Mechanistic Study of CRABP2: Accelerating Lung Cancer Migration and Metastasis through Regulation of the ROS/Src Signaling Pathway.

Objective: This study aims to establish a theoretical foundation for the clinical treatment of lung cancer by investigating the regulatory role of CRABP2 in the ROS/Src signaling pathway, specifically in accelerating the migration and metastasis of lung cancer. Methods: Lung cancer mouse models were established using BALB/c-nu mice, randomly assigned to the control group (NC group) and the experimental group (mimic group). Tumor volume was precisely observed. The impact of CRABP2 on lung cancer migration and metastasis was analyzed through hematoxylin and eosin (H&E) staining and histochemical staining observation. Protein expression analysis was employed to assess CRABP2, ESR1, NOX1, NOX4, p-Src, and p-FAK levels, shedding light on the underlying mechanism. CRABP2's influence on lung cancer migration and metastasis was further investigated using scratch and Transwell experiments. Results: The findings revealed that the mimic group, with enhanced CRABP2 expression, exhibited a higher proliferation rate and increased migration and metastasis capabilities in lung cancer. Protein expression analysis demonstrated that CRABP2 and ESR1 positively influenced the ROS/Src pathway, promoting lung cancer migration and metastasis. Scratch and Transwell's experiments supported the fact that CRABP2 significantly accelerated lung cancer migration and metastasis. Conclusions: CRABP2 plays a crucial role in expediting lung cancer migration and metastasis by upregulating ESR1 expression, consequently activating the ROS/Src pathway. This study introduces a novel therapeutic avenue for the clinical treatment of lung cancer, offering a theoretical framework for advancing lung cancer treatment strategies.

Drought Eliminates the Difference in Root Trait Plasticity and Mycorrhizal Responsiveness of Two Semiarid Grassland Species with Contrasting Root System.

Root traits and arbuscular mycorrhizal (AM) fungi are important in determining the access of plants to soil resources. However, whether plants with different root systems (i.e., taproot vs. fibrous-root) exhibit different root trait plasticity and mycorrhizal responsiveness under drought remains largely unexplored. Tap-rooted Lespedeza davurica and fibrous-rooted Stipa bungeana were grown in monocultures in sterilized and live soils, followed by a drought treatment. Biomass, root traits, root colonization by AM fungi, and nutrient availability were evaluated. Drought decreased biomass and root diameter but increased the root:shoot ratio (RSR), specific root length (SRL), soil NO3--N, and available P for the two species. Under control and drought conditions, soil sterilization significantly increased the RSR, SRL, and soil NO3--N for L. davurica, but this only occurs under drought condition for S. bungeana. Soil sterilization significantly reduced AM fungal root colonization of both species, but drought significantly increased it in live soil. In water-abundant conditions, tap-rooted L. davurica may depend more on AM fungi than fibrous-rooted S. bungeana; however, under drought conditions, AM fungi are of equal importance in favoring both plant species to forage soil resources. These findings provide new insights for understanding the resource utilization strategies under climate change.

Integration of Deep Learning and Sequential Metabolism to Rapidly Screen Dipeptidyl Peptidase (DPP)-IV Inhibitors from Gardenia jasminoides Ellis.

Traditional Chinese medicine (TCM) possesses unique advantages in the management of blood glucose and lipids. However, there is still a significant gap in the exploration of its pharmacologically active components. Integrated strategies encompassing deep-learning prediction models and active validation based on absorbable ingredients can greatly improve the identification rate and screening efficiency in TCM. In this study, the affinity prediction of 11,549 compounds from the traditional Chinese medicine system's pharmacology database (TCMSP) with dipeptidyl peptidase-IV (DPP-IV) based on a deep-learning model was firstly conducted. With the results, Gardenia jasminoides Ellis (GJE), a food medicine with homologous properties, was selected as a model drug. The absorbed components of GJE were subsequently identified through in vivo intestinal perfusion and oral administration. As a result, a total of 38 prototypical absorbed components of GJE were identified. These components were analyzed to determine their absorption patterns after intestinal, hepatic, and systemic metabolism. Virtual docking and DPP-IV enzyme activity experiments were further conducted to validate the inhibitory effects and potential binding sites of the common constituents of deep learning and sequential metabolism. The results showed a significant DPP-IV inhibitory activity (IC50 53 ± 0.63 µg/mL) of the iridoid glycosides' potent fractions, which is a novel finding. Genipin 1-gentiobioside was screened as a promising new DPP-IV inhibitor in GJE. These findings highlight the potential of this innovative approach for the rapid screening of active ingredients in TCM and provide insights into the molecular mechanisms underlying the anti-diabetic activity of GJE.

