RESUMO
A high throughput screen performed to identify catalytic inhibitors of the oncogenic fusion form of cAMP-dependent protein kinase A catalytic subunit alpha (J-PKAcα) found an individual fraction from an organic extract of the marine soft coral Acrozoanthus australiae as active. Bioassay-guided isolation led to the identification of a 2-amino adenine alkaloid acroamine A (1), the first secondary metabolite discovered from this genus and previously reported as a synthetic product. As a naturally occurring protein kinase inhibitor, to unambiguously assign its chemical structure using modern spectroscopic and spectrometric techniques, five N-methylated derivatives acroamines A1-A5 (2-6) were semisynthesized. Three additional brominated congeners A6-A8 (7-9) were also semisynthesized to investigate the structure-activity relationship of the nine compounds as J-PKAcα inhibitors. Compounds 1-9 were tested for J-PKAcα and wild-type PKA inhibitory activities, which were observed exclusively in acroamine A (1) and its brominated analogs (7-9) achieving moderate potency (IC50 2-50 µM) while none of the N-methylated analogs exhibited kinase inhibition.
RESUMO
Merkel cell carcinoma (MCC) is a rare and aggressive cutaneous cancer. Two new prenylated indole 2,5-diketopiperazine alkaloids, brevianamides E1 (1) and E2 (2), were isolated from a Penicillium fungus. Both compounds showed moderate cytotoxic activity against select MCC cell lines (i.e., MCC13, MKL-1, UISO, and WaGa) in the low micromolar range. The relative and absolute configurations of 1 and 2 were determined by combined approaches, including NOESY spectroscopy, DFT ECD and DP4 plus calculations, and Marfey's reaction. Literature research and the comparison of NMR and ECD data led to the structure revision of three previously reported natural analogues, notoamides K and P and asperversiamide L. The structurally unstable 1 and 2 underwent steady interconversion under neutral aqueous conditions. Investigation of the degradation of 2 in acidic methanol solutions led to the identification of a new methoxylated derivative (6) and two new ring-opened products (7 and 8) with the rearranged, elongated, 4-methylpent-3-ene side chain. The facile transformation of 2 to 7 and 8 was promoted by the intrinsic impurity (i.e., formaldehyde) of HPLC-grade methanol through the aza-Cope rearrangement.
Assuntos
Dicetopiperazinas , Penicillium , Penicillium/química , Dicetopiperazinas/farmacologia , Dicetopiperazinas/química , Estrutura Molecular , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Ensaios de Seleção de Medicamentos AntitumoraisRESUMO
Pyrrole-containing natural products form a large group of structurally diverse compounds that occur in both terrestrial and marine organisms. In the present study the formation of trideuteromethylated artifacts of pyrrole-containing natural products was investigated, focusing on the discorhabdins. Three deuterated discorhabdins, 1, 3, and 5, were identified to be isolation procedure artifacts caused by the presence of DMSO-d6 during NMR sample preparation and handling. Three additional semisynthetic derivatives, 7-9, were made during the investigation of the mechanism of formation, which was shown to be driven by trideuteromethyl radicals in the presence of water, methanol, TFA, and traces of iron in the deuterated solvent. Generation of trideuteromethylated artifacts was also confirmed for other classes of pyrrole-containing metabolites, namely, makaluvamines, tambjamines, and dibromotryptamines, which had also been dissolved in DMSO-d6 during the structure elucidation process. Semisynthetic discorhabdins were assessed for antiproliferative activity against a panel of human tumor cell lines, and 14-trideuteromethyldiscorhabdin L (3) averaged low micromolar potency.
