ABSTRACT
BACKGROUND: Invasive deep brain stimulation (DBS) has been shown to be effective in treating patients with Parkinson's disease (PD), yet its clinical use is limited to patients at the advanced stage of the disease. Transcranial temporal interference stimulation (tTIS) may be a novel nonneurosurgical and safer alternative, yet its therapeutic potential remains unexplored. OBJECTIVE: This pilot study aims to examine the feasibility and safety of tTIS targeting the right globus pallidus internus (GPi) for motor symptoms in patients with PD. METHODS: Twelve participants with mild PD completed this randomized, double-blind, and sham-controlled experiment. Each of them received either 20-minute or sham tTIS of the right GPi. Before and immediately after the stimulation, participants completed the Movement Disorder Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS-III) in the "medication-on" state to assess the motor symptoms. The blinding efficacy and side effects were also assessed. RESULTS: tTIS was well tolerated by participants, with only mild, transient adverse effects reported. tTIS significantly reduced MDS-UPDRS-III scores by 6.64 points (14.7%), particularly in bradykinesia (23.5%) and tremor (15.3%). The left side showed more significant alleviation in motor symptoms, particularly bradykinesia, compared to the right side. Participants with severer bradykinesia and tremor before stimulation experienced greater improvement after tTIS. CONCLUSION: This pilot study suggests that the tTIS, as a novel noninvasive DBS approach, is feasible and safe for alleviating motor symptoms in mild PD, especially bradykinesia and tremor. Future larger-scale and more definitive studies are needed to confirm the benefits. © 2024 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
ABSTRACT
Traditionally, hybrid promoters are constructed, in Saccharomyces cerevisiae, by joining the core region and the upstream activating sequences from different native promoters. Here, we describe a new design that makes use of the core promoters from foreign organisms: viruses, humans, and the yeast Schizosaccharomyces pombe. With this approach, we realized a library of 59 new constitutive promoters that span over nine folds in gene expression.
Subject(s)
Promoter Regions, Genetic , Saccharomyces cerevisiae , Promoter Regions, Genetic/genetics , Saccharomyces cerevisiae/genetics , Gene Library , Schizosaccharomyces/genetics , Humans , Genetic Engineering/methodsABSTRACT
Solid-state lithium metal batteries (SSLMBs) have gained significant attention in energy storage research due to their high energy density and significantly improved safety. But there are still certain problems with lithium dendrite growth, interface stability, and room-temperature practicality. Nature continually inspires human development and intricate design strategies to achieve optimal structural applications. Innovative solid-state electrolytes (SSEs), inspired by diverse natural species, have demonstrated exceptional physical, chemical, and mechanical properties. This review provides an overview of typical bionic-structured materials in SSEs, particularly those mimicking plant and animal structures, with a focus on their latest advancements in applications of solid-state lithium metal batteries. Commencing from plant structures encompassing roots, trunks, leaves, flowers, fruits, and cellular levels, the detailed influence of biomimetic strategies on SSE design and electrochemical performance are presented in this review. Subsequently, the recent progress of animal-inspired nanostructures in SSEs is summarized, including layered structures, surface morphologies, and interface compatibility in both two-dimensional (2D) and three-dimensional (3D) aspects. Finally, we also evaluate the current challenges and provide a concise outlook on future research directions. We anticipate that the review will provide useful information for future reference regarding the design of bionic-structured materials in SSEs.
ABSTRACT
The liver is the central organ for digestion and detoxification and has unique metabolic and regenerative capacities. The hepatobiliary system originates from the foregut endoderm, in which cells undergo multiple events of cell proliferation, migration, and differentiation to form the liver parenchyma and ductal system under the hierarchical regulation of transcription factors. Studies on liver development and diseases have revealed that SRY-related high-mobility group box 9 (SOX9) plays an important role in liver embryogenesis and the progression of hepatobiliary diseases. SOX9 is not only a master regulator of cell fate determination and tissue morphogenesis, but also regulates various biological features of cancer, including cancer stemness, invasion, and drug resistance, making SOX9 a potential biomarker for tumor prognosis and progression. This review systematically summarizes the latest findings of SOX9 in hepatobiliary development, homeostasis, and disease. We also highlight the value of SOX9 as a novel biomarker and potential target for the clinical treatment of major liver diseases.
