RESUMO
The high affinity of the biotin-streptavidin interaction has made this non-covalent coupling an indispensable strategy for the immobilization and enrichment of biomolecular affinity reagents. However, the irreversible nature of the biotin-streptavidin bond renders surfaces functionalized using this strategy permanently modified and not amenable to regeneration strategies that could increase assay reusability and throughput. To increase the utility of biotinylated targets, we here introduce a method for reversibly immobilizing biotinylated thrombin-binding aptamers onto a Ni-nitrilotriacetic acid (Ni-NTA) sensor chip using 6xHis-tagged streptavidin as a regenerable capture ligand. This approach enabled the reproducible immobilization of aptamers and measurements of aptamer-protein interaction in a surface plasmon resonance assay. The immobilized aptamer surface was stable during five experiments over two days, despite the reversible attachment of 6xHis-streptavidin to the Ni-NTA surface. In addition, we demonstrate the reproducibility of this immobilization method and the affinity assays performed using it. Finally, we verify the specificity of the biotin tag-streptavidin interaction and assess the efficiency of a straightforward method to regenerate and reuse the surface. The method described here will allow researchers to leverage the versatility and stability of the biotin-streptavidin interaction while increasing throughput and improving assay efficiency.
Assuntos
Aptâmeros de Nucleotídeos , Biotina , Ácido Nitrilotriacético , Estreptavidina , Ressonância de Plasmônio de Superfície , Estreptavidina/química , Biotina/química , Aptâmeros de Nucleotídeos/química , Ácido Nitrilotriacético/química , Ácido Nitrilotriacético/análogos & derivados , Técnicas Biossensoriais/métodos , Trombina/química , Compostos OrganometálicosRESUMO
The Nicoya OpenSPR is a benchtop surface plasmon resonance (SPR) instrument. As with other optical biosensor instruments, it is suitable for the label-free interaction analysis of a diverse set of biomolecules, including proteins, peptides, antibodies, nucleic acids, lipids, viruses, and hormones/cytokines. Supported assays include affinity/kinetics characterization, concentration analysis, yes/no assessment of binding, competition studies, and epitope mapping. OpenSPR exploits localized SPR detection in a benchtop platform and can be connected with an autosampler (XT) to perform automated analysis over an extended time period. In this review article, we provide a comprehensive survey of the 200 peer-reviewed papers published between 2016 and 2022 that use the OpenSPR platform. We highlight the range of biomolecular analytes and interactions that have been investigated using the platform, provide an overview on the most common applications for the instrument, and point out some representative research that highlights the flexibility and utility of the instrument.
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Anticorpos , Bioensaio , Citocinas , Interpretação Estatística de Dados , CinéticaRESUMO
Surface plasmon resonance (SPR) is a popular real-time technique for the measurement of binding affinity and kinetics, and bench-top instruments combine affordability and ease of use with other benefits of the technique. Biomolecular ligands labeled with the 6xHis tag can be immobilized onto sensing surfaces presenting the Ni2+-nitrilotriacetic acid (NTA) functional group. While Ni-NTA immobilization offers many advantages, including the ability to regenerate and reuse the sensors, its use can lead to signal variability between experimental replicates. We report here a study of factors contributing to this variability using the Nicoya OpenSPR as a model system and suggest ways to control for those factors, increasing the reproducibility and rigor of the data. Our model ligand/analyte pairs were two ovarian cancer biomarker proteins (MUC16 and HE4) and their corresponding monoclonal antibodies. We observed a broad range of non-specific binding across multiple NTA chips. Experiments run on the same chips had more consistent results in ligand immobilization and analyte binding than experiments run on different chips. Further assessment showed that different chips demonstrated different maximum immobilizations for the same concentration of injected protein. We also show a variety of relationships between ligand immobilization level and analyte response, which we attribute to steric crowding at high ligand concentrations. Using this calibration to inform experimental design, researchers can choose protein concentrations for immobilization corresponding to the linear range of analyte response. We are the first to demonstrate calibration and normalization as a strategy to increase reproducibility and data quality of these chips. Our study assesses a variety of factors affecting chip variability, addressing a gap in knowledge about commercially available sensor chips. Controlling for these factors in the process of experimental design will minimize variability in analyte signal when using these important sensing platforms.
