ABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Signal transducer and activator of transcription 3 (STAT3) has a critical role in regulating cell fate, inflammation and immunity1,2. Cytokines and growth factors activate STAT3 through kinase-mediated tyrosine phosphorylation and dimerization3,4. It remains unknown whether other factors promote STAT3 activation through different mechanisms. Here we show that STAT3 is post-translationally S-palmitoylated at the SRC homology 2 (SH2) domain, which promotes the dimerization and transcriptional activation of STAT3. Fatty acids can directly activate STAT3 by enhancing its palmitoylation, in synergy with cytokine stimulation. We further identified ZDHHC19 as a palmitoyl acyltransferase that regulates STAT3. Cytokine stimulation increases STAT3 palmitoylation by promoting the association between ZDHHC19 and STAT3, which is mediated by the SH3 domain of GRB2. Silencing ZDHHC19 blocks STAT3 palmitoylation and dimerization, and impairs the cytokine- and fatty-acid-induced activation of STAT3. ZDHHC19 is frequently amplified in multiple human cancers, including in 39% of lung squamous cell carcinomas. High levels of ZDHHC19 correlate with high levels of nuclear STAT3 in patient samples. In addition, knockout of ZDHHC19 in lung squamous cell carcinoma cells significantly blocks STAT3 activity, and inhibits the fatty-acid-induced formation of tumour spheres as well as tumorigenesis induced by high-fat diets in an in vivo mouse model. Our studies reveal that fatty-acid- and ZDHHC19-mediated palmitoylation are signals that regulate STAT3, which provides evidence linking the deregulation of palmitoylation to inflammation and cancer.
Subject(s)
Acyltransferases/metabolism , Fatty Acids/metabolism , Lipoylation , Lung Neoplasms/metabolism , STAT3 Transcription Factor/metabolism , Acyltransferases/antagonists & inhibitors , Acyltransferases/chemistry , Acyltransferases/deficiency , Animals , Carcinogenesis , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Conserved Sequence , Cysteine/metabolism , Disease Models, Animal , Heterografts , Humans , Inflammation/metabolism , Inflammation/pathology , Interferon-gamma/immunology , Interferon-gamma/metabolism , Interleukin-6/immunology , Interleukin-6/metabolism , Lung Neoplasms/pathology , Mice , Mice, SCID , Neoplasm Transplantation , Phosphorylation , Protein Multimerization , STAT3 Transcription Factor/chemistry , Signal Transduction , src Homology DomainsABSTRACT
BACKGROUND: Although statins are a class I recommendation for prevention of atherosclerotic cardiovascular disease and its complications, their use is suboptimal. Differential underuse may mediate disparities in cardiovascular health for systematically marginalized persons. OBJECTIVE: To estimate disparities in statin use by race-ethnicity-gender and to determine whether these potential disparities are explained by medical appropriateness of therapy and structural factors. DESIGN: Cross-sectional analysis. SETTING: National Health and Nutrition Examination Survey from 2015 to 2020. PARTICIPANTS: Persons eligible for statin therapy based on 2013 and 2018 American College of Cardiology/American Heart Association blood cholesterol guidelines. MEASUREMENTS: The independent variable was race-ethnicity-gender. The outcome of interest was use of a statin. Using the Institute of Medicine framework for examining unequal treatment, we calculated adjusted prevalence ratios (aPRs) to estimate disparities in statin use adjusted for age, disease severity, access to health care, and socioeconomic status relative to non-Hispanic White men. RESULTS: For primary prevention, we identified a lower prevalence of statin use that was not explained by measurable differences in disease severity or structural factors among non-Hispanic Black men (aPR, 0.73 [95% CI, 0.59 to 0.88]) and non-Mexican Hispanic women (aPR, 0.74 [CI, 0.53 to 0.95]). For secondary prevention, we identified a lower prevalence of statin use that was not explained by measurable differences in disease severity or structural factors for non-Hispanic Black men (aPR, 0.81 [CI, 0.64 to 0.97]), other/multiracial men (aPR, 0.58 [CI, 0.20 to 0.97]), Mexican American women (aPR, 0.36 [CI, 0.10 to 0.61]), non-Mexican Hispanic women (aPR, 0.57 [CI, 0.33 to 0.82), non-Hispanic White women (aPR, 0.69 [CI, 0.56 to 0.83]), and non-Hispanic Black women (aPR, 0.75 [CI, 0.57 to 0.92]). LIMITATION: Cross-sectional data; lack of geographic, language, or statin-dose data. CONCLUSION: Statin use disparities for several race-ethnicity-gender groups are not explained by measurable differences in medical appropriateness of therapy, access to health care, and socioeconomic status. These residual disparities may be partially mediated by unobserved processes that contribute to health inequity, including bias, stereotyping, and mistrust. PRIMARY FUNDING SOURCE: National Institutes of Health.