ABSTRACT
The effects of adipocyterich microenvironment (ARM) on chemoresistance have garnered increasing interest. Ovarian cancer (OVCA) is a representative adipocyterich associated cancer. In the present study, epithelial OVCA (EOC) was used to investigate the influence of ARM on chemoresistance with the aim of identifying novel targets and developing novel strategies to reduce chemoresistance. Bioinformatics analysis was used to explore the effects of ARMassociated mechanisms contributing to chemoresistance and treated EOC cells, primarily OVCAR3 cells, with human adipose tissue extracts (HATES) from the peritumoral adipose tissue of patients were used to mimic ARM in vitro. Specifically, the peroxisome proliferatoractivated receptor γ (PPARγ) antagonist GW9662 and the ABC transporter G family member 2 (ABCG2) inhibitor KO143, were used to determine the underlying mechanisms. Next, the effect of HATES on the expression of PPARγ and ABCG2 in OVCAR3 cells treated with cisplatin (DDP) and paclitaxel (PTX) was determined. Additionally, the association between PPARγ, ABCG2 and chemoresistance in EOC specimens was assessed. To evaluate the effect of inhibiting PPARγ, using DDP, a nude mouse model injected with OVCAR3shPPARγ cells and a C57BL/6 model injected with ID8 cells treated with GW9662 were established. Finally, the factors within ARM that contributed to the mechanism were determined. It was found that HATES promoted chemoresistance by increasing ABCG2 expression via PPARγ. Expression of PPARγ/ABCG2 was related to chemoresistance in EOC clinical specimens. GW9662 or knockdown of PPARγ improved the efficacy of chemotherapy in mice. Finally, angiogenin and oleic acid played key roles in HATES in the upregulation of PPARγ. The present study showed that the introduction of ARMeducated PPARγ attenuated chemoresistance in EOC, highlighting a potentially novel therapeutic adjuvant to chemotherapy and shedding light on a means of improving the efficacy of chemotherapy from the perspective of ARM.
Subject(s)
Anilides , Ovarian Neoplasms , Animals , Female , Humans , Mice , Adipocytes/metabolism , Apoptosis , ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 2/metabolism , Carcinoma, Ovarian Epithelial/drug therapy , Cell Line, Tumor , Drug Resistance, Neoplasm/genetics , Mice, Inbred C57BL , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , PPAR gamma/genetics , PPAR gamma/metabolism , Tumor Microenvironment , Up-RegulationABSTRACT
BACKGROUND: Hepatocellular carcinoma (HCC) is the sixth incidence of cancer and the third leading cause of cancer mortality in the world. Facing the ever-increasing population of HCC patients, there is still an urgent need to find good diagnostic and prognostic markers to explore new therapeutic targets. Phosphotriesterase-related (PTER) protein, an expressed protein in the liver and injured or ploycystic kidneys, was reported to be correlated with serum aspartate aminotransferase (AST) and alanine transaminase (ALT). Our study aimed to investigate the expression of PTER protein in HCC patients and the association between PTER protein expression with clinicopathological features of HCC. METHODS: Western blot analysis and immunohistochemistry (IHC) were performed in paired para-tumor and liver tumor tissues and HCC tissue microarray (TMA) to detect PTER protein expression. Correlation between PTER protein and prognostic factors were analyzed through univariate and multivariate analysis. RESULTS: We identified that PTER protein was significantly up-regulated in HCC tumors. Our data revealed that high PTER protein expression was associated with aggressive clinicopathological features of HCC, such as advanced tumor staging, vascular invasion, recurrence, and shorter overall survival (OS) and disease-free survival (DFS) time. Besides, in multivariate analyses, PTER protein was an independent predictor for OS (P=0.004) and DFS (P=0.013) for HCC patients. Meanwhile, the prognosis of patients with high PTER protein is much worse than those with low PTER protein expression. CONCLUSIONS: PTER protein expression is raised in HCC tissues and may be a potential prognostic predictor for HCC patients.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Humans , Disease-Free Survival , PrognosisABSTRACT
