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The poxvirus-derived protein vCCI (viral CC chemokine inhibitor) binds almost all members of the CC chemokine family with nanomolar affinity, inhibiting their pro-inflammatory actions. Understanding the affinity and specificity of vCCI could lead to new anti-inflammatory therapeutics. CCL17, also known as TARC, is unusual among CC chemokines by having only micromolar binding to vCCI. We have used sequence analysis and molecular simulations to determine the cause of this weak binding, which identified several locations in CCL17 where mutations seemed likely to improve binding to vCCI. Based on the aforementioned analysis, we expressed and tested multiple mutants of CCL17. We found two single point mutants V44K and Q45R that increased binding affinity to vCCI by 2-3-fold and, in combination, further improved affinity by 7-fold. The CCL17 triple mutant G17R/V44K/Q45R yielded a Kd of 0.25 ± 0.13 µM, a 68-fold improvement in affinity compared to the complex with wild-type CCL17. A quadruple mutant G17R/V44K/Q45R/R57W showed high affinity (0.59 ± 0.09 µM) compared to the wild type but lower affinity than the triple mutant. This work demonstrates that sequence comparisons and molecular simulations can predict chemokine mutations that increase the level of binding to vCCI, an important first step in developing engineered chemokine inhibitors useful for anti-inflammatory therapy.
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The designability of orthogonal coiled coil (CC) dimers, which draw on well-established design rules, plays a pivotal role in fueling the development of CCs as synthetically versatile assembly-directing motifs for the fabrication of bionanomaterials. Here, we aim to expand the synthetic CC toolkit through establishing a "minimalistic" set of orthogonal, de novo CC peptides that comprise 3.5 heptads in length and a single buried Asn to prescribe dimer formation. The designed sequences display excellent partner fidelity, confirmed via circular dichroism (CD) spectroscopy and Ni-NTA binding assays, and are corroborated in silico using molecular dynamics (MD) simulation. Detailed analysis of the MD conformational data highlights the importance of interhelical E@g-N@a interactions in coordinating an extensive 6-residue hydrogen bonding network that "locks" the interchain Asn-Asn' contact in place. The enhanced stability imparted to the Asn-Asn' bond elicits an increase in thermal stability of CCs up to ~15°C and accounts for significant differences in stability within the collection of similarly designed orthogonal CC pairs. The presented work underlines the utility of MD simulation as a tool for constructing de novo, orthogonal CCs, and presents an alternative handle for modulating the stability of orthogonal CCs via tuning the number of interhelical E@g-N@a contacts. Expansion of CC design rules is a key ingredient for guiding the design and assembly of more complex, intricate CC-based architectures for tackling a variety of challenges within the fields of nanomedicine and bionanotechnology.
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Simulación de Dinámica Molecular , Péptidos , Secuencia de Aminoácidos , Estructura Secundaria de Proteína , Péptidos/química , Dominios Proteicos , Dicroismo CircularRESUMEN
Environmental management often requires making decisions despite system uncertainty. One such example is mudflat mediation in flood control reservoirs. Reservoir mudflats limit development of diverse fish assemblages due to the lack of structural habitat provided by plants. Seeding mudflats with agricultural plants may mimic floodplain wetlands once inundated and provide fish habitat and achieve habitat management objectives. However, planting success is uncertain because of unpredictable water level fluctuations that affect plant survival and growth. Decision support tools can account for uncertainty that influences decision outcomes and reduce the risk in reservoir mudflat planting decisions. We used Bayesian decision networks and sensitivity analyses to quantify uncertainty surrounding mudflat plantings as supplemental fish habitat in four northwest Mississippi reservoirs. When averaged across all uncertainty, planting was the optimal decision only in Enid Lake. Response profiles indicated planting decisions depended on elevation contours within Enid, Sardis, and Grenada reservoirs. No planting was optimal at all elevations for Arkabutla Lake. These results provide a quantified basis for establishing best management practices and identify key system states that influence decision outcomes. The process used in this study to evaluate planting decisions can be applied to any reservoir by modifying reservoir dependent inputs to evaluate planting decisions to provide supplemental fish habitat.