Subject(s)
Atherosclerosis , Cardiovascular Diseases , Healthcare Disparities , Hydroxymethylglutaryl-CoA Reductase Inhibitors , Adult , Female , Humans , Male , Atherosclerosis/drug therapy , Atherosclerosis/epidemiology , Atherosclerosis/ethnology , Atherosclerosis/prevention & control , Black or African American , Cardiovascular Diseases/drug therapy , Cross-Sectional Studies , Ethnicity , Hispanic or Latino , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Nutrition Surveys , United States/epidemiology , White , Healthcare Disparities/ethnology , Healthcare Disparities/statistics & numerical dataABSTRACT
Interleukin 6 (IL-6) is known to regulate the CD4 T cell function by inducing gene expression of a number of cytokines through activation of Stat3 transcription factor. Here, we reveal that IL-6 strengthens the mechanics of CD4 T cells. The presence of IL-6 during activation of mouse and human CD4 T cells enhances their motility (random walk and exploratory spread), resulting in an increase in travel distance and higher velocity. This is an intrinsic effect of IL-6 on CD4 T-cell fitness that involves an increase in mitochondrial Ca2+ Although Stat3 transcriptional activity is dispensable for this process, IL-6 uses mitochondrial Stat3 to enhance mitochondrial Ca2+-mediated motility of CD4 T cells. Thus, through a noncanonical pathway, IL-6 can improve competitive fitness of CD4 T cells by facilitating cell motility. These results could lead to alternative therapeutic strategies for inflammatory diseases in which IL-6 plays a pathogenic role.
Subject(s)
Cell Movement/physiology , Interleukin-6/metabolism , STAT3 Transcription Factor/metabolism , Animals , CD4-Positive T-Lymphocytes/metabolism , Calcium/metabolism , Cell Differentiation/drug effects , Cell Movement/drug effects , Cytokines/metabolism , Female , Lymphocyte Activation/drug effects , Male , Mice , Mice, Inbred C57BL , Mitochondria/metabolism , STAT3 Transcription Factor/physiology , Signal Transduction/drug effectsABSTRACT
Cancer is often characterized by aberrant gene expression patterns caused by the inappropriate activation of transcription factors. Signal transducer and activator of transcription 3 (STAT3) is a key transcriptional regulator of many protumorigenic processes and is persistently activated in many types of human cancer. However, like many transcription factors, STAT3 has proven difficult to target clinically. To address this unmet clinical need, we previously developed a cell-based assay of STAT3 transcriptional activity and performed an unbiased and high-throughput screen of small molecules known to be biologically active in humans. We identified the antimicrobial drug pyrimethamine as a novel and specific inhibitor of STAT3 transcriptional activity. Here, we show that pyrimethamine does not significantly affect STAT3 phosphorylation, nuclear translocation, or DNA binding at concentrations sufficient to inhibit STAT3 transcriptional activity, suggesting a potentially novel mechanism of inhibition. To identify the direct molecular target of pyrimethamine and further elucidate the mechanism of action, we used a new quantitative proteome profiling approach called proteome integral solubility alteration coupled with a metabolomic analysis. We identified human dihydrofolate reductase as a target of pyrimethamine and demonstrated that the STAT3-inhibitory effects of pyrimethamine are the result of a deficiency in reduced folate downstream of dihydrofolate reductase inhibition, implicating folate metabolism in the regulation of STAT3 transcriptional activity. This study reveals a previously unknown regulatory node of the STAT3 pathway that may be important for the development of novel strategies to treat STAT3-driven cancers.
