genes and Genomes enrichment analysis, and gene set enrichment analysis. A 6-lncRNA panel of HCC consisting of RHPN1-AS1, LINC01224, CTD-2510F5.4, RP1-228H13.5, LINC01011, and RP11-324I22.4 was eventually identified, and show good performance in predicting the survivals of patients with HCC and distinguishing the immunomodulation of TIME of high- and low-risk patients. Functional analysis also suggested that this 6-lncRNA panel may play an essential role in promoting tumor progression and immune regulation of TIME. In this study, 6 potential lncRNAs were identified as the prognostic biomarkers in HCC, and the regulatory mechanisms involved in HCC were initially explored.

An advanced network pharmacology study to explore the novel molecular mechanism of Compound Kushen Injection for treating hepatocellular carcinoma by bioinformatics and experimental verification.

BACKGROUND: Compound Kushen Injection (CKI) is a Chinese patent drug that exerts curative effects in the clinical treatment of hepatocellular carcinoma (HCC). This study aimed to explore the targets and potential pharmacological mechanisms of CKI in the treatment of HCC. METHODS: In this study, network pharmacology was used in combination with molecular biology experiments to predict and verify the molecular mechanism of CKI in the treatment of HCC. The constituents of CKI were identified by UHPLC-MS/MS and literature search. The targets corresponding to these compounds and the targets related to HCC were collected based on public databases. To screen out the potential hub targets of CKI in the treatment of HCC, a compound-HCC target network was constructed. The underlying pharmacological mechanism was explored through the subsequent enrichment analysis. Interactive Gene Expression Profiling Analysis and Kaplan-Meier plotter were used to examine the expression and prognostic value of hub genes. Furthermore, the effects of CKI on HCC were verified through molecular docking simulations and cell experiments in vitro. RESULTS: Network analysis revealed that BCHE, SRD5A2, EPHX2, ADH1C, ADH1A and CDK1 were the key targets of CKI in the treatment of HCC. Among them, only CDK1 was highly expressed in HCC tissues, while the other 5 targets were lowly expressed. Furthermore, the six hub genes were all closely related to the prognosis of HCC patients in survival analysis. Molecular docking revealed that there was an efficient binding potential between the constituents of CKI and BCHE. Experiments in vitro proved that CKI inhibited the proliferation of HepG2 cells and up-regulated SRD5A2 and ADH1A, while down-regulated CDK1 and EPHX2. CONCLUSIONS: This study revealed and verified the targets of CKI on HCC based on network pharmacology and experiments and provided a scientific reference for further mechanism research.

Role of Pyroptosis and Ferroptosis in the Progression of Atherosclerotic Plaques.

Pyroptosis is a special way of programmed cell death which is dependent on the activation of cysteinyl aspartate specific proteinase 1 (Caspase-1) and Caspase-4/5/11. Ferroptosis is an iron-dependent cell death that characterized by the intra-cellular lipid peroxidation-mediated membrane damage. Pyroptosis or ferroptosis in macrophages, smooth muscle cells, and vascular endothelial cells are believed to be closely related to the progression of atherosclerotic plaques. Therefore, we discuss the role of pyroptosis and ferroptosis in the development of atherosclerotic plaques and may provide new strategies for the treatment of atherosclerosis.

Triglyceride-glucose index and estimated 10-year risk of a first hard cardiovascular event.

Background: Whether Triglyceride-glucose (TyG) index is associated with 10-year risk of a first hard atherosclerotic cardiovascular disease (ASCVD) event in the United States remains unclear. Methods: In this cross-sectional study, the participants, ranged from 40 to 79 years old, were from the National Health and Nutrition Examination Survey (NHANES) between 1999 and 2018. TyG index was the independent variable and 10-year risk of a first hard ASCVD was the dependent variable. The other variables, such as age, gender, race, body mass index (BMI), hypertension treatment states, smoking states and low-density lipoprotein cholesterol (LDL-C) et al. were considered as the potential confounding factors. Multivariate linear regression models and smooth curve fittings were used to evaluate the association between TyG index and 10-year risk of a first hard ASCVD event. Results: A total of 2,142 participants were included in the analysis. The results showed that TyG index was associated with an increased 10-year risk of a first hard ASCVD event [ß = 2.208, 95% (1.716, 2.700), P < 0.00001]. The association had statistical significance in both men [ß = 3.862 95% CI (3.274, 4.450), P < 0.00001] and women [ß = 1.067, 95% CI (0.286, 1.849), P = 0.00756)] according to subgroup analysis. Smooth curve fittings revealed that TyG index was linearly associated with 10-year risk of ASCVD in both male and female. Conclusion: Triglyceride-glucose index was associated with an increased 10-year risk of a first hard ASCVD event in the United States, suggesting it is necessary to monitor and control an appropriate range of TyG index.

