Eriodictyol regulates white adipose tissue browning and hepatic lipid metabolism in high fat diet-induced obesity mice via activating AMPK/SIRT1 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Blossom of Citrus aurantium L. var. amara Engl. (CAVA) has been popularly consumed as folk medicine and dietary supplement owing to its various beneficial effects and especially anti-obesity potential. Our previous study predicted that eriodictyol was probably one of the key active compounds of the total flavonoids from blossom of CAVA. However, effects of eriodictyol in anti-obesity were still elusive. AIM OF THE STUDY: This study was performed to explore the precise role of eriodictyol in white adipose tissue (WAT) browning and hepatic lipid metabolism, and simultaneously, to verify the impact of eriodictyol on the total flavonoids of CAVA in losing weight. MATERIALS AND METHODS: The pancreas lipase assay was conducted and oleic acid-induced HepG2 cells were established to preliminarily detect the lipid-lowering potential of eriodictyol. Then, high fat diet-induced obesity (DIO) mouse model was established for in vivo studies. The biochemical indicators of mice were tested by commercial kits. The histopathological changes of WAT and liver in mice were tested by H&E staining, Oil Red O staining and Sirius Red staining. Immunohistochemical, Western blot assay, as well as RT-qPCR analysis were further performed. Additionally, molecular docking assay was used to simulate the binding of eriodictyol with potential target proteins. RESULTS: In vitro studies showed that eriodictyol intervention potently inhibited pancreatic lipase activity and reversed hepatic steatosis in oleic acid-induced HepG2 cells. Consistently, long-term medication of eriodictyol also effectively prevented obesity and improved lipid and glucose metabolism in diet-induced obesity mice. Obesity-induced histopathological changes in iWAT, eWAT and BAT, and abnormal expression levels of IL-10, IL-6 and TNF-α in iWAT of DIO mice were also significantly reversed by eriodictyol treatment. Eriodictyol administration significantly and potently promoted browning of iWAT by increasing expression levels of thermogenic marker protein of UCP1, as well as brown adipocyte-specific genes of PGC-1α, SIRT1 and AMPKα1. Further assays revealed that eriodictyol enhanced mitochondrial function, as shown by an increase in compound IV activity and the expression of tricarboxylic acid cycle-related genes. Besides, eriodictyol addition markedly reversed hepatic damages and hepatic inflammation, and enhanced hepatic lipid metabolism in DIO mice, as evidenced by its regulation on p-ACC, CPT1-α, UCP1, PPARα, PGC-1α, SIRT1 and p-AMPKα expression. Molecular docking results further validated that AMPK/SIRT1 pathway was probably the underlying mechanisms by which eriodictyol acted. CONCLUSION: Eriodictyol exhibited significant anti-obesity effect, which was comparable to that of the total flavonoids from blossom of CAVA. These findings furnished theoretical basis for the application of eriodictyol in weight loss.

The relationship and clinical significance of lactylation modification in digestive system tumors.

Lactylation, an emerging post-translational modification, plays a pivotal role in the initiation and progression of digestive system tumors. This study presents a comprehensive review of lactylation in digestive system tumors, underscoring its critical involvement in tumor development and progression. By focusing on metabolic reprogramming, modulation of the tumor microenvironment, and the molecular mechanisms regulating tumor progression, the potential of targeting lactylation as a therapeutic strategy is highlighted. The research reveals that lactylation participates in gene expression regulation and cell signaling by affecting the post-translational states of histones and non-histone proteins, thereby influencing metabolic pathways and immune evasion mechanisms in tumor cells. Furthermore, this study assesses the feasibility of lactylation as a therapeutic target, providing insights for clinical treatment of gastrointestinal cancers. Future research should concentrate on elucidating the mechanisms of lactylation, developing efficient lactylation inhibitors, and validating their therapeutic efficacy in clinical trials, which could transform current cancer treatment and immunotherapy approaches. In summary, this review emphasizes the crucial role of lactylation in tumorigenesis and progression through a detailed analysis of its molecular mechanisms and clinical significance.

Bibliometric analysis reveals the research hotspots and trends of nasopharyngeal carcinoma immunotherapy.