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Ecosistema , Peces , Animales , Teorema de Bayes , Ambiente , InundacionesRESUMEN
Approaches for white crappie, Pomoxis annularis sperm cryopreservation have led to interest in applying similar methods to black-stripe black crappie, Pomoxis nigromaculatus. Their rarity in wild populations makes them a preferred phenotype for hatchery use. Sperm cryopreservation procedures were compared between black-stripe black crappie and white crappie for sperm motility and egg fertilization rate. There was no difference in black-stripe black crappie sperm motility after thawing between 5% dimethyl sulfoxide (DMSO, 45% motility) and 10% methanol (50% motility). However, fertilization rates were higher (p < .001) for sperm cryoprotected with 5% DMSO (38 ± 8%) than 10% methanol (22 ± 7%). Hatchery use requires sperm-to-egg ratios and fertilizing potential of single doses (i.e., 0.5 ml straw). Using black-stripe black crappie sperm (2.5 × 108 sperm/ml; 5% DMSO), the highest fertilization (27%) was found using single straws with 785 eggs (0.25 ml); total sperm:egg ratio: 159,000:1; motile sperm:egg ratio: 71,700:1. Therefore, sperm of two Pomoxis species could be cryopreserved using 350 mOsmol/kg Hanks' balanced salt solution as an extender, 5% DMSO as a cryoprotectant, cooling at 40°C/min, and thawing for 8 s at 40°C to maintain sperm motility and fertility. Basic protocols can be generalized within a genus if variables such as sperm concentration, process timing, and sample volumes are controlled.
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Two different methods, metagenetics and free-otolith identification, were used to identify prey in the stomach contents of 531 Gymnura lessae captured by trawling in Mobile Bay, Alabama 2016-2018. Both methods were found to produce analogous results and were therefore combined into a single complete dataset. All prey were teleosts; the families Sciaenidae and Engraulidae were the most important prey (prey specie index of relative importance 89.3% IPSRI ). Multivariate analyses indicated that the diet of G. lessae varied with sex and seasonality. Specifically, variability was probably due to morphologically larger females consuming larger teleost prey species compared with males, whereas seasonal variability was probably due to changes in the available prey community composition. The findings indicate that both metagenetics and free otolith identification, used independently or complementarily, offer robust means of characterising dietary habits for teleost-specialised species such as G. lessae, which may play an important role in the structure and maintenance of coastal food webs such as those in Mobile Bay.
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Conducta Alimentaria/fisiología , Membrana Otolítica , Rajidae/fisiología , Animales , Dieta/veterinaria , Femenino , Cadena Alimentaria , Contenido Digestivo , Masculino , MetagenómicaRESUMEN
Certain viruses have the ability to subvert the mammalian immune response, including interference in the chemokine system. Poxviruses produce the chemokine binding protein vCCI (viral CC chemokine inhibitor; also called 35K), which tightly binds to CC chemokines. To facilitate the study of vCCI, we first provide a protocol to produce folded vCCI from Escherichia coli (E. coli.) It is shown here that vCCI binds with unusually high affinity to viral Macrophage Inflammatory Protein-II (vMIP-II), a chemokine analog produced by the virus, human herpesvirus 8 (HHV-8). Fluorescence anisotropy was used to investigate the vCCI:vMIP-II complex and shows that vCCI binds to vMIP-II with a higher affinity than most other chemokines, having a Kd of 0.06 ± 0.006 nM. Nuclear magnetic resonance (NMR) chemical shift perturbation experiments indicate that key amino acids used for binding in the complex are similar to those found in previous work. Molecular dynamics were then used to compare the vCCI:vMIP-II complex with the known vCCI:Macrophage Inflammatory Protein-1ß/CC-Chemokine Ligand 4 (MIP-1ß/CCL4) complex. The simulations show key interactions, such as those between E143 and D75 in vCCI/35K and R18 in vMIP-II. Further, in a comparison of 1 µs molecular dynamics (MD) trajectories, vMIP-II shows more overall surface binding to vCCI than does the chemokine MIP-1ß. vMIP-II maintains unique contacts at its N-terminus to vCCI that are not made by MIP-1ß, and vMIP-II also makes more contacts with the vCCI flexible acidic loop (located between the second and third beta strands) than does MIP-1ß. These studies provide evidence for the basis of the tight vCCI:vMIP-II interaction while elucidating the vCCI:MIP-1ß interaction, and allow insight into the structure of proteins that are capable of broadly subverting the mammalian immune system.