Assuntos
Produtos Biológicos , Dimetil Sulfóxido , Humanos , Dimetil Sulfóxido/química , Pirróis/química , Produtos Biológicos/farmacologia , Artefatos , Solventes/químicaRESUMO
The DNAJB1-PRKACA oncogenic gene fusion results in an active kinase enzyme, J-PKAcα, that has been identified as an attractive antitumor target for fibrolamellar hepatocellular carcinoma (FLHCC). A high-throughput assay was used to identify inhibitors of J-PKAcα catalytic activity by screening the NCI Program for Natural Product Discovery (NPNPD) prefractionated natural product library. Purification of the active agent from a single fraction of an Aplidium sp. marine tunicate led to the discovery of two unprecedented alkaloids, aplithianines A (1) and B (2). Aplithianine A (1) showed potent inhibition against J-PKAcα with an IC50 of â¼1 µM in the primary screening assay. In kinome screening, 1 inhibited wild-type PKA with an IC50 of 84 nM. Further mechanistic studies including cocrystallization and X-ray diffraction experiments revealed that 1 inhibited PKAcα catalytic activity by competitively binding to the ATP pocket. Human kinome profiling of 1 against a panel of 370 kinases revealed potent inhibition of select serine/threonine kinases in the CLK and PKG families with IC50 values in the range â¼11-90 nM. An efficient, four-step total synthesis of 1 has been accomplished, enabling further evaluation of aplithianines as biologically relevant kinase inhibitors.
Assuntos
Produtos Biológicos , Carcinoma Hepatocelular , Humanos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases , Carcinoma Hepatocelular/patologia , Serina , Proteínas de Choque Térmico HSP40/química , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismoRESUMO
A library of naturally occurring and semi-synthetic discorhabdins was assessed for their effects on Merkel cell carcinoma (MCC) cell viability. The set included five new natural products and semi-synthetic compounds whose structures were elucidated with NMR, HRMS, and ECD techniques. Several discorhabdins averaged sub-micromolar potency against the MCC cell lines tested and most of the active compounds showed selectivity towards virus-positive MCC cell lines. An investigation of structure-activity relationships resulted in an expanded understanding of the crucial structural features of the discorhabdin scaffold. Mechanistic cell death assays suggested that discorhabdins, unlike many other MCC-active small molecules, do not induce apoptosis, as shown by the lack of caspase activation, annexin V staining, and response to caspase inhibition. Similarly, discorhabdin treatment failed to increase MCC intracellular calcium and ROS levels. In contrast, the rapid loss of cellular reducing potential and mitochondrial membrane potential suggested that discorhabdins induce mitochondrial dysfunction leading to non-apoptotic cell death.
Assuntos
Alcaloides , Carcinoma de Célula de Merkel , Neoplasias Cutâneas , Humanos , Carcinoma de Célula de Merkel/tratamento farmacológico , Morte Celular , Relação Estrutura-Atividade , Alcaloides/farmacologia , Caspases , Neoplasias Cutâneas/tratamento farmacológicoRESUMO
The recent demonstration that adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) plays an oncogenic role in a number of important cancers has led to a renaissance in drug development interest targeting this kinase. We therefore have established a suite of biochemical, cell-based, and structural biology assays for identifying and evaluating new pharmacophores for PKA inhibition. This discovery process started with a 384-well high-throughput screen of more than 200,000 substances, including fractionated natural product extracts. Identified active compounds were further prioritized in biochemical, biophysical, and cell-based assays. Priority lead compounds were assessed in detail to fully characterize several previously unrecognized PKA pharmacophores including the generation of new X-ray crystallography structures demonstrating unique interactions between PKA and bound inhibitor molecules.