Subject(s)
Anti-Infective Agents , Pyrimethamine , STAT3 Transcription Factor , Tetrahydrofolate Dehydrogenase , Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Cell Line, Tumor , Folic Acid/metabolism , Humans , Proteome/metabolism , STAT3 Transcription Factor/antagonists & inhibitors , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , Tetrahydrofolate Dehydrogenase/genetics , Tetrahydrofolate Dehydrogenase/metabolismABSTRACT
BACKGROUND: Higher circulating prolactin has been associated with increased breast cancer risk. Prolactin binding to the prolactin receptor (PRLR) can activate the transcription factor STAT5, thus, we examined the association between plasma prolactin and breast cancer risk by tumor expression of PRLR, STAT5, and the upstream kinase JAK2. METHODS: Using data from 745 cases and 2454 matched controls in the Nurses' Health Study, we conducted polytomous logistic regression to examine the association between prolactin (> 11 ng/mL vs. ≤ 11 ng/mL) measured within 10 years of diagnosis and breast cancer risk by PRLR (nuclear [N], cytoplasmic [C]), phosphorylated STAT5 (pSTAT5; N, C), and phosphorylated JAK2 (pJAK2; C) tumor expression. Analyses were conducted separately in premenopausal (n = 168 cases, 765 controls) and postmenopausal women (n = 577 cases, 1689 controls). RESULTS: In premenopausal women, prolactin levels > 11 ng/mL were positively associated with risk of tumors positive for pSTAT5-N (OR 2.30, 95% CI 1.02-5.22) and pSTAT5-C (OR 1.64, 95% CI 1.01-2.65), but not tumors that were negative for these markers (OR 0.98, 95% CI 0.65-1.46 and OR 0.73, 95% CI 0.43-1.25; p-heterogeneity = 0.06 and 0.02, respectively). This was stronger when tumors were positive for both pSTAT5-N and pSTAT5-C (OR 2.88, 95% CI 1.14-7.25). No association was observed for PRLR or pJAK2 (positive or negative) and breast cancer risk among premenopausal women. Among postmenopausal women, plasma prolactin levels were positively associated with breast cancer risk irrespective of PRLR, pSTAT5, or pJAK2 expression (all p-heterogeneity ≥ 0.21). CONCLUSION: We did not observe clear differences in the association between plasma prolactin and breast cancer risk by tumor expression of PRLR or pJAK2, although associations for premenopausal women were observed for pSTAT5 positive tumors only. While additional studies are needed, this suggests that prolactin may act on human breast tumor development through alternative pathways.
Subject(s)
Breast Neoplasms , Prolactin , Female , Humans , Breast Neoplasms/epidemiology , Prolactin/blood , STAT5 Transcription FactorABSTRACT
Leukemias bearing fusions of the AF10/MLLT10 gene are associated with poor prognosis, and therapies targeting these fusion proteins (FPs) are lacking. To understand mechanisms underlying AF10 fusion-mediated leukemogenesis, we generated inducible mouse models of acute myeloid leukemia (AML) driven by the most common AF10 FPs, PICALM/CALM-AF10 and KMT2A/MLL-AF10, and performed comprehensive characterization of the disease using transcriptomic, epigenomic, proteomic, and functional genomic approaches. Our studies provide a detailed map of gene networks and protein interactors associated with key AF10 fusions involved in leukemia. Specifically, we report that AF10 fusions activate a cascade of JAK/STAT-mediated inflammatory signaling through direct recruitment of JAK1 kinase. Inhibition of the JAK/STAT signaling by genetic Jak1 deletion or through pharmacological JAK/STAT inhibition elicited potent antioncogenic effects in mouse and human models of AF10 fusion AML. Collectively, our study identifies JAK1 as a tractable therapeutic target in AF10-rearranged leukemias.
Subject(s)
Carcinogenesis , Gene Rearrangement , Janus Kinases , MAP Kinase Signaling System/genetics , Neoplasm Proteins , STAT Transcription Factors , Transcription Factors , Animals , Carcinogenesis/genetics , Carcinogenesis/metabolism , Female , Humans , Janus Kinases/genetics , Janus Kinases/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , STAT Transcription Factors/genetics , STAT Transcription Factors/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , U937 CellsABSTRACT
In response to tumor necrosis factor (TNF), NF-κB enters the nucleus and promotes inflammatory and stress-responsive gene transcription. Because NF-κB deregulation is associated with disease, one might expect strict control of NF-κB localization. However, nuclear NF-κB levels exhibit considerable cell-to-cell variability, even in unstimulated cells. To resolve this paradox and determine how transcription-inducing signals are encoded, we quantified single-cell NF-κB translocation dynamics and transcription in the same cells. We show that TNF-induced transcription correlates best with fold change in nuclear NF-κB, not absolute nuclear NF-κB abundance. Using computational modeling, we find that an incoherent feedforward loop, from competition for binding to κB motifs, could provide memory of the preligand state necessary for fold-change detection. Experimentally, we observed three gene-specific transcriptional patterns that our model recapitulates by modulating competition strength alone. Fold-change detection buffers against stochastic variation in signaling molecules and explains how cells tolerate variability in NF-κB abundance and localization.
