RESUMEN
The objective of this study was to evaluate the efficacy of various decontamination methods and subsequent sterilization on contaminated and inoculated diamond-coated burs. One hundred forty new diamond-coated burs and 120 extracted human molars were utilized in this study. The burs were divided into 7 groups (n = 20): 1, positive control; 2, negative control; 3, new, unused burs; and 4 to 7, burs subjected to various combinations of cleaning methods (manual cleaning, use of a cleaning stone, and/or ultrasonic cleaning) after contamination. In all of the groups except group 3, the burs were sterilized and used to abrade the enamel and dentin of the sterilized extracted teeth. In groups 1 and 4 to 7, the burs were subsequently inoculated with 1 of the following microorganisms: Enterococcus faecalis (ATCC 19433), Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 15442), or Geobacillus stearothermophilus (ATCC 7953). Twenty-four hours after inoculation, the burs in group 2 and groups 4 to 7 were subjected to the cleaning treatments and sterilized with steam. The burs in all 7 groups were then cultured for bacterial contamination. No growth of any bacterial type was observed in any of the groups except the positive control group. The use of a cleaning stone in combination with manual or ultrasonic cleaning resulted in the least amount of remaining tooth debris on the diamond-coated burs. The contaminated and inoculated diamond-coated burs tested in this study were successfully sterilized, and the tested bacteria were eliminated. If using a diamond-coated bur multiple times, practitioners should consider utilizing debridement with a cleaning stone followed by either manual or ultrasonic cleaning and then by a single cycle of steam sterilization.
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Descontaminación , Diamante , Humanos , Diente Molar , Esterilización , Propiedades de SuperficieRESUMEN
Background. Our aim was to compare the emerging technique of single-incision laparoscopic surgery complete mesocolic excision (SILS CME) colectomy with the standard multiport laparoscopic CME (MPL CME) colectomy. Methods. MEDLINE (PubMed), Scopus, EMBASE, Ovid, and the Cochrane library were searched. Studies comparing the SILS CME with MPL CME in adults with colon adenocarcinoma were included. The Jadad and Newcastle Ottawa Scales were used to critically appraise the studies. The presence of statistical heterogeneity or publication bias was examined. Results. Seven studies (3 randomized) with a total number of 1344 patients were included (546 SILS CME and 798 MPL CME). No difference was found in anastomotic leakage (odds ratio [OR] = 0.79 [0.31 to 2.03]; P = .63), number of lymph nodes (weighted mean difference [WMD] = 0.85 [-0.97 to 2.66]; P = .36), hospital stay (WMD = 0.01 [-0.19 to 0.20]; P = .96), overall survival (hazard ratio [HR] = 1.19 [0.29 to 4.80]; P = .81), and disease-free survival (HR = 1.30 [0.30 to 5.61]; P = .72). Skin incision was shorter in SILS CME group (WMD = -3.02 [-3.25 to -2.80]; P < .00001) but with no difference in pain reported in postoperative day 1 (standardized mean difference [SMD] = -0.21 [-0.50 to 0.09]; P = .17) or day 2 (SMD = 0.16 [-0.52 to 0.84]; P = .64). Conclusions. SILS CME, although technically more demanding, has equivalent short- and long-term outcomes when compared with MPL CME. Potential benefits in cosmesis or postoperative pain need to be further explored by high-quality randomized controlled trials.