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Quimiocina CXCL2/química , Polarización de Fluorescencia , Herpesvirus Humano 8/química , Simulación de Dinámica Molecular , Complejos Multiproteicos/química , Virus Vaccinia/química , Proteínas Virales/química , Quimiocina CXCL2/genética , Herpesvirus Humano 8/genética , Complejos Multiproteicos/genética , Estructura Cuaternaria de Proteína , Virus Vaccinia/genética , Proteínas Virales/genéticaRESUMEN
Adult Chinook salmon (Oncorhynchus tshawytscha) migrate from salt water to freshwater streams to spawn. Immune responses in migrating adult salmon are thought to diminish in the run up to spawning, though the exact mechanisms for diminished immune responses remain unknown. Here we examine both adaptive and innate immune responses as well as pathogen burdens in migrating adult Chinook salmon in the Upper Willamette River basin. Messenger RNA transcripts encoding antibody heavy chain molecules slightly diminish as a function of time, but are still present even after fish have successfully spawned. In contrast, the innate anti-bacterial effector proteins present in fish plasma rapidly decrease as spawning approaches. Fish also were examined for the presence and severity of eight different pathogens in different organs. While pathogen burden tended to increase during the migration, no specific pathogen signature was associated with diminished immune responses. Transcript levels of the immunosuppressive cytokines IL-10 and TGF beta were measured and did not change during the migration. These results suggest that loss of immune functions in adult migrating salmon are not due to pathogen infection or cytokine-mediated immune suppression, but is rather part of the life history of Chinook salmon likely induced by diminished energy reserves or hormonal changes which accompany spawning.
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Migración Animal/fisiología , Salmón/inmunología , Inmunidad Adaptativa , Animales , Femenino , Proteínas de Peces/inmunología , Inmunidad Innata , Interleucina-10/inmunología , Masculino , Estaciones del Año , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
Throughout history, coronaviruses have posed challenges to both public health and the global economy; nevertheless, methods to combat them remain rudimentary, primarily due to the absence of experiments to understand the function of various viral components. Among these, membrane (M) proteins are one of the most elusive because of their small size and challenges with expression. Here, we report the development of an expression system to produce tens to hundreds of milligrams of M protein per liter of Escherichia coli culture. These large yields render many previously inaccessible structural and biophysical experiments feasible. Using cryo-electron microscopy and atomic force microscopy, we image and characterize individual membrane-incorporated M protein dimers and discover membrane thinning in the vicinity, which we validated with molecular dynamics simulations. Our results suggest that the resulting line tension, along with predicted induction of local membrane curvature, could ultimately drive viral assembly and budding.
