The clinical and genetic aspects of six individuals with GH1 variants and isolated growth hormone deficiency type II.

Background: Isolated growth hormone deficiency type II (IGHD II) is an autosomal dominant disorder characterized by a GH1 gene variant resulting in a significant reduction in growth hormone (GH) secretion and a subsequent decrease of plasma insulin-like growth factor 1 (IGF-1) levels and eventual growth impairment. Objective: This study aimed to identify causative variants in six Chinese families with IGHD II, exploring both clinical and genetic characteristics. Methods: Detailed clinical data, including clinical presentations, physical charateristics, medical and family histories, as well as genetic test results, were systematically examined. Results: Six children, comprising four males and two females, with a mean age of 4.64 ± 1.15 years, exhibited short stature with a mean height of -3.95 ± 1.41 SDS. Four of them had a family history of short stature, while one patient presented with pulmonary hypertension. All children demonstrated GH deficiency in growth hormone stimulation tests (mean peak GH value: 2.83 ± 2.46 ng/mL). Exome sequencing for the six patients and targeted gene sequencing for their family members revealed heterozygous variants in the GH1 gene, including Exon2-5del, c.334T>C, c.291 + 1G>A, c.291 + 2T>A, 1.5 kb deletion, and 1.7 kb deletion, with four variants being novel. Four patients underwent human recombinant growth hormone (rhGH) replacement therapy, initiating treatment at a mean age of 4.6 ± 0.7 years. The mean height increase in patients was 1.21 ± 0.3 SDS in the first six months of treatment and 1.79 ± 0.15 SDS in the first year. Conclusion: Our findings contribute to expanding the genotypic and phenotypic spectra of individuals with IGHD II.

Targeted Degradation of SOS1 Exhibits Potent Anticancer Activity and Overcomes Resistance in KRAS-Mutant Tumors and BCR-ABL-Positive Leukemia.

SOS1 is an essential guanine nucleotide exchange factor for RAS that also plays a critical role in the activation of the small GTPase RAC mediated by BCR-ABL in leukemogenesis. Despite this, small molecule inhibitors targeting SOS1 have shown limited efficacy in clinical trials for KRAS mutant cancers, and their potential as a therapeutic approach for chronic myeloid leukemia (CML) remains largely unexplored. In this study, we developed a potent SOS1 PROTAC SIAIS562055, which was designed by connecting a CRBN ligand to an analogue of the SOS1 inhibitor BI-3406. SIAIS562055 exhibited sustained degradation of SOS1 and inhibition of downstream ERK pathways, resulting in superior anti-proliferative activity compared to small molecule inhibitors. SIAIS562055 also potentiated the activity of both KRAS inhibitors in KRAS-mutant cancers and ABL inhibitors in BCR-ABL+ CML. In KRAS-mutant xenografts, SIAIS562055 displayed promising antitumor potency as a monotherapy and enhanced ERK inhibition and tumor regression when combined with KRAS inhibitors, overcoming acquired resistance. In CML cells, SIAIS562055 promoted the active uptake of BCR-ABL inhibitors by upregulating the carnitine/organic cation transporter SLC22A4. SIAIS562055 and BCR-ABL inhibitors synergistically enhanced inhibition of ABL phosphorylation and downstream signaling, demonstrating robust antitumor activities in both mouse xenografts and primary CML patient samples. In summary, this study suggests that PROTAC-mediated SOS1 degradation represents an effective therapeutic strategy for treating not only KRAS-mutant cancers but also BCR-ABL-harboring leukemia.

Occurrence and distribution of emerging contaminants in wastewater treatment plants: A globally review over the past two decades.