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Colectomía , Neoplasias del Colon , Laparoscopía , Mesocolon/cirugía , Anciano , Anciano de 80 o más Años , Colectomía/métodos , Colectomía/mortalidad , Neoplasias del Colon/mortalidad , Neoplasias del Colon/patología , Neoplasias del Colon/cirugía , Supervivencia sin Enfermedad , Femenino , Humanos , Laparoscopía/métodos , Laparoscopía/mortalidad , Ganglios Linfáticos/patología , Masculino , Persona de Mediana Edad , Complicaciones PosoperatoriasRESUMEN
Intrinsically disordered regions are present in one-third of eukaryotic proteins and are overrepresented in cellular processes such as signaling, suggesting that intrinsically disordered proteins (IDPs) may have a functional advantage over folded proteins. Upon interacting with a partner macromolecule, a subset of IDPs can fold and bind to form a well-defined three-dimensional conformation. For example, disordered BH3-only proteins bind promiscuously to a large number of homologous BCL-2 family proteins, where they fold to a helical structure in a groove on the BCL-2-like protein surface. As two protein chains are involved in the folding reaction, and the structure is only formed in the presence of the partner macromolecule, this raises the question of where the folding information is encoded. Here, we examine these coupled folding and binding reactions to determine which component determines the folding and binding pathway. Using Φ value analysis to compare transition state interactions between the disordered BH3-only proteins PUMA and BID and the folded BCL-2-like proteins A1 and MCL-1, we found that, even though the BH3-only protein is disordered in isolation and requires a stabilizing partner to fold, its folding and binding pathway is encoded in the IDP itself; the reaction is not templated by the folded partner. We suggest that, by encoding both its transition state and level of residual structure, an IDP can evolve a specific kinetic profile, which could be a crucial functional advantage of disorder.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Pliegue de Proteína , Proteínas Supresoras de Tumor/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/química , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/química , Cristalografía por Rayos X , Proteínas Intrínsecamente Desordenadas/química , Cinética , Ratones , Modelos Moleculares , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/química , Unión Proteica , Conformación Proteica , Transducción de Señal , Termodinámica , Proteínas Supresoras de Tumor/químicaRESUMEN
The realization of the therapeutic potential of targeting the M1 muscarinic acetylcholine receptor (mAChR) for the treatment of cognitive decline in Alzheimer's disease has prompted the discovery of M1 mAChR ligands showing efficacy in alleviating cognitive dysfunction in both rodents and humans. Among these is GSK1034702 (7-fluoro-5-methyl-3-[1-(oxan-4-yl)piperidin-4-yl]-1H-benzimidazol-2-one), described previously as a potent M1 receptor allosteric agonist, which showed procognitive effects in rodents and improved immediate memory in a clinical nicotine withdrawal test but induced significant side effects. Here we provide evidence using ligand binding, chemical biology and functional assays to establish that rather than the allosteric mechanism claimed, GSK1034702 interacts in a bitopic manner at the M1 mAChR such that it can concomitantly span both the orthosteric and an allosteric binding site. The bitopic nature of GSK1034702, together with the intrinsic agonist activity and a lack of muscarinic receptor subtype selectivity reported here, all likely contribute to the adverse effects of this molecule in clinical trials. Although they impart beneficial effects on learning and memory, we conclude that these properties are undesirable in a clinical candidate due to the likelihood of adverse side effects. Rather, our data support the notion that "pure" positive allosteric modulators showing selectivity for the M1 mAChR with low levels of intrinsic activity would be preferable to provide clinical efficacy with low adverse responses.
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Acetilcolina/metabolismo , Agonistas Muscarínicos/farmacología , Receptor Muscarínico M1/metabolismo , Receptores Muscarínicos/metabolismo , Regulación Alostérica/efectos de los fármacos , Sitio Alostérico/efectos de los fármacos , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Animales , Bencimidazoles/farmacología , Sitios de Unión/efectos de los fármacos , Células CHO , Línea Celular , Ensayos Clínicos como Asunto , Cricetinae , Cricetulus , Humanos , Aprendizaje/efectos de los fármacos , Masculino , Memoria/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Unión Proteica/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
In the search for improved symptomatic treatment options for neurodegenerative and neuropsychiatric diseases, muscarinic acetylcholine M1 receptors (M1 mAChRs) have received significant attention. Drug development efforts have identified a number of novel ligands, some of which have advanced to the clinic. However, a significant issue for progressing these therapeutics is the lack of robust, translatable, and validated biomarkers. One valuable approach to assessing target engagement is to use positron emission tomography (PET) tracers. In this study we describe the pharmacological characterization of a selective M1 agonist amenable for in vivo tracer studies. We used a novel direct binding assay to identify nonradiolabeled ligands, including LSN3172176, with the favorable characteristics required for a PET tracer. In vitro functional and radioligand binding experiments revealed that LSN3172176 was a potent partial agonist (EC50 2.4-7.0 nM, Emax 43%-73%), displaying binding selectivity for M1 mAChRs (Kd = 1.5 nM) that was conserved across species (native tissue Kd = 1.02, 2.66, 8, and 1.03 at mouse, rat, monkey, and human, respectively). Overall selectivity of LSN3172176 appeared to be a product of potency and stabilization of the high-affinity state of the M1 receptor, relative to other mAChR subtypes (M1 > M2, M4, M5 > M3). In vivo, use of wild-type and mAChR knockout mice further supported the M1-preferring selectivity profile of LSN3172176 for the M1 receptor (78% reduction in cortical occupancy in M1 KO mice). These findings support the development of LSN3172176 as a potential PET tracer for assessment of M1 mAChR target engagement in the clinic and to further elucidate the function of M1 mAChRs in health and disease.