Subject(s)
Models, Statistical , NF-kappa B/metabolism , RNA, Messenger/metabolism , Transcription, Genetic , Tumor Necrosis Factor-alpha/metabolism , Binding Sites , Binding, Competitive , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , Computer Simulation , Gene Expression Regulation , HeLa Cells , Humans , Ligands , Molecular Imaging , NF-kappa B/genetics , Protein Binding , Protein Interaction Domains and Motifs , Protein Transport , RNA, Messenger/genetics , Signal Transduction , Single-Cell Analysis , Tumor Necrosis Factor-alpha/geneticsABSTRACT
BACKGROUND: Malignant pleural mesothelioma (MPM) is a highly aggressive cancer with a dismal prognosis. There is increasing interest in targeting chromatin regulatory pathways in difficult-to-treat cancers. In preliminary studies, we found that KDM4A (lysine-specific histone demethylase 4) was overexpressed in MPM. METHODS: KDM4A protein expression was determined by immunohistochemistry or immunoblotting. Functional inhibition of KDM4A by targeted knockdown and small molecule drugs was correlated to cell growth using cell lines and a xenograft mouse model. Gene expression profiling was performed to identify KDM4A-dependent signature pathways. RESULTS: Levels of KDM4A were found to be significantly elevated in MPM patients compared to normal mesothelial tissue. Inhibiting the enzyme activity efficiently reduced cell growth in vitro and reduced tumour growth in vivo. KDM4A inhibitor-induced apoptosis was further enhanced by the BH3 mimetic navitoclax. KDM4A expression was associated with pathways involved in cell growth and DNA repair. Interestingly, inhibitors of the DNA damage and replication checkpoint regulators CHK1 (prexasertib) and WEE1 (adavosertib) within the DNA double-strand break repair pathway, cooperated in the inhibition of cell growth. CONCLUSIONS: The results establish a novel and essential role for KDM4A in growth in preclinical models of MPM and identify potential therapeutic approaches to target KDM4A-dependent vulnerabilities.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Jumonji Domain-Containing Histone Demethylases/genetics , Jumonji Domain-Containing Histone Demethylases/metabolism , Mesothelioma, Malignant/pathology , Up-Regulation , Aniline Compounds/administration & dosage , Aniline Compounds/pharmacology , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , Humans , Mesothelioma, Malignant/drug therapy , Mesothelioma, Malignant/genetics , Mesothelioma, Malignant/metabolism , Mice , Pyrazines/administration & dosage , Pyrazines/pharmacology , Pyrazoles/administration & dosage , Pyrazoles/pharmacology , Pyrimidinones/administration & dosage , Pyrimidinones/pharmacology , Sulfonamides/administration & dosage , Sulfonamides/pharmacology , Up-Regulation/drug effects , Xenograft Model Antitumor AssaysABSTRACT
The signal transducer and activator of transcription 3 (STAT3) protein is constitutively activated in several cancers. STAT3 activity can be blocked by inhibiting its Src Homology 2 (SH2) domain, but phosphotyrosine and its isosteres have poor bioavailability. In this work, we develop peptide-based inhibitors of STAT3-SH2 by combining chemical strategies that have proven effective for targeting other SH2 domains. These strategies include a STAT3-specific selectivity sequence, non-hydrolyzable phosphotyrosine isosteres, and a high-efficiency cell-penetrating peptide. Peptides that combined these three strategies had substantial biological stability and cytosolic delivery, as measured using highly quantitative cell-based assays. However, these peptides did not inhibit STAT3 activity in cells. By comparing in vitro binding affinity, cell penetration, and proteolytic stability, this work explores the delicate balance of factors that contribute to biological activity for peptidic inhibitors of STAT3.