Emerging contaminants are pervasive in aquatic environments globally, encompassing pharmaceuticals, personal care products, steroid hormones, phenols, biocides, disinfectants and various other compounds. Concentrations of these contaminants are detected ranging from ng/L to µg/L. Even at trace levels, these contaminants can pose significant risks to ecosystems and human health. This article systematically summarises and categorizes data on the concentrations of 54 common emerging contaminants found in the influent and effluent of wastewater treatment plants across various geographical regions: North America, Europe, Oceania, Africa, and Asia. It reviews the occurrence and distribution of these contaminants, providing spatial and causal analyses based on data from these regions. Notably, the maximum concentrations of the pollutants observed vary significantly across different regions. The data from Africa, in particular, show more frequent detection of pharmaceutical maxima in wastewater treatment plants.

A novel experimental mouse model of diabetic nonalcoholic steatohepatitis: A critical role for acid-sensitive Ion Channel 1a.

BACKGROUND: A two-way relationship exists between type 2 diabetes (T2DM) and human nonalcoholic steatohepatitis (NASH). Several diabetic NASH models have the disadvantages of long cycles or inconsistent with the actual incidence of human disease, which would be costly and time-consuming to investigate disease pathogenesis and develop drugs. Therefore, there is an urgent need to establish a diabetic NASH mouse model. METHODS: The combination between Fructose-palmitate-cholesterol diet (FPC) and Streptozotocin (STZ) (FPC+STZ) was used to construct diabetic NASH mouse model. The in vivo effects of silencing acid-sensitive Ion Channel 1a (ASIC1a) were examined with an adeno-associated virus 9 (AAV9) carrying ASIC1a short hairpin RNA (shRNA) in FPC+STZ model. RESULTS: The mice fed with FPC for 12 weeks had insulin resistance, hyperinsulinemia, lipid accumulation, and increased hepatic levels of inflammatory factors. However, it still did not develop remarkable liver fibrosis. Most interestingly, noticeable fibrotic scars were observed in the liver of mice from FPC+STZ group. Furthermore, insulin therapy significantly ameliorated FPC+STZ-induced NASH-related liver fibrosis, indicating that hyperglycemia is of great significance in NASH development and progression. Importantly, ASIC1a was found to be involved in the pathogenesis of diabetic NASH as demonstrated that silencing ASIC1a in HSCs significantly ameliorated FPC+STZ-induced NASH fibrosis. Mechanistically, ASIC1a interacted with Poly Adp-adenosine ribose polymerase (PARP1) to promote HSC activation by inducing autophagy. CONCLUSION: A FPC diet combined with an injection of STZ induces a diabetic NASH mouse model in a shorter period. Targeting ASIC1a may provide a novel therapeutic target for the treatment of diabetic NASH.

Reducing overconfident errors in molecular property classification using Posterior Network.

Deep-learning-based classification models are increasingly used for predicting molecular properties in drug development. However, traditional classification models using the Softmax function often give overconfident mispredictions for out-of-distribution samples, highlighting a critical lack of accurate uncertainty estimation. Such limitations can result in substantial costs and should be avoided during drug development. Inspired by advances in evidential deep learning and Posterior Network, we replaced the Softmax function with a normalizing flow to enhance the uncertainty estimation ability of the model in molecular property classification. The proposed strategy was evaluated across diverse scenarios, including simulated experiments based on a synthetic dataset, ADMET predictions, and ligand-based virtual screening. The results demonstrate that compared with the vanilla model, the proposed strategy effectively alleviates the problem of giving overconfident but incorrect predictions. Our findings support the promising application of evidential deep learning in drug development and offer a valuable framework for further research.

Shenqi Fuzheng injection alleviates chemotherapy-induced cachexia by restoring glucocorticoid signaling in hypothalamus.

Image 1.

Identification and validation of SLCO4C1 as a biological marker in hepatocellular carcinoma based on anoikis classification features.