Gemcitabine is a nucleoside analog that has been successfully used in the treatment of multiple cancers. However, intrinsic or acquired resistance reduces the chemotherapeutic potential of gemcitabine. Here, we revealed a previously unappreciated mechanism by which phosphatase and tensin homolog (PTEN), one of the most frequently mutated genes in human cancers, dominates the decision-making process that is central to the regulation of gemcitabine efficacy in cholangiocarcinoma (CCA). By investigating a gemcitabine-treated CCA cohort, we found that PTEN deficiency was correlated with the improved efficacy of gemcitabine-based chemotherapy. Using cell-based drug sensitivity assays, cell line-derived xenograft, and patient-derived xenograft models, we further confirmed that PTEN deficiency or genetic-engineering down-regulation of PTEN facilitated gemcitabine efficacy both in vitro and in vivo. Mechanistically, PTEN directly binds to and dephosphorylates the C terminus of the catalytic subunit of protein phosphatase 2A (PP2Ac) to increase its enzymatic activity, which further dephosphorylates deoxycytidine kinase (DCK) at Ser74 to diminish gemcitabine efficacy. Therefore, PTEN deficiency and high phosphorylation of DCK predict a better response to gemcitabine-based chemotherapy in CCA. We speculate that the combination of PP2A inhibitor and gemcitabine in PTEN-positive tumors could avoid the resistance of gemcitabine, which would benefit a large population of patients with cancer receiving gemcitabine or other nucleoside analogs.
Subject(s)
Bile Duct Neoplasms , Cholangiocarcinoma , Humans , Phosphorylation , Gemcitabine , Nucleosides , Bile Ducts, Intrahepatic , PTEN PhosphohydrolaseABSTRACT
Introduction: Cholangiocarcinoma (CCA) is a malignant tumor of the biliary epithelium with a poor prognosis. The lack of biomarkers to predict therapeutic response and prognosis is one of the major challenges for CCA treatment. Tertiary lymphoid structures (TLS) provide a local and pivotal microenvironment for tumor immune responses. The prognostic value and clinical relevance of TLS in CCA remain unclear. We aimed to explore the characteristics and clinical significance of TLS in CCA. Methods: We investigated the prognostic value and clinical relevance of TLS in CCA using a surgery cohort containing 471 CCA patients (cohort 1) and an immunotherapy cohort containing 100 CCA patients (cohort 2). Hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining were used to evaluate the maturity of TLS. Multiplex IHC (mIHC) was employed to characterize the composition of TLS. Results: Different maturity of TLS were observed in CCA tissue sections. Strong staining of the four-gene signature including PAX5, TCL1A, TNFRSF13C, and CD79A were found in TLS regions. A high density of intra-tumoral TLS (T-score high) were significantly correlated with longer overall survival (OS) both in CCA cohort 1 (p = 0.002) and cohort 2 (p = 0.01), whereas a high density of peri-tumoral TLS (P-score high) were associated with shorter OS in these two cohorts (p = 0.003 and p = 0.03, respectively). Conclusion: The established four-gene signature efficiently identified the TLS in CCA tissues. The abundance and spatial distribution of TLS were significantly correlated with the prognosis and immune checkpoint inhibitors (ICIs) immunotherapy response of CCA patients. The presence of intra-tumoral TLS are positive prognostic factors for CCA, which provide a theoretical basis for the future diagnosis and treatment of CCA.