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Projetos de Pesquisa , Ressonância de Plasmônio de Superfície , Ligantes , Reprodutibilidade dos Testes , Ressonância de Plasmônio de Superfície/métodos , Ácido Nitrilotriacético/química , Anticorpos MonoclonaisRESUMO
Developing a mass spectrometry-based assay for the ovarian cancer biomarker CA125 (MUC16) is a desirable goal, because it may enable detection of molecular regions that are not recognized by antibodies and are therefore analytically silent in the current immunoassay. Additionally, the ability to characterize the CA125 proteoforms expressed by individuals may offer clinical insight. Enrichment of CA125 from malignant ascites may provide a high-quality source of this important ovarian cancer biomarker, but a reliable strategy for such enrichment is currently lacking. Beginning with crude ascites isolated from three individual patients with high grade serous ovarian cancer, we enriched for MUC16 using filtration, ion exchange, and size exclusion chromatography and then performed bottom-up proteomics on the isolated proteins. This approach of enrichment and analysis reveals that the peptides detected via mass spectrometry map to the SEA domain and C-loop regions within the tandem repeat domains of CA125 and that peptide abundance correlates with clinical CA125 counts.
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Biomarcadores Tumorais , Neoplasias Ovarianas , Ascite/diagnóstico , Biomarcadores Tumorais/genética , Antígeno Ca-125 , Feminino , Humanos , Espectrometria de Massas , Proteínas de Membrana , Neoplasias Ovarianas/diagnósticoRESUMO
The development of novel affinity probes for cancer biomarkers may enable powerful improvements in analytical methods for detecting and treating cancer. In this report, we describe our use of capillary electrophoresis (CE) as the separation mechanism in the process of selecting DNA aptamers with affinity for the ovarian cancer biomarker HE4. Rather than the conventional use of cloning and sequencing as the last step in the aptamer selection process, we used high-throughput sequencing on an Illumina platform. This data-rich approach, combined with a bioinformatics pipeline based on freely available computational tools, enabled the entirety of the selection process-and not only its endpoint-to be characterized. Affinity probe CE and fluorescence anisotropy assays demonstrate the binding affinity of a set of aptamer candidates identified through this bioinformatics approach. Graphical Abstract A population of candidate aptamers is sequenced on an Illumina platform, enabling the process by which aptamers are selected over multiple SELEX rounds to be characterized. Bioinformatics tools are used to identify enrichment of selected aptamers and groupings into clusters based on sequence and structural similarity. A subset of sequenced aptamers may be intelligently chosen for in vitro testing.