RESUMO
Silver(I) ions have the propensity of undergoing reduction to form metallic silver within olefin/paraffin separation systems when they are subjected to hydrogen at elevated temperatures. Ionic liquids (ILs) are versatile solvents known for their low vapor pressure, high thermal stability, and structural tunability and have been shown to minimize hydrogen-induced reduction of silver(I) ions when employed as solvents. In the development of robust separation platforms that employ silver(I) ions, it is essential to deploy reliable approaches capable of measuring and assessing the factors that lower the overall separation performance. In this study, silver(I) ions dissolved in an imidazolium-based IL are subjected to mixed gas streams composed of hydrogen, nitrogen, and methane under varying temperatures. Using inverse gas chromatography, a total of 44 columns with stationary phases containing four different concentrations of silver(I) bis[(trifluoromethyl)sulfonyl]imide ([Ag+][NTf2 -]) dissolved in the 1-decyl-3-methylimidazolium ([C10MIM+]) [NTf2 -] IL were used to measure partition coefficients of olefins and paraffins, as well as aromatics, esters, and ketones. Upon exposing the stationary phases to mixed gases at elevated temperatures, olefin partitioning between the silver(I) ion pseudophase and the two other phases (i.e., carrier gas and IL stationary phase) was observed to decrease over time, while partitioning between the IL stationary phase and carrier gas remained unchanged. It was found that exposure gases composed of 5.0 to 85.0 mol % hydrogen and temperatures ranging from 95 to 130 °C resulted in a remarkable acceleration of silver(I) ion reduction and an approximate 36.4-61.3% decrease in olefin partitioning between the silver(I) ion pseudophase and both the carrier gas and IL stationary phase after 60 h. While binary mixtures of hydrogen and nitrogen resulted in a continuous decrease in silver(I) ion-olefin complexation capability, a ternary gas mixture produced varied silver(I) ion reduction kinetics.
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Glyphosate is a globally applied herbicide yet it has been relatively undetectable in-field samples outside of gold-standard techniques. Its presumed nontoxicity toward humans has been contested by the International Agency for Research on Cancer, while it has been detected in farmers' urine, surface waters and crop residues. Rapid, on-site detection of glyphosate is hindered by lack of field-deployable and easy-to-use sensors that circumvent sample transportation to limited laboratories that possess the equipment needed for detection. Herein, the flavoenzyme, glycine oxidase, immobilized on platinum-decorated laser-induced graphene (LIG) is used for selective detection of glyphosate as it is a substrate for GlyOx. The LIG platform provides a scaffold for enzyme attachment while maintaining the electronic and surface properties of graphene. The sensor exhibits a linear range of 10-260 µ m, detection limit of 3.03 µ m, and sensitivity of 0.991 nA µ m -1. The sensor shows minimal interference from the commonly used herbicides and insecticides: atrazine, 2,4-dichlorophenoxyacetic acid, dicamba, parathion-methyl, paraoxon-methyl, malathion, chlorpyrifos, thiamethoxam, clothianidin, and imidacloprid. Sensor function is further tested in complex river water and crop residue fluids, which validate this platform as a scalable, direct-write, and selective method of glyphosate detection for herbicide mapping and food analysis.
RESUMO
Seven new peptaibols named tolypocladamides A-G have been isolated from an extract of the fungus Tolypocladium inflatum, which inhibits the interaction between Raf and oncogenic Ras in a cell-based high-throughput screening assay. Each peptaibol contains 11 amino acid residues, an octanoyl or decanoyl fatty acid chain at the N-terminus, and a leucinol moiety at the C-terminus. The peptaibol sequences were elucidated on the basis of 2D NMR and mass spectral fragmentation analyses. Amino acid configurations were determined by advanced Marfey's analyses. Tolypocladamides A-G caused significant inhibition of Ras/Raf interactions with IC50 values ranging from 0.5 to 5.0 µM in a nanobioluminescence resonance energy transfer (NanoBRET) assay; however, no interactions were observed in a surface plasmon resonance assay for binding of the compounds to wild type or G12D mutant Ras constructs or to the Ras binding domain of Raf. NCI 60 cell line testing was also conducted, and little panel selectivity was observed.