VEGF-B targeting by aryl hydrocarbon receptor mediates the migration and invasion of choriocarcinoma stem-like cells.

Unlike other members of the VEGF family, the function of VEGF-B in tumor progression remains to be elucidated. Thus, the present study aimed to determine the function of VEGF-B in human choriocarcinoma cells by investigating its detailed effects and molecular mechanisms. VEGF-B and aryl hydrocarbon receptor (AhR) expression were evaluated by reverse transcription-quantitative PCR analysis and western blot analysis in JEG-3 cells and choriocarcinoma stem-like cells (CSLCs) and their proliferation, migration, and invasion after the transfection of short hairpin RNA VEGF-B, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; AhR agonist) treatment or StemRegenin 1 (SR1; AhR antagonist) treatment were examined by cell proliferation assay, wound healing assay and Transwell assay. In addition, luciferase reporter analysis and bioinformatics data mining were used to investigate the association between VEGF-B and AhR. Upregulation of VEGF-B and AhR expression was observed in CSLCs. Following VEGF-B knockdown or SR1 treatment, the proliferative, migratory, and invasive abilities of CSLCs were significantly decreased, contrary to the findings after TCDD treatment. It was also found that AhR enhanced VEGF-B transcriptional activity by binding to the relative promoter region. These observations indicated that VEGF-B may be an oncogene that promotes choriocarcinoma cell migration and invasion targeted by AhR. Therefore, targeting VEGF-B may provide a novel therapeutic opportunity for choriocarcinoma.

Physiologically based pharmacokinetic combined BTK occupancy modeling for optimal dosing regimen prediction of acalabrutinib in patients alone, with different CYP3A4 variants, co-administered with CYP3A4 modulators and with hepatic impairment.

PURPOSE: To develop a mathematical model combined between physiologically based pharmacokinetic and BTK occupancy (PBPK-BO) to simultaneously predict pharmacokinetic (PK) and pharmacodynamic (PD) changes of acalabrutinib (ACA) and active metabolite ACP-5862 in healthy humans as well as PD in patients. Next, to use the PBPK-BO to determine the optimal dosing regimens in patients alone, with different CYP3A4 variants, when co-administration with four CYP3A4 modulators and in patients with hepatic impairment, respectively. METHODS: The PBPK-BO model was built using physicochemical and biochemical properties of ACA and ACP-5862 and then verified by observed PK and PD data from healthy humans and patients. Finally, the model was applied to determine optimal dosing regimens in various clinical situations. RESULTS: The simulations demonstrated that 100 mg ACA twice daily (BID) was the optimal dosing regimen in patients alone. Additionally, dosage regimens might be reduced to 50 mg BID in patients with five CYP3A4 variants. Moreover, the dosing regimen should be modified to 100 mg (even to 50 mg) once daily (QD) when co-administration with erythromycin or clarithromycin, and be increased to 200 mg BID with rifampicin, and but be avoided co-administration with itraconazole. Furthermore, dosage regimen simulations showed that optimal dosing might be decreased to 50 mg BID in patients with mild and moderate hepatic impairment, and be avoided taking ACA in severely hepatically impaired patients. CONCLUSION: This PBPK-BO model can predict PK and PD in healthy humans and patients and also predict the optimal dosing regimens in various clinical situations.

Physiologically based pharmacokinetic combined JAK2 occupancy modelling to simulate PK and PD of baricitinib with kidney transporter inhibitors and in patients with hepatic/renal impairment.