BACKGROUND: Hepatocellular carcinoma (HCC) exhibits a high degree of invasiveness and is closely associated with rapid disease progression. Multiple lines of evidence indicate a strong correlation between anoikis resistance and tumor progression, invasion, and metastasis. Nevertheless, the classification of anoikis in HCC and the investigation of novel biological target mechanisms in this context continue to pose challenges, requiring further exploration. METHODS: Combined with HCC samples from TCGA, GEO and ICGC databases, cluster analysis was conducted on anoikis genes, revealing novel patterns among different subtypes. Significant gene analysis of different gene subtypes was performed using WCGNA. The anoikis prognostic risk model was established by Lasso-Cox. Go, KEGG, and GSEA were applied to investigate pathway enrichment primarily observed in risk groups. We compared the disparities in immune infiltration, TMB, tumor microenvironment (TME), and drug sensitivity between the two risk groups. RT-qPCR and Western blotting were performed to validate the expression levels of SLCO4C1 in HCC. The biological functions of SLCO4C1 in HCC cells were assessed through various experiments, including CCK8 assay, colony formation assay, invasion migration assay, wound healing assay, and flow cytometry analysis. RESULTS: HCC was divided into 2 anoikis subtypes, and the subtypeB had a better prognosis. An anoikis prognostic model based on 12 (COPZ2, ACTG2, IFI27, SPP1, EPO, SLCO4C1, RAB26, STC2, RAC3, NQO1, MYCN, HSPA1B) risk genes is important for survival and prognosis. Significant differences were observed in immune cell infiltration, TME, and drug sensitivity analysis between the risk groups. SLCO4C1 was downregulated in HCC. SLCO4C1 downregulation promoted the proliferation, invasion, migration, and apoptosis of HCC cells. The tumor-suppressive role of SLCO4C1 in HCC has been confirmed. CONCLUSIONS: Our study presents a novel anoikis classification method for HCC that reveals the association between anoikis features and HCC. The anoikis feature is a critical biomarker bridging tumor cell death and tumor immunity. In this study, we provided the first evidence of SLCO4C1 functioning as a tumor suppressor in HCC.

CDC123 promotes Hepatocellular Carcinoma malignant progression by regulating CDKAL1.

The cell proliferation protein 123 (CDC123) is involved in the synthesis of the eukaryotic initiation factor 2 (eIF2), which regulates eukaryotic translation. Although CDC123 is considered a candidate oncogene in breast cancer, its expression and role in Hepatocellular Carcinoma (HCC) remain unknown. Herein, we obtained the CDC123 RNA-seq and clinical prognostic data from the TCGA database. The mRNA level revealed that CDC123 was highly expressed in HCC patients, and Kaplan-Meier analysis implied better prognoses in HCC patients with low CDC123 expression (P < 0.001). The multivariate Cox analysis revealed that the CDC123 level was an independent prognostic factor (P < 0.001). We further confirmed a high CDC123 expression in HCC cell lines. Additionally, we found that CDC123 knockdown in HCC cell lines significantly inhibited cellular proliferation, invasion, and migration. Moreover, CDC123 was co-expressed with the CDK5 Regulatory Subunit-Associated Protein 1 Like 1 (CDKAL1), whose mRNA level was decreased after silencing CDC123. Therefore, we hypothesized that CDC123 promotes HCC progression by regulating CDKAL1.

Energy preservation for skeletal muscles: Shenqi Fuzheng injection prevents tissue wasting and restores bioenergetic profiles in a mouse model of chemotherapy-induced cachexia.