Assuntos
Antineoplásicos , Hypocreales , Aminoácidos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Hypocreales/química , Peptaibols/farmacologiaRESUMO
Merkel cell carcinoma (MCC) is a rare, but aggressive skin cancer the incidence of which has increased significantly in recent years. The majority of MCCs have incorporated Merkel cell polyomavirus (VP-MCC) while the remainder are virus-negative (VN-MCC). Although a variety of therapeutic options have shown promise in treating MCC, there remains a need for additional therapeutics as well as probes for better understanding MCC. A high-throughput screening campaign was used to assess the ability of > 25,000 synthetic and natural product compounds as well as > 20,000 natural product extracts to affect growth and survival of VN-MCC and VP-MCC cell lines. Sixteen active compounds were identified that have mechanisms of action reported in the literature along with a number of compounds with unknown mechanisms. Screening results with pure compounds suggest a range of potential targets for MCC including DNA damage, inhibition of DNA or protein synthesis, reactive oxygen species, and proteasome inhibition as well as NFκB inhibition while also suggesting the importance of zinc and/or copper binding. Many of the active compounds, particularly some of the natural products, have multiple reported targets suggesting that this strategy might be a particularly fruitful approach. Processing of several active natural product extracts resulted in the identification of additional MCC-active compounds. Based on these results, further investigations focused on natural products sources, particularly of fungal origin, are expected to yield further potentially useful modulators of MCC.
Assuntos
Antineoplásicos , Produtos Biológicos , Carcinoma de Célula de Merkel , Neoplasias Cutâneas , Antineoplásicos/química , Antineoplásicos/farmacologia , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Carcinoma de Célula de Merkel/tratamento farmacológico , Carcinoma de Célula de Merkel/metabolismo , Carcinoma de Célula de Merkel/patologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologiaRESUMO
Activating mutations in RAS are found in approximately 30% of human cancers, resulting in the delivery of a persistent signal to critical downstream effectors that drive tumorigenesis. RAS-driven malignancies respond poorly to conventional cancer treatments and inhibitors that target RAS directly are limited; therefore, the identification of new strategies and/or drugs to disrupt RAS signaling in tumor cells remains a pressing therapeutic need. Taking advantage of the live-cell bioluminescence resonance energy transfer (BRET) methodology, we describe the development of a NanoBRET screening platform to identify compounds that modulate binding between activated KRAS and the CRAF kinase, an essential effector of RAS that initiates ERK cascade signaling. Using this strategy, libraries containing synthetic compounds, targeted inhibitors, purified natural products, and natural product extracts were evaluated. These efforts resulted in the identification of compounds that inhibit RAS/RAF binding and in turn suppress RAS-driven ERK activation, but also compounds that have the deleterious effect of enhancing the interaction to upregulate pathway signaling. Among the inhibitor hits identified, the majority were compounds derived from natural products, including ones reported to alter KRAS nanoclustering (ophiobolin A), to impact RAF function (HSP90 inhibitors and ROS inducers) as well as some with unknown targets and activities. These findings demonstrate the potential for this screening platform in natural product drug discovery and in the development of new therapeutic agents to target dysregulated RAS signaling in human disease states such as cancer.
Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Fibroblastos/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Proteínas ras/agonistas , Proteínas ras/antagonistas & inibidores , Animais , Fibroblastos/metabolismo , Humanos , Ligantes , Nanotecnologia/métodos , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas ras/metabolismoRESUMO
An extract of a Sinularia sp. soft coral showed inhibitory activity against the E3-ubiquitin ligase casitas B-lineage lymphoma proto-oncogene B (Cbl-b). Subsequent bioassay-guided separation of the extract provided a series of terpenoid-derived spermidine and spermine amides that were named sinularamides A-G (1-7). Compounds 1-7 represent new natural products; however, sinularamide A (1) was previously reported as a synthetic end product. The structures of sinularamides A-G (1-7) were elucidated by analysis of spectroscopic and spectrometric data from NMR, IR, and HRESIMS experiments and by comparison with literature data. All of the isolated compounds showed Cbl-b inhibitory activities with IC50 values that ranged from approximately 6.5 to 33 µM.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Antozoários/química , Proteínas Proto-Oncogênicas c-cbl/antagonistas & inibidores , Espermidina/farmacologia , Espermina/farmacologia , Terpenos/farmacologia , Animais , Estrutura Molecular , Palau , Espermidina/isolamento & purificação , Espermina/isolamento & purificação , Terpenos/isolamento & purificaçãoRESUMO
The transfer of the small protein ubiquitin to a target protein is an intricately orchestrated process called ubiquitination that results in modulation of protein function or stability. Proper regulation of ubiquitination is essential, and dysregulation of this process is implicated in several human diseases. An example of a ubiquitination cascade that is a central signaling node in important disease-associated pathways is that of CBLB [a human homolog of a viral oncogene Casitas B-lineage lymphoma (CBL) from the Cas NS-1 murine retrovirus], a RING finger ubiquitin ligase (E3) whose substrates include a number of important cell-signaling kinases. These include kinases important in immune function that act in the T cell receptor and costimulatory pathways, the Tyro/Axl/MerTK (TAM) receptor family in natural killer (NK) cells, as well as growth factor receptor kinases like epidermal growth factor receptor (EGFR). Loss of CBLB has been shown to increase innate and adaptive antitumor immunity. This suggests that small-molecule modulation of CBLB E3 activity could enhance antitumor immunity in patients. To explore the hypothesis that enzymatic inhibition of E3s may result in modulation of disease-related signaling pathways, we established a high-throughput screen of >70,000 chemical entities for inhibition of CBLB activity. Although CBLB was chosen as a proof-of-principle target for inhibitor discovery, we demonstrate that our assay is generalizable to monitoring the activity of other ubiquitin ligases. We further extended our observed in vitro inhibition with additional cell-based models of CBLB activity. From these studies, we demonstrate that a class of natural product-based alkaloids, known as methyl ellipticiniums (MEs), is capable of inhibiting ubiquitin ligases intracellularly.
Assuntos
Descoberta de Drogas/métodos , Inibidores Enzimáticos/química , Técnicas In Vitro , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/química , Animais , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Metilação , Camundongos , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Ubiquitinação/efeitos dos fármacosRESUMO
Bioassay-guided separation of an extract from a Dictyosporium sp. isolate led to the identification of six new compounds, 1-6, together with five known compounds, 7-11. The structures of the new compounds were primarily established by extensive 1D and 2D NMR experiments. The absolute configurations of compounds 3-6 were determined by comparison of their experimental electronic circular dichroism (ECD) spectra with DFT quantum mechanical calculated ECD spectra. Compounds 3-5 possess novel structural scaffolds, and biochemical studies revealed that oxepinochromenones 1 and 7 inhibited the activity of MALT1 protease.
Assuntos
Inibidores Enzimáticos/isolamento & purificação , Fungos/metabolismo , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologiaAssuntos
Membrana Celular/metabolismo , Imagem Óptica/métodos , Animais , Bactérias/química , Linhagem Celular Tumoral , Membrana Celular/ultraestrutura , Corantes Fluorescentes/química , Humanos , Microscopia de Fluorescência/métodos , Nanoestruturas , Receptores de Superfície Celular/metabolismo , Análise Espectral Raman/métodosRESUMO
Cysteine residues (Cys) in the membrane proximal region are common post-translational modification (PTM) sites in transmembrane proteins. Herein, the effects of a highly conserved membrane proximal α-subunit Cys1368 on the diffusion properties of αPS2CßPS integrins are reported. Sequence alignment shows that this cysteine is palmitoylated in human α3 and α6 integrin subunits. Replacing Cys1368 in wild-type integrins with valine (Val1368) putatively blocks a PTM site and alters integrins' ligand binding and diffusion characteristics. Both fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT) diffusion measurements show Val1368 integrins are more mobile compared to wild-type integrins. Approximately 33% and 8% more Val1368 integrins are mobile as measured by FRAP and SPT, respectively. The mobile Val1368 integrins also exhibit less time-dependent diffusion, as measured by FRAP. Tandem mass spectrometry data suggest that Cys1368 contains a redox or palmitoylation PTM in αPS2CßPS integrins. This membrane proximal Cys may play an important role in the diffusion of other alpha subunits that contain this conserved residue.