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Tomografía de Emisión de Positrones/métodos , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/metabolismo , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Humanos , Cinética , Ratones , Trazadores Radiactivos , Ratas , Reproducibilidad de los ResultadosRESUMEN
Appropriate integration of cellular signals requires a delicate balance of ligand-target binding affinities. Increasing the level of residual structure in intrinsically disordered proteins (IDPs), which are overrepresented in these cellular processes, has been shown previously to enhance binding affinities and alter cellular function. Conserved proline residues are commonly found flanking regions of IDPs that become helical upon interacting with a partner protein. Here, we mutate these helix-flanking prolines in p53 and MLL and find opposite effects on binding affinity upon an increase in free IDP helicity. In both cases, changes in affinity were due to alterations in dissociation, not association, rate constants, which is inconsistent with conformational selection mechanisms. We conclude that, contrary to previous suggestions, helix-flanking prolines do not regulate affinity by modulating the rate of complex formation. Instead, they influence binding affinities by controlling the lifetime of the bound complex.
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N-Metiltransferasa de Histona-Lisina/química , Proteínas Intrínsecamente Desordenadas/química , Proteínas de la Membrana/química , Proteína de la Leucemia Mieloide-Linfoide/química , Fosfoproteínas/química , Prolina/química , Proteína p53 Supresora de Tumor/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Clonación Molecular , Secuencia Conservada , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Modelos Moleculares , Mutación , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Prolina/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Intrinsically disordered proteins (IDPs) are characterized by a lack of persistent structure. Since their identification more than a decade ago, many questions regarding their functional relevance and interaction mechanisms remain unanswered. Although most experiments have taken equilibrium and structural perspectives, fewer studies have investigated the kinetics of their interactions. Here we review and highlight the type of information that can be gained from kinetic studies. In particular, we show how kinetic studies of coupled folding and binding reactions, an important class of signaling event, are needed to determine mechanisms.
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Proteínas Reguladoras de la Apoptosis/química , Proteína de Unión a CREB/química , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/química , Proteínas Intrínsecamente Desordenadas/química , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/química , Proteínas Proto-Oncogénicas/química , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Cinética , Simulación de Dinámica Molecular , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Unión Proteica , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Electricidad Estática , TermodinámicaRESUMEN
RalA and RalB are members of the Ras family of small G proteins and are activated downstream of Ras via RalGEFs. The RalGEF-Ral axis represents one of the major effector pathways controlled by Ras and as such is an important pharmacological target. RalA and RalB are approximately 80% identical at the amino acid level; despite this, they have distinct roles both in normal cells and in the disease state. We have used our structure of RalB-RLIP76 to guide an analysis of Ral-effector interaction interfaces, creating panels of mutant proteins to probe the energetics of these interactions. The data provide a physical mechanism that underpins the effector selective mutations commonly employed to dissect Ral G protein function. Comparing the energetic landscape of the RalB-RLIP76 and RalB-Sec5 complexes reveals mutations in RalB that lead to differential binding of the two effector proteins. A panel of RLIP76 mutants was used to probe the interaction between RLIP76 and RalA and -B. Despite 100% sequence identity in the RalA and -B contact residues with RLIP76, differences still exist in the energetic profiles of the two complexes. Therefore, we have revealed properties that may account for some of the functional separation observed with RalA and RalB at the cellular level. Our mutations, in both the Ral isoforms and RLIP76, provide new tools that can be employed to parse the complex biology of Ral G protein signaling networks. The combination of these thermodynamic and structural data can also guide efforts to ablate RalA and -B activity with small molecules and peptides.