BACKGROUND: Energy deficiency is the characteristic of chemotherapy-induced cachexia (CIC) which is manifested by muscle wasting. glycolysis, tricarboxylic acid (TCA) cycle, and lipid metabolism are central to muscle bioenergy production, which is vulnerable to chemotherapy during cancer treatment. Recent investigations have spotlighted the potential of Shenqi Fuzheng injection (SQ), a Chinese proprietary medicine comprising Radix Codonopsis and Radix Astragali, in alleviating CIC. However, the specific effects of SQ on muscle energy metabolism remains less explored. PURPOSE AND METHODS: Here, we integrated transcriptomics, spatial metabolomics, gas chromatography-mass spectrometry targeted quantitative analysis, and transmission electron microscopy techniques, combined with Seahorse live-cell metabolic analysis to reveal the changes in genes and pathways related to energy metabolism in the CIC model and SQ's protective effects at molecular and functional levels. RESULTS: Our data showed that chemotherapeutic agents caused glycolysis imbalance, which further leads to metabolic derangements of TCA cycle intermediates. SQ maintained glycolysis balance by facilitating pyruvate fluxing to mitochondria for more efficient bioenergy production, which involved a dual effect on promoting functions of mitochondrial pyruvate dehydrogenase complexes and inhibiting lactate dehydrogenase for lactate production. As a result of the sustained pyruvate level achieved by SQ administration, glycolysis balance was maintained, which further led to the preservation of mitochondrial integrity and function of electron transport chain, thereby, ensuring the normal operation of the TCA cycle and the proper synthesis of adenosine triphosphate (ATP). The above results were further validated using the Seahorse live-cell assay. CONCLUSION: In conclusion, our study highlights SQ as a promising strategy for CIC management, emphasizing its ability to harmonize the homeostasis of the muscle bioenergetic profile. Beyond its therapeutic implications, this study also offers a novel perspective for the development of innovative treatments in the realm of herbal medicine.

Single-cell and genetic multi-omics analysis combined with experiments confirmed the signature and potential targets of cuproptosis in hepatocellular carcinoma.

Background: Cuproptosis, as a recently discovered type of programmed cell death, occupies a very important role in hepatocellular carcinoma (HCC) and provides new methods for immunotherapy; however, the functions of cuproptosis in HCC are still unclear. Methods: We first analyzed the transcriptome data and clinical information of 526 HCC patients using multiple algorithms in R language and extensively described the copy number variation, prognostic and immune infiltration characteristics of cuproptosis related genes (CRGs). Then, the hub CRG related genes associated with prognosis through LASSO and Cox regression analyses and constructed a prognostic prediction model including multiple molecular markers and clinicopathological parameters through training cohorts, then this model was verified by test cohorts. On the basis of the model, the clinicopathological indicators, immune infiltration and tumor microenvironment characteristics of HCC patients were further explored via bioinformation analysis. Then, We further explored the key gene biological function by single-cell analysis, cell viability and transwell experiments. Meantime, we also explored the molecular docking of the hub genes. Results: We have screened 5 hub genes associated with HCC prognosis and constructed a prognosis prediction scoring model. And the model results showed that patients in the high-risk group had poor prognosis and the expression levels of multiple immune markers, including PD-L1, CD276 and CTLA4, were higher than those patients in the low-risk group. We found a significant correlation between risk score and M0 macrophages and memory CD4+ T cells. And the single-cell analysis and molecular experiments showed that BEX1 were higher expressed in HCC tissues and deletion inhibited the proliferation, invasion and migration and EMT pathway of HCC cells. Finally, it was observed that BEX1 could bind to sorafenib to form a stable conformation. Conclusion: The study not only revealed the multiomics characteristics of CRGs in HCC but also constructed a new high-accuracy prognostic prediction model. Meanwhile, BEX1 were also identified as hub genes that can mediate the cuproptosis of hepatocytes as potential therapeutic targets for HCC.

Prognosis signature for predicting the survival and immunotherapy response in esophageal carcinoma based on cellular senescence-related genes.