Immunotherapy is a promising strategy for nasopharyngeal carcinoma (NPC), a common and aggressive malignancy with poor prognosis. However, the literature lacks a comprehensive and objective overview of the current research status and trends of immunotherapy-related fields in NPC. We performed a bibliometric analysis of 513 original articles and reviews in English on immunotherapy for NPC from the Web of Science Core Collection database, using CiteSpace and Bibliometrix software tools. We visualized the development trend of publications, the distribution of countries/regions, the co-occurrence of keywords, the collaboration and citation of authors, the citation of journals, the evolution of topics, and the thematic map. We found that the publication volume increased sharply after 2017, with China as the main contributor and leader, the US as an important partner, and the Netherlands as a potential innovator. The research focused on immune checkpoint inhibitors and cell therapy, which were also the hotspots of clinical trials. Tumor microenvironment, immune infiltration, multicenter studies, and novel immunotherapy were the frontier topics and the key challenges for future research. CD137l-DC, lymphoma, and chimeric antigen receptor were emerging topics with good prospects. Our study provides a valuable insight into the research status and trends of immunotherapy for NPC, which may guide future research directions and clinical applications.

Antioxidant, Antimicrobial, and Anti-Inflammatory Effects of Liriodendron chinense Leaves.

Liriodendron chinense has been widely utilized in traditional Chinese medicine to treat dispelling wind and dampness and used for alleviating cough and diminishing inflammation. However, the antioxidant, antimicrobial, and anti-inflammatory effects of L. chinense leaves and the key active constituents remained elusive. So, we conducted some experiments to support the application of L. chinense in traditional Chinese medicine by investigating the antioxidant, antibacterial, and anti-inflammatory abilities, and to identify the potential key constituents responsible for the activities. The ethanol extract of L. chinense leaves (LCLE) was isolated and extracted, and assays measuring ferric reducing antioxidant power, total reducing power, DPPH•, ABTS•+, and •OH were used to assess its in vitro antioxidant capacities. Antimicrobial activities of LCLE were investigated by minimal inhibitory levels, minimum antibacterial concentrations, disc diffusion test, and scanning electron microscope examination. Further, in vivo experiments including macro indicators examination, histopathological examination, and biochemical parameters measurement were conducted to investigate the effects of LCLE on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. LCLE was further isolated and purified through column chromatography, and LPS-induced RAW264.7 cells were constructed to assess the diminished inflammation potential of the identified chemical composites. ABTS•+ and •OH radicals were extensively neutralized by the LCLE treatment. LCLE administration also presented broad-spectrum antimicrobial properties, especially against Staphylococcus epidermidis by disrupting cell walls. LPS-induced ALI in mice was significantly ameliorated by LCLE intervention, as evidenced by the histological changes in the lung and liver tissues as well as the reductions of nitric oxide (NO), TNF-α, and IL-6 production. Furthermore, three novel compounds including fragransin B2, liriodendritol, and rhamnocitrin were isolated, purified, and identified from LCLE. These three compounds exhibited differential regulation on NO accumulation and IL-10, IL-1ß, IL-6, TNF-α, COX-2, and iNOS mRNA expression in RAW264.7 cells induced by LPS. Fragransin B2 was more effective in inhibiting TNF-α mRNA expression, while rhamnocitrin was more powerful in inhibiting IL-6 mRNA expression. LCLE had significant antioxidant, antimicrobial, and anti-inflammatory effects. Fragransin B2, liriodendritol, and rhamnocitrin were probably key active constituents of LCLE, which might act synergistically to treat inflammatory-related disorders. This study provided a valuable view of the healing potential of L. chinense leaves in curing inflammatory diseases.

Artificial intelligence-based analysis of tumor-infiltrating lymphocyte spatial distribution for colorectal cancer prognosis.

BACKGROUND: Artificial intelligence (AI) technology represented by deep learning has made remarkable achievements in digital pathology, enhancing the accuracy and reliability of diagnosis and prognosis evaluation. The spatial distribution of CD3 + and CD8 + T cells within the tumor microenvironment has been demonstrated to have a significant impact on the prognosis of colorectal cancer (CRC). This study aimed to investigate CD3 CT (CD3 + T cells density in the core of the tumor [CT]) prognostic ability in patients with CRC by using AI technology. METHODS: The study involved the enrollment of 492 patients from two distinct medical centers, with 358 patients assigned to the training cohort and an additional 134 patients allocated to the validation cohort. To facilitate tissue segmentation and T-cells quantification in whole-slide images (WSIs), a fully automated workflow based on deep learning was devised. Upon the completion of tissue segmentation and subsequent cell segmentation, a comprehensive analysis was conducted. RESULTS: The evaluation of various positive T cell densities revealed comparable discriminatory ability between CD3 CT and CD3-CD8 (the combination of CD3 + and CD8 + T cells density within the CT and invasive margin) in predicting mortality (C-index in training cohort: 0.65 vs. 0.64; validation cohort: 0.69 vs. 0.69). The CD3 CT was confirmed as an independent prognostic factor, with high CD3 CT density associated with increased overall survival (OS) in the training cohort (hazard ratio [HR] = 0.22, 95% confidence interval [CI]: 0.12-0.38, P <0.001) and validation cohort (HR = 0.21, 95% CI: 0.05-0.92, P = 0.037). CONCLUSIONS: We quantify the spatial distribution of CD3 + and CD8 + T cells within tissue regions in WSIs using AI technology. The CD3 CT confirmed as a stage-independent predictor for OS in CRC patients. Moreover, CD3 CT shows promise in simplifying the CD3-CD8 system and facilitating its practical application in clinical settings.