Subject(s)
Bile Duct Neoplasms , Cholangiocarcinoma , Tertiary Lymphoid Structures , Humans , Tumor Microenvironment , Prognosis , Cholangiocarcinoma/genetics , Cholangiocarcinoma/therapy , Immunotherapy , Bile Duct Neoplasms/therapy , Bile Ducts, Intrahepatic/pathologyABSTRACT
BACKGROUND & AIMS: In eukaryotes, the ubiquitin-proteasome system and the autophagy-lysosome pathway are essential for maintaining cellular proteostasis and associated with cancer progression. Our previous studies have demonstrated that phosphatase and tensin homolog (PTEN), one of the most frequently mutated genes in human cancers, limits proteasome abundance and determines chemosensitivity to proteasome inhibitors in cholangiocarcinoma (CCA). However, whether PTEN regulates the lysosome pathway remains unclear. METHODS: We tested the effects of PTEN on lysosome biogenesis and exosome secretion using loss- and gain-of-function strategies in CCA cell lines. Using in vitro dephosphorylation assays, we explored the regulatory mechanism between PTEN and the key regulator of lysosome biogenesis, transcription factor EB (TFEB). Using the migration assays, invasion assays, and trans-splenic liver metastasis mouse models, we evaluated the function of PTEN deficiency, TFEB-mediated lysosome biogenesis, and exosome secretion on tumor metastasis. Moreover, we investigated the clinical significance of PTEN expression and exosome secretion by retrospective analysis. RESULTS: PTEN facilitated lysosome biogenesis and acidification through its protein phosphatase activity to dephosphorylate TFEB at Ser211. Notably, PTEN deficiency increased exosome secretion by reducing lysosome-mediated degradation of multi-vesicular bodies, which further facilitated the proliferation and invasion of CCA. TFEB agonist curcumin analog C1 restrained the metastatic phenotype caused by PTEN deficiency in mouse models, and we highlighted the correlation between PTEN deficiency and exosome secretion in clinical cohorts. CONCLUSIONS: In CCA, PTEN deficiency impairs lysosome biogenesis to facilitate exosome secretion and cancer metastasis in a TFEB phosphorylation-dependent manner.
Subject(s)
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Cholangiocarcinoma , Exosomes , PTEN Phosphohydrolase , Animals , Humans , Mice , Autophagy , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Cholangiocarcinoma/metabolism , Disease Models, Animal , Exosomes/metabolism , Lysosomes/physiology , Proteasome Endopeptidase Complex , PTEN Phosphohydrolase/metabolism , Retrospective StudiesABSTRACT
Bacterial infection and massive blood loss are major challenges for global public health. Herein, a series of tannic acid encapsulated O-carboxymethyl chitosan (CMC) based hydrogels were prepared using a facile approach for both hemorrhage control and effective anti-bacterium. The results indicated that the tannic acid-cosslinked CMC hydrogels had excellent mechanical property, swelling ability as well as great cytocompatibility. Comparably, with increasing tannic acid loading, the bleeding control and antibacterial performance against both E. coli and S. aureus were improved simultaneously, especially for the 5% tannic acid-cosslinked CMC hydrogel. Moreover, the prepared CMC hydrogel loading with tannic acid could induce hemocytes and platelets aggregation, promote the blood clotting and achieve bleeding control in vivo due to the interconnected fibrous web structure and the chemical activation (the phenol group of tannic acid). Thus, the resultant CMC hydrogel enabled the maintenance of high bioavailability of tannic acid and synchronization with the interconnected fibrous structure of CMC hydrogels, which was expected to be a promising candidate for robust and safe hemostatic dressings.