Background: Cellular senescence occurs throughout life and can play beneficial roles in a variety of physiological processes, including embryonic development, tissue repair, and tumor suppression. However, the relationship between cellular senescence-related genes (CSRGs) and immunotherapy in esophageal carcinoma (ECa) remains poorly defined. Methods: The data set used in the analysis was retrieved from TCGA (Research Resource Identifier (RRID): SCR_003193), GEO (RRID: SCR_005012), and CellAge databases. Data processing, statistical analysis, and diagram formation were conducted in R software (RRID: SCR_001905) and GraphPad Prism (RRID: SCR_002798). Based on CSRGs, we used the TCGA database to construct a prognostic signature for ECa and then validated it in the GEO database. The predictive efficiency of the signature was evaluated using receiver operating characteristic (ROC) curves, Cox regression analysis, nomogram, and calibration curves. According to the median risk score derived from CSRGs, patients with ECa were divided into high- and low-risk groups. Immune infiltration and immunotherapy were also analyzed between the two risk groups. Finally, the hub genes of the differences between the two risk groups were identified by the STRING (RRID: SCR_005223) database and Cytoscape (RRID: SCR_003032) software. Results: A six-gene risk signature (DEK, RUNX1, SMARCA4, SREBF1, TERT, and TOP1) was constructed in the TCGA database. Patients in the high-risk group had a worse overall survival (OS) was disclosed by survival analysis. As expected, the signature presented equally prognostic significance in the GSE53624 cohort. Next, the Area Under ROC Curve (AUC=0.854) and multivariate Cox regression analysis (HR=3.381, 2.073-5.514, P<0.001) also proved that the risk signature has a high predictive ability. Furthermore, we can more accurately predict the prognosis of patients with ECa by nomogram constructed by risk score. The result of the TIDE algorithm showed that ECa patients in the high-risk group had a greater possibility of immune escape. At last, a total of ten hub genes (APOA1, MUC5AC, GC, APOA4, AMBP, FABP1, APOA2, SOX2, MUC8, MUC17) between two risk groups with the highest interaction degrees were identified. By further analysis, four hub genes (APOA4, AMBP, FABP1, and APOA2) were related to the survival differences of ECa. Conclusions: Our study reveals comprehensive clues that a novel signature based on CSRGs may provide reliable prognosis prediction and insight into new therapy for patients with ECa.

Excessive bowel volume loss during anus-preserving surgery for rectal cancer affects the bowel function after operation: A prospective observational cohort study (Bas-1611).

Background: Bowel volume loss during anus-preserving surgery (APS) may result in low anterior resection syndrome (LARS). We conducted this prospective observational cohort study to measure the incidence of LARS after surgery and evaluate the relationship between bowel volume loss and bowel function. Methods: Patients with R0 resectable rectal cancer who consented to several bowel function surveys through telephone interviews after the operation were included. Enrolled patients underwent standard APS for rectal cancer, and three length indexes, viz. length of excised bowel, length of the distal margin and length of the proximal margin (LPM) of fresh bowel specimens, were measured in vitro. Results: The three measured variables of the specimens showed a positively skewed distribution. Patient interviews revealed a trend of gradual improvement in bowel function. Univariate analyses revealed that longer LPM was associated with a significantly negative impact on bowel function at all time points. In multivariate analysis, LPM was found to be a significant risk factorstatistically significant, but its impact was not as strong as that of radiotherapy and low-middle tumour. Furthermore, there was no significant difference in the lymph node detection rate between <10-cm and ≥10-cm LPM groups. Conclusion: In APS for rectal cancer, bowel volume loss is an important factor causing postoperative bowel dysfunction. Controlling LPM to <10 cm may help improve postoperative bowel function.

Disulfidptosis classification of hepatocellular carcinoma reveals correlation with clinical prognosis and immune profile.