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Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/isolamento & purificação , Eletroforese Capilar/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias Ovarianas/genética , Proteínas/genética , Técnicas de Cultura Celular por Lotes , Cromatografia de Afinidade/métodos , Feminino , Marcadores Genéticos/genética , Humanos , Neoplasias Ovarianas/diagnóstico , Técnica de Seleção de Aptâmeros/métodos , Proteína 2 do Domínio Central WAP de Quatro DissulfetosRESUMO
Over three decades have passed since the first report on the expression of CA125 by ovarian tumors. Since that time our understanding of ovarian cancer biology has changed significantly to the point that these tumors are now classified based on molecular phenotype and not purely on histological attributes. However, CA125 continues to be, with the recent exception of HE4, the only clinically reliable diagnostic marker for ovarian cancer. Many large-scale clinical trials have been conducted or are underway to determine potential use of serum CA125 levels as a screening modality or to distinguish between benign and malignant pelvic masses. CA125 is a peptide epitope of a 3-5 million Da mucin, MUC16. Here we provide an in-depth review of the literature to highlight the importance of CA125 as a prognostic and diagnostic marker for ovarian cancer. We focus on the increasing body of literature describing the biological role of MUC16 in the progression and metastasis of ovarian tumors. Finally, we consider previous and on-going efforts to develop therapeutic approaches to eradicate ovarian tumors by targeting MUC16. Even though CA125 is a crucial marker for ovarian cancer, the exact structural definition of this antigen continues to be elusive. The importance of MUC16/CA125 in the diagnosis, progression and therapy of ovarian cancer warrants the need for in-depth research on the biochemistry and biology of this mucin. A renewed focus on MUC16 is likely to culminate in novel and more efficient strategies for the detection and treatment of ovarian cancer.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Antígeno Ca-125/genética , Imunoterapia , Proteínas de Membrana/genética , Neoplasias Ovarianas/terapia , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/imunologia , Antígeno Ca-125/imunologia , Ensaios Clínicos como Assunto , Progressão da Doença , Feminino , Expressão Gênica , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/imunologia , Metástase Neoplásica , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Prognóstico , Proteínas/genética , Proteínas/imunologia , Proteína 2 do Domínio Central WAP de Quatro DissulfetosRESUMO
The biomarker CA125, a peptide epitope located in several tandem repeats of the mucin MUC16, is the gold standard for monitoring regression and recurrence of high-grade serous ovarian cancer in response to therapy. However, the CA125 epitope along with several structural features of the MUC16 molecule are ill defined. One central aspect still unresolved is the number of tandem repeats in MUC16 and how many of these repeats contain the CA125 epitope. Studies from the early 2000s assembled short DNA reads to estimate that MUC16 contained 63 repeats.Here, we conduct Nanopore long-read sequencing of MUC16 transcripts from three primary ovarian tumors and established cell lines (OVCAR3, OVCAR5, and Kuramochi) for a more exhaustive and accurate estimation and sequencing of the MUC16 tandem repeats.The consensus sequence derived from these six sources was confirmed by proteomics validation and agrees with recent additions to the NCBI database. We propose a model of MUC16 containing 19-not 63-tandem repeats. In addition, we predict the structure of the tandem repeat domain using the deep learning algorithm, AlphaFold.The predicted structure displays an SEA domain and unstructured linker region rich in proline, serine, and threonine residues in all 19 tandem repeats. These studies now pave the way for a detailed characterization of the CA125 epitope. Sequencing and modeling of the MUC16 tandem repeats along with their glycoproteomic characterization, currently underway in our laboratories, will help identify novel epitopes in the MUC16 molecule that improve on the sensitivity and clinical utility of the current CA125 assay. SIGNIFICANCE: Despite its crucial role in clinical management of ovarian cancer, the exact molecular sequence and structure of the biomarker, CA125, are not defined. Here, we combine long-read sequencing, mass spectrometry, and in silico modeling to provide the foundational dataset for a more complete characterization of the CA125 epitope.
Assuntos
Neoplasias Ovarianas , Humanos , Feminino , Neoplasias Ovarianas/diagnóstico , Biomarcadores Tumorais/genética , Proteínas de Membrana/genética , Apoptose , Linhagem Celular Tumoral , Antígeno Ca-125/genética , Epitopos/genética , Modelos MolecularesRESUMO
The enrichment of trace proteins from human fluid samples is of great importance in diverse clinical and industrial applications. In clinical diagnostics, such enrichment may enable detection of trace proteins that serve as biomarkers of disease. Affinity-based approaches, such as immunoaffinity pulldown, are widely used to enrich trace proteins, but this strategy relies on the availability and performance of antibodies that act on all proteoforms in an unbiased manner. Our prior work to characterize MUC16 (the mucin protein that carries the ovarian cancer biomarker CA125) by mass spectrometry successfully overcame the reliance on affinity-based enrichment and was used to enrich this biomarker from ascites of individual ovarian cancer patients, however, this strategy was not demonstrated on clinically relevant volumes of serum, a biofluid that is more accessible than ascites. The present work developed a non-affinity-based chromatographic method to enrich MUC16 from serum. The enriched MUC16 sample was further processed using a Midi Top 14 abundant protein depletion column. Peptides identified using bottom-up proteomics yielded 1-8% coverage of MUC16. Additionally, MUC16 was detected in samples containing less than the clinical cut-off level of CA125 (35 U mL-1), suggesting that this strategy of enrichment and bottom-up proteomics can enable analysis of CA125 from the serum of individuals with early-stage ovarian cancer and those whose tumors express CA125 (MUC16) at low levels.