Subject(s)
Peptides/metabolism , STAT3 Transcription Factor/antagonists & inhibitors , Alanine/analogs & derivatives , Alanine/chemistry , Cell Line, Tumor , Cell Survival/drug effects , Cytosol/metabolism , Humans , Naphthalenes/chemistry , Peptides/chemical synthesis , Peptides/chemistry , Peptides/pharmacology , Peptides, Cyclic/chemistry , Protein Binding , Protein Stability , STAT3 Transcription Factor/chemistry , STAT3 Transcription Factor/metabolism , src Homology DomainsABSTRACT
The transcription factor signal activator and transducer or transcription (STAT3), which regulates genes controlling proliferation, survival, and invasion, is activated inappropriately in many human cancers, including breast cancer. Activation of STAT3 can lead to both malignant cellular behavior and suppression of immune cell function in the tumor microenvironment. Through a chemical-biology screen, pyrimethamine (PYR), an FDA approved anti-microbial drug, was identified as an inhibitor of STAT3 function at concentrations known to be achieved safely in humans. We report that PYR shows therapeutic activity in two independent mouse models of breast cancer, with both direct tumor inhibitory and immune stimulatory effects. PYR-inhibited STAT3 activity in TUBO and TM40D-MB metastatic breast cancer cells in vitro and inhibited tumor cell proliferation and invasion into Matrigel basement membrane matrix. In tumor-transplanted mice, PYR had both direct and indirect tumor inhibitory effects. Tumor-bearing mice treated with PYR showed reduced STAT3 activation in tumor cells, attenuated tumor growth, and reduced tumor-associated inflammation. In addition, expression of Lamp1 by tumor infiltrating CD8+ T cells was elevated, indicating enhanced release of cytotoxic granules. These findings suggest that PYR may have beneficial effects in the treatment of breast cancer.
Subject(s)
Adjuvants, Immunologic/therapeutic use , Anti-Infective Agents/therapeutic use , Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy , CD8-Positive T-Lymphocytes/immunology , Pyrimethamine/therapeutic use , STAT3 Transcription Factor/metabolism , Animals , Cell Line, Tumor , Cell Proliferation , Cytotoxicity, Immunologic , Disease Models, Animal , Female , Humans , Lysosomal Membrane Proteins/genetics , Lysosomal Membrane Proteins/metabolism , Mice , Mice, Inbred BALB C , Neoplasm Transplantation , Pyrimethamine/pharmacology , STAT3 Transcription Factor/antagonists & inhibitors , Tumor Escape , United StatesABSTRACT
The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) is frequently activated inappropriately in a wide range of hematological and solid cancers, but clinically available therapies targeting STAT3 are lacking. Using a computational strategy to identify compounds opposing the gene expression signature of STAT3, we discovered atovaquone (Mepron), an antimicrobial approved by the US Food and Drug Administration, to be a potent STAT3 inhibitor. We show that, at drug concentrations routinely achieved clinically in human plasma, atovaquone inhibits STAT3 phosphorylation, the expression of STAT3 target genes, and the viability of STAT3-dependent hematological cancer cells. These effects were also observed with atovaquone treatment of primary blasts isolated from patients with acute myelogenous leukemia or acute lymphocytic leukemia. Atovaquone is not a kinase inhibitor but instead rapidly and specifically downregulates cell-surface expression of glycoprotein 130, which is required for STAT3 activation in multiple contexts. The administration of oral atovaquone to mice inhibited tumor growth and prolonged survival in a murine model of multiple myeloma. Finally, in patients with acute myelogenous leukemia treated with hematopoietic stem cell transplantation, extended use of atovaquone for Pneumocystis prophylaxis was associated with improved relapse-free survival. These findings establish atovaquone as a novel, clinically accessible STAT3 inhibitor with evidence of anticancer efficacy in both animal models and humans.