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Isoformas de Proteínas/química , Termodinámica , Proteínas de Unión al GTP ral/química , Transportadoras de Casetes de Unión a ATP/química , Proteínas Activadoras de GTPasa/química , Proteínas de Transporte Vesicular/químicaRESUMEN
Membraneless organelles, or biomolecular condensates, enable cells to compartmentalize material and processes into unique biochemical environments. While specific, attractive molecular interactions are known to stabilize biomolecular condensates, repulsive interactions, and the balance between these opposing forces, are largely unexplored. Here, we demonstrate that repulsive and attractive electrostatic interactions regulate condensate stability, internal mobility, interfaces, and selective partitioning of molecules both in vitro and in cells. We find that signaling ions, such as calcium, alter repulsions between model Ddx3 and Ddx4 condensate proteins by directly binding to negatively charged amino acid sidechains and effectively inverting their charge, in a manner fundamentally dissimilar to electrostatic screening. Using a polymerization model combined with generalized stickers and spacers, we accurately quantify and predict condensate stability over a wide range of pH, salt concentrations, and amino acid sequences. Our model provides a general quantitative treatment for understanding how charge and ions reversibly control condensate stability.
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Orgánulos , Proteínas , Orgánulos/metabolismo , Proteínas/metabolismo , ADN Helicasas/metabolismo , ARN Helicasas DEAD-box/metabolismo , Iones/análisis , Iones/metabolismoRESUMEN
An inherent tension between religion and psychotherapy has inhibited the conversation between the two paradigms in determining the most effective approaches to improving mental health outcomes for people of faith. Preliminary research has suggested that the intersection between the two may prove fruitful in providing mental health interventions. As a part of a broader big-data study sponsored by the Bridges Consortium of Brigham Young University and underwritten by the John Templeton Foundation, the present study evaluated the effectiveness of the spiritually integrated strategies of the modality Gestalt Pastoral Care (GPC) focusing on two goals: (a) determining the overall clinical effectiveness of GPC, not previously scientifically validated, and (b) evaluating the effectiveness of GPC in the reduction of symptoms most commonly seen in clients using empirically validated clinical measures. Using a practice-based research design, 324 participants, engaging in individual session format and/or multiperson retreat format, were followed up to 1 year using measures including the Clinically Adaptive Multidimensional Outcome Survey (CAMOS), the Clinical Outcomes in Routine Evaluation (CORE-10), the Primary Care PTSD Screen for Diagnostic and Statistical Manual of Mental Disorders [DSM]-5(PC-Post-traumatic Stress Disorder [PTSD]-5), and the Spiritual Index of Well-Being (SIWB). A series of paired t-tests compared differences from the first session (pretreatment) to last session (posttreatment) and showed significant improvements in all of the clinical outcomes. These data indicate a slightly stronger relationship between symptom reduction and delivery in the retreat setting. (PsycInfo Database Record (c) 2022 APA, all rights reserved).