Subject(s)
Chitosan , Staphylococcus aureus , Hydrogels/pharmacology , Hydrogels/chemistry , Escherichia coli , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Chitosan/chemistry , Tannins/pharmacology , Tannins/chemistry , HemostasisABSTRACT
Cholangiocarcinoma (CCA) is an epithelial malignancy with a dismal prognosis owing to limited treatment options. Here, we identified several compound candidates against CCA using a high-throughput drug screen with approved or emerging oncology drugs, among which kinesin spindle protein (KSP) inhibitors showed potent cytotoxic effects on CCA cells. Treatment with KSP inhibitors SB743921 and ARRY520 caused significant tumor suppression in CCA xenograft models in vivo. Mechanistically, KSP inhibitors led to the formation of abnormal monopolar spindles, which further resulted in the mitotic arrest and cell death of CCA cells both in vivo and in vitro. KEGG pathway analysis of transcriptional data confirmed this finding. Moreover, our clinical data as well as the TCGA database showed KIF11 expression was abundant in most CCA tumor specimens and associated with poor outcomes of CCA patients. Our results demonstrate that the therapeutic regimen of KSP inhibitors could be a promising treatment strategy in CCA.
Subject(s)
Antineoplastic Agents , Bile Duct Neoplasms , Cholangiocarcinoma , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Bile Duct Neoplasms/drug therapy , Bile Duct Neoplasms/genetics , Bile Ducts, Intrahepatic , Cholangiocarcinoma/drug therapy , Cholangiocarcinoma/genetics , Humans , Kinesins/geneticsABSTRACT
Oncogene-derived metabolic reprogramming is important for anabolic growth of cancer cells, which is now considered to be not simply rely on glycolysis. Pentose phosphate pathway and tricarboxylic acid cycle also play pivotal roles in helping cancer cells to meet their anabolic and energy demands. The present work focused on gankyrin, a relatively specific oncogene in hepatocellular carcinoma (HCC), and its impact on glycolysis and mitochondrial homeostasis. Metabolomics, RNA-seq analysis, and subsequent conjoint analysis illustrated that gankyrin regulated the pentose phosphate pathway (PPP), tricarboxylic acid (TCA) cycle, and mitochondrial function and homeostasis, which play pivotal roles in tumor development. Mechanistically, gankyrin was found to modulate HCC metabolic reprogramming via TIGAR. Gankyrin positively regulated the transcription of TIGAR through Nrf2, which bound to the antioxidant response elements (AREs) in the promoter of TIGAR. Interestingly, TIGAR feedback regulated the transcription of Nrf2 and subsequently gankyrin by promoting nuclear importation of PGC1α. The loop between gankyrin, Nrf2, and TIGAR accelerated glucose metabolism toward the PPP and TCA cycle, which provided vital building blocks, such as NADPH, ATP, and ribose of tumor and further facilitated the progression of HCC.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Humans , Carcinoma, Hepatocellular/pathology , Citric Acid Cycle , Liver Neoplasms/pathology , Glycolysis , Glucose/metabolismABSTRACT
NRF2 is the master transcriptional activator of cytoprotective genes and Kelch-like ECH-associated protein 1 (Keap1), a biosensor for electrophiles and oxidation, promotes NRF2 degradation in unstressed conditions. SQSTM1/p62, an oncogenic protein aberrantly accumulated in hepatocellular carcinoma (HCC), binds and sequestrates Keap1, leading to the prevention of NRF2 degradation. Here, we show that p15INK4b-related sequence/regulation of nuclear pre-mRNA domain-containing protein 1A (RPRD1A) is highly expressed in HCC tumors and correlated with aggressive clinicopathological features. RPRD1A competitively interacts with TRIM21, an E3 ubiquitin ligase of p62, resulting in the decrease of p62 ubiquitination and the increased sequestration for Keap1. Therefore, RPRD1A enhances the nuclear translocation of NRF2, which induces gene expression for counteracting oxidative stress, maintaining cancer cells survival, and promoting HCC development. Moreover, disturbing the redox homeostasis of cancer cells by genetic knockdown of RPRD1A sensitizes cancer cells to platinum-induced cell death. Our study reveals RPRD1A is involved in the oxidative stress defense program and highlights the therapeutic benefits of targeting pathways that support antioxidation.