Chemical Composition and Antibacterial and Antiulcerative Colitis Activities of Essential Oil from Pruni Semen.

This study was sought to investigate the chemical composition and antibacterial and antiulcerative colitis (UC) effects of essential oil from Pruni Semen (PSEO). A GC-MS assay showed that the major compounds in PSEO were products of amygdalin hydrolysis, which possessed great antibacterial and anti-inflammatory potential. In vitro antibacterial experiments demonstrated that PSEO treatment inhibited activity of four kinds of intestinal pathogens probably by disrupting the cell wall. Further in vivo studies showed that PSEO administration significantly improved physiological indexes, attenuated histopathological characteristics, and inhibited proinflammatory cytokine production in dextran sulfate sodium (DSS)-induced UC mice. Network pharmacology and molecular docking results predicted that PSEO might prevent UC via regulating the PI3K/AKT pathway. Western blotting and immunofluorescence assays were further conducted for verification, and the results evidenced that PSEO intervention significantly regulated the PI3K/AKT pathway and the expression of its downstream proteins in DSS-induced mice. PSEO might provide a new dietary strategy for UC treatment.

An artificial intelligence-based ecological index for prognostic evaluation of colorectal cancer.

BACKGROUND AND OBJECTIVE: In the tumor microenvironment (TME), the dynamic interaction between tumor cells and immune cells plays a critical role in predicting the prognosis of colorectal cancer. This study introduces a novel approach based on artificial intelligence (AI) and immunohistochemistry (IHC)-stained whole-slide images (WSIs) of colorectal cancer (CRC) patients to quantitatively assess the spatial associations between tumor cells and immune cells. To achieve this, we employ the Morisita-Horn ecological index (Mor-index), which allows for a comprehensive analysis of the spatial distribution patterns between tumor cells and immune cells within the TME. MATERIALS AND METHODS: In this study, we employed a combination of deep learning technology and traditional computer segmentation methods to accurately segment the tumor nuclei, immune nuclei, and stroma nuclei within the tumor regions of IHC-stained WSIs. The Mor-index was used to assess the spatial association between tumor cells and immune cells in TME of CRC patients by obtaining the results of cell nuclei segmentation. A discovery cohort (N = 432) and validation cohort (N = 137) were used to evaluate the prognostic value of the Mor-index for overall survival (OS). RESULTS: The efficacy of our method was demonstrated through experiments conducted on two datasets comprising a total of 569 patients. Compared to other studies, our method is not only superior to the QuPath tool but also produces better segmentation results with an accuracy of 0.85. Mor-index was quantified automatically by our method. Survival analysis indicated that the higher Mor-index correlated with better OS in the discovery cohorts (HR for high vs. low 0.49, 95% CI 0.27-0.77, P = 0.0014) and validation cohort (0.21, 0.10-0.46, < 0.0001). CONCLUSION: This study provided a novel AI-based approach to segmenting various nuclei in the TME. The Mor-index can reflect the immune status of CRC patients and is associated with favorable survival. Thus, Mor-index can potentially make a significant role in aiding clinical prognosis and decision-making.

CircRNA.0007127 triggers apoptosis through the miR-513a-5p/CASP8 axis in K-562 cells.