PURPOSE: Our aim is to build a physiologically based pharmacokinetic and JAK2 occupancy model (PBPK-JO) to simultaneously predict pharmacokinetic (PK) and pharmacodynamic (PD) changes of baricitinib (BAR) in healthy humans when co-administrated with kidney transporters OAT3 and MATE2-K inhibitors, and in patients with hepatic and renal impairment. METHODS: Probenecid and vandetanib were selected as OAT3 and MATE2-K competitive inhibitors, respectively. The PBPK-JO model was built using physicochemical and biochemical properties of BAR, and then verified by observed clinical PK. Finally, the model was applied to determine optimal dosing regimens in various clinical situations. RESULTS: Here, we have successfully simulated PK and JAK2 occupancy profiles in humans by PBPK-JO model. Moreover, this modelling reproduced every observed PK data, and every mean relative deviation (MRD) was below 2. The simulation suggested that PK of BAR had a significant change (2.22-fold increase), however PD only had a slight increase of 1.14-fold. Additionally, the simulation also suggested that vandetanib was almost unlikely to affect the PK and PD of BAR. In simulations of hepatic and renal impairment patients, the predictions suggested that significant changes in the PK and PD of BAR occurred. However, there was a lower fold increase in JAK2 occupancy than in PK in patients relative to healthy individuals. CONCLUSION: Administration dose adjustment of BAR when co-administrated with OAT3 inhibitors or in patients with hepatic or renal impairment should combine PK and PD changes of BAR, instead of only considering PK alteration.

Prognostic value of microRNAs in patients with small cell lung cancer: a meta-analysis.

BACKGROUND: An increasing number of studies have shown that microRNAs play an important role in the occurrence and development of small cell lung cancer, which mainly manifest as oncogenic and tumor inhibition. Therefore, microRNAs may affect the survival of patients with small cell lung cancer. In this meta-analysis, we will evaluate the role of microRNAs in the overall survival of patients with small cell lung cancer, which may provide valuable information for the treatment of small cell lung cancer. METHODS: We searched the PubMed, Embase, and Web of Science online databases to determine the effect of microRNAs on the prognosis of patients with small cell lung cancer. The data and characteristics of each study were extracted, and the hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated to estimate the effect. RESULTS: A total of 7 articles, involving 427 subjects and 15 studies, were included in this meta-analysis. The pooled HR of the relationship between the microRNA expression level and the overall survival rate of small cell lung cancer patients was 1.25 (95% CI: 1.06-1.47). There was a significant difference in the prognostic value of oncogenic and tumor inhibition microRNAs among patients with small cell lung cancer, with pooled HRs of 1.60 (95% CI: 1.35-1.90) and 0.42 (95% CI: 0.30-0.57), respectively. CONCLUSIONS: MicroRNAs have a significant impact on the overall survival of small cell lung cancer patients, suggesting that microRNAs can be used as potential prognostic markers and may provide treatment strategies for small cell lung cancer patients. TRIAL REGISTRATION: The protocol was registered on PROSPERO website with the registration number of CRD42022334363. The relevant registration information can be obtained from the website https://www.crd.york.ac.uk/prospero/#searchadvanced .

Evaluating the Properties of Ginger Protease-Degraded Collagen Hydrolysate and Identifying the Cleavage Site of Ginger Protease by Using an Integrated Strategy and LC-MS Technology.

(1) Methods: An integrated strategy, including in vitro study (degree of hydrolysis (DH) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity) and in vivo study (absorption after oral administration in rats), was developed to evaluate the properties of the fish skin gelatin hydrolysates prepared using different proteases (pepsin, alkaline protease, bromelain, and ginger protease). Meanwhile, in order to identify the hydrolysis site of ginger protease, the peptides in the ginger protease-degraded collagen hydrolysate (GDCH) were comprehensively characterized by liquid chromatography/tandem mass spectrometry (LC-MS) method. (2) Results: The GDCH exhibited the highest DH (20.37%) and DPPH radical scavenging activity (77.73%), and in vivo experiments showed that the GDCH was more efficiently absorbed by the gastrointestinal tract. Further oral administration experiments revealed that GDCH was not entirely degraded to free amino acids and can be partially absorbed as dipeptides and tripeptides in intact forms, including Pro-Hyp, Gly-Pro-Hyp, and X-Hyp-Gly tripeptides. LC-MS results determined the unique substrate specificity of ginger protease recognizing Pro and Hyp at the P2 position based on the amino acids at the P2 position from the three types of tripeptides (Gly-Pro-Y, X-Hyp-Gly, and Z-Pro-Gly) and 136 identified peptides (>4 amino acids). Interestingly, it suggested that ginger protease can also recognize Ala in the P2 position. (3) Conclusions: This study comprehensively evaluated the properties of GDCH by combining in vitro and in vivo strategies, and is the first to identify the cleavage site of ginger protease by LC-MS technique. It provides support for the follow-up study on the commercial applications of ginger protease and bioactivities of the hydrolysate produced by ginger protease.

