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Pharmacokinetics and pharmacodynamics of many biologics are influenced by their complex binding to biological receptors. Biologics consist of diverse groups of molecules with different binding kinetics to its receptors including IgG with simple one-to-one drug receptor bindings, bispecific antibody (BsAb) that binds to two different receptors, and antibodies that can bind to six or more identical receptors. As the binding process is typically much faster than elimination (or internalization) and distribution processes, quasi-equilibrium (QE) binding models are commonly used to describe drug-receptor binding kinetics of biologics. However, no general QE modeling framework is available to describe complex binding kinetics for diverse classes of biologics. In this paper, we describe novel approaches of using differential algebraic equations (DAE) to solve three QE multivalent drug-receptor binding (QEMB) models. The first example describes the binding kinetics of three-body equilibria of BsAb that binds to 2 different receptors for trimer formation. The second example models an engineered IgG variant (Multabody) that can bind to 24 identical target receptors. The third example describes an IgG with modified neonatal Fc receptor (FcRn) binding affinity that competes for the same FcRn receptor as endogenous IgG. The model parameter estimates were obtained by fitting the model to all data simultaneously. The models allowed us to study potential roles of cooperative binding on bell-shaped drug exposure-response relationships of BsAb, and concentration-depended distribution of different drug-receptor complexes for Multabody. This DAE-based QEMB model platform can serve as an important tool to better understand complex binding kinetics of diverse classes of biologics.
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Determining a drug dosing recommendation with a PKPD model can be a laborious and complex task. Recently, an optimal dosing algorithm (OptiDose) was developed to compute the optimal doses for any pharmacometrics/PKPD model for a given dosing scenario. In the present work, we reformulate the underlying optimal control problem and elaborate how to solve it with standard commands in the software NONMEM. To demonstrate the potential of the OptiDose implementation in NONMEM, four relevant but substantially different optimal dosing tasks are solved. In addition, the impact of different dosing scenarios as well as the choice of the therapeutic goal on the computed optimal doses are discussed.
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Algoritmos , Programas InformáticosRESUMEN
INTRODUCTION: We previously characterized associations between brain imaging measurements of amyloid-ß (Aß) plaque burden and apolipoprotein E (APOE) ε4 gene dose in a small number of cognitively unimpaired late-middle-aged APOE ε4 homozygotes (HMs), heterozygotes (HTs), and noncarriers (NCs). We now characterize cross-sectional Aß plaque, tau tangle, and cortical atrophy (neurodegeneration) measurements, classifications, and associations with age in a larger number of unimpaired HMs, HTs, and NCs over a wider age range. METHODS: We analyzed 11 C Pittsburgh compound B (Aß) positron emission tomography (PET), flortaucipir (tau) PET, and volumetric magnetic resonance imaging data from 164 study participants of age 47-86 years, including 26 APOE ε4 HMs, 48 HTs, and 90 NCs matched for age and sex. RESULTS: Aß PET measurements rose, plateaued at the respective ages of 68 and 76, and then declined with age in unimpaired HM and HT groups. Compared with NCs, these two groups began to have significantly higher Aß PET measurements at ages 62 and 70, respectively, and no longer had significantly higher measurements by ages 71 and 78, respectively. They began to have significantly higher entorhinal cortex tau PET measurements at ages 66 and 70, respectively, and no longer had significantly higher measurements by ages 74 and 78, respectively. Brain atrophy measurements tended to decline slowly with age in all three genetic groups. Their elevated tau PET measurements were attributable to those with positive Aß PET scans. 41.0%, 18.0%, and 5.0% of the 47- to 70-year-old HMs, HTs, and NCs and 25.0%, 79.0%, and 38.0% of the 71- to 86-year-old HMs, HTs, and NCs had positive Aß PET scans, and the long-term recall memory scores are significantly higher in the older HMs than in HT and NC groups, suggesting resistance to Aß deposition in those HMs who remained unimpaired at older ages. CONCLUSIONS: This study provides information about Aß plaque burden, tau tangle burden, and neurodegeneration in cognitively unimpaired persons at three levels of genetic risk for AD. Unimpaired APOE ε4 HMs can be studied before their 70s to evaluate the understanding of factors, processes, and interventions involved in the predisposition to and prevention of AD, and after their 70s, to discover factors, processes, and interventions involved in the resilience or resistance to and prevention of AD.