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COVID-19 , Membrana Dobles de Lípidos , Humanos , Membrana Dobles de Lípidos/química , SARS-CoV-2/metabolismo , Microscopía por Crioelectrón , Proteínas de la Matriz Viral/metabolismo , Proteínas de la Membrana , Escherichia coli/metabolismoRESUMEN
Nuclear pore complexes (NPCs) gate the only conduits for nucleocytoplasmic transport in eukaryotes. Their gate is formed by nucleoporins containing large intrinsically disordered domains with multiple phenylalanine-glycine repeats (FG domains). In combination, these are hypothesized to form a structurally and chemically homogeneous network of random coils at the NPC center, which sorts macromolecules by size and hydrophobicity. Instead, we found that FG domains are structurally and chemically heterogeneous. They adopt distinct categories of intrinsically disordered structures in non-random distributions. Some adopt globular, collapsed coil configurations and are characterized by a low charge content. Others are highly charged and adopt more dynamic, extended coil conformations. Interestingly, several FG nucleoporins feature both types of structures in a bimodal distribution along their polypeptide chain. This distribution functionally correlates with the attractive or repulsive character of their interactions with collapsed coil FG domains displaying cohesion toward one another and extended coil FG domains displaying repulsion. Topologically, these bipartite FG domains may resemble sticky molten globules connected to the tip of relaxed or extended coils. Within the NPC, the crowding of FG nucleoporins and the segregation of their disordered structures based on their topology, dimensions, and cohesive character could force the FG domains to form a tubular gate structure or transporter at the NPC center featuring two separate zones of traffic with distinct physicochemical properties.
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Proteínas de Complejo Poro Nuclear/química , Secuencia de Aminoácidos , Electroforesis en Gel de Poliacrilamida , Glicina/química , Datos de Secuencia Molecular , Fenilalanina/química , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
Chemokines are small proteins that are critical for immune function, being primarily responsible for the activation and chemotaxis of leukocytes. As such, many viruses, as well as parasitic arthropods, have evolved systems to counteract chemokine function in order to maintain virulence, such as binding chemokines, mimicking chemokines, or producing analogs of transmembrane chemokine receptors that strongly bind their targets. The focus of this review is the large group of chemokine binding proteins (CBP) with an emphasis on those produced by mammalian viruses. Because many chemokines mediate inflammation, these CBP could possibly be used pharmaceutically as anti-inflammatory agents. In this review, we summarize the structural properties of a diverse set of CBP and describe in detail the chemokine binding properties of the poxvirus-encoded CBP called vCCI (viral CC Chemokine Inhibitor). Finally, we describe the current and emerging capabilities of combining computational simulation, structural analysis, and biochemical/biophysical experimentation to understand, and possibly re-engineer, protein-protein interactions.
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Proteínas Portadoras , Poxviridae , Animales , Proteínas Portadoras/metabolismo , Quimiocinas , Humanos , Mamíferos/metabolismo , Poxviridae/química , Poxviridae/metabolismo , Unión Proteica , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
BACKGROUND: Molecular dynamics (MD) simulation is a powerful technique for sampling the meta-stable and transitional conformations of proteins and other biomolecules. Computational data clustering has emerged as a useful, automated technique for extracting conformational states from MD simulation data. Despite extensive application, relatively little work has been done to determine if the clustering algorithms are actually extracting useful information. A primary goal of this paper therefore is to provide such an understanding through a detailed analysis of data clustering applied to a series of increasingly complex biopolymer models. RESULTS: We develop a novel series of models using basic polymer theory that have intuitive, clearly-defined dynamics and exhibit the essential properties that we are seeking to identify in MD simulations of real biomolecules. We then apply spectral clustering, an algorithm particularly well-suited for clustering polymer structures, to our models and MD simulations of several intrinsically disordered proteins. Clustering results for the polymer models provide clear evidence that the meta-stable and transitional conformations are detected by the algorithm. The results for the polymer models also help guide the analysis of the disordered protein simulations by comparing and contrasting the statistical properties of the extracted clusters. CONCLUSIONS: We have developed a framework for validating the performance and utility of clustering algorithms for studying molecular biopolymer simulations that utilizes several analytic and dynamic polymer models which exhibit well-behaved dynamics including: meta-stable states, transition states, helical structures, and stochastic dynamics. We show that spectral clustering is robust to anomalies introduced by structural alignment and that different structural classes of intrinsically disordered proteins can be reliably discriminated from the clustering results. To our knowledge, our framework is the first to utilize model polymers to rigorously test the utility of clustering algorithms for studying biopolymers.