BACKGROUND: Circular RNAs (circRNAs) are covalently closed single-stranded RNAs with multiple biological functions. CircRNA.0007127 is derived from the carbon catabolite repression 4-negative on TATA-less (CCR4-NOT) complex subunit 2 (CNOT2), which was found to regulate tumor cell apoptosis through caspase pathway. METHODS: Potential circRNA.0007127 target microRNAs (miRNAs) were analyzed by miRanda, TargetScan, and RNAhybrid software, and the miRNAs with binding sites for apoptosis-related genes were screened. The roles of circRNA.0007127 and its downstream target, microRNA (miR)|-513a-5p, were validated by quantitative real-time polymerase chain reaction (qPCR), flow cytometry, mitochondrial membrane potential, immunofluorescence, western blot, and caspase-8 (CASP8) protein activity in vitro in H2O2-induced K-562 cells. The circRNA.|0007127|‒|miR-513a-5p and CASP8|‒|miR-513a-5p interactions were verified by luciferase reporter assays. RESULTS: Silencing circRNA.0007127 decreased cell apoptosis by inhibiting CASP8 pathway activation in K-562 cells. Compared with the control group, the expression of CASP8 was reduced by 50% and the 43-kD fragment of CASP8 protein was significantly reduced (P≤0.05). The luciferase reporting assay showed that circRNA.0007127 combined with miR-513a-5p or CASP8, with extremely significant differences (P≤0.001). The overexpression of miR-513a-5p inhibited the gene expression level of CASP8 in a human myeloid leukemia cell model (75% change) and the level of a 43-kD fragment of CASP8 protein (P≤0.01). The rescue experiment showed that cotransfection with circRNA.0007127 small-interfering RNA (siRNA) and the miR-513a-5p inhibitor increased CASP8 gene expression and the apoptosis rate, suggesting that the miR-513a-5p inhibitor is a circRNA.0007127 siRNA antagonist. CONCLUSIONS: CircRNA.0007127 regulates K-562 cell apoptosis through the miR-513a-5p/CASP8 axis, which can serve as a novel powerful molecular target for K-562 cells.

Dextran sulfate prevents excess aggregation of human pluripotent stem cells in 3D culture by inhibiting ICAM1 expression coupled with down-regulating E-cadherin through activating the Wnt signaling pathway.

BACKGROUND: Human pluripotent stem cells (hPSCs) have great potential in applications for regenerative medicine and drug development. However, 3D suspension culture systems for clinical-grade hPSC large-scale production have been a major challenge. Accumulating evidence has demonstrated that the addition of dextran sulfate (DS) could prevent excessive adhesion of hPSCs from forming larger aggregates in 3D suspension culture. However, the signaling and molecular mechanisms underlying this phenomenon remain elusive. METHODS: By using a cell aggregate culture assay and separating big and small aggregates in suspension culture systems, the potential mechanism and downstream target genes of DS were investigated by mRNA sequence analysis, qRT-PCR validation, colony formation assay, and interference assay. RESULTS: Since cellular adhesion molecules (CAMs) play important roles in hPSC adhesion and aggregation, we assumed that DS might prevent excess adhesion through affecting the expression of CAMs in hPSCs. As expected, after DS treatment, we found that the expression of CAMs was significantly down-regulated, especially E-cadherin (E-cad) and intercellular adhesion molecule 1 (ICAM1), two highly expressed CAMs in hPSCs. The role of E-cad in the adhesion of hPSCs has been widely investigated, but the function of ICAM1 in hPSCs is hardly understood. In the present study, we demonstrated that ICAM1 exhibited the capacity to promote the adhesion in hPSCs, and this adhesion was suppressed by the treatment with DS. Furthermore, transcriptomic analysis of RNA-seq revealed that DS treatment up-regulated genes related to Wnt signaling resulting in the activation of Wnt signaling in which SLUG, TWIST, and MMP3/7 were highly expressed, and further inhibited the expression of E-cad. CONCLUSION: Our results demonstrated that DS played an important role in controlling the size of hPSC aggregates in 3D suspension culture by inhibiting the expression of ICAM1 coupled with the down-regulation of E-cad through the activation of the Wnt signaling pathway. These results represent a significant step toward developing the expansion of hPSCs under 3D suspension condition in large-scale cultures.

3D hESC exosomes enriched with miR-6766-3p ameliorates liver fibrosis by attenuating activated stellate cells through targeting the TGFßRII-SMADS pathway.