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BACKGROUND: Despite its importance in the clinical management of ovarian cancer, the CA125 biomarker-located on the mucin protein MUC16-is still not completely understood. Questions remain about MUC16's function and structure, specifically the identity and location of the CA125 epitopes. OBJECTIVE: The goal of this study was to characterize the interaction of individual recombinant repeats from the tandem repeat domain of MUC16 with antibodies used in the clinical CA125 II test. METHODS: Using E. coli expression, we isolated nine repeats from the putative antigenic domain of CA125. Amino acid composition of recombinant repeats was confirmed by high-resolution mass spectrometry. We characterized the binding of four antibodies-OC125, M11, "OC125-like," and "M11-like"-to nine recombinant repeats using Western blotting, indirect enzyme-linked immunosorbent assay (ELISA), and localized surface plasmon resonance (SPR) spectroscopy. RESULTS: Each recombinant repeat was recognized by a different combination of CA125 antibodies. OC125 and "OC125-like" antibodies did not bind the same set of recombinant repeats, nor did M11 and "M11-like" antibodies. CONCLUSIONS: Characterization of the interactions between MUC16 recombinant repeats and CA125 antibodies will contribute to ongoing efforts to identify the CA125 epitopes and improve our understanding of this important biomarker.
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BACKGROUND: Despite its importance in the clinical management of ovarian cancer, the CA125 biomarker - located on the mucin protein MUC16 - is still not completely understood. Questions remain about MUC16's function and structure, specifically the identity and location of the CA125 epitopes. OBJECTIVE: The goal of this study was to characterize the interaction of individual recombinant repeats from the tandem repeat domain of MUC16 with antibodies used in the clinical CA125 II test. METHODS: Using E. coli expression, we isolated nine repeats from the putative antigenic domain of CA125. Amino acid composition of recombinant repeats was confirmed by high-resolution mass spectrometry. We characterized the binding of four antibodies - OC125, M11, "OC125-like," and "M11-like" - to nine recombinant repeats using Western blotting, indirect enzyme-linked immunosorbent assay (ELISA), and localized surface plasmon resonance (SPR) spectroscopy. RESULTS: Each recombinant repeat was recognized by a different combination of CA125 antibodies. OC125 and "OC125-like" antibodies did not bind the same set of recombinant repeats, nor did M11 and "M11-like" antibodies. CONCLUSIONS: Characterization of the interactions between MUC16 recombinant repeats and CA125 antibodies will contribute to ongoing efforts to identify the CA125 epitopes and improve our understanding of this important biomarker.
Assuntos
Anticorpos , Humanos , Anticorpos/imunologia , Proteínas Recombinantes/imunologia , Ensaio de Imunoadsorção Enzimática , Ressonância de Plasmônio de SuperfícieRESUMO
N-linked glycosylation is an important post-translational modification that is difficult to identify and quantify in traditional bottom-up proteomics experiments. Enzymatic deglycosylation of proteins by peptide:N-glycosidase F (PNGase F) prior to digestion and subsequent mass spectrometry analysis has been shown to improve coverage of various N-linked glycopeptides, but the inclusion of this step may add up to a day to an already lengthy sample preparation process. An efficient way to integrate deglycosylation with bottom-up proteomics would be a valuable contribution to the glycoproteomics field. Here, we demonstrate a proteomics workflow in which deglycosylation and proteolytic digestion of samples occur simultaneously using suspension trapping (S-Trap). This approach adds no time to standard digestion protocols. Applying this sample preparation strategy to a human serum sample, we demonstrate improved identification of potential N-glycosylated peptides in deglycosylated samples compared with non-deglycosylated samples, identifying 156 unique peptides that contain the N-glycosylation motif (asparagine-X-serine/threonine), the deamidation modification characteristic of PNGase F, and an increase in peptide intensity over a control sample. We expect that this rapid sample preparation strategy will assist in the identification and quantification of both known and potential glycoproteins. Data are available via ProteomeXchange with the identifier PXD037921.