Investigation on the mechanisms of guiqi huoxue capsule for treating cervical spondylosis based on network pharmacology and molecular docking.

BACKGROUND: Guiqi huoxue capsule (GQHXC) is a patented Chinese medicine used for treating a liver and kidney deficiency and blood stasis syndrome due to qi deficiency. It is caused by cervical spondylosis (cervical spondylotic radiculopathy (CSR), mixed cervical spondylosis mainly composed of nerve root type). Its underlying mechanisms need, however, to be further clarified. METHODS: In this study, collecting compounds, predicting therapeutic targets, constructing networks, and analyzing biological functions and pathways were based on network pharmacology analysis. In addition, molecular docking verification was engaged to assess the binding potential of selected target-compound pairs. RESULTS: We established 5 networks: compound-putative target network of GQHXC, protein-protein interaction (PPI) network related to CSR, compound-CSR target network, potential therapeutic targets PPI network, and herb-compound-target-pathway network. Network analysis indicated that 7 targets (tumor necrosis factor [TNF], interleukin 6 [IL6], nitric oxide synthase 3 [NOS3], Interleukin-8 [CXCL8], prostaglandin-endoperoxide synthase 2 [PTGS2], vascular endothelial growth factor A [VEGFA], and AP-1 transcription factor subunit [JUN]) might be the therapeutic targets of GQHXC in CSR. Moreover, molecular docking verification showed that TNF, IL6, NOS3, CXCL8, PTGS2, VEGFA, and JUN had a good is interaction with the corresponding compounds. Furthermore, enrichment analysis indicated that GQHXC might exert a curative role in CSR by regulating some important pathways, such as TNF signaling pathway, NF-kappa B signaling pathway, AGE-RAGE signaling pathway in diabetic complications, and so on. CONCLUSION: Our study preliminarily explained the underlying mechanisms of GQHXC for treating CSR, and molecular docking verification was adopted as an additional verification. These findings laid a valuable foundation for experimental research and further application of GQHXC in the clinical treatment of CSR.

High Throughput Transcriptome Data Analysis and Computational Verification Reveal Immunotherapy Biomarkers of Compound Kushen Injection for Treating Triple-Negative Breast Cancer.

BACKGROUND: Although notable therapeutic and prognostic benefits of compound kushen injection (CKI) have been found when it was used alone or in combination with chemotherapy or radiotherapy for triple-negative breast cancer (TNBC) treatment, the effects of CKI on TNBC microenvironment remain largely unclear. This study aims to construct and validate a predictive immunotherapy signature of CKI on TNBC. METHODS: The UPLC-Q-TOF-MS technology was firstly used to investigate major constituents of CKI. RNA sequencing data of CKI-perturbed TNBC cells were analyzed to detect differential expression genes (DEGs), and the GSVA algorithm was applied to explore significantly changed pathways regulated by CKI. Additionally, the ssGSEA algorithm was used to quantify immune cell abundance in TNBC patients, and these patients were classified into distinct immune infiltration subgroups by unsupervised clustering. Then, prognosis-related genes were screened from DEGs among these subgroups and were further overlapped with the DEGs regulated by CKI. Finally, a predictive immunotherapy signature of CKI on TNBC was constructed based on the LASSO regression algorithm to predict mortality risks of TNBC patients, and the signature was also validated in another TNBC cohort. RESULTS: Twenty-three chemical components in CKI were identified by UPLC-Q-TOF-MS analysis. A total of 3692 DEGs were detected in CKI-treated versus control groups, and CKI significantly activated biological processes associated with activation of T, natural killer and natural killer T cells. Three immune cell infiltration subgroups with 1593 DEGs were identified in TNBC patients. Then, two genes that can be down-regulated by CKI with hazard ratio (HR) > 1 and 26 genes that can be up-regulated by CKI with HR < 1 were selected as key immune- and prognosis-related genes regulated by CKI. Lastly, a five-gene prognostic signature comprising two risky genes (MARVELD2 and DYNC2I2) that can be down-regulated by CKI and three protective genes (RASSF2, FERMT3 and RASSF5) that can be up-regulated by CKI was developed, and it showed a good performance in both training and test sets. CONCLUSIONS: This study proposes a predictive immunotherapy signature of CKI on TNBC, which would provide more evidence for survival prediction and treatment guidance in TNBC as well as a paradigm for exploring immunotherapy biomarkers in compound medicines.