BACKGROUND: Exosomes secreted from stem cells exerted salutary effects on the fibrotic liver. Herein, the roles of exosomes derived from human embryonic stem cell (hESC) in anti-fibrosis were extensively investigated. Compared with two-dimensional (2D) culture, the clinical and biological relevance of three-dimensional (3D) cell spheroids were greater because of their higher regeneration potential since they behave more like cells in vivo. In our study, exosomes derived from 3D human embryonic stem cells (hESC) spheroids and the monolayer (2D) hESCs were collected and compared the therapeutic potential for fibrotic liver in vitro and in vivo. RESULTS: In vitro, PKH26 labeled-hESC-Exosomes were shown to be internalized and integrated into TGFß-activated-LX2 cells, and reduced the expression of profibrogenic markers, thereby regulating cellular phenotypes. TPEF imaging indicated that PKH26-labeled-3D-hESC-Exsomes possessed an enhanced capacity to accumulate in the livers and exhibited more dramatic therapeutic potential in the injured livers of fibrosis mouse model. 3D-hESC-Exosomes decreased profibrogenic markers and liver injury markers, and improved the level of liver functioning proteins, eventually restoring liver function of fibrosis mice. miRNA array revealed a significant enrichment of miR-6766-3p in 3D-hESC-Exosomes, moreover, bioinformatics and dual luciferase reporter assay identified and confirmed the TGFßRII gene as the target of miR-6766-3p. Furthermore, the delivery of miR-6766-3p into activated-LX2 cells decreased cell proliferation, chemotaxis and profibrotic effects, and further investigation demonstrated that the expression of target gene TGFßRII and its downstream SMADs proteins, especially phosphorylated protein p-SMAD2/3 was also notably down-regulated by miR-6766-3p. These findings unveiled that miR-6766-3p in 3D-hESC-Exosomes inactivated SMADs signaling by inhibiting TGFßRII expression, consequently attenuating stellate cell activation and suppressing liver fibrosis. CONCLUSIONS: Our results showed that miR-6766-3p in the 3D-hESC-Exosomes inactivates smads signaling by restraining TGFßRII expression, attenuated LX2 cell activation and suppressed liver fibrosis, suggesting that 3D-hESC-Exosome enriched-miR-6766-3p is a novel anti-fibrotic therapeutics for treating chronic liver disease. These results also proposed a significant strategy that 3D-Exo could be used as natural nanoparticles to rescue liver injury via delivering antifibrotic miR-6766-3p.

Gene excavation and expression analysis of CYP and UGT related to the post modifying stage of gypenoside biosynthesis in Gynostemma pentaphyllum (Thunb.) Makino by comprehensive analysis of RNA and proteome sequencing.

Previous studies have revealed that gypenosides produced from Gynostemma pentaphyllum (Thunb.) Makino are mainly dammarane-type triterpenoid saponins with diverse structures and important biological activities, but the mechanism of diversity for gypenoside biosynthesis is still unclear. In this study, a combination of isobaric tags for relative and absolute quantification (iTRAQ) proteome analysis and RNA sequencing transcriptome analysis was performed to identify the proteins and genes related to gypenoside biosynthesis. A total of 3925 proteins were identified by proteomic sequencing, of which 2537 were quantified. Seventeen cytochrome P450 (CYP) and 11 uridine 5'-diphospho-glucuronosyltransferase (UDP-glucuronosyltransferase, UGT) candidate genes involved in the side chain synthesis and modification of gypenosides were found. Seven putative CYPs (CYP71B19, CYP77A3, CYP86A7, CYP86A8, CYP89A2, CYP90A1, CYP94A1) and five putative UGTs (UGT73B4, UGT76B1, UGT74F2, UGT91C1 and UGT91A1) were selected as candidate structural modifiers of triterpenoid saponins, which were cloned for gene expression analysis. Comprehensive analysis of RNA sequencing and proteome sequencing showed that some CYPs and UGTs were found at both the transcription and translation levels. In this study, an expression analysis of 7 CYPs and 5 UGTs that contributed to gypenoside biosynthesis and distribution in G. pentaphyllum was performed, providing consistent results that will inspire more future research on vital genes/proteins involved in gypenoside biosynthesis.

Predicting Neoadjuvant Chemoradiotherapy Response in Locally Advanced Rectal Cancer Using Tumor-Infiltrating Lymphocytes Density.

PURPOSE: Accumulating evidence revealed the predictive value of tumor-infiltrating lymphocytes (TILs) for neoadjuvant chemoradiotherapy (nCRT) response in solid tumors. This study quantified TILs density using hematoxylin and eosin (H&E) stained whole-slide images (WSIs) and investigated the predictive value of TILs density on nCRT response in locally advanced rectal cancer (LARC) patients. PATIENTS AND METHODS: Two hundred and ten patients diagnosed with LARC were enrolled in this study. The density of TILs in the stroma region was quantified by a semi-automatic method in WSIs. Patients were stratified into low-TILs and high-TILs groups using the median value as the threshold. The tumor regression grade (TRG) was used to assess the response to nCRT in tumor resected specimens. Based on TRG, patients were classified into major-responder (TRG 0-1) and non-responder (TRG 2-3) groups. RESULTS: The TILs density was significantly correlated with the nCRT response. Specifically, patients with high-TILs tend to have a higher major-responder rate than the low-TILs group (63.8% vs 47.6%, P = 0.026). Univariate analysis showed the TILs density was a predictor for the nCRT response (high vs low, odds ratio [OR] =1.94, 95% confidence interval 1.12-3.37, P = 0.019), and multivariate analysis further confirmed the correlation (adjusted odds ratio [AOR] = 2.41, 1.28-4.56, P = 0.007). CONCLUSION: Patients with a high-TIL density have a higher major-responder rate than the low-TILs group, indicating patients with a strong immune response benefit more from nCRT. This semi-automatic method can facilitate the individualized preoperative prediction of the TRG for LARC patients before nCRT.