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Amiloide/metabolismo , Apolipoproteína E4/genética , Encéfalo/fisiología , Voluntarios Sanos/estadística & datos numéricos , Neuroimagen , Proteínas tau/metabolismo , Anciano , Alelos , Cognición , Estudios Transversales , Femenino , Humanos , Estudios Longitudinales , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Pruebas Neuropsicológicas/estadística & datos numéricos , Placa Amiloide , Tomografía de Emisión de PositronesRESUMEN
The ability of the replisome to seamlessly coordinate both high fidelity and translesion DNA synthesis requires a means to regulate recruitment and binding of enzymes from solution. Co-occupancy of multiple DNA polymerases within the replisome has been observed primarily in bacteria and is regulated by posttranslational modifications in eukaryotes, and both cases are coordinated by the processivity clamp. Because of the heterotrimeric nature of the PCNA clamp in some archaea, there is potential to occupy and regulate specific polymerases at defined subunits. In addition to specific PCNA and polymerase interactions (PIP site), we have now identified and characterized a novel protein contact between the Y-family DNA polymerase and the B-family replication polymerase (YB site) bound to PCNA and DNA from Sulfolobus solfataricus. These YB contacts are essential in forming and stabilizing a supraholoenzyme (SHE) complex on DNA, effectively increasing processivity of DNA synthesis. The SHE complex can not only coordinate polymerase exchange within the complex but also provides a mechanism for recruitment of polymerases from solution based on multiequilibrium processes. Our results provide evidence for an archaeal PCNA 'tool-belt' recruitment model of multienzyme function that can facilitate both high fidelity and translesion synthesis within the replisome during DNA replication.
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Proteínas Arqueales/metabolismo , Reparación del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/genética , Western Blotting , ADN de Archaea/química , ADN de Archaea/genética , ADN de Archaea/metabolismo , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/metabolismo , Cinética , Modelos Moleculares , Conformación de Ácido Nucleico , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Espectrometría de Fluorescencia , Sulfolobus solfataricus/enzimología , Sulfolobus solfataricus/genética , Sulfolobus solfataricus/metabolismoRESUMEN
DNA ligases have broad application in molecular biology, from traditional cloning methods to modern synthetic biology and molecular diagnostics protocols. Ligation-based detection of polynucleotide sequences can be achieved by the ligation of probe oligonucleotides when annealed to a complementary target sequence. In order to achieve a high sensitivity and low background, the ligase must efficiently join correctly base-paired substrates, while discriminating against the ligation of substrates containing even one mismatched base pair. In the current study, we report the use of capillary electrophoresis to rapidly generate mismatch fidelity profiles that interrogate all 256 possible base-pair combinations at a ligation junction in a single experiment. Rapid screening of ligase fidelity in a 96-well plate format has allowed the study of ligase fidelity in unprecedented depth. As an example of this new method, herein we report the ligation fidelity of Thermus thermophilus DNA ligase at a range of temperatures, buffer pH and monovalent cation strength. This screen allows the selection of reaction conditions that maximize fidelity without sacrificing activity, while generating a profile of specific mismatches that ligate detectably under each set of conditions.
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Proteínas Bacterianas/química , Emparejamiento Base , ADN Ligasas/química , Ensayos Analíticos de Alto Rendimiento , Thermus thermophilus/química , Proteínas Bacterianas/aislamiento & purificación , Disparidad de Par Base , ADN Ligasa (ATP) , ADN Ligasas/aislamiento & purificación , Fluoresceína/química , Colorantes Fluorescentes/química , Concentración de Iones de Hidrógeno , Sensibilidad y Especificidad , Especificidad por Sustrato , Temperatura , Thermus thermophilus/enzimologíaRESUMEN
DNA ligases, essential to both in vivo genome integrity and in vitro molecular biology, catalyze phosphodiester bond formation between adjacent 3'-OH and 5'-phosphorylated termini in dsDNA. This reaction requires enzyme self-adenylylation, using ATP or NAD+ as a cofactor, and AMP release concomitant with phosphodiester bond formation. In this study, we present the first fast time scale binding kinetics of T4 DNA ligase to both nicked substrate DNA (nDNA) and product-equivalent non-nicked dsDNA, as well as binding and release kinetics of AMP. The described assays utilized a fluorescein-dT labeled DNA substrate as a reporter for ligase·DNA interactions via stopped-flow fluorescence spectroscopy. The analysis revealed that binding to nDNA by the active adenylylated ligase occurs in two steps, an initial rapid association equilibrium followed by a transition to a second bound state prior to catalysis. Furthermore, the ligase binds and dissociates from nicked and nonsubstrate dsDNA rapidly with initial association affinities on the order of 100 nM regardless of enzyme adenylylation state. DNA binding occurs through a two-step mechanism in all cases, confirming prior proposals of transient binding followed by a transition to a productive ligase·nDNA (Lig·nDNA) conformation but suggesting that weaker nonproductive "closed" complexes are formed as well. These observations demonstrate the mechanistic underpinnings of competitive inhibition by rapid binding of nonsubstrate DNA, and of substrate inhibition by blocking of the self-adenylylation reaction through nick binding by deadenylylated ligase. Our analysis further reveals that product release is not the rate-determining step in turnover.