Assuntos
Cisteína/metabolismo , Cadeias alfa de Integrinas/química , Cadeias alfa de Integrinas/genética , Substituição de Aminoácidos , Animais , Proteínas de Bactérias/genética , Bioquímica/métodos , Cisteína/química , Difusão , Drosophila/genética , Proteínas de Drosophila/química , Recuperação de Fluorescência Após Fotodegradação/métodos , Cadeias alfa de Integrinas/metabolismo , Proteínas Luminescentes/genética , Modificação Traducional de Proteínas , Valina/química , Valina/metabolismoRESUMO
BACKGROUND: Ninety percent of perinatal hepatitis B virus (HBV) infections result in chronic HBV (CHBV), which carries 25% risk of premature death from progressive liver injury, cirrhosis, and liver cancer. In 1990, the Centers for Disease Control and Prevention (CDC) funded Perinatal Hepatitis B Prevention Programs (PHBPP) to ensure postexposure prophylaxis for exposed infants and accelerate elimination of perinatal CHBV in the United States. From 2000 to 2009, the annual rates of perinatal CHBV reported by PHBPP (0.8%-2.4%) were consistently lower than expected rates from CDC models (3.0%-4.1%), suggesting that rates of CHBV might be higher among infants whose outcomes were not identified by PHBPP. To better understand the factors impacting modeled expected number and rates of perinatal CHBV, we examined historic CDC models, applied updated inputs to the 2009 CDC model, and performed sensitivity analyses over a range of parameter values. METHODS: Models employed estimates of the annual number of births to hepatitis B surface antigen (HBsAg)-positive pregnant women, and data from PHBPP and National Immunization Surveys. Published literature provided prenatal HBsAg screening rates, efficacy of postexposure prophylaxis (PEP), and perinatal HBV transmission rates. RESULTS: The updated 2009 model predicted 952 perinatal CHBV infections, equivalent to a baseline rate of 3.84%, among infants of HBsAg-positive women. Sensitivity analyses yielded a possible range of perinatal CHBV rates between 0.60% and 15.41%. The proportion of infants receiving timely PEP, the efficacy of PEP, and perinatal HBV transmission rate were major "drivers" of CHBV rates. Three-way sensitivity analysis yielded possible perinatal CHBV rates between 0.79% and 13.64%. CONCLUSIONS: Modeling provided useful programmatic goals for achieving elimination of perinatal CHBV in the United States. Limitations of data inputs likely contributed to discrepancies between predicted and reported rates.
Assuntos
Hepatite B/epidemiologia , Transmissão Vertical de Doenças Infecciosas , Complicações Infecciosas na Gravidez/virologia , Feminino , Antígenos de Superfície da Hepatite B/sangue , Vírus da Hepatite B , Humanos , Gravidez , Complicações Infecciosas na Gravidez/epidemiologia , Estados Unidos/epidemiologiaRESUMO
OBJECTIVE: To analyze the cost-effectiveness of the national Perinatal Hepatitis B Prevention Program (PHBPP) over the lifetime of the 2009 US birth cohort and compare the costs and outcomes of the program to a scenario without PHBPP support. PHBPP's goals are to ensure all infants born to hepatitis B (HepB) surface antigen-positive women receive timely postexposure prophylaxis, complete HepB vaccine series, and obtain serologic testing after series completion. METHODS: A decision analytic tree and a long-term Markov model represented the risk of perinatal and childhood infections under different prevention alternatives, and the long-term health and economic consequences of HepB infection. Outcome measures were the number of perinatal infections and childhood infections from infants born to HepB surface antigen-positive women, quality-adjusted life-years (QALYs), lifetime costs, and incremental cost per QALY gained. The health outcomes and total costs of each strategy were compared incrementally. Costs were evaluated from the health care system perspective and expressed in US dollars at a 2010 price base. RESULTS: In all analyses, the PHBPP increased QALYs and led to higher reductions in the number of perinatal and childhood infections than no PHBPP, with a cost-effectiveness ratio of $2602 per QALY. In sensitivity analyses, the cost-effectiveness ratio was robust to variations in model inputs, and there were instances where the program was both more effective and cost saving. CONCLUSIONS: This study indicated that the current PHBPP represents a cost-effective use of resources, and ensuring the program reaches all pregnant women could present additional public health benefits.