Design of an efficient photocatalyst: a type II heterojunction for enhanced hydrogen production driven by visible light.

Solar hydrogen production, which is an eco-friendly method to obtain energy, is still far away from wide commercialization due to the lack of an efficient catalyst. Effective calculations can reduce trial and error costs and provide mechanistic explanations while exploring efficient catalysts. Herein, a type II heterojunction Mg-containing-porphyrin/g-C3N4 is proven to be an efficient photocatalyst by using a combination of DFT and many-body Green's function theory. Our results show that the heterojunction can significantly enhance the absorption of visible light and realize the separation of photogenerated electrons and holes after excitation. Subsequently, water absorbing on the excited surface decomposes into H+ and OH- easily, and then produces H2 and O2 with reduced free energy. Our investigation and explanation can provide theoretical support for designing photonic devices based on porphyrin and g-C3N4, and deepen the understanding of how H2O splits into H2.

Integrated Bioinformatic Analysis of Competing Endogenous RNA Network of Choriocarcinoma.

BACKGROUND Numerous studies have demonstrated that noncoding RNAs are involved in choriocarcinoma (CC). The competing endogenous RNA (ceRNA) network plays an important role in the occurrence and development of carcinoma. However, the involvement of the ceRNA network in CC remains unclear. The current study aimed to investigate the regulatory mechanism of ceRNA in CC. MATERIAL AND METHODS We downloaded the messenger RNAs (mRNAs) expression profiles (GSE20510 and GSE65654) and microRNAs (miRNAs) expression profiles (GSE32346 and GSE130489) from GEO datasets. The limma package of R software was used to identify differentially expressed RNAs (DERNAs). Then, we performed functional annotation of the differentially expressed mRNAs (DEmRNAs). TargetScan, miRDB, miRWalk, and Starbase were used to construct a CC-specific ceRNA network and select key molecules. RESULTS The results identified a total of 177 DEmRNAs and 189 differentially expressed miRNAs (DEmiRNAs) between the trophoblast and CC cell line samples. Ten differentially expressed lncRNAs (DElncRNAs) were obtained based on experimental studies. The DEmRNAs were mainly enriched in cell proliferation, positive regulation of the apoptotic process, and cell death. A total of 10 genes were ascertained as hub genes. Based on DEmRNAs, DEmiRNAs, and DElncRNAs, a CC-specific ceRNA network was established. Five DElncRNAs, 15 DEmiRNAs, and 45 DEmRNAs were identified. In addition, LINC00261, MEG3, MALAT1, H19, and OGFRP1 were identified as 5 key lncRNAs in choriocarcinoma. CONCLUSIONS This study provides novel insights into CC mechanisms and identified potential therapeutic targets for CC.

Long noncoding RNA H19 promotes chemotherapy resistance in choriocarcinoma cells.