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ADN Ligasas/metabolismo , ADN/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Cinética , Unión ProteicaRESUMEN
BACKGROUND: Older age is a major risk factor for severe COVID-19 disease which has been associated with a variety of neurologic complications, both acutely and chronically. OBJECTIVE: We sought to determine whether milder COVID-19 disease in older vulnerable individuals is also associated with cognitive and behavioral sequelae. METHODS: Neuropsychological, behavioral, and clinical outcomes before and after contracting COVID-19 disease, were compared in members of two ongoing longitudinal studies, the Arizona APOE Cohort and the national Alzheimer's Disease Research Center (ADRC). RESULTS: 152 APOE and 852 ADRC cohort members, mean age overall roughly 70 years, responded to a survey that indicated 21 APOE and 57 ADRC members had contracted COVID-19 before their ensuing (post-COVID) study visit. The mean interval between test sessions that preceded and followed COVID was 2.2 years and 1.2 years respectively for the APOE and ADRC cohorts. The magnitude of change between the pre and post COVID test sessions did not differ on any neuropsychological measure in either cohort. There was, however, a greater increase in informant (but not self) reported cognitive change in the APOE cohort (pâ=â0.018), but this became nonsignificant after correcting for multiple comparisons. CONCLUSION: Overall members of both cohorts recovered well despite their greater age-related vulnerability to more severe disease.
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Enfermedad de Alzheimer , COVID-19 , Disfunción Cognitiva , Humanos , Anciano , Pruebas Neuropsicológicas , COVID-19/complicaciones , Cognición , Estudios Longitudinales , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/psicología , Apolipoproteínas E/genética , Apolipoproteína E4 , Disfunción Cognitiva/etiologíaRESUMEN
We have biochemically and kinetically characterized the polymerase and exonuclease activities of the third B-family polymerase (Dpo3) from the hyperthermophilic Crenarchaeon, Sulfolobus solfataricus (Sso). We have established through mutagenesis that despite incomplete sequence conservation, the polymerase and exonuclease active sites are functionally conserved in Dpo3. Using pre-steady-state kinetics, we can measure the fidelity of nucleotide incorporation by Dpo3 from the polymerase active site alone to be 10(3)-10(4) at 37 °C. The functional exonuclease proofreading active site will increase fidelity by at least 10(2), making Dpo3 comparable to other DNA polymerases in this family. Additionally, Dpo3's exonuclease activity is modulated by temperature, where a loss of promiscuous degradation activity can be attributed to a reorganization of the exonuclease domain when it is bound to primer-template DNA at high temperatures. Unexpectedly, the DNA binding affinity is weak compared with those of other DNA polymerases of this family. A comparison of the fidelity, polymerization kinetics, and associated functional exonuclease domain with those previously reported for other Sso polymerases (Dpo1 and Dpo4) illustrates that Dpo3 is a potential player in the proper maintenance of the archaeal genome.