A Multidimensional Bayesian Network Meta-Analysis of Chinese Herbal Injections for Treating Non-small Cell Lung Cancer With Gemcitabine and Cisplatin.

Introduction: As non-small cell lung cancer (NSCLC) seriously threatens human health, several clinical studies have reported that Chinese herbal injections (CHIs) in combination with and gemcitabine plus cisplatin (GP) are beneficial. This multidimensional network meta-analysis aimed to compare the clinical efficacy and safety of different CHIs in combination with GP against NSCLC. Methods: Randomized controlled trials (RCTs) for the treatment of NSCLC were retrieved from seven electronic databases from inception to April 30, 2020. Study selection and data extraction were based on a priori criteria. Data analysis was performed using Stata 13.0, WinBUGS 14.0 software. Multidimensional cluster analysis was performed using the "scatterplot3d" package in R 3.6.1 software. Results: This network meta-analysis included 71 eligible RCTs and 10 Chinese herbal injections. Delisheng injection and Kangai injection had the highest probability in terms of clinical effectiveness rate (94.60%) and gastrointestinal reactions (82.62%) when combined with GP compared with the other interventions. Compound Kushen injection combined with GP ranked ahead of the other interventions in terms of performance status (73.36%) and abnormal liver function (87.17%). Shenmai injection combined with GP had the highest probability in terms of leukopenia (94.59%) and thrombocytopenia (99.18%). Conclusion: The current evidence revealed that CHIs combined with GP have a better impact on patients with NSCLC than GP alone. Aidi injection, Compound kushen injection, and Kanglaite injection deserve more attention of clinicians when combined with GP in patients with NSCLC. Additionally, due to the limitations of this network meta-analysis, further well-designed, large-sample, multicenter RCTs are required to support our findings adequately.

Investigation on the clinical efficacy and mechanism of compound kushen injection in treating esophageal cancer based on multi-dimensional network meta-analysis and in vitro experiment.

ETHNOPHARMACOLOGICAL RELEVANCE: Esophageal cancer, as a high incidence of gastrointestinal cancer, has an indelible impact on human life and health. The combination of Chinese herbal injections and chemotherapy is commonly applied in the treatment of Esophageal cancer. AIM OF THE STUDY: This study aimed to confirm the clinical advantage of Compound Kushen Injection to treat esophageal cancer and explore its molecular mechanism. METHODS: The network meta-analysis method was used for the clinical evaluation of anti-tumor Chinese herbal injections. Initially, several electronic databases were searched to identify randomized controlled trials regarding Chinese herbal injections to treat esophageal cancer from their inception to September 5, 2020. Then, WinBugs and Stata software was used to calculate and analyze the outcome indicators, including total clinical efficiency, improvement of quality of life and adverse reactions. Furthermore, the surface under the cumulative ranking curve and three-dimensional cluster analysis were used to rank the efficacy and safety of Chinese herbal injections about each outcome. Cell Counting Kit-8 assay was used to observe the effect of Compound Kushen Injection on the proliferation of esophageal cancer cells. Real-Time Quantitative PCR and Western Blot analysis were used to detect the mRNA and protein expression of EGFR and AURKA in ESCA cells. RESULTS: The surface under the cumulative ranking curve of Compound Kushen Injection combined with chemotherapy in total clinical efficiency, quality of life, reduction of nausea and vomiting were ranking at 89.1%, 81.8% and 92.4%, respectively. Compound Kushen Injection was determined as the dominant variety in the treatment of esophageal cancer which can inhibit the proliferation of esophageal cancer cells and downregulate the overexpression of EGFR and AURKA mRNA and protein. CONCLUSION: In this study, network meta-analysis was applied to confirm that Compound Kushen Injection has a curative effect on esophageal cancer and is superior to other anti-tumor Chinese herbal injections. Combined with the network pharmacology and in vitro experiment, the mechanism of Compound Kushen Injection inhibiting the proliferation of esophageal cancer cells by regulating the abnormal expression of EGFR and AURKA was revealed.