Choriocarcinoma (CC) is a trophoblast tumor prone to early distant organ metastases. At present, the main treatment for CC is chemotherapy, but chemotherapy resistance readily occurs and leads to treatment failure. H19 is a long noncoding RNA, and its abnormal expression has been found in various tumors, including CC. H19 is also considered to be related to the drug resistance mechanism of the same cancers. To investigate the role of H19 in drug-resistant CC cells, the following experiments were designed. We used human CC cell line JEG-3 to establish cell lines resistant to methotrexate and 5-fluorouracil (JEG-3/MTX and JEG-3/5-FU) and detected the expression of H19 in JEG-3, JEG-3/MTX, JEG-3/5-FU cells, JEG-3 with MTX, and JEG-3 with 5-FU. We found that the expression of H19 in the JEG-3/MTX and JEG-3/5-FU cells were significantly higher than that in JEG-3 cells. JEG-3 cells were treated with MTX or 5-FU for and quantitative real-time polymerase chain reaction assay revealed that H19 messenger RNA expression increased. Furthermore, after H19 was knocked out, the drug resistance index of the JEG-3/MTX and JEG-3/5-FU cells decreased; the proliferation, migration, and invasion ability diminished significantly; and apoptosis increased significantly. Finally, we detected the total and phosphorylation protein expression of phosphatidylinositol-3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) in the JEG-3/MTX and JEG-3/5-FU cells. The total protein of PI3K, AKT, and mTOR in the H19 knockout resistant cells showed no significant change relative to those in the H19 non-knockout resistant cells, whereas the phosphorylated proteins of PI3K, AKT, and mTOR were significantly decreased. Phosphorylated proteins of PI3K, AKT, and mTOR in the JEG-3/MTX and JEG-3/5-FU cells were significantly higher than that in JEG-3 cells. After using inhibition of phosphorylated PI3K/AKT/mTOR, the proliferation, migration, and invasion ability of the JEG-3/MTX and JEG-3/5-FU cells diminished significantly; and apoptosis increased significantly. On the basis of the above experiments, we concluded that H19 is related to the drug resistance of CC, and the knockout of H19 can reduce the drug resistance of resistant CC cells; and decrease the proliferative, migratory, and invasive ability; and increase the apoptosis. PI3K/AKT/mTOR pathway might be involved in H19-mediated effects. H19 is expected to be a therapeutic target for the treatment of drug-resistant chorionic carcinoma.

Curcumol Controls Choriocarcinoma Stem-Like Cells Self-Renewal via Repression of DNA Methyltransferase (DNMT)- and Histone Deacetylase (HDAC)-Mediated Epigenetic Regulation.

BACKGROUND Cancer stem cells (CSCs), in choriocarcinoma and other carcinomas, possess the ability of self-renewal and multilineage differentiation potential. We previous isolated choriocarcinoma cancer stem-like cells (CSLCs), which hold the stemness characteristics of CSCs. Epigenetic modifications have emerged as drivers in tumorigenesis, but the mechanisms of CSCs are largely unknown, and new drug therapies are needed to break the persistence of CSCs. MATERIAL AND METHODS Quantitative real-time PCR (qRT-PCR) and Western blot analysis were performed to detect the expression of DNMTs, HDACs, and stemness-genes. DNMTs and HDACs silencing and overexpressing lentivirus were transfected into JEG-3 cells to investigate the epigenetic functions in CSLCs. In vivo expression of curcumol effects of CSLCs on DNMTs and HDACs were analyzed by immunohistochemistry. RESULTS Expression of DNMT1, DNMT3b, HDAC1, and HDAC3 were increased in choriocarcinoma CSLCs. Consistent with the inhibitory effect of 5-AzaC and TSA on CSLCs, DNMT/HDAC knockdown displayed significant repression of self-renewal in CSLCs. Curcumol inhibited the stemness ability of CSLCs in vitro and in vivo, and the inhibitory effect we observed was mediated in part through repressing activity of DNMTs and HDACs. Importantly, curcumol showed a better effect than DNMT and HDAC inhibitors combined in eliminating CSLCs. CONCLUSIONS These findings indicate that DNMT- and HDAC-mediated epigenetic regulation plays an important role in the biology of choriocarcinoma CSLCs, and curcumol has the potential to be a new drug to fight CSLCs, warranting further investigation of epigenetic-based therapies.

Isolation, Culture and Identification of Choriocarcinoma Stem-Like Cells from the Human Choriocarcinoma Cell-Line JEG-3.

BACKGROUND/AIMS: Cancer stem cells (CSCs) exhibit enhanced proliferative capacity and resistance to chemotherapy; however, choriocarcinoma CSCs have not yet been reported. In this study the human choriocarcinoma cell line JEG-3 was cultured in serum free media, and the characteristics of suspension and parental adherent JEG-3 cells were compared. METHODS: Cell proliferation, colony-formation, soft agar clonogenicity, and transwell invasion assays were performed in vitro, and tumor xenografts in BALB/c nude mice were used to evaluate stem cell properties. RESULTS: In serum-supplemented medium (SSM), JEG-3 cells were 4.51 ± 1.71% CD44+, 7.67 ± 2.67% CD133+, and 13.85 ± 2.95% ABCG2+. In serum-free medium (SFM), the expression of these markers increased to 53.08 ± 3.15%, 47.40 ± 2.67%, and 78.70 ± 7.16%, respectively. Moreover, suspension JEG-3 cells exhibited enhanced colony-formation capability as well as invasive and proliferative ability in vitro, alongside enhanced tumorigenic properties in vivo. Suspension JEG-3 cells also exhibited resistance to the chemotherapeutic drugs methotrexate, fluorouracil and etoposide. When seeded in serum supplemented medium, suspension JEG-3 cells readopted an adherent phenotype and continued to differentiate with no significant difference in the morphology between suspension and parent cells. CONCLUSION: In this study, choriocarcinoma stem-like cells (CSLCs) were isolated from the human choriocarcinoma JEG-3 cell line by SFM culture and characterized.