Assuntos
Hepatite B/economia , Hepatite B/prevenção & controle , Transmissão Vertical de Doenças Infecciosas/economia , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Programas Nacionais de Saúde/economia , Análise Custo-Benefício , Árvores de Decisões , Feminino , Humanos , Recém-Nascido , Gravidez , Complicações Infecciosas na Gravidez/prevenção & controle , Anos de Vida Ajustados por Qualidade de Vida , Estados UnidosRESUMO
Integrins are ubiquitous adhesion receptors that are important for signaling and integrating the extracellular matrix and cytoskeleton. The role of cytoplasmic proteins vinculin, focal adhesion kinase (FAK), integrin-linked kinase (ILK), and membrane proteins epidermal growth factor receptor (EGFR) and Notch in altering αPS2CßPS integrin lateral diffusion was measured using single particle tracking (SPT) and RNA interference (RNAi). SPT measures heterogeneous diffusion properties, and RNAi selectively reduces the concentration of a target protein. After systematically reducing the concentration of vinculin, FAK, ILK, EGFR, or Notch, there was a 31 to 80% increase in the mobile integrin fraction, indicating that these five targeted proteins (or assemblies that contain these proteins) are responsible for immobilizing a fraction of the integrins when all proteins are present at native concentrations. The average diffusion coefficient of all mobile integrins did not change after any of the RNAi treatments, and the percentage of Brownian, directed, or anomalous/constrained trajectories relative to total mobile trajectories did not change after vinculin or EGFR RNAi. However, the fraction of anomalous/constrained trajectories relative to the total mobile trajectories increased 9 to 19% after FAK, ILK, and Notch RNAi, when the concentration of these proteins was reduced. In the case of FAK, ILK, and Notch, native concentrations of these proteins simultaneously increase the immobile fraction of integrins but decrease the diffusion constraints to those integrins that remain mobile. Comparisons of single receptor and ensemble measurements of diffusion and what is known about the effect of these proteins in altering integrin clustering are discussed.
Assuntos
Citoplasma/metabolismo , Integrinas/metabolismo , Proteínas de Membrana/metabolismo , Animais , Linhagem Celular , Difusão , Drosophila , Receptores ErbB/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Vinculina/metabolismoRESUMO
The role of three membrane proteins in altering the diffusion and clustering of integrin receptors has been measured. Integrins are membrane proteins responsible for integrating intracellular and extracellular signaling events and anchoring cells to the extracellular matrix. The methodology used to elucidate the role of other membrane proteins in altering integrin diffusion and clustering combines fluorescence microscopy with RNA interference (RNAi), which is a technique to reduce the expression of a target protein. The three RNAi-targeted membrane proteins were epidermal growth factor receptor (EGFR), platelet-derived growth factor/vascular endothelial growth factor-related receptor (Pvr), and Notch. Real-time polymerase chain reaction or quantitative immunocytochemistry was used to measure a reduction in mRNA or protein concentration after RNAi treatment, respectively. Fluorescence recovery after photobleaching showed that reducing the concentration of EGFR or Notch results in less constrained integrin diffusion and, in the case of Notch RNAi, 4 % more mobile integrins. Fluorescence resonance energy transfer measurements performed before and after RNAi treatments indicate that clustering decreases for wild-type integrin, but increases for a high-ligand-affinity integrin mutant after reducing the expression of EGFR, Pvr, or Notch. A model to explain the measured changes after reducing the expression of these three membrane proteins involving cholesterol-enriched nanodomains is proposed.