Revealing the Common Mechanisms of Scutellarin in Angina Pectoris and Ischemic Stroke Treatment via a Network Pharmacology Approach.

OBJECTIVE: To investigate the shared mechanisms of scutellarin in angina pectoris (AP) and ischemic stroke (IS) treatment. METHODS: A network pharmacology approach was used to detect the potential mechanisms of scutellarin in AP and IS treatment by target prediction, protein-protein interaction (PPI) data collection, network construction, network analysis, and enrichment analysis. Furthermore, molecular docking simulation was employed to analyze the interaction between scutellarin and core targets. RESULTS: Two networks were established, including a disease-target network and a PPI network of scutellarin targets against AP and IS. Network analysis showed that 14 targets, namely, AKT1, VEGFA, JUN, ALB, MTOR, ESR1, MAPK8, HSP90AA1, NOS3, SERPINE1, FGA, F2, FOXO3, and STAT1, might be the therapeutic targets of scutellarin in AP and IS. Among them, NOS3 and F2 were recognized as the core targets. Additionally, molecular docking simulation confifirmed that scutellarin exhibited a relatively high potential for binding to the active sites of NOS3 and F2. Furthermore, enrichment analysis indicated that scutellarin might exert a therapeutic role in both AP and IS by regulating several important pathways, such as coagulation cascades, mitogen-activated protein kinase (MAPK) signaling pathway, phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway, Toll-like receptor signaling pathway, hypoxia inducible factor-1 (HIF-1) signaling pathway, forkhead box O (FoxO) signaling pathway, tumor necrosis factor (TNF) signaling pathway, adipocytokine signaling pathway, insulin signaling pathway, insulin resistance, and estrogen signaling pathway. CONCLUSIONS: The shared underlying mechanisms of scutellarin on AP and IS treatment might be strongly associated with its vasorelaxant, anticoagulant, anti-inflammatory, and antioxidative effects as well as its effect on improving lipid metabolism.

Pro-Angiogenic Role of LncRNA HULC in Microvascular Endothelial Cells via Sequestrating miR-124.

BACKGROUND/AIMS: HULC is a multifunctional lncRNA that has pro-angiogenic function in various cancers. The present study was designed to see the role of lncRNA HULC in normal endothelial cells angiogenesis. METHODS: Cell viability, apoptosis, migration, tube formation and expression levels of angiogenesis-related proteins were respectively assessed in human microvascular endothelial HMEC-1 cells after lncRNA HULC was silenced by shRNA transfection. Cross-regulation between lncRNA HULC and miR-124, and between miR-124 and MCL-1 were detected by qRT-PCR, sequence analysis, and luciferase reporter assay. RESULTS: Silence of lncRNA HULC significantly reduced viability, migration, tube formation and protein levels of VEGF, VEGFR2, CD144 and eNOS in HMEC-1 cells. Meanwhile, silence of lncRNA HULC induced apoptosis in HMEC-1 cells, as Bcl-2 was down-regulated, Bax was up-regulated, and caspase-3 and -9 were cleaved. miR-124 expression was negatively regulated by lncRNA HULC, and HULC worked as a molecular sponge for miR-124, in having miR-124 exhausted. Besides, MCL-1 was a target gene of miR-124. Rescue assay results showed that the effects of lncRNA HULC silence on HMEC-1 cells growth, migration and angiogenesis were abolished by miR-124 suppression. Similarly, the effects of miR-124 on HMEC-1 cells were abolished by MCL-1 overexpression. Furthermore, MCL-1 activated PI3K/AKT and JAK/STAT signaling pathways. CONCLUSION: These findings suggest a pro-angiogenic role of lncRNA HULC in endothelial cells. The pro-angiogenic actions of lncRNA HULC may be through sponging miR-124, preventing MCL-1 from degradation by miR-124.

Silence of lncRNA UCA1 Represses the Growth and Tube Formation of Human Microvascular Endothelial Cells Through miR-195.