Mitigating Effect of Matricin Against Benzo(a)pyrene-induced Lung Carcinogenesis in Experimental Mice Model.

BACKGROUND: Lung cancer is a life-threatening disease that is still prevalent worldwide. This study aims to evaluate the effects of matricin, a sesquiterpene, on the carcinogenic agent benzo(a)pyrene [B(a)P]-induced lung cancer in Swiss albino mice. METHODS: Lung cancer was induced by oral administration of B(a)P at 50 mg/kg b. wt. in model Swiss-albino mice (group II) as well in experimental group III, and treated with matricin (100 mg/kg b. wt.) in group III. Upon completion of treatment for 18 weeks, the changes in body weight, tumor formation, enzymatic and non-enzymatic antioxidant levels (GSH, SOD, GPx, GR, QR, CAT), lipid peroxidation (LPO) level, pro-inflammatory cytokines (TNF-α, IL-6, IL-1ß), immunoglobulin levels (IgG, IgM), apoptosis markers (Bax, Bcl-xL), tumor markers (carcinoembryogenic antigen (CEA), neuron-specific enolase (NSE)), and histopathological (H&E) alterations were determined. RESULTS: The results indicate that B(a)P caused a significant increase of tumor formation in the lungs, increased tumor markers and inflammatory cytokines in serum, and depletion of enzymatic/ non-enzymatic antioxidants and immunoglobulins, compared to the untreated control group. Matricin treatment significantly reversed the changes caused by B(a)P as evidenced by the biochemical and histopathological assays. CONCLUSION: The changes caused by matricin clearly indicate the cancer-preventive effects of matricin against B(a)P-induced lung cancer in animal models, which can be attributed to the antioxidant activity, immunomodulation, and mitigation of the NF-kß pathway.

Prognostic role of CRABP2 in lung cancer: a meta-analysis.

BACKGROUND: The prognostic value of cellular retinoic acid-binding protein 2 (CRABP2), in lung cancer patients remains to be uncertained. Therefore, our research attempted to assess the relationship between CRABP2 and survival analysis in lung cancer patients through meta-analysis. METHOD: Related literature retrieved from Cochrane Library, Ovid, Embase, PubMed, the CNKI, and the Web of Science. The latest update of the search was May 1, 2023. The outcome indicators included as effective measures in the study were hazard ratio (HR), and 95% confidence interval (CI). The Stata 12.0 software was used to analyze the data. RESULTS: A total of4 studies were finally enrolled in our meta-analysis. The increased plasma level of CRABP2 predicted poor OS in lung cancer patient with a combined HR of 1.14 (95% CI: 1.00-1.30), and were not associated with poor PFS with combined HR: 1.15% CI: 0.63-2.09) in lung cancer patients. CONCLUSIONS: Our meta-analysis found the increased plasma level of CRABP2 was associated with poor OS independently in NSCLC patients. The plasma CRABP2 level may be an indicator of biological aggressiveness of the tumor. Our research was promising regarding the feasibility and utility of plasma CRABP2 as a novel prognostic biomarker in NSCLC, and the findings warrant further investigation.

Canopy FGF signaling regulator 3 affects prognosis, immune infiltration, and PI3K/AKT pathway in colon adenocarcinoma.