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Cuidado Pastoral , Trastornos por Estrés Postraumático , Humanos , Psicoterapia/métodos , Religión , Espiritualidad , Trastornos por Estrés Postraumático/psicologíaRESUMEN
Nicotinamide adenine dinucleotide (NAD+) is an essential cofactor required for proper functioning of all cells and its decline is correlated with advancing age and disease. This randomized, triple-blind, placebo-controlled, crossover pilot study assessed the efficacy and safety of a combination of nicotinamide with D-ribose (RiaGev) for NAD metabolome enhancement and related benefits in healthy middle-aged adults. Supplementing with 1520 mg RiaGev twice daily for 7 days significantly increased the NAD+ metabolome in blood, especially NADP+ by 27% compared to the placebo group (p = 0.033) and over the baseline (p = 0.007). Increases in glutathione and high energy phosphates were also observed in the blood. Seven-day supplementation with RiaGev significantly (p = 0.013) reduced overall blood glucose without significant changes in insulin secretion (p = 0.796), suggesting an improved insulin sensitivity and glucose tolerance. The waking salivary cortisol of the subjects steadily and significantly decreased (p = 0.026) in the RiaGev group in contrast to the placebo. Subjects in the RiaGev group showed less fatigue, improved mental concentration and motivation over the baseline (p = 0.015, 0.018, and 0.012, respectively) as observed through the Checklist Individual Strength (CIS) questionnaire. There were no clinically relevant adverse events, or alterations in hematology, electrolytes, liver, and kidney markers pre- and post-supplementation. RiaGev appears to be safe and efficacious in increasing NAD+ metabolome in healthy middle-aged adults, as shown by this study.
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NAD , Niacinamida , Adulto , Humanos , Metaboloma , Persona de Mediana Edad , NAD/metabolismo , Proyectos Piloto , RibosaRESUMEN
Intracellular compartmentalization through liquid-liquid phase separation is an emerging organizing principle of cell biology. These compartments, such as the nucleolus and stress granules, are collectively known as membraneless organelles or biomolecular condensates. In vitro studies of many protein components of biomolecular condensates, such as the intrinsically disordered regions of Ddx4, FUS, and Laf-1 proteins, have revealed much about the driving forces of the phase separation process. A common approach is to investigate how the temperature at which a protein solution forms condensates-the transition temperature-responds to changes in the solution composition. We describe a method to measure the in vitro transition temperature of a sub-10 µL sample of a phase-separating solution using transmitted light microscopy. Through monitoring changes in transition temperature with solution conditions, this approach allows the impact of additional biomolecules and additives to be quantitatively assessed and permits the construction of phase diagrams.
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Proteínas Intrínsecamente Desordenadas/química , Microscopía/métodos , Transición de Fase , Termometría/métodos , Temperatura de Transición , ARN Helicasas DEAD-box/química , Humanos , Procesamiento de Imagen Asistido por Computador , Microscopía/instrumentación , Nefelometría y Turbidimetría , Concentración Osmolar , Soluciones , Termometría/instrumentaciónRESUMEN
The purpose of this study was to investigate the manufacturer's claims regarding a novel needleless intraligamentary local anesthesia injection device (Numbee, BioDent) to provide effective single tooth anesthesia. Investigators compared the Numbee with a traditional inferior alveolar nerve block (IANB) during a restorative procedure on mandibular teeth. A randomized, split-mouth design was conducted with 15 adult subjects receiving an IANB on one side and a Numbee injection on the same tooth type on the contralateral side. Subjects recorded injection pain using the Visual Analog Scale (VAS) and their preferred injection technique. Anesthesia was considered profound with 2 consecutive electric pulp tester readings of 80. If subjects became symptomatic during the restorative procedure, rescue anesthesia was administered. The difference in VAS scores for injection pain between the Numbee and the IANB was not significant (p = .078). For the IANB, the incidence of profound anesthesia was 46%, and required rescue anesthesia was 20%. For the Numbee, the incidence of profound anesthesia was 0%, and required rescue anesthesia was 60%. Subject preference was evenly split (50/50%) between the 2 techniques. The IANB outperformed the Numbee device for achieving profound anesthesia and requiring less rescue anesthesia.