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Algoritmos , Simulación de Dinámica Molecular , Proteínas/química , Biopolímeros/química , Análisis por Conglomerados , Simulación por Computador , Modelos Moleculares , Conformación Molecular , Proteínas/metabolismoRESUMEN
The nuclear pore complex (NPC) provides the sole aqueous conduit for macromolecular exchange between the nucleus and the cytoplasm of cells. Its diffusion conduit contains a size-selective gate formed by a family of NPC proteins that feature large, natively unfolded domains with phenylalanine-glycine repeats (FG domains). These domains of nucleoporins play key roles in establishing the NPC permeability barrier, but little is known about their dynamic structure. Here we used molecular modeling and biophysical techniques to characterize the dynamic ensemble of structures of a representative FG domain from the yeast nucleoporin Nup116. The results showed that its FG motifs function as intramolecular cohesion elements that impart order to the FG domain and compact its ensemble of structures into native premolten globular configurations. At the NPC, the FG motifs of nucleoporins may exert this cohesive effect intermolecularly as well as intramolecularly to form a malleable yet cohesive quaternary structure composed of highly flexible polypeptide chains. Dynamic shifts in the equilibrium or competition between intra- and intermolecular FG motif interactions could facilitate the rapid and reversible structural transitions at the NPC conduit needed to accommodate passing karyopherin-cargo complexes of various shapes and sizes while simultaneously maintaining a size-selective gate against protein diffusion.
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Glicina/química , Proteínas de Complejo Poro Nuclear/química , Fenilalanina/química , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas/fisiología , Transporte Activo de Núcleo Celular/fisiología , Secuencias de Aminoácidos/fisiología , Modelos Moleculares , Peso Molecular , Poro Nuclear/química , Poro Nuclear/metabolismo , Poro Nuclear/ultraestructura , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/ultraestructura , Unión Proteica/fisiología , Estructura Cuaternaria de Proteína , Secuencias Repetitivas de Aminoácido/fisiología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructura , TermodinámicaRESUMEN
Carboxylesterases metabolize numerous exogenous and endogenous ester-containing compounds including the chemotherapeutic agent CPT-11, anti-influenza viral agent oseltamivir, and many agrochemicals. Trifluoromethyl ketone (TFK)-containing compounds with a sulfur atom beta to the ketone moiety are some of the most potent carboxylesterase and amidase inhibitors identified to date. This study examined the effects of alkyl chain length (i.e., steric effects) and sulfur oxidation state upon TFK inhibitor potency (IC50) and binding kinetics (k(i)). The selective carboxylesterase inhibitor benzil was used as a non-TFK containing control. These effects were examined using two commercial esterases (porcine and rabbit liver esterase) and two human recombinant esterases (hCE-1 and hCE-2) as well as human recombinant fatty acid amide hydrolase (FAAH). In addition, the inhibition mechanism was examined using a combination of 1H NMR, X-ray crystallography, and ab initio calculations. Overall, the data show that while sulfur oxidation state profoundly affects both inhibitor potency and binding kinetics, the steric effects dominate and override the contributions of sulfur oxidation. In addition, the data suggest that inclusion of a sulfur atom beta to the ketone contributes an increase (approximately 5-fold) in inhibitor potency due to effects upon ketone hydration and/or intramolecular hydrogen bond formation. These results provide further information on the nature of the TFK binding interaction and will be useful in increasing our understanding of this basic biochemical process.