A new mode of cell death, disulfidptosis, has been discovered. Clinical prognostic significance of disulfidptosis related pattern in hepatocellular carcinoma(HCC). In this study, a risk score model was established based on disulfidptosis model to analyze the role of risk score in clinical prognosis, immune cell infiltration, drug sensitivity and immunotherapy response. Disulfidptosis subtype were constructed based on the transcriptional profiles of 15 disulfidptosis-related genes(DRGs). All 601 samples were defined as high risk group(HRG) and low risk group(LRG) based on the disulfidptosis risk score. Drug sensitivity and response to immunotherapy were calculated by immunophenotypic score(IPS), tumor prediction, tumor immune dysfunction and rejection(TIDE). RT-qPCR was used to determine the mRNA level of disulfidptosis prognostic gene. Risk groups was identified as potential predictors of immune cell infiltration, drug sensitivity, and immunotherapy responsiveness. HRG may benefit from immunotherapy. Classification is very effective in predicting the prognosis and therapeutic effect of patients, and provides a reference for accurate individualized treatment. This study suggests that new biomarkers related to Disulfidptosis can be used in clinical diagnosis of liver cancer to predict prognosis and treatment targets.

A compounds annotation strategy using targeted molecular networking for offline two-dimensional liquid chromatography-mass spectrometry analysis: Yupingfeng as a case study.

Component overlapping and long-time consumption hinder the data processing of offline two-dimensional liquid chromatography mass spectrometry (offline 2D-LC MS) system. Although molecular networking has been commonly employed in data processing of liquid chromatography mass spectrometry (LC-MS), its application in offline 2D-LC MS is challenged by voluminous and redundant data. In light of this, for the first time, a data deduplication and visualization strategy combining hand-in-hand alignment with targeted molecular networking (TMN) for compounds annotation of offline 2D-LC MS data was developed and applied to the chemical profile of Yupingfeng (YPF), a classical traditional Chinese medicine (TCM) prescription, as a case study. Firstly, an offline 2D-LC MS system was constructed for the separation and data acquisition of YPF extract. Then the data of 12 fractions derived from YPF were deconvoluted and aligned as a whole data file by hand-in-hand alignment, resulting in a 49.2% reduction in component overlapping (from 17951 to 9112 ions) and an improvement in the MS2 spectrum quality of precursor ions. Subsequently, the MS2-similarity adjacency matrix of focused parent ions was computed by a self-building Python script, which realized the construction of an innovative TMN. Interestingly, the TMN was found to be able to efficiently distinguish and visualize the co-elution, in-source fragmentations and multi-type adduct ions in a clustering network. Consequently, a total of 497 compounds were successfully identified depending on only seven TMN analysis guided by product ions filtering (PIF) and neutral loss filtering (NLF) for the targeted compounds in YPF. This integrated strategy improved the efficiency of targeted compound discovery in offline 2D-LC MS data, also shown a huge scalability in accurate compound annotation of complex samples. In conclusion, our study developed available concepts and tools while providing a research paradigm for efficient and rapid compound annotation in complex samples such as TCM prescriptions, with YPF as an example.

Kupffer cell pyroptosis mediated by METTL3 contributes to the progression of alcoholic steatohepatitis.

Chronic alcohol consumption is a major risk factor for alcoholic steatohepatitis (ASH). Previous studies have shown that direct injury of hepatocytes is the key factor in its occurrence and development. However, our study shows that the role of Kupffer cells in ASH cannot be ignored. We isolated Kupffer cells from the livers of ASH mice and found that alcohol consumption induced Kupffer cell pyroptosis and increased the release of interleukin-1ß (IL-1ß). Furthermore, we screened the related m6A enzyme methyltransferase-like 3 (METTL3) from liver Kupffer cells, and found that silencing METTL3 alleviated inflammatory cytokine eruption by Kupffer cell pyroptosis in ASH mice. In vitro, we silenced METTL3 with lentivirus in BMDMs and RAW264.7 cells and confirmed that METTL3 could reduce pyroptosis by influencing the splicing of pri-miR-34A. Together, our results revealed a critical role of KC pyroptosis in ASH and highlighted the mechanism by which METLL3 relieves cell pyroptosis, which could be a promising therapeutic strategy for ASH.

Associations of Obesity With Growth and Puberty in Children: A Cross-Sectional Study in Fuzhou, China.