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Proteínas Arqueales/química , ADN Polimerasa III/química , Sulfolobus solfataricus/enzimología , Secuencia de Aminoácidos , Proteínas Arqueales/metabolismo , Dominio Catalítico , ADN Polimerasa III/metabolismo , Replicación del ADN , Genoma Arqueal , Cinética , Datos de Secuencia Molecular , Polimerizacion , Alineación de SecuenciaRESUMEN
Many therapeutic antibodies act as antagonists to competitively block cellular signaling pathways. We describe here an approach for the therapeutic use of monoclonal antibodies based on context-dependent attenuation to reduce pathologically high activity while allowing homeostatic signaling in biologically important pathways. Such attenuation is achieved by modulating the kinetics of a ligand binding to its various receptors and regulatory proteins rather than by complete blockade of signaling pathways. The anti-interleukin-1beta (IL-1beta) antibody XOMA 052 is a potent inhibitor of IL-1beta activity that reduces the affinity of IL-1beta for its signaling receptor and co-receptor but not for its decoy and soluble inhibitory receptors. This mechanism shifts the effective dose response of the cytokine so that the potency of IL-1beta bound by XOMA 052 is 20-100-fold lower than that of IL-1beta in the absence of antibody in a variety of in vitro cell-based assays. We propose that by decreasing potency of IL-1beta while allowing binding to its clearance and inhibitory receptors, XOMA 052 treatment will attenuate IL-1beta activity in concert with endogenous regulatory mechanisms. Furthermore, the ability to bind the decoy receptor may reduce the potential for accumulation of antibody.target complexes. Regulatory antibodies like XOMA 052, which selectively modulate signaling pathways, may represent a new mechanistic class of therapeutic antibodies.
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Anticuerpos Monoclonales/farmacología , Interleucina-1beta/fisiología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Bioingeniería , Fibroblastos/citología , Fibroblastos/fisiología , Células HeLa/efectos de los fármacos , Células HeLa/fisiología , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Humanos , Interleucina-1/fisiología , Interleucina-1beta/efectos de los fármacos , Riñón/efectos de los fármacos , Riñón/fisiología , Cinética , Ligandos , Luciferasas/genética , Pulmón/citología , Pulmón/fisiología , FN-kappa B/fisiología , Fosfoproteínas/efectos de los fármacos , Fosfoproteínas/metabolismo , Receptores de Interleucina-1/efectos de los fármacos , Receptores de Interleucina-1/fisiología , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
This NONMEM tutorial shows how to evaluate and optimize clinical trial designs, using algorithms developed in design software, such as PopED and PFIM 4.0. Parameter precision and model parameter estimability is obtained by assessing the Fisher Information Matrix (FIM), providing expected model parameter uncertainty. Model parameter identifiability may be uncovered by very large standard errors or inability to invert an FIM. Because evaluation of FIM is more efficient than clinical trial simulation, more designs can be investigated, and the design of a clinical trial can be optimized. This tutorial provides simple and complex pharmacokinetic/pharmacodynamic examples on obtaining optimal sample times, doses, or best division of subjects among design groups. Robust design techniques accounting for likely variability among subjects are also shown. A design evaluator and optimizer within NONMEM allows any control stream first developed for trial design exploration to be subsequently used for estimation of parameters of simulated or clinical data, without transferring the model to another software. Conversely, a model developed in NONMEM could be used for design optimization. In addition, the $DESIGN feature can be used on any model file and dataset combination to retrospectively evaluate the model parameter uncertainty one would expect given that the model generated the data, particularly if outliers of the actual data prevent a reasonable assessment of the variance-covariance. The NONMEM trial design feature is suitable for standard continuous data, whereas more elaborate trial designs or with noncontinuous data-types can still be accomplished in optimal design dedicated software like PopED and PFIM.
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Algoritmos , Ensayos Clínicos como Asunto/métodos , Modelos Estadísticos , Simulación por Computador , Humanos , Modelos Biológicos , Proyectos de InvestigaciónRESUMEN
Parameter estimation of a nonlinear model based on maximizing the likelihood using gradient-based numerical optimization methods can often fail due to premature termination of the optimization algorithm. One reason for such failure is that these numerical optimization methods cannot distinguish between the minimum, maximum, and a saddle point; hence, the parameters found by these optimization algorithms can possibly be in any of these three stationary points on the likelihood surface. We have found that for maximization of the likelihood for nonlinear mixed effects models used in pharmaceutical development, the optimization algorithm Broyden-Fletcher-Goldfarb-Shanno (BFGS) often terminates in saddle points, and we propose an algorithm, saddle-reset, to avoid the termination at saddle points, based on the second partial derivative test. In this algorithm, we use the approximated Hessian matrix at the point where BFGS terminates, perturb the point in the direction of the eigenvector associated with the lowest eigenvalue, and restart the BFGS algorithm. We have implemented this algorithm in industry standard software for nonlinear mixed effects modeling (NONMEM, version 7.4 and up) and showed that it can be used to avoid termination of parameter estimation at saddle points, as well as unveil practical parameter non-identifiability. We demonstrate this using four published pharmacometric models and two models specifically designed to be practically non-identifiable.