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Amidohidrolasas/metabolismo , Hidrolasas de Éster Carboxílico/metabolismo , Cetonas/química , Azufre/química , Amidohidrolasas/antagonistas & inhibidores , Animales , Hidrolasas de Éster Carboxílico/antagonistas & inhibidores , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Humanos , Cinética , Modelos Moleculares , Oxidación-Reducción , Unión Proteica , Conejos , Relación Estructura-Actividad , PorcinosRESUMEN
The understanding of mutagenic potency has been primarily approached using "quantitative structure-activity relationships" (QSAR). Often this method allows the prediction of mutagenic potency of the compound based on its structure. But it does not give the underlying reason why the mutagenic activities differ. We have taken a set of heterocyclic amine structures and used molecular dynamic calculations to dock these molecules into the active site of a computational model of the cytochrome P4501A2 enzyme. The calculated binding strength using Boltzman distribution constants was then compared to the QSAR value (HF/6-31G* optimized structures) and the Ames/Salmonella mutagenic potency. Further understanding will only come from knowing the complete set of mutagenic determinants. These include the nitrenium ion half-life, DNA adduct half-life, efficiency of repair of the adduct, and ultimately fixation of the mutation through cellular processes. For two isomers, PhIP and 3-Me-PhIP, we showed that for the 100-fold difference in the mutagenic potency a 5-fold difference can be accounted for by differences in the P450 oxidation. The other factor of 20 is not clearly understood but is downstream from the oxidation step. The application of QSAR (chemical characteristics) to biological principles related to mutagenesis is explored in this report.
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Aminas/efectos adversos , Alimentos/efectos adversos , Compuestos Heterocíclicos/efectos adversos , Imidazoles/efectos adversos , Mutágenos , Simulación por Computador , Reparación del ADN , Isomerismo , Modelos Biológicos , Estructura Molecular , Relación Estructura-Actividad CuantitativaRESUMEN
PURPOSE: The selection and characterization of anti-MUC-1 single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer molecules designed for optimal blood clearance and tumor penetration. The mucin MUC-1 was chosen as an antigen because it is abundantly expressed on epithelial cancers in an aberrantly glycosylated form, making it structurally and antigenically distinct from MUC-1 expressed on normal cells. EXPERIMENTAL DESIGN: A previously constructed anti-MUC-1 phage display library from hyperimmunized mice, with 5 x 10(5) calculated variants, was screened for the selection of anti-MUC-1 scFvs. Selection criteria were high binding to a MUC-1 peptide containing 4 tandem repeats of 20 amino acids and to MUC-1-positive MCF-7 (human breast cancer) cell lysates in ELISA. RESULTS: Six anti-MUC-1 scFv clones were selected and characterized. Nucleotide sequencing showed that four of them were full length scFv genes (variable heavy chain + variable light chain), whereas the remaining two contained either a variable heavy chain or a variable light chain alone. Their binding affinities (K(a)) range between 8 x 10(7) and 10(9) M(-1). Immunohistopathology demonstrated reactivity with breast cancer cells (MCF-7 and BT20) and human breast biopsy tissue. Molecular modeling revealed high structural similarity of the anti-MUC-1 scFvs with the X-ray-determined structure of the anti-CEA scFv (MFE-23). CONCLUSIONS: In vitro antigen binding was demonstrated for the selected anti-MUC-1 scFvs. The binding affinities of these scFvs are in a promising range for efficient in vivo antigen binding. These anti-MUC-1 scFvs will be evaluated as antigen-binding modules in new multifunctional agents for the detection and therapy of cancer.