RESUMO
Reversed-phase liquid chromatography (RPLC) is widely used to reduce sample complexity prior to mass spectrometry (MS) analysis in bottom-up proteomics. Improving peptide separation in complex samples enables lower-abundance proteins to be identified. Multidimensional separations that combine orthogonal separation modes improve protein and peptide identifications over RPLC alone. Here we report a preparative capillary electrophoresis (CE) fractionation method that combines CE and RPLC separations. Using this method, we demonstrate improved protein and peptide identification in a tryptic digest of E. coli cell lysate, with 132 ± 33% more protein identifications and 185 ± 65% more peptide identifications over non-fractionated samples. Fractionation enables detection of lower-abundance proteins in this complex sample. We demonstrate improved coverage of ovarian cancer biomarker MUC16 isolated from conditioned cell media, with 6.73% sequence coverage using CE fractionation compared to 2.74% coverage without preparative fractionation. This new method will allow researchers performing bottom-up proteomics to harness the advantages of CE separations while using widely available LC-MS/MS instrumentation.
Assuntos
Proteômica , Espectrometria de Massas em Tandem , Cromatografia Líquida , Eletroforese Capilar/métodos , Escherichia coli , Peptídeos/química , Proteínas/química , Proteômica/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
The ability to functionalize gold nanoparticle surfaces with target ligands is integral to developing effective nanosystems for biomedical applications, ranging from point-of-care diagnostic devices to site-specific cancer therapies. By forming strong covalent bonds with gold, thiol functionalities can easily link molecules of interest to nanoparticle surfaces. Unfortunately, thiols are inherently prone to oxidative degradation in many biologically relevant conditions, which limits their broader use as surface ligands in commercial assays. Recently, N-heterocyclic carbene (NHC) ligands emerged as a promising alternative to thiols since initial reports demonstrated their remarkable stability against ligand displacement and stronger metal-ligand bonds. This work explores the long-term stability of NHC-functionalized gold nanoparticles suspended in five common biological media: phosphate-buffered saline, tris-glycine potassium buffer, tris-glycine potassium magnesium buffer, cell culture media, and human serum. The NHCs on gold nanoparticles were probed with surface-enhanced Raman spectroscopy (SERS) and X-ray photoelectron spectroscopy (XPS). SERS is useful for monitoring the degradation of surface-bound species because the resulting vibrational modes are highly sensitive to changes in ligand adsorption. Our measurements indicate that imidazole-based NHCs remain stable on gold nanoparticles over the 21 days of examination in all tested environments, with no observed change in the molecule's SERS signature, XPS response, or UV-vis plasmon band.
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The uropygial secretions of some bird species contain volatile and semivolatile compounds that are hypothesized to serve as chemical signals. The abundance of secretion components varies with age and season, although these effects have not been investigated in many species. We used solid-phase microextraction headspace sampling and solvent extraction coupled with gas chromatography-mass spectrometry to detect and identify volatile and semivolatile chemical compounds in uropygial secretions of gray catbirds (Dumetella carolinensis). We identified linear and branched saturated carboxylic acids from acetic (C2) through hexacosanoic (C26); linear alcohols from decanol (C10) through docosanol (C22); one aromatic aldehyde; one monounsaturated carboxylic acid; two methyl ketones; and a C28 ester. We tested for the effect of age on signal strength and found that juvenile birds produced greater amounts of volatile C4 through C7 acids and semivolatile C20 through C26 acids, although the variation among individuals was large. Adult birds displayed small concentrations and minimal individual variation among volatile compounds, but produced significantly higher levels of long-chain linear alcohols than juvenile birds. We tested for the effects of season/location by sampling adult catbirds at their Ohio breeding grounds and at their Florida wintering grounds and found that the heaviest carboxylic acids are significantly more abundant in secretions from birds sampled during winter at the Florida site, whereas methyl ketones are more abundant in birds sampled during summer on the Ohio breeding grounds. We observed no effect of sex on semivolatile compounds, but we found a significant effect of sex on levels of carboxylic acids (C4 through C7) for juvenile birds only.