The combination of dextran sulphate and polyvinyl alcohol prevents excess aggregation and promotes proliferation of pluripotent stem cells in suspension culture.

OBJECTIVES: For clinical applications of cell-based therapies, a large quantity of human pluripotent stem cells (hPSCs) produced in standardized and scalable culture processes is required. Currently, microcarrier-free suspension culture shows potential for large-scale expansion of hPSCs; however, hPSCs tend to aggregate during culturing leading to a negative effect on cell yield. To overcome this problem, we developed a novel protocol to effectively control the sizes of cell aggregates and enhance the cell proliferation during the expansion of hPSCs in suspension. MATERIALS AND METHODS: hPSCs were expanded in suspension culture supplemented with polyvinyl alcohol (PVA) and dextran sulphate (DS), and 3D suspension culture of hPSCs formed cell aggregates under static or dynamic conditions. The sizes of cell aggregates and the cell proliferation as well as the pluripotency of hPSCs after expansion were assessed using cell counting, size analysis, real-time quantitative polymerase chain reaction, flow cytometry analysis, immunofluorescence staining, embryoid body formation, teratoma formation and transcriptome sequencing. RESULTS: Our results demonstrated that the addition of DS alone effectively prevented hPSC aggregation, while the addition of PVA significantly enhanced hPSC proliferation. The combination of PVA and DS not only promoted cell proliferation of hPSCs but also produced uniform and size-controlled cell aggregates. Moreover, hPSCs treated with PVA, or DS or a combination, maintained the pluripotency and were capable of differentiating into all three germ layers. mRNA-seq analysis demonstrated that the combination of PVA and DS significantly promoted hPSC proliferation and prevented cell aggregation through improving energy metabolism-related processes, regulating cell growth, cell proliferation and cell division, as well as reducing the adhesion among hPSC aggregates by affecting expression of genes related to cell adhesion. CONCLUSIONS: Our results represent a significant step towards developing a simple and robust approach for the expansion of hPSCs in large scale.

Establishment of a 3D model of tumor-driven angiogenesis to study the effects of anti-angiogenic drugs on pericyte recruitment.

Hepatocellular carcinoma (HCC), as a well-vascularized tumor, has attracted increasing attention in antiangiogenic therapies. Notably, emerging studies reveal that the long-term administration of antiangiogenic drugs induces hypoxia in tumors. Pericytes, which play a vital role in vascular stabilization and maturation, have been documented to be associated with antiangiogenic drug-induced tumor hypoxia. However, the role of antiangiogenic agents in regulating pericyte behavior still remains elusive. In this study, by using immunostaining analysis, we first demonstrated that tumors obtained from HCC patients were highly angiogenic, in which vessels were irregularly covered by pericytes. Therefore, we established a new 3D model of tumor-driven angiogenesis by culturing endothelial cells, pericytes, cancer stem cells (CSCs) and mesenchymal stem cells (MSCs) with microcarriers in order to investigate the effects and mechanisms exerted by antiangiogenic agents on pericyte recruitment during tumor angiogenesis. Interestingly, microcarriers, as supporting matrices, enhanced the interactions between tumor cells and the extracellular matrix (ECM), promoted malignancy of tumor cells and increased tumor angiogenesis within the 3D model, as determined by qRT-PCR and immunostaining. More importantly, we showed that zoledronic acid (ZA) reversed the inhibited pericyte recruitment, which was induced by sorafenib (Sora) treatment, through fostering the expression and activation of ErbB1/ErbB2 and PDGFR-ß in pericytes, in both an in vitro 3D model and an in vivo xenograft HCC mouse model. Hence, our model provides a more pathophysiologically relevant platform for the assessment of therapeutic effects of antiangiogenic compounds and identification of novel pharmacological targets, which might efficiently improve the benefits of antiangiogenic treatment for HCC patients.

ITGB1 Drives Hepatocellular Carcinoma Progression by Modulating Cell Cycle Process Through PXN/YWHAZ/AKT Pathways.