Subject(s)
Antineoplastic Agents/pharmacology , Atovaquone/pharmacology , Drug Discovery , Gene Expression Regulation, Neoplastic/drug effects , STAT3 Transcription Factor/antagonists & inhibitors , Animals , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Apoptosis/genetics , Atovaquone/chemistry , Atovaquone/therapeutic use , Cell Line, Tumor , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Survival/drug effects , Cell Survival/genetics , Cytokine Receptor gp130/metabolism , Disease Models, Animal , Down-Regulation/drug effects , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Mice , Phosphorylation/drug effects , Phosphotyrosine/metabolism , STAT3 Transcription Factor/metabolism , Treatment OutcomeABSTRACT
Maturation of dendritic cells (DCs) is required to induce T cell immunity, whereas immature DCs can induce immune tolerance. Although the transcription factor STAT5 is suggested to participate in DC maturation, its role in this process remains unclear. In this study, we investigated the effect of STAT5 inhibition on LPS-induced maturation of human monocyte-derived DCs (Mo-DCs). We inhibited STAT5 by treating Mo-DCs with JQ1, a selective inhibitor of BET epigenetic readers, which can suppress STAT5 function. We found that JQ1 inhibits LPS-induced STAT5 phosphorylation and nuclear accumulation, thereby attenuating its transcriptional activity in Mo-DCs. The diminished STAT5 activity results in impaired maturation of Mo-DCs, as indicated by defective upregulation of costimulatory molecules and CD83, as well as reduced secretion of IL-12p70. Expression of constitutively activated STAT5 in JQ1-treated Mo-DCs overcomes the effects of JQ1 and enhances the expression of CD86, CD83, and IL-12. The activation of STAT5 in Mo-DCs is mediated by GM-CSF produced following LPS stimulation. Activated STAT5 then leads to increased expression of both GM-CSF and GM-CSFR, triggering an autocrine loop that further enhances STAT5 signaling and enabling Mo-DCs to acquire a more mature phenotype. JQ1 decreases the ability of Mo-DCs to induce allogeneic CD4(+) and CD8(+) T cell proliferation and production of proinflammatory cytokines. Furthermore, JQ1 leads to a reduced generation of inflammatory CD8(+) T cells and decreased Th1 differentiation. Thus, JQ1 impairs LPS-induced Mo-DC maturation by inhibiting STAT5 activity, thereby generating cells that can only weakly stimulate an adaptive-immune response. Therefore, JQ1 could have beneficial effects in treating T cell-mediated inflammatory diseases.
Subject(s)
Azepines/pharmacology , Cell Differentiation/drug effects , Dendritic Cells/cytology , Dendritic Cells/drug effects , STAT5 Transcription Factor/antagonists & inhibitors , Triazoles/pharmacology , Antigens, Surface/metabolism , Cell Differentiation/immunology , Cytokines/biosynthesis , Dendritic Cells/immunology , Dendritic Cells/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Healthy Volunteers , Humans , Immunophenotyping , Inflammation Mediators/metabolism , Janus Kinases/antagonists & inhibitors , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Models, Biological , Monocytes/cytology , Monocytes/drug effects , Monocytes/immunology , Monocytes/metabolism , Phenotype , Protein Interaction Domains and Motifs , STAT5 Transcription Factor/metabolism , Signal Transduction , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolismABSTRACT
The need to increase the peak wet weather secondary treatment capacity of the City of Akron, Ohio, Water Reclamation Facility (WRF) provided the opportunity to test an integrated methodology for maximizing the peak wet weather secondary treatment capacity of activated sludge systems. An initial investigation, consisting of process modeling of the secondary treatment system and computational fluid dynamics (CFD) analysis of the existing relatively shallow secondary clarifiers (3.3 and 3.7 m sidewater depth in 30.5 m diameter units), indicated that a significant increase in capacity from 416 000 to 684 000 m3/d or more was possible by adding step feed capabilities to the existing bioreactors and upgrading the existing secondary clarifiers. One of the six treatment units at the WRF was modified, and an extensive 2-year testing program was conducted to determine the total peak wet weather secondary treatment capacity achievable. The results demonstrated that a peak wet weather secondary treatment capacity approaching 974 000 m3/d is possible as long as secondary clarifier solids and hydraulic loadings could be separately controlled using the step feed capability provided. Excellent sludge settling characteristics are routinely experienced at the City of Akron WRF, raising concerns that the identified peak wet weather secondary treatment capacity could not be maintained should sludge settling characteristics deteriorate for some reason. Computational fluid dynamics analysis indicated that the impact of the deterioration of sludge settling characteristics could be mitigated and the identified peak wet weather secondary treatment capacity maintained by further use of the step feed capability provided to further reduce secondary clarifier solids loading rates at the identified high surface overflow rates. The results also demonstrated that effluent limits not only for total suspended solids (TSS) and five-day carbonaceous biochemical oxygen demand (cBOD5) could be maintained, but also for ammonia-nitrogen and total phosphorous (TP). Although hydraulic limitations in other parts of the WRP prevent this full capacity to be realized, the City is proceeding to implement the modifications identified using this integrated methodology.