Assuntos
Glândulas Exócrinas/metabolismo , Passeriformes/fisiologia , Fatores Etários , Aldeídos/análise , Animais , Ácidos Carboxílicos/análise , Glândulas Exócrinas/química , Álcoois Graxos/análise , Feminino , Florida , Cromatografia Gasosa-Espectrometria de Massas , Cetonas/análise , Masculino , Ohio , Estações do Ano , Microextração em Fase Sólida , VolatilizaçãoRESUMO
Aptamers have many useful attributes including specific binding to molecular targets. After aptamers are identified, their target binding must be characterized. Fluorescence anisotropy (FA) is one technique that can be used to characterize affinity and to optimize aptamer-target interactions. Efforts to make FA assays more efficient by reducing assay volume and time from mixing to measurement may save time and resources by minimizing consumption of costly reagents. Here, we use thrombin and two thrombin-binding aptamers as a model system to show that plate-based FA experiments can be performed in volumes as low as 2 µL per well with 20 minute incubations with minimal loss in assay precision. We demonstrate that the aptamer-thrombin interaction is best modelled with the Hill equation, indicating cooperative binding. The miniaturization of this assay has implications in drug development, as well as in the efficiency of aptamer selection workflows by allowing for higher throughput aptamer analysis.
Assuntos
Aptâmeros de Nucleotídeos , Aptâmeros de Nucleotídeos/metabolismo , Bioensaio , Polarização de Fluorescência , Ligação Proteica , TrombinaRESUMO
A highly conserved region of 21 amino acids flanked by cysteine residues, contained within a larger repeated domain, has been proposed to be the antibody-binding site in the ovarian cancer biomarker CA125 (MUC16). In this study solid-phase peptide synthesis with Fmoc protection chemistry was used to assemble a 21-mer peptide corresponding to the most frequently occurring antibody binding sequence in CA125. Potentially significant sequence variants were also synthesized. Peptide secondary structure was investigated using Fourier transform infrared spectroscopy, revealing the consensus sequence peptide to be largely unstructured at physiological pH whether the cysteine residues were reduced or were oxidized to form an intramolecular disulfide bond. Substitution of serine for proline at position 8 (P8S) results in ß-sheet formation in peptides involved in intramolecular disulfide bonds. This ß-sheet structure does not persist in peptides incapable of intramolecular disulfide bonding because of sequence nor in peptides treated with the reducing agent dithiothreitol. In CA125, P8S is predicted to occur in â¼25% of repeat domains, suggesting that this structural motif is a non-negligible contributor to overall structure and function. These findings suggest that future structural characterization efforts of CA125 should be especially mindful of the amino acid sequence and oxidation state of the protein.
Assuntos
Biomarcadores Tumorais/química , Biomarcadores Tumorais/síntese química , Antígeno Ca-125/química , Epitopos de Linfócito B/química , Proteínas de Membrana/química , Sequência de Aminoácidos , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
A dendrochemical study of cottonwood trees (Populus deltoides) was conducted across a childhood cancer cluster in eastern Sandusky County (Ohio, USA). The justification for this study was that no satisfactory explanation has yet been put forward, despite extensive local surveys of aerosols, groundwater, and soil. Concentrations of eight trace metals were measured by ICP-MS in microwave-digested 5-year sections of increment cores, collected during 2012 and 2013. To determine whether the onset of the first cancer cases could be connected to an emergence of any of these contaminants, cores spanning the period 1970-2009 were taken from 51 trees of similar age, inside the cluster and in a control area to the west. The abundance of metals in cottonwood tree annual rings served as a proxy for their long-term, low-level accumulation from the same sources whereby exposure of the children may have occurred. A spatial analysis of cumulative metal burdens (lifetime accumulation in the tree) was performed to search for significant 'hotspots', employing a scan statistic with a mask of variable radius and center. For Cd, Cr, and Ni, circular hotspots were found that nearly coincide with the cancer cluster and are similar in size. No hotspots were found for Co, Cu, and Pb, while As and V were largely below method detection limits. Whereas our results do not implicate exposure to metals as a causative factor, we conclude that, after 1970, cottonwood trees have accumulated more Cd, Cr, and Ni, inside the childhood cancer cluster than elsewhere in Sandusky County.