Subject(s)
Carcinoma, Hepatocellular/genetics , Liver Neoplasms/genetics , RNA-Binding Proteins/metabolism , Carcinoma, Hepatocellular/pathology , Cell Cycle Proteins , Disease Progression , Female , Humans , Liver Neoplasms/pathology , Male , Middle Aged , NF-E2-Related Factor 2/metabolism , Repressor Proteins , Ribonucleoproteins , Signal TransductionABSTRACT
To ensure safety, it is necessary to test the connected vehicle (CV) technology before application. The goal of this study is to provide a case reference for the testing of the connected vehicle technology. The connected vehicle technology test platform is built based on the driving simulator. Taking fog zone, tunnel zone, and work zone as analysis cases, drivers were invited to participate in driving simulation experiments, related data was collected, and the impact of connected vehicle technology on driving behavior and safety was analyzed. The results of the fog zone imply that drivers have a high degree of compliance with the connected vehicle technology. However, it also increases the visual workload of drivers to a certain extent. The results of the tunnel zone indicate that the connected vehicle technology can enhance driving safety by enabling drivers to remain cautious. The results of the work zone demonstrate that the connected vehicle technology is able to promote drivers' ability of controlling speed and lane-changing. Overall, the results show that the connected vehicle technology has a positive effect on enhancing driving behavior and safety. The research framework and the development of the connected vehicle technology test platform based on the driving simulator given in the paper are dynamic and reproducible, which provides a reference for researchers in related fields, and the case analysis in this paper enriches the research of connected vehicle technology.
Subject(s)
Accidents, Traffic , Automobile Driving , Accidents, Traffic/prevention & control , Computer Simulation , Humans , TechnologyABSTRACT
Puerarin has the anti-Alzheimer's disease (AD) activity,which can reverse nerve injury induced by Aßand inhibit neuronal apoptosis.However,its potential pharmacodynamic mechanism still needs to be further researched.The occurrence and development of AD is due to the change of multiple metabolic links in the body,which leads to the destruction of balance.Puerarin may act on multiple targets and multiple metabolic processes to achieve therapeutic purposes.Quantitative proteomic analysis provides a new choice to understand the mechanism as completely as possible.This research adopted SH-SY5Y cells induced by Aß_(1-42)to establish AD cell model,and Aßimmunofluorescence detection showed that Aßdecreased significantly after puerarin intervention.The mechanism of puerarin reversing SH-SY5Y cell injured by Aß_(1-42)was further explored by using label-free non-labeled quantitative technology and Western blot detection based on bioinformatics analysis result.The results showed that most of the differential proteins were related to biological processes such as cellular component organization or biogenesis,cellular component organization and cellular component biogenesis,and they mainly participated in the top ten pathways of P value such as pathogenic Escherichia coli infection,m TOR signaling pathway,regulation of autophagy,regulation of actin cytoskeleton,spliceosome,hepatocellular carcinoma,tight junction,non-small cell lung cancer,apoptosis and gap junction.Annexin V/PI flow cytometry and TUNEL were used to detect apoptosis,and the results showed that Aßdecreased significantly and the rate of apoptosis decreased significantly after puerarin intervention.Western blot analysis found that the protein expression level of autophagy related protein LC3â ¡was up-regulated after Aßinduction,and the degree of this up-regulation was further enhanced in puerarin intervention group.The trend of the ratio of LC3â ¡/LC3â among groups was the same as the protein expression level of LC3â ¡ï¼the protein expression level of p62 in the control group,AD model group and puerarin intervention group decreased successively.Protein interaction network analysis showed that CAP1 was correlated with TUBA1B,HSP90AB2P,DNM1L,TUBA1A and ERK1/2,and the correlation between CAP1 and ERK1/2 was the highest among them.Western blot showed that the expressions of p-ERK1/2,Bax and CAP1 were significantly down-regulated and the protein expression level of Bcl-2 was significantly up-regulated after puerarin intervention.Therefore,puerarin might improve the SH-SY5Y cells injured by Aß_(1-42)through the interaction of multiple biological processes and pathways in cells multiple locations,and CAP1 might play an important role among them.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Isoflavones , Lung Neoplasms , Amyloid beta-Peptides , Apoptosis , Cell Line, Tumor , Humans , Isoflavones/pharmacology , ProteomicsABSTRACT
Synthetic gene circuits are made of DNA sequences, referred to as transcription units, that communicate by exchanging proteins or RNA molecules. Proteins are, mostly, transcription factors that bind promoter sequences to modulate the expression of other molecules. Promoters are, therefore, key components in genetic circuits. In this review, we focus our attention on the construction of artificial promoters for the yeast S. cerevisiae, a popular chassis for gene circuits. We describe the initial techniques and achievements in promoter engineering that predated the start of the Synthetic Biology epoch of about 20 years. We present the main applications of synthetic promoters built via different methods and discuss the latest innovations in the wet-lab engineering of novel promoter sequences.