Subject(s)
Drainage, Sanitary/methods , Waste Disposal Facilities , Waste Disposal, Fluid/methods , Water Purification , Rain , Water MovementsABSTRACT
Bioactive phytochemicals can suppress the growth of malignant cells, and investigation of the mechanisms responsible can assist in the identification of novel therapeutic strategies for cancer therapy. Ginger has been reported to exhibit potent anti-cancer effects, although previous reports have often focused on a narrow range of specific compounds. Through a direct comparison of various ginger compounds, we determined that gingerenone A selectively kills cancer cells while exhibiting minimal toxicity toward normal cells. Kinase array screening revealed JAK2 and S6K1 as the molecular targets primarily responsible for gingerenone A-induced cancer cell death. The effect of gingerenone A was strongly associated with relative phosphorylation levels of JAK2 and S6K1, and administration of gingerenone A significantly suppressed tumor growth in vivo. More importantly, the combined inhibition of JAK2 and S6K1 by commercial inhibitors selectively induced apoptosis in cancer cells, whereas treatment with either agent alone did not. These findings provide rationale for dual targeting of JAK2 and S6K1 in cancer for a combinatorial therapeutic approach.
Subject(s)
Apoptosis/drug effects , Diarylheptanoids/pharmacology , Enzyme Inhibitors/pharmacology , Janus Kinase 2/antagonists & inhibitors , Ribosomal Protein S6 Kinases, 70-kDa/antagonists & inhibitors , Cell Line, Tumor , Humans , Janus Kinase 2/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/metabolismSubject(s)
Antineoplastic Agents/therapeutic use , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Neoplasm Proteins/antagonists & inhibitors , Pyrimethamine/therapeutic use , STAT3 Transcription Factor/antagonists & inhibitors , Adult , Aged , Aged, 80 and over , Antineoplastic Agents/administration & dosage , B-Lymphocytes/drug effects , B-Lymphocytes/metabolism , Cell Line, Tumor , Dose-Response Relationship, Drug , Drug Repositioning , Drug Screening Assays, Antitumor , Female , Gene Expression Profiling , Gene Expression Regulation, Leukemic/drug effects , Humans , Male , Middle Aged , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Progression-Free Survival , Pyrimethamine/administration & dosage , Pyrimethamine/pharmacologyABSTRACT
The performance characteristics of relatively shallow (3.3 and 3.7 m sidewater depth in 30.5 m diameter) activated sludge secondary clarifiers were extensively evaluated during a 2-year testing program at the City of Akron Water Reclamation Facility (WRF), Ohio, USA. Testing included hydraulic and solids loading stress tests, and measurement of sludge characteristics (zone settling velocity (ZSV), dispersed and flocculated total suspended solids), and the results were used to calibrate computational fluid dynamic (CFD) models of the various clarifiers tested. The results demonstrated that good performance could be sustained at surface overflow rates in excess of 3 m/h, as long as the clarifier influent mixed liquor suspended solids (MLSS) concentration was controlled to below critical values. The limiting solids loading rate (SLR) was significantly lower than the value predicted by conventional solids flux analysis based on the measured ZSV/MLSS relationship. CFD analysis suggested that this resulted because mixed liquor entering the clarifier was being directed into the settled sludge blanket, diluting it and also creating a 'thin' concentration sludge blanket that overlays the thicker concentration sludge blanket typically expected. These results indicate the need to determine the allowable SLR for shallow clarifiers using approaches other than traditional solids flux analysis. A combination of actual testing and CFD analyses are demonstrated here to be effective in doing so.