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Fragmentos de Inmunoglobulinas/química , Mucina-1/química , Neoplasias/terapia , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Unión Competitiva , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Biblioteca de Genes , Glicosilación , Humanos , Región Variable de Inmunoglobulina/química , Inmunohistoquímica , Cinética , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos/química , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Most pathogen detection tests are imperfect, with a sensitivity < 100%, thereby resulting in the potential for a false negative, where a pathogen is present but not detected. False negatives in a sample inflate the number of non-detections, negatively biasing estimates of pathogen prevalence. Histological examination of tissues as a diagnostic test can be advantageous as multiple pathogens can be examined and providing important information on associated pathological changes to the host. However, it is usually less sensitive than molecular or microbiological tests for specific pathogens. Our study objectives were to 1) develop a hierarchical occupancy model to examine pathogen prevalence in spring Chinook salmon Oncorhynchus tshawytscha and their distribution among host tissues 2) use the model to estimate pathogen-specific test sensitivities and infection rates, and 3) illustrate the effect of using replicate within host sampling on sample sizes required to detect a pathogen. We examined histological sections of replicate tissue samples from spring Chinook salmon O. tshawytscha collected after spawning for common pathogens seen in this population: Apophallus/echinostome metacercariae, Parvicapsula minibicornis, Nanophyetus salmincola/ metacercariae, and Renibacterium salmoninarum. A hierarchical occupancy model was developed to estimate pathogen and tissue-specific test sensitivities and unbiased estimation of host- and organ-level infection rates. Model estimated sensitivities and host- and organ-level infections rates varied among pathogens and model estimated infection rate was higher than prevalence unadjusted for test sensitivity, confirming that prevalence unadjusted for test sensitivity was negatively biased. The modeling approach provided an analytical approach for using hierarchically structured pathogen detection data from lower sensitivity diagnostic tests, such as histology, to obtain unbiased pathogen prevalence estimates with associated uncertainties. Accounting for test sensitivity using within host replicate samples also required fewer individual fish to be sampled. This approach is useful for evaluating pathogen or microbe community dynamics when test sensitivity is <100%.
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Interacciones Huésped-Patógeno , Micrococcaceae/patogenicidad , Modelos Biológicos , Salmón/microbiología , Trematodos/patogenicidad , Animales , Microbiota , Salmón/parasitologíaRESUMEN
Replication and related processes in eukaryotic cells require replication factor C (RFC) to load a molecular clamp for DNA polymerase in an ATP-driven process, involving multiple molecular interactions. The detailed understanding of this mechanism is hindered by the lack of data regarding structure, mutual arrangement, and dynamics of the players involved. In this study, we analyzed interactions that take place during loading onto DNA of either the PCNA clamp or the Rad9-Rad1-Hus1 checkpoint complex, using computationally derived molecular models. Combining the modeled structures for each RFC subunit with known structural, biochemical, and genetic data, we propose detailed models of how two of the RFC subunits, RFC1 and RFC3, interact with the C-terminal regions of PCNA. RFC1 is predicted to bind PCNA similarly to the p21-PCNA interaction, while the RFC3-PCNA binding is proposed to be similar to the E. coli delta-beta interaction. Additional sequence and structure analysis, supported by experimental data, suggests that RFC5 might be the third clamp loader subunit to bind the equivalent PCNA region. We discuss functional implications stemming from the proposed model of the RFC1-PCNA interaction and compare putative clamp-interacting regions in RFC1 and its paralogs, Rad17 and Ctf18. Based on the individual intermolecular interactions, we propose RFC and PCNA arrangement that places three RFC subunits in association with each of the three C-terminal regions in PCNA. The two other RFC subunits are positioned at the two PCNA interfaces, with the third PCNA interface left unobstructed. In addition, we map interactions at the level of individual subunits between the alternative clamp loader/clamp system, Rad17-RFC(2-5)/Rad9-Rad1-Hus1. The proposed models of interaction between two clamp/clamp loader pairs provide both structural framework for interpretation of existing experimental data and a number of specific findings that can be subjected to direct experimental testing.