BACKGROUND: Colon adenocarcinoma (COAD) is a malignant tumor of the digestive system. The mechanisms underlying COAD development and progression are still largely unknown. AIM: To identify the role of canopy FGF signaling regulator 3 (CNPY3) in the development and progression of COAD by using bioinformatic tools and functional experiments. METHODS: Bioinformatic data were downloaded from public databases. The associations of clinicopathological features, survival, and immune function with the expression of CNPY3 were analyzed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses and Gene Set Enrichment Analysis were used to explore the related pathways. Then, quantitative real-time PCR and immunohistochemistry were used for validation of CNPY3 expression in clinical samples and tumor cell lines. Cell lines with CNPY3 knockdown were constructed to further analyze gene functions. The functional experiments included proliferation, invasion, migration and apoptosis assays. RESULTS: In both the TCGA cohort and the merged dataset, elevated CNPY3 expression was observed in tumor tissues. High CNPY3 expression correlated with adverse survival and compromised immune functions. Functional enrichment analysis suggested that the pro-oncogenic properties of CNPY3 might be linked to the PI3K-AKT signaling pathway. CNPY3 expression was validated at both the RNA and protein levels. Functional assays indicated that cell proliferation, invasion, and migration were inhibited and cell apoptosis was promoted after CNPY3 knockdown. Additionally, Western blot results revealed the downregulation of key proteins in the PI3K/AKT pathway following CNPY3 knockdown. PI3K/AKT pathway activator reversed the decrease in proliferation, invasion, and migration and the increase in apoptosis. Notably, CNPY3 knockdown still affected the cells when the pathway was inhibited. CONCLUSION: This study showed that CNPY3 is upregulated in COAD and might regulate COAD development and progression by the PI3K/AKT pathway. Thus, CNPY3 might be a promising therapeutic target.

Synthetic enhancers including TFREs improve transgene expression in CHO cells.

The human cytomegalovirus major immediate early gene (CMV) promoter is currently the most preferred promoter for recombinant therapeutic proteins (RTPs) production in CHO cells. To enhance the production of RTPs, five synthetic enhancers including multiple transcription factor regulatory elements (TFREs) were evaluated to enhance recombinant protein level in transient and stably transfected CHO cells. Compared with the control, four elements can enhance the report genes expression under both two transfected states. Further, the function of these four enhancers on human serum albumin (HSA) were investigated. We found that the transient expression can increase by up to 1.5 times, and the stably expression can maximum increase by up to 2.14 times. The enhancement of transgene expression was caused by the boost of their corresponding mRNA levels. Transcriptomics analysis was performed and found that transcriptional activation and cell cycle regulation genes were involved. In conclusion, optimization of enhancers in the CMV promoter could increase the production yield of transgene in transfected CHO cells, which has significance for developing high-yield CHO cell expression system.

Multi-omics reveals bufadienolide Q-markers of Bufonis Venenum based on antitumor activity and cardiovascular toxicity in zebrafish.

BACKGROUND: Bufonis Venenum (BV) is a traditional animal-based Chinese medicine with therapeutic effects against cancer. However, its clinical use is significantly restricted due to associated cardiovascular risks. BV's value in China's market is typically assessed based on "content priority," focusing on indicator components. However, these components of BV possess both antitumor activity and toxicity, and the correlation between the antitumor activity and toxicity of BV has not yet been elucidated. PURPOSE: This study employs an integrated multi-omics approach to identify bufadienolide Q-markers and explore the correlation between BV's antitumor activity and toxicity. The aim is to establish a more comprehensive method for BV's quality. METHODS: Normal zebrafish and HepG2 xenograft zebrafish were chosen as activity and toxicity evaluation models. Ultra-high performance liquid chromatography (UHPLC) coupled with a linear ion trap orbitrap (LTQ-Orbitrap) mass spectrometry was used to quantify eight batches of BV and key "toxic and effective" components were screened out. Transcriptomic and metabolomic analyses were performed to elucidate the regulatory mechanisms underlying the antitumor activity and cardiovascular toxicity of the key components in BV. RESULTS: Eight key "toxic and effective" compounds were identified: resibufogenin, cinobufagin, arenobufagin, bufotalin, bufalin, gamabufotalin, desacetylcinobufagin, and telocinobufagin. The findings showed that bufalin and cinobufagin interfered with calcium homeostasis through CaV and CaSR, induced cardiotoxicity, and upregulated CASP9 to activate myocardial cell apoptosis. However, desacetylcinobufagin exhibited greater potential in terms of anti-tumor effects. Combining the results of untargeted and targeted metabolomics revealed that desacetylcinobufagin could have a callback effect on differential lipids and correct abnormal energy and amino acid metabolism caused by cancer, similar to cinobufagin and bufalin. Microscale thermophoresis (MST) ligand binding measurements also showed that the binding of desacetylcinobufagin to GPX4 has a more potent ability to induce ferroptosis in tumor cells compared to cinobufagin. CONCLUSION: An innovative evaluation method based on the zebrafish was developed to investigate the relationship between the toxicity and efficacy of BV. This study identified toxicity and activity Q-markers and explored the mechanism between the two effects of BV. The research data could offer valuable insights into the efficacy of BV. Additionally, desacetylcinobufagin, an active ingredient with low toxicity, was found to enhance the quality of BV.