Objectives: To investigate the associations of obesity with growth and puberty in children. Methods: From November 2017 to December 2019, height, weight, and Tanner stages of 26,879 children aged 3-18 years in Fuzhou, China were assessed. Results: The obese group was significantly taller than the non-obese group after age 4 years for both genders, yet there was no significant difference in height between obese and non-obese group after 15.5 years old for boys and 12.5 years old for girls. The inflection points of significant growth deceleration in obese and non-obese groups were 14.4 and 14.6 years old for boys, and 11.8 and 12.8 years old for girls, respectively. The proportions of testicular development in boys with obesity and non-obesity were 7.96% and 5.08% at 8.5-8.9 years old, respectively, while the proportions of breast development in girls were 17.19% and 3.22% at age 7.5-7.9 years old, respectively. Conclusion: Children with obesity were taller in early childhood, earlier onset of puberty and earlier cessation of growth than children with non-obesity of the same age. However, there was sex dimorphism on the effect of obesity on the incidence of precocious puberty.

Dissecting chemical markers for controlling "Paozhi" method of traditional Chinese medicine with untargeted metabolomics: Vinegar-baked Euphorbia kansui as a case study.

The processing of Traditional Chinese Medicine requires the appropriate parameters, while the specific chemical markers are still absent to obtain the optimized processing. In this study, we used vinegar-baked Euphorbia kansui as a case to dissect the chemical markers for the baking process using untargeted metabolomics. The robust chemical markers were selected based on the three rules, correlation, significant difference, and controllability. All the differential features were categorized based on their mass defects. After the differential analysis, 310 differential compounds were screened out and could be mainly divided into six categories: diacylglycerols and triacylglycerols demonstrated increasing trends with the baking time in the discriminant model, while ingenane-type diterpenes, jatrophane-type diterpenes, fatty acid esters, and fatty acids had decreasing trends. It was unexpected to find that the diterpenes did not correlate with the baking time. Only very few compounds meet the three rules. They were validated with a high-performance liquid chromatography method. Finally, only 13-Hydroxy-9,11-octadecadienoic acid and its isomer 9-Hydroxy-10,12-octadecadienoic acid could be used further to differentiate the commercial vinegar-baked Euphorbia kansui. It would be of interest to evaluate whether these two compounds could be utilized as markers to control more processing methods in future studies.

Determination of the intermediates in glycolysis and tricarboxylic acid cycle with an improved derivatization strategy using gas chromatography-mass spectrometry in complex samples.

Traditional Chinese medicine (TCM) is recognized as a complex matrix, and improved analytical methods are crucial to extract the key indicators and depict the interaction and alteration of the complex matrix. Shenqi Fuzheng Injection (SQ), a water extract of Radix Codonopsis and Radix Astragali, has demonstrated preventative effects on myotube atrophy induced by chemotherapeutic agents. To achieve the improved analytical capability of complex biological samples, we established a highly reproducible, sensitive, specific, and robust gas chromatography-tandem mass spectrometry (GC-MS) method to detect glycolysis and tricarboxylic acid (TCA) cycle intermediates with optimized factors in the extraction and derivatization process. Our method detected fifteen metabolites and covered most intermediate metabolites in glycolysis and TCA cycles, including glucose, glucose-6-phosphate, fructose-6-phosphate, dihydroxyacetone phosphate, 3-diphosphoglycerate, phosphoenolpyruvate, pyruvate, lactate, citrate, cis-aconitate, isocitrate, α-ketoglutarate, succinate, fumarate, and malate. Through methodological verification of the method, it was found that the linear correlation coefficients of each compound in the method were greater than 0.98, all of which had lower limits of quantification, the recovery rate was 84.94-104.45%, and the accuracy was 77.72-104.92%. The intraday precision was 3.72-15.37%, the interday precision was 5.00-18.02%, and the stability was 7.85-15.51%. Therefore, the method has good linearity, accuracy, precision, and stability. The method was further applied to study the attenuating effects of the SQ in a chemotherapeutic agents-induced C2C12 myotube atrophy model to evaluate the changes in the tricarboxylic acid cycle and glycolytic products under the action by the complex systems of TCM and disease model. Our study provided an improved method to explore TCM's pharmacodynamic constituents and action mechanisms.