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Proteínas de Unión al ADN/metabolismo , Modelos Moleculares , Secuencia de Aminoácidos , Sitios de Unión , Proteínas de Ciclo Celular/metabolismo , Biología Computacional , Datos de Secuencia Molecular , Proteínas Nucleares , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Proteína de Replicación C , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ProteínaRESUMEN
Carboxylesterases are important enzymes responsible for the hydrolysis and metabolism of numerous pharmaceuticals and xenobiotics. These enzymes are potently inhibited by trifluoromethyl ketone containing (TFK) inhibitors. We demonstrated that the ketone hydration state was affected by the surrounding chemical moieties and was related to inhibitor potency, with inhibitors that favored the gem-diol conformation exhibiting greater potency. Ab initio calculations were performed to determine the energy of hydration of the ketone, and the values were correlated with esterase inhibition data for a series of carboxylesterase inhibitors. This system was examined in three different mammalian models (human liver microsomes, murine liver microsomes, and commercial porcine liver esterase) and in an insect enzyme preparation (juvenile hormone esterase). In all cases, the extent of ketone hydration was strongly correlated with biological potency. Our results showed a very strong correlation with the extent of hydration, accounting for 94% of activity for human liver microsome esterase inhibition (p < 0.01). The atomic charge on the carbon atom of the carbonyl group in the TFK also strongly correlated with inhibitor potency, accounting for 94% of inhibition activity in human liver microsomes (p < 0.01). In addition, we provide crystallographic evidence of intramolecular hydrogen bonding in sulfur-containing inhibitors and relate these data to gem-diol formation. This study provides insight into the mechanism of carboxylesterase inhibition and raises the possibility that inhibitors that too strongly favor the gem-diol configuration have decreased potency due to low rate of ketone formation.
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Hidrolasas de Éster Carboxílico/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Cetonas/química , Animales , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/metabolismo , Cristalografía por Rayos X , Inhibidores Enzimáticos/metabolismo , Femenino , Humanos , Enlace de Hidrógeno , Técnicas In Vitro , Cetonas/metabolismo , Masculino , Ratones , Microsomas Hepáticos/enzimología , Relación Estructura-Actividad Cuantitativa , Especificidad de la Especie , Porcinos , Termodinámica , Agua/químicaRESUMEN
To investigate the mechanism(s) of bisalkylation by isophosphoramide mustard (IPM), IPM-beta,beta,beta',beta'-d(4) was synthesized and the products of its reaction with thiosulfate (at pD 7.0) were analyzed by NMR. By both (1)H and (13)C NMR, the distribution of deuterium in the products was consistent with bisalkylation through sequential aziridinyl intermediates [(NCH(2)CD(2)S):(NCD(2)CH(2)S) = 53:47]. Under the given reaction conditions, label scrambling as a result of thiosulfate acting as a leaving group was ruled out through control experiments. The data gave a calculated kinetic isotope effect of 0.97 per deuterium. For the initial aziridine species formed from IPM, ab initio quantum chemical calculations gave a hybridization value of sp(2.4)(-)(2.5) for each of the C-H bonds of the reaction centers, and this correlated with the observed inverse isotope effect. Other structure and bond order data were also determined for this aziridine intermediate and related compounds.
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Carcinogenic heterocyclic amines are produced from overcooked foods and are highly mutagenic in most short-term test systems. One of the most abundant of these amines, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), induces breast, colon and prostate tumors in rats. Human dietary epidemiology studies suggest a strong correlation between either meat consumption or well-done muscle meat consumption and cancers of the colon, breast, stomach, lung and esophagus. For over 20 years our laboratory has helped define the human exposure to these dietary carcinogens. In this report we describe how various environmental exposures may modulate the risk from exposure to heterocyclic amines, especially PhIP. To assess the impact of foods on PhIP metabolism in humans, we developed an LC/MS/MS method to analyze the four major PhIP urinary metabolites following the consumption of a single portion of grilled chicken. Adding broccoli to the volunteers' diet altered the kinetics of PhIP metabolism. At the cellular level we have found that PhIP itself stimulates a significant estrogenic response in MCF-7 cells, but even more interestingly, co-incubation of the cells with herbal teas appear to enhance the response. Numerous environmental chemicals found in food or the atmosphere can impact the exposure, metabolism, and cell proliferation response of heterocyclic amines.