BACKGROUND/AIMS: Recent studies have suggested that several lncRNAs contribute to the angiogenic function of endothelial cells. Herein, we set out to reveal whether lncRNA UCA1 has functions in endothelial angiogenesis. METHODS: The expression levels of lncRNA UCA1, miR-195 and CCND1 in human microvascular endothelial HMEC-1 cells were altered by transfection. Subsequently, cell viability, migration, tube formation and apoptosis of HMEC-1 cells were respectively assessed. The cross-talk between lncRNA UCA1, miR-195, CCND1, and MEK/ERK and mTOR signaling pathways were investigated by performing qRT-PCR and Western blotting. RESULTS: Silence of lncRNA UCA1 repressed HMEC-1 cells viability, migration, tube formation, and induced apoptosis. Meanwhile, silence of lncRNA UCA1 significantly up-regulated miR-195 expression. These alterations induced by lncRNA UCA1 were further enhanced by miR-195 overexpression, while were attenuated by miR-195 suppression. Moreover, silence of lncRNA UCA1 deactivated MEK/ERK and mTOR signaling pathways via a miR-195-dependent regulation. And the deactivation of MEK/ERK and mTOR signaling pathways led to a down-regulation of CCND1. CONCLUSION: This study demonstrates that silence of lncRNA UCA1 largely represses microvascular endothelial cells growth and tube formation. Silence of lncRNA UCA1 exerts its function possibly via up-regulation of miR-195, which in turn inactivates MEK/ERK and mTOR signaling pathways, and ultimately represses CCND1 expression.

Preliminary comparative study on the effect of different chinese drugs for strengthening Pi in antagonizing diet induced obesity.

OBJECTIVE: To observe the difference in fatty degree, glucose-lipid disorder and adipose-hormones between diet induced obesity (DIO) rats and diet induced obesity resistance (DIO-R) rats, and to explore the effect and acting mechanism of Chinese drugs for strengthening Pi (CD-SP) and those for both strengthening Pi and dissolving phlegm (CD-SPDP) in inhibiting obesity. METHODS: Excepting eight rats allocated in the blank control group, the other 54 rats were fed with high-lipid forage for 12 weeks to establish models of obesity. Finally, 30 DIO rats and 8 DIO-R rats (shown by their body weight) were obtained. The DIO rats were divided into three groups, which were given gastric perfusion, respectively, with normal saline (Group A), CD-SP (Group B), and CD-SPDP (Group C). Fourteen weeks later, the animals' body weight (BW), length (BL), blood levels of fasting insulin (FIn), fasting glucose (FBG), triglyceride (TG), cholesterol (TC), leptin (LP), neuropeptide Y (NPY), C-reactive protein (CRP), tumor necrosis factor-alpha(TNF-alpha), adiponectin (AN), and resistin (RS) were measured; insulin resistance index (IRI) was calculated, and the degree of obesity and lipid content in abdominal cavity of rats were estimated. Moreover, the levels of LP, CRP, TNF-alpha, AN and RS in homogenate of rats' adipose tissues (ATH) were determined. RESULTS: After 12 weeks of high-lipid diet, the BW of DIO rats was higher than that of normal or DIO-R rats. After a 14-week continuous high-lipid diet feeding, in DIO rats, BW, lipid coefficient (LC), and IRI were significantly increased (P<0.01); serum levels of TNF-alpha, LP and AN were lower, NPY was higher, while the ATH levels of LP and AN were lower and TNF-alpha was higher in DIO rats than in DIO-R rats (P<0.05 or P<0.01); blood levels of FBG and lipids in DIO rats showed an increasing trend but was statistically insignificant (P>0.05); no significant difference was found in serum levels of CRP and RS due to the overly high data dispersion. Comparisons of the 3 DIO groups showed that BW, body weight index (BWI), LC and IRI were significantly lowered after treatment (P<0.01) in Group C, while these indexes were not significantly different between Group A and B; the serum levels of TNF-alpha, LP, and AN increased, NPY decreased in Group B and C, ATH levels of LP and AN increased, and TNF-alpha decreased in the two groups; but NPY, LP, and AN in blood and ATH were higher in Group C than those in Group B (P<0.05 or P<0.01). CONCLUSION: CD-SPDP could inhibit DIO and IR, showing that the effect is better than that of CD-SP, and its mechanism is related to promotion of LP and AN secretion and elevation of serum NPY.