ABSTRACT
Promoters are fundamental components of synthetic gene circuits. They are DNA segments where transcription initiation takes place. New constitutive and regulated promoters are constantly engineered in order to meet the requirements for protein and RNA expression into different genetic networks. In this work, we constructed and optimized new synthetic constitutive promoters for the yeast Saccharomyces cerevisiae. We started from foreign (e.g., viral) core promoters as templates. They are, usually, unfunctional in yeast but can be activated by extending them with a short sequence, from the CYC1 promoter, containing various transcription start sites (TSSs). Transcription was modulated by mutating the TATA box composition and varying its distance from the TSS. We found that gene expression is maximized when the TATA box has the form TATAAAA or TATATAA and lies between 30 and 70 nucleotides upstream of the TSS. Core promoters were turned into stronger promoters via the addition of a short UAS. In particular, the 40 nt bipartite UAS from the GPD promoter can enhance protein synthesis considerably when placed 150 nt upstream of the TATA box. Overall, we extended the pool of S. cerevisiae promoters with 59 new samples, the strongest overcoming the native TEF2 promoter.
Subject(s)
Genetic Engineering , Promoter Regions, Genetic , Saccharomyces cerevisiae/genetics , 5' Untranslated Regions , Base Sequence , Gene Expression Regulation, Fungal , Genes, Reporter , Mutation , TATA Box , Transcription Initiation SiteABSTRACT
Gallbladder cancer (GBC) is an aggressive malignancy of biliary tract with poor prognosis. Although several studies have shown the frequency of relevant genetic alterations, there are few genetic models or translational studies that really benefit for GBC treatment in the era of precision medicine. By targeted sequencing and immunohistochemistry staining, we identified that phosphate and tension homology deleted on chromosome ten (PTEN) was frequently altered in GBC specimens, and loss of PTEN expression was independently correlated with poor survival outcomes. Further drug screening assays revealed proteasome inhibitor bortezomib as a promising agent for GBC treatment, and knockdown of PTEN increased bortezomib efficacy both in vivo and in vitro. Therapeutic evaluation of patient derived xenografts (PDXs) strongly supported the utilization of bortezomib in PTEN deficient GBC. Mechanically, functional PTEN inhibited ARE-dependent transcriptional activity, the same machinery regulating the transcription of proteasome subunits, thus PTEN suppressed proteasome activity and bortezomib sensitivity. Through siRNA screening, we identified the ARE-related transcriptional suppressor BACH1 involved in PTEN-mediated proteasome inhibition and regulated by PTEN-AKT1 axis. In summary, our study indicates that proteasome activity represents a prime therapeutic target in PTEN-deficient GBC tumors, which is worthy of further clinical validation.