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Algoritmos , Química Farmacéutica/métodos , Química Farmacéutica/estadística & datos numéricos , Dinámicas no LinealesRESUMEN
In this second tutorial on NONMEM, the examples of typical pharmacokinetic/pharmacodynamic modeling problems that occur in the pharmaceutical field will be presented, which the reader can use as a template for his or her own modeling endeavors. Each of the problems presented is challenging in some way, and the logic behind setting up each problem is discussed. Logical concepts of the problem itself as well as the technical aspect of how to set it up in NONMEM are described and demonstrated. The concepts behind the various estimation algorithms will first be described to allow the user a better understanding of how to use them.
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In this tutorial, the various components of NONMEM will be described, and the basic steps of setting up NONMEM control stream files and data files will be demonstrated. Some basic concepts of nonlinear mixed effects modeling will be discussed, along with simple examples demonstrating how to use NONMEM to perform population analysis of clinical data.
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BACKGROUND: Brain imaging measurements can provide evidence of possible preclinical Alzheimer's disease (AD). Their ability to predict individual imminent clinical conversion remains unclear. OBJECTIVE: To investigate the ability of pre-specified volumetric magnetic resonance imaging (MRI) and fluorodeoxyglucose positron emission tomography (FDG-PET) measurements to predict which cognitively unimpaired older participants would subsequently progress to amnestic mild cognitive impairment (aMCI) within 2 years. METHODS: From an apolipoprotein E4 (APOE4) enriched prospective cohort study, 18 participants subsequently progressed to the clinical diagnosis of aMCI or probable AD dementia within 1.8±0.8 years (progressors); 20 participants matched for sex, age, education, and APOE allele dose remained cognitively unimpaired for at least 4 years (nonprogressors). A complementary control group not matched for APOE allele dose included 35 nonprogressors. Groups were compared on baseline FDG-PET and MRI measures known to be preferentially affected in the preclinical and clinical stages of AD and by voxel-wise differences in regional gray matter volume and glucose metabolism. Receiver Operating Characteristic, binary logistic regression, and leave-one-out procedures were used to predict clinical outcome for the a priori measures. RESULTS: Compared to non-progressors and regardless of APOE-matching, progressors had significantly reduced baseline MRI and PET measurements in brain regions preferentially affected by AD and reduced hippocampal volume was the strongest predictor of an individual's imminent progression to clinically significant memory decline (79% sensitivity/78% specificity among APOE-matched cohorts). CONCLUSION: Regional MRI and FDG-PET measurements may be useful in predicting imminent progression to clinically significant memory decline.
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Enfermedad de Alzheimer/diagnóstico por imagen , Encéfalo/diagnóstico por imagen , Disfunción Cognitiva/diagnóstico por imagen , Anciano , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Apolipoproteínas E/genética , Encéfalo/metabolismo , Encéfalo/patología , Disfunción Cognitiva/genética , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/patología , Progresión de la Enfermedad , Femenino , Fluorodesoxiglucosa F18 , Humanos , Estudios Longitudinales , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Imagen Multimodal , Tomografía de Emisión de Positrones , Pronóstico , Estudios Prospectivos , Radiofármacos , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Carriers of the APOE ε4 allele are at increased risk of developing Alzheimer's disease (AD), and have been shown to have reduced cerebral metabolic rate of glucose (CMRgl) in the same brain areas frequently affected in AD. These individuals also exhibit reduced plasma levels of apolipoprotein E (apoE) attributed to a specific decrease in the apoE4 isoform as determined by quantification of individual apoE isoforms in APOE ε4 heterozygotes. Whether low plasma apoE levels are associated with structural and functional brain measurements and cognitive performance remains to be investigated. METHODS: Using quantitative mass spectrometry we quantified the plasma levels of total apoE and the individual apoE3 and apoE4 isoforms in 128 cognitively normal APOE ε3/ε4 individuals included in the Arizona APOE cohort. All included individuals had undergone extensive neuropsychological testing and 25 had in addition undergone FDG-PET and MRI to determine CMRgl and regional gray matter volume (GMV). RESULTS: Our results demonstrated higher apoE4 levels in females versus males and an age-dependent increase in the apoE3 isoform levels in females only. Importantly, a higher relative ratio of apoE4 over apoE3 was associated with GMV loss in the right posterior cingulate and with reduced CMRgl bilaterally in the anterior cingulate and in the right hippocampal area. Additional exploratory analysis revealed several negative associations between total plasma apoE, individual apoE isoform levels, GMV and CMRgl predominantly in the frontal, occipital and temporal areas. Finally, our results indicated only weak associations between apoE plasma levels and cognitive performance which further appear to be affected by sex. CONCLUSIONS: Our study proposes a sex-dependent and age-dependent variation in plasma apoE isoform levels and concludes that peripheral apoE levels are associated with GMV, CMRgl and possibly cognitive performance in cognitively healthy individuals with a genetic predisposition to AD.