Integrin ß1 (ITGB1), which acts as an extracellular matrix (ECM) receptor, has gained increasing attention as a therapeutic target for the treatment of hepatocellular carcinoma (HCC). However, the underpinning mechanism of how ITGB1 drives HCC progression remains elusive. In this study, we first found that ITGB1 expression was significantly higher in HCC tissues than in normal controls by bioinformatics analysis. Furthermore, bioinformatics analysis revealed that paxillin (PXN) and 14-3-3 protein zeta (YWHAZ) are the molecules participating in ITGB1-regulated HCC tumor cell cycle progression. Indeed, immunohistochemistry (IHC) revealed that ITGB1, paxillin, and YWHAZ were strongly upregulated in paired HCC tissue compared with adjacent normal tissues. Notably, the inhibition of ITGB1 expression by small interfering RNA (siRNA) resulted in the downregulated expression of PXN and YWHAZ in primary HCC cells, as assessed by western blot and immunostaining. In addition, ITGB1 knockdown markedly impaired the aggressive behavior of HCC tumor cells and delayed cell cycle progression as determined by cell migration assay, drug-resistance analysis, colony formation assay, quantitative real-time polymerase chain reaction (qRT-PCR), and cell cycle analysis as well as cell viability measurements. More importantly, we proved that xenograft ITGB1high tumors grew more rapidly than ITGB1low tumors. Altogether, our study showed that the ITGB1/PXN/YWHAZ/protein kinase B (AKT) axis enhances HCC progression by accelerating the cell cycle process, which offers a promising approach to halt HCC tumor growth.

Proteomic analysis on the regulation of DOPA-melanin synthesis in Talaromyces marneffei.

BACKGROUND: Yeast form of T.marneffei can produce DOPA-melanin which perform an important role in the pathogen surviving in macrophage. So far, the proteomic associated with melanin synthesis remain unclearly in T.marneffei. METHODS: The whole yeast cell proteins were extracted from T.marneffei cultured with or without l-DOPA. Using two-dimensional gel electrophoresis combined with MALDI-TOF mass spectrometry, distinguished proteins were identified between T.marneffei cultured with or without l-DOPA. Furthermore, geldanamycin were used to assess the inhibition effect on T.marneffei melanin production in vitro. RESULTS: 16 distinguished proteins were identified in DOPA-melanized yeast cells, as well as 15 triple-up-expressed proteins and 7 triple-down-expressed proteins in comparison with non DOPA-melanized yeast cells. Of note, proteins differentially expressed proteins were predominantly heat shock proteins. HSP90/60/70 genes expressions increased significantly demonstrated by q-RT-PCR, which was consistent with the proteomics changes. GO analysis showed that the majority of differentially expressed proteins including HSPs(especially HSP90) were found enriched in stress response, cellular process, protein folding, stimuli response and biological process. KEGG pathway analysis showed that proteins were enriched predominantly in phagosome. HSP90 inhibitor(Geldanamycin) inhibited the brown-black pigment production of T.marneffei yeast grown on brain heart infusion agar, as well as the inhibition effect was observed by transmission electron microscope. CONCLUSIONS: The results demonstrates that HSP90 palys an essential role in T.marneffei DOPA-melanin synthesis pathway.

Positive Selection of Squalene Synthase in Cucurbitaceae Plants.

Triterpenoid saponins are secondary metabolites synthesized through isoprenoid pathways in plants. Cucurbitaceae represent an important plant family in which many species contain cucurbitacins as secondary metabolites synthesized through isoprenoid and triterpenoid pathways. Squalene synthase (SQS) is required for the biosynthesis of isoprenoids, but the forces driving the evolution of SQS remain undetermined. In this study, 10 SQS cDNA sequences cloned from 10 species of Cucurbitaceae and 49 sequences of SQS downloaded from GenBank and UniProt databases were analyzed in a phylogenetic framework to identify the evolutionary forces for functional divergence. Through phylogenetic construction and positive selection analysis, we found that SQS sequences are under positive selection. The sites of positive selection map to functional and transmembrane domains. 180L, 189S, 194S, 196S, 265I, 289P, 389P, 390T, 407S, 408A, 410R, and 414N were identified as sites of positive selection that are important during terpenoid synthesis and map to transmembrane domains. 196S and 407S are phosphorylated and influence SQS catalysis and triterpenoid accumulation. These results reveal that positive selection is an important evolutionary force for SQS in plants. This provides new information into the molecular evolution of SQS within the Cucurbitaceae family.

Positive selection and functional divergence of farnesyl pyrophosphate synthase genes in plants.