Subject(s)
Bortezomib/administration & dosage , Down-Regulation , Gallbladder Neoplasms/drug therapy , Mutation , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , Adult , Aged , Animals , Bortezomib/pharmacology , Cell Line, Tumor , Female , Gallbladder Neoplasms/genetics , Gallbladder Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , HEK293 Cells , Humans , Male , Mice , Middle Aged , Proteasome Endopeptidase Complex/metabolism , Survival Analysis , Xenograft Model Antitumor Assays , Young AdultABSTRACT
BACKGROUND AND AIMS: Cancer cell survival depends on the balance between reactive oxygen species production and scavenging, which is regulated primarily by NRF2 during tumorigenesis. Here, we demonstrate that deletion of RBP5-mediating protein (RMP) in an autonomous mouse model of intrahepatic cholangiocarcinoma (ICC) delays tumor progression. APPROACH AND RESULTS: RMP-overexpressing tumor cells exhibited enhanced tolerance to oxidative stress and apoptosis. Mechanistically, RMP competes with NRF2 for binding to the Kelch domain of KEAP1 (Kelch-like ECH-associated protein 1) through the E**E motif, leading to decreased NRF2 degradation via ubiquitination, thus increasing NRF2 nuclear translocation and downstream transactivation of antioxidant genes. This RMP-KEAP1-NRF2 axis promotes ICC tumorigenesis, metastasis, and drug resistance. Consistent with these findings, the RMP level in human ICC is positively correlated with the protein level of NRF2 and is associated with poor prognosis. CONCLUSION: These findings reveal that RMP is involved in the oxidative stress defense program and could be exploited for targeted cancer therapies.
Subject(s)
Carcinogenesis , Cholangiocarcinoma/metabolism , Kelch-Like ECH-Associated Protein 1/metabolism , NF-E2-Related Factor 2/metabolism , Repressor Proteins/metabolism , Animals , Apoptosis , Carcinogenesis/drug effects , Carcinogenesis/metabolism , Cell Line , Cell Transformation, Neoplastic/metabolism , Cholangiocarcinoma/pathology , Drug Resistance, Neoplasm , Humans , Mice , Oxidative StressABSTRACT
Neuroinflammation is closely related with the pathogenesis and progress of neurodegenerative diseases including Alzheimer's disease (AD). Loganin, an iridoid glycoside obtained from traditional Chinese medicine Cornus officinalis, has properties of inhibiting inflammation and improving memory. The present study was aimed to investigate effects of loganin on Aß-induced inflammation and to explore the underlying mechanisms. BV-2 microglia cells were stimulated with 10 µM Aß1-42 for 24 h to induce inflammatory damage. According to results of CCK-8 assay, the doses of loganin in present work were 10 and 30 µM. We found that treatment with loganin could inhibit Aß1-42 -induced microglia activation. Furthermore, loganin treatment prevented the over-production of Tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6), Macrophage Chemotactic Protein 1(MCP-1), Nitric oxide (NO), Prostaglandin E2 (PGE2) and the up-regulation of inducible nitric oxide synthase (iNOS) and Cyclooxygenase 2 (COX-2) in Aß1-42 -stimulated BV-2 cells. Results from Western blots demonstrated that loganin inhibited Aß1-42 -induced elevation in Toll-like receptor 4 (TLR4), Myeloid Differentiation Factor 88 (MyD88) and TNF receptor-associated factor 6 (TRAF6). Loganin treatment also attenuated the increased phosphorylation level of IRAK4 caused by Aß1-42 . Additionally, loganin alleviated nuclear translocation of NF-κB p65 subunit in Aß1-42 -stimulated BV-2 cells, and this phenomenon could be reversed by TLR4 agonist LPS. Further, the anti-inflammatory effects of loganin were attenuated when TLR4 signaling pathway was re-activated by LPS. Taken together, our data indicated that loganin could attenuate inflammatory response induced by Aß in BV-2 microglia cells, partially through deactivating the TLR4/TRAF6/NF-κB axis.