Subject(s)
Sewage/analysis , Waste Disposal, Fluid/methods , Flocculation , Models, Theoretical , OhioABSTRACT
This review discusses extracellular vesicles (EVs), which are submicron-scale, anuclear, phospholipid bilayer membrane enclosed vesicles that contain lipids, metabolites, proteins, and RNA (micro and messenger). They are shed from many, if not all, cell types and are present in biological fluids and conditioned cell culture media. The term EV, as coined by the International Society of Extracellular Vesicles (ISEV), encompasses exosomes (30-100 nm in diameter), microparticles (100-1000 nm), apoptotic blebs, and other EV subsets. EVs have been implicated in cell-cell communication, coagulation, inflammation, immune response modulation, and disease progression. Multiple studies report that EV secretion from disease-affected cells contributes to disease progression, e.g., tumor niche formation and cancer metastasis. EVs are attractive sources of biomarkers due to their biological relevance and relatively noninvasive accessibility from a range of physiological fluids. This review is focused on the molecular profiling of the protein and lipid constituents of EVs, with emphasis on mass-spectrometry-based "omic" analytical techniques. The challenges in the purification and molecular characterization of EVs, including contamination of isolates and limitations in sample quantities, are discussed along with possible solutions. Finally, the review discusses the limited but growing investigation of post-translational modifications of EV proteins and potential strategies for future in-depth molecular characterization of EVs.
Subject(s)
Extracellular Vesicles/chemistry , Lipids/chemistry , Mass Spectrometry/methods , Proteomics , Animals , Culture Media, Conditioned , Electrophoresis, Gel, Two-Dimensional , HumansABSTRACT
Kidney injury molecule-1 (KIM-1)/T cell Ig and mucin domain-containing protein-1 (TIM-1) is upregulated more than other proteins after AKI, and it is highly expressed in renal damage of various etiologies. In this capacity, KIM-1/TIM-1 acts as a phosphatidylserine receptor on the surface of injured proximal tubular epithelial cells, mediating phagocytosis of apoptotic cells, and it may also act as a costimulatory molecule for immune cells. Despite recognition of KIM-1 as an important therapeutic target for kidney disease, the regulators of KIM-1 transcription in the kidney remain unknown. Using a bioinformatics approach, we identified upstream regulators of KIM-1 after AKI. In response to tubular injury in rat and human kidneys or oxidant stress in human proximal tubular epithelial cells (HPTECs), KIM-1 expression increased significantly in a manner that corresponded temporally and regionally with increased phosphorylation of checkpoint kinase 1 (Chk1) and STAT3. Both ischemic and oxidant stress resulted in a dramatic increase in reactive oxygen species that phosphorylated and activated Chk1, which subsequently bound to STAT3, phosphorylating it at S727. Furthermore, STAT3 bound to the KIM-1 promoter after ischemic and oxidant stress, and pharmacological or genetic induction of STAT3 in HPTECs increased KIM-1 mRNA and protein levels. Conversely, inhibition of STAT3 using siRNAs or dominant negative mutants reduced KIM-1 expression in a kidney cancer cell line (769-P) that expresses high basal levels of KIM-1. These observations highlight Chk1 and STAT3 as critical upstream regulators of KIM-1 expression after AKI and may suggest novel approaches for therapeutic intervention.
Subject(s)
Acute Kidney Injury/metabolism , Cell Adhesion Molecules/metabolism , Membrane Glycoproteins/metabolism , Protein Kinases/metabolism , Receptors, Virus/metabolism , STAT3 Transcription Factor/metabolism , Acute Kidney Injury/genetics , Acute Kidney Injury/therapy , Animals , Cell Adhesion Molecules/genetics , Cell Line , Checkpoint Kinase 1 , Computational Biology , DNA Damage , Gene Expression Regulation , Hepatitis A Virus Cellular Receptor 1 , Humans , Kidney/metabolism , Male , Membrane Glycoproteins/genetics , Oxidative Stress , Phosphorylation , Promoter Regions, Genetic , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , Rats , Rats, Wistar , Receptors, Virus/genetics , Reperfusion Injury/genetics , Reperfusion Injury/metabolism , STAT3 Transcription Factor/antagonists & inhibitors , STAT3 Transcription Factor/geneticsABSTRACT
The transcription factor STAT3 drives the expression of genes promoting cellular proliferation, survival, and pluripotency. The description of STAT3 mutations and their clinical correlates in myeloid neoplasms, such as acute myeloid leukemia and myelodysplastic syndromes, raises new insights into both the pathogenesis and the targeted therapy of these diseases.