Ovarian seromucinous carcinoma: an independent epithelial ovarian cancer?

BACKGROUND: 2020 World Health Organization Classification of Female Genital Tumors removed ovarian seromucinous carcinoma as a distinct entity and recategorized it as ovarian endometrioid carcinoma with mucinous differentiation according to its pathological features. The aim of this study was to find whether ovarian seromucinous carcinoma truly represented a distinct category of ovarian tumors or an analogue of ovarian endometrioid carcinoma. METHODS: Twelve patients diagnosed with ovarian seromucinous carcinoma and received surgery at the Xiangya Hospital from January 2010 to December 2019 were included in this study. Clinicopathological features such as clinical symptoms, serological indicators, surgical information, postoperative findings, chemotherapy sensitivity, follow-up information, HE staining and IHC staining images and other clinicopathologic features were collected. Using t-test and Kaplan Meier to perform statistical analysis. Pathological review was conducted using the 2014 World Health Organization criteria. All pathological diagnoses were reviewed by two experienced pathologists. RESULTS: The age of 12 patients diagnosed with ovarian seromucinous carcinoma ranged from 23 to 68 years, with a median age of 46.8 years. Serum level of CA125 was elevated in 10 patients, and CA125/CEA ratio was less than 25 in 6 patients. Eleven patients underwent radical ovarian cancer surgery, and one patient underwent fertility preservation surgery. The progression free survival and overall survival of ovarian seromucinous carcinoma is 46.8 months and 50.2 months. Kaplan-Meier survival curve showed that the prognosis of ovarian seromucinous carcinoma and ovarian endometrioid carcinoma was significantly different (P = 0.03). The prognosis of ovarian seromucinous carcinoma and ovarian mucinous carcinoma was similar. CONCLUSION: Although ovarian seromucinous carcinoma and ovarian endometrioid carcinoma are similar in pathologic morphology, their clinical features and prognosis are significantly different. The signs, serum biomarker and prognosis of the ovarian seromucinous carcinoma are similar with ovarian mucinous carcinoma. Therefore, ovarian seromucinous carcinoma is not suitable to be directly classified as ovarian endometrioid carcinoma.

Effects of Different Livestock Grazing on Foliar Fungal Diseases in an Alpine Grassland on the Qinghai-Tibet Plateau.

In grassland ecosystems, the occurrence and transmission of foliar fungal diseases are largely dependent on grazing by large herbivores. However, whether herbivores that have different body sizes differentially impact foliar fungal diseases remains largely unexplored. Thus, we conducted an 8-year grazing experiment in an alpine grassland on the Qinghai-Tibet Plateau in China and tested how different types of livestock (sheep (Ovis aries), yak (Bos grunniens), or both)) affected foliar fungal diseases at the levels of both plant population and community. At the population level, grazing by a single species (yak or sheep) or mixed species (sheep and yak) significantly decreased the severity of eight leaf spot diseases. Similarly, at the community level, both single species (yak or sheep) and mixed grazing by both sheep and yak significantly decreased the community pathogen load. However, we did not find a significant difference in the community pathogen load among different types of livestock. These results suggest that grazing by large herbivores, independently of livestock type, consistently decreased the prevalence of foliar fungal diseases at both the plant population and community levels. We suggest that moderate grazing by sheep or yak is effective to control the occurrence of foliar fungal diseases in alpine grasslands. This study advances our knowledge of the interface between disease ecology, large herbivores, and grassland science.