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Apolipoproteína E3/sangre , Apolipoproteína E3/genética , Apolipoproteína E4/sangre , Apolipoproteína E4/genética , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Factores de Edad , Anciano , Cognición , Estudios de Cohortes , Femenino , Predisposición Genética a la Enfermedad , Glucosa/metabolismo , Sustancia Gris/diagnóstico por imagen , Sustancia Gris/metabolismo , Humanos , Imagen por Resonancia Magnética , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Pruebas Neuropsicológicas , Tamaño de los Órganos , Tomografía de Emisión de Positrones , Factores SexualesRESUMEN
DNA ligases catalyze the repair of phosphate backbone breaks in DNA, acting with highest activity on breaks in one strand of duplex DNA. Some DNA ligases have also been observed to ligate two DNA fragments with short complementary overhangs or blunt-ended termini. In this study, several wild-type DNA ligases (phage T3, T4, and T7 DNA ligases, Paramecium bursaria chlorella virus 1 (PBCV1) DNA ligase, human DNA ligase 3, and Escherichia coli DNA ligase) were tested for their ability to ligate DNA fragments with several difficult to ligate end structures (blunt-ended termini, 3'- and 5'- single base overhangs, and 5'-two base overhangs). This analysis revealed that T4 DNA ligase, the most common enzyme utilized for in vitro ligation, had its greatest activity on blunt- and 2-base overhangs, and poorest on 5'-single base overhangs. Other ligases had different substrate specificity: T3 DNA ligase ligated only blunt ends well; PBCV1 DNA ligase joined 3'-single base overhangs and 2-base overhangs effectively with little blunt or 5'- single base overhang activity; and human ligase 3 had highest activity on blunt ends and 5'-single base overhangs. There is no correlation of activity among ligases on blunt DNA ends with their activity on single base overhangs. In addition, DNA binding domains (Sso7d, hLig3 zinc finger, and T4 DNA ligase N-terminal domain) were fused to PBCV1 DNA ligase to explore whether modified binding to DNA would lead to greater activity on these difficult to ligate substrates. These engineered ligases showed both an increased binding affinity for DNA and increased activity, but did not alter the relative substrate preferences of PBCV1 DNA ligase, indicating active site structure plays a role in determining substrate preference.
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ADN Ligasas/metabolismo , Roturas del ADN de Doble Cadena , Electroforesis Capilar , HumanosRESUMEN
DNA ligases are essential both to in vivo replication, repair and recombination processes, and in vitro molecular biology protocols. Prior characterization of DNA ligases through gel shift assays has shown the presence of a nick site to be essential for tight binding between the enzyme and its dsDNA substrate, with no interaction evident on dsDNA lacking a nick. In the current study, we observed a significant substrate inhibition effect, as well as the inhibition of both the self-adenylylation and nick-sealing steps of T4 DNA ligase by non-nicked, non-substrate dsDNA. Inhibition by non-substrate DNA was dependent only on the total DNA concentration rather than the structure; with 1 µg/mL of 40-mers, 75-mers, or circular plasmid DNA all inhibiting ligation equally. A >15-fold reduction in T4 DNA ligase self-adenylylation rate when in the presence of high non-nicked dsDNA concentrations was observed. Finally, EMSAs were utilized to demonstrate that non-substrate dsDNA can compete with nicked dsDNA substrates for enzyme binding. Based upon these data, we hypothesize the inhibition of T4 DNA ligase by non-nicked dsDNA is direct evidence for a two-step nick-binding mechanism, with an initial, nick-independent, transient dsDNA-binding event preceding a transition to a stable binding complex in the presence of a nick site.