Assuntos
Monitoramento Ambiental , Neoplasias/epidemiologia , Populus/química , Oligoelementos/análise , Criança , Humanos , Ohio/epidemiologia , Solo/química , Análise Espacial , Inquéritos e Questionários , Árvores/químicaRESUMO
The thrombin-binding 15mer and 29mer ssDNA aptamers are a widely used model system. Despite their ubiquity, controversies persist regarding the nature of the aptamer-protein interactions. Reported affinities vary widely; the role of metal ions in binding is unclear; the structure of the complex is contested. We interrogated the effects of instrument, buffer, and mathematical model on apparent affinities of thrombin aptamers for their target. Instrumental method had a pronounced effect on affinity constants for the 15mer and marginal effect the apparent affinity of the 29mer. Buffer composition and ionic environment did not have significant effects. Affinity probe capillary electrophoresis experiments revealed distinct peaks from samples of 29mer aptamer and thrombin, supporting the model of a 1 aptamer:2 protein complex. Fits to high quality data with five mathematical models further support this stoichiometry, as the binding of both aptamers was best described by the Hill equation with Hill coefficients > 1. Our results indicate that the instrumental method and mathematical model influence apparent affinity of thrombin aptamers and that both aptamers bind thrombin in a 1 aptamer: 2 protein stoichiometry through an induced fit mechanism.
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Culex pipiens serves as the endemic vector of West Nile virus (WNV) in eastern North America, where house sparrows (HOSP, Passer domesticus) serve as a reservoir host. We tested the hypotheses that: (1) Attraction of Cx. pipiens to HOSP is influenced by bird age and (2) that age-specific variation in chemical profiles of bird uropygial gland secretions informs this choice. We conducted mosquito choice trials in an olfactometer and found that Cx. pipiens were more often attracted to adult sparrows over nestlings, however, they demonstrated no preference for adults over fledglings. Using gas chromatography-mass spectrometry we observed age-specific differences in the semi-volatile chemical profiles of house sparrow uropygial gland secretions. Contrary to our hypothesis, we found no significant difference in mosquito feeding preference between the secretions of adults and those of either nestlings or fledglings. We suggest that other chemical cues influence the feeding preference of Cx. pipiens, either independently of uropygial gland secretions, or synergistically with them.
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Culex pipiens, the endemic mosquito vector of West Nile virus in eastern North America, is responsible for maintenance of the virus in avian reservoir hosts, the most important of which appears to be the American robin. One reason for the greater involvement of robins is believed to be the feeding preference of Cx. pipiens, however, the basis of this preference is not understood. We tested the hypothesis that the species-specific chemical profile of avian uropygial gland secretions are used by Cx. pipiens as cues to locate birds and, therefore, may contribute to the observed feeding preferences. We used gas chromatography-mass spectrometry to identify the semi-volatile components of the uropygial gland secretions of American robins and two other common reservoir host species, the house sparrow and European starling. We found that the chemical composition of the robin secretions was different from those of the sparrows and starlings. Through behavioral choice trials conducted in a dual-port olfactometer, we also found that Cx. pipiens did not prefer the secretions of robins over the other two species. Surprisingly, however, we found that Cx pipiens were more often attracted to live starlings over robins and to the secretions of starlings over those of robins.