BACKGROUND: Farnesyl pyrophosphate synthase (FPS) belongs to the short-chain prenyltransferase family, and it performs a conserved and essential role in the terpenoid biosynthesis pathway. However, its classification, evolutionary history, and the forces driving the evolution of FPS genes in plants remain poorly understood. RESULTS: Phylogeny and positive selection analysis was used to identify the evolutionary forces that led to the functional divergence of FPS in plants, and recombinant detection was undertaken using the Genetic Algorithm for Recombination Detection (GARD) method. The dataset included 68 FPS variation pattern sequences (2 gymnosperms, 10 monocotyledons, 54 dicotyledons, and 2 outgroups). This study revealed that the FPS gene was under positive selection in plants. No recombinant within the FPS gene was found. Therefore, it was inferred that the positive selection of FPS had not been influenced by a recombinant episode. The positively selected sites were mainly located in the catalytic center and functional areas, which indicated that the 98S and 234D were important positively selected sites for plant FPS in the terpenoid biosynthesis pathway. They were located in the FPS conserved domain of the catalytic site. We inferred that the diversification of FPS genes was associated with functional divergence and could be driven by positive selection. CONCLUSIONS: It was clear that protein sequence evolution via positive selection was able to drive adaptive diversification in plant FPS proteins. This study provides information on the classification and positive selection of plant FPS genes, and the results could be useful for further research on the regulation of triterpenoid biosynthesis.

Genetic association of NOS1 exon18, NOS1 exon29, ABCB1 1236C/T, and ABCB1 3435C/T polymorphisms with the risk of Parkinson's disease: A meta-analysis.

BACKGROUND: Parkinson's disease (PD) is the second most frequent neurodegenerative disorder. Previous publications have investigated the association of NOS1 and ABCB1 polymorphisms with PD risk. However, those studies have provided some contradictory results. METHODS: Literature searches were performed using PubMed, Embase, PDgene, China National Knowledge Infrastructure database, and Google Scholar. Odds ratios (ORs) with 95% confidence intervals (CIs) were applied to evaluate the strength of association. RESULTS: The analysis results indicated that NOS1 exon18 polymorphism was associated with developing PD in 4 genetic models (allelic: OR = 1.25, 95%CI 1.09-1.44, P = 0.001; homozygous: OR = 1.79, 95%CI 1.32-2.45, P < 0.001; recessive: OR = 1.70, 95%CI 1.26-2.28, P < 0.001; dominant: OR = 1.22, 95%CI 1.02-1.46, P = 0.03), whereas exon29 polymorphism was not correlated to PD susceptibility. In addition, ABCB1 1236C/T polymorphism was related to PD in the recessive (OR = 0.80, 95%CI 0.66-0.97, P = 0.025) and overdominant (OR = 1.21, 95%CI 1.03-1.43, P = 0.02) models, which might indicate the opposite effects of 2 minor variants of this locus on Parkinson's disease. However, this associated result was not robust enough to withstand statistically significant correction. On the other hand, no association was found between ABCB1 3435C/T polymorphism and the predisposition to PD in 5 genetic models, and such an absence of relationship was further confirmed by subgroup analysis in Caucasians and Asians. Whether the polymorphisms of these 4 loci were linked to PD or not, our study provided some interesting findings that differ from the previous results with regard to their genetic susceptibility. CONCLUSION: The NOS1 exon18 and ABCB1 1236C/T variants might play a role in the risk of Parkinson's disease, whereas NOS1 exon29 and ABCB1 3435C/T polymorphisms might not contribute to PD susceptibility.

Genetic Association of CHAT rs3810950 and rs2177369 Polymorphisms with the Risk of Alzheimer's Disease: A Meta-Analysis.

Choline acetyltransferase (CHAT) rs3810950 and rs2177369 polymorphisms have been implicated in susceptibility to Alzheimer's disease (AD). Due to the inconsistent results from previous studies, a meta-analysis was performed to estimate the association between these polymorphisms and AD risk more precisely. Pooled results of our meta-analysis indicated CHAT rs2177369 polymorphism was correlated with decreasing AD risk in one of five genetic models (dominant: OR = 0.77, 95% CI: 0.62-0.96), while rs3810950 mutant was associated with AD development in three models (allelic: OR = 1.18, 95% CI: 1.01-1.37, homozygous: OR = 1.63, 95% CI: 1.09-2.42, and recessive: OR = 1.65, 95% CI: 1.20-2.26). In subgroup analysis by ethnicity, the association between CHAT rs3810950 polymorphism and AD risk was just found in the recessive model (OR = 1.47, 95% CI: 1.05-2.07) among Caucasians, while four genetic models (allelic: OR = 1.23, 95% CI: 1.01-1.48; homozygous: OR = 2.24, 95% CI: 1.48-3.39; dominant: OR = 1.21, 95% CI: 1.06-1.40; and recessive: OR = 2.18, 95% CI: 1.45-3.29) assumed this association in Asians. In conclusion, our meta-analysis indicated CHAT rs2177369 polymorphism might play a protective role in AD, while rs3810950 variant was a risk factor for AD but its single heterozygous mutations might not influence susceptibility to AD.