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ADN Ligasas/química , ADN/química , Catálisis , Replicación del ADN , Técnicas In Vitro , CinéticaRESUMEN
Alzheimers disease (AD) is a progressive neurodegenerative disease most prevalent in the elderly. Distinguishing disease-related memory decline from normal age-related memory decline has been clinically difficult due to the subtlety of cognitive change during the preclinical stage of AD. In contrast, sensitive biomarkers derived from in vivo neuroimaging data could improve the early identification of AD. In this study, we employed a morphometric analysis in the hippocampus and lateral ventricle. A novel group-wise template-based segmentation algorithm was developed for ventricular segmentation. Further, surface multivariate tensor-based morphometry and radial distance on each surface point were computed. Using Hotellings T2 test, we found significant morphometric differences in both hippocampus and lateral ventricle between stable and clinically declining subjects. The left hemisphere was more severely affected than the right during this early disease stage. Hippocampal and ventricular morphometry has significant potential as an imaging biomarker for onset prediction and early diagnosis of AD.
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Alzheimer's disease (AD) is a progressive brain disease. Accurate diagnosis of AD and its prodromal stage, mild cognitive impairment, is crucial for clinical trial design. There is also growing interests in identifying brain imaging biomarkers that help evaluate AD risk presymptomatically. Here, we applied a recently developed multivariate tensor-based morphometry (mTBM) method to extract features from hippocampal surfaces, derived from anatomical brain MRI. For such surface-based features, the feature dimension is usually much larger than the number of subjects. We used dictionary learning and sparse coding to effectively reduce the feature dimensions. With the new features, an Adaboost classifier was employed for binary group classification. In tests on publicly available data from the Alzheimers Disease Neuroimaging Initiative, the new framework outperformed several standard imaging measures in classifying different stages of AD. The new approach combines the efficiency of sparse coding with the sensitivity of surface mTBM, and boosts classification performance.
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PURPOSE: To determine the feasibility of administration, safety, toxicity, immunogenicity, pharmacokinetics, maximum tolerated dose, and biodistribution of ING-1, a high-affinity, Human-Engineered monoclonal antibody (heMAb) to the Mr 40,000 epithelial cell adhesion molecule Ep-CAM, in patients with advanced adenocarcinomas. EXPERIMENTAL DESIGN: ING-1 was initially administered to patients as a 1-hour intravenous infusion every 3 weeks. Toxicity and pharmacokinetic data led to the evaluation of a weekly schedule. The distribution of iodine-131 (131I)-labeled ING-1 was studied. RESULTS: Twenty-five patients received 82 courses of ING-1. Minimal toxicity was initially observed at the 0.03-, 0.10-, and 0.30-mg/kg dose levels. A patient dosed at 1.0 mg/kg developed acute pancreatitis with severe abdominal pain, nausea, and vomiting. A patient dosed at 0.3 mg/kg had an asymptomatic amylase and lipase elevation to 502 units/L and 1,627 units/L, respectively. Both patients made uncomplicated recoveries. No other dose-limiting toxicities were observed. Regardless of dose, the volume of distribution (mean +/- SEM) was 46.6 +/- 1.6 mL/kg. ING-1 clearance decreased with increasing dose. To minimize toxicity and increase dose intensity, we then administered ING-1 weekly. No significant toxicity was observed in 7 patients dosed at 0.1 mg/kg. Studies of 131I-labeled ING-1 biodistribution showed radiolocalization to colorectal and prostate cancers. A patient with colorectal cancer had an 80% decrement in the levels of carcinoembryonic antigen. CONCLUSION: The recommended dose for ING-1 is 0.10 mg/kg by intravenous infusion weekly. The absence of severe toxicity at this dose, low immunogenicity, and preliminary evidence of ING-1 tumor localization and antitumor efficacy support the further clinical development of this antibody to treat Ep-CAM-positive malignant diseases.