Multi-omics profiling of PC-3 cells reveals bufadienolides-induced lipid metabolic remodeling by regulating long-chain lipids synthesis and hydrolysis.

INTRODUCTION: Lipid metabolism participates in various biological processes such as proliferation, apoptosis, migration, invasion, and maintenance of membrane homeostasis of prostate tumor cells. Bufadienolides, the active ingredients of Chansu, show a robust anti-proliferative effect against prostate cancer cells in vitro, but whether bufadienolides could regulate the lipid metabolism in prostate cancer has not been evaluated. OBJECTIVES: Our study explored the regulatory effects of bufadienolides on lipid metabolism in human prostate carcinoma cells (PC-3). METHODS: Untargeted lipidomics and transcriptomics were combined to study the effect of different bufadienolides interventions on lipid and gene changes of PC-3 cells. The key genes related to lipid metabolism and prostate cancer development were verified by qPCR and western blotting. RESULTS: Lipidomic analysis showed that the active bufadienolides significantly downregulated the content of long-chain lipids of PC-3 cells. Based on transcriptomic and qPCR analyses, many genes related to lipid metabolism were significantly regulated by active bufadienolides, such as ELOVL6, CYP2E1, GAL3ST1, CERS1, PLA2G10, PLD1, SPTLC3, and GPX2. Bioinformatics analysis of the Cancer Genome Atlas database and literature retrieval showed that elongation of very long-chain fatty acids protein 6 (ELOVL6) and phospholipase D1 (PLD1) might be important regulatory genes. Western blot analysis revealed that active bufadienolides could downregulate PLD1 protein levels which might promote anti-prostate cancer effect. CONCLUSIONS: All these findings support that bufadienolides might induce lipid metabolic remodeling by regulating long-chain lipids synthesis and phospholipid hydrolysis to achieve an anti-prostate cancer effect, and PLD1 would probably be the key protein.

Notch3 signaling promotes colorectal tumor growth by enhancing immunosuppressive cells infiltration in the microenvironment.

BACKGROUND: Macrophage infiltration in the tumor microenvironment participates in the regulation of tumor progression. Previous studies have found that Notch signaling pathway is involved in regulating the progression of colorectal cancer (CRC), however, the specific mechanism is still unclear. METHODS: The correlation between Notch signaling pathway and macrophage infiltration was investigated in TCGA database and verified in clinical samples of patients with CRC using immunohistochemistry. Gene Set Enrichment Analysis was used to find out genes related to Notch3 expression. Colony formation assay, and flow cytometry were utilized to test tumor growth and immune cell infiltration in vitro and in vivo. RESULTS: Using bioinformatics analysis and clinical sample validation, we found that Notch3 was highly expressed in colon tumor tissues compared to adjacent normal tissues, and it participated in regulating the recruitment of macrophages to the tumor microenvironment. Furthermore, we found that the Notch3 expression was positively correlated with the expression of macrophage recruitment-related cytokines in colon tumor tissues. Finally, we demonstrated that depletion of Notch3 had no significant effect on the growth of colon tumor cells in vitro, while, attenuated the growth of colon cancer tumors in vivo. Simultaneous, immunosuppressive cells, macrophages and myeloid-derived suppressor cell (MDSC) infiltration were dramatically reduced in the tumor microenvironment. CONCLUSION: Our study illustrated that Notch3 could facilitate the progression of CRC by increasing the infiltration of macrophages and MDSCs to promote the immunosuppressive tumor microenvironment. Targeting Notch3 specifically is a potentially effective treatment for CRC.