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Anestesia Dental , Bloqueo Nervioso , Adulto , Anestesia Dental/efectos adversos , Anestesia Local , Anestésicos Locales , Método Doble Ciego , Humanos , Nervio Mandibular , Diente Molar , Bloqueo Nervioso/efectos adversos , JeringasRESUMEN
Intrinsically disordered proteins are abundant in signaling processes such as transcription. Suitable binding and unbinding rates of proteins with their partners are critical for allowing them to perform their biological roles. Understanding how these are achieved, and indeed designing strategies for intervening or modulating related biological processes, therefore requires kinetic studies. In this chapter, we describe stopped-flow-based methods for determining association and dissociation rate constants for pairs of macromolecular binding partners. We describe how to select the simplest appropriate model to describe the interaction, and highlight cases where it is possible to distinguish between induced fit and conformational selection binding mechanisms. Finally, we go on to describe methods for examining the role of electrostatic forces in binding processes, and for describing the transition state for binding processes that have folding associated with them.
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Proteínas Intrínsecamente Desordenadas/metabolismo , Espectrometría de Fluorescencia/instrumentación , Algoritmos , Animales , Diseño de Equipo , Humanos , Proteínas Intrínsecamente Desordenadas/química , Cinética , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Espectrometría de Fluorescencia/métodos , Electricidad EstáticaRESUMEN
In the original version of this Article, the Acknowledgement section omitted financial support from the Deutsche Forschungsgemeinschaft grant SFB 958/A4. This error has now been corrected in both the PDF and HTML versions of the Article.
RESUMEN
BACKGROUND: Germ-line mutations in the base-excision-repair gene MYH have been associated with recessive inheritance of multiple colorectal adenomas. Tumors from affected persons displayed excess somatic transversions of a guanine-cytosine pair to a thymine-adenine pair (G:C-->T:A) in the APC gene. METHODS: We screened for germ-line MYH mutations in 152 patients with multiple (3 to 100) colorectal adenomas and 107 APC-mutation-negative probands with classic familial adenomatous polyposis (>100 adenomas). Subgroups were analyzed for changes in the related genes MTH1 and OGG1. Adenomas were tested for somatic APC mutations. RESULTS: Six patients with multiple adenomas and eight patients with polyposis had biallelic germline MYH variants. Missense and protein-truncating mutations were found, and the spectrums of mutations were very similar in the two groups of patients. In the tumors of carriers of biallelic mutations, all somatic APC mutations were G:C-->T:A transversions. In the group with multiple adenomas, about one third of patients with more than 15 adenomas had biallelic MYH mutations. In the polyposis group, no patient with biallelic MYH mutations had severe disease (>1000 adenomas), but three had extracolonic disease. No clearly pathogenic MTH1 or OGG1 mutations were identified. CONCLUSIONS: Germ-line MYH mutations predispose persons to a recessive phenotype, multiple adenomas, or polyposis coli. For patients with about 15 or more colorectal adenomas--especially if no germ-line APC mutation has been identified and the family history is compatible with recessive inheritance--genetic testing of MYH is indicated for diagnosis and calculation of the level of risk in relatives. Clinical care of patients with biallelic MYH mutations should be similar to that of patients with classic or attenuated familial adenomatous polyposis.
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Adenoma/genética , Poliposis Adenomatosa del Colon/genética , Neoplasias Colorrectales/genética , ADN Glicosilasas , Enzimas Reparadoras del ADN , Genes APC , Mutación de Línea Germinal , N-Glicosil Hidrolasas/genética , Adolescente , Adulto , Anciano , Estudios de Casos y Controles , Niño , ADN-Formamidopirimidina Glicosilasa , Femenino , Humanos , Pérdida de Heterocigocidad , Masculino , Persona de Mediana Edad , Monoéster Fosfórico Hidrolasas/genéticaRESUMEN
Understanding and control of structures and rates involved in protein ligand binding are essential for drug design. Unfortunately, atomistic molecular dynamics (MD) simulations cannot directly sample the excessively long residence and rearrangement times of tightly binding complexes. Here we exploit the recently developed multi-ensemble Markov model framework to compute full protein-peptide kinetics of the oncoprotein fragment 25-109Mdm2 and the nano-molar inhibitor peptide PMI. Using this system, we report, for the first time, direct estimates of kinetics beyond the seconds timescale using simulations of an all-atom MD model, with high accuracy and precision. These results only require explicit simulations on the sub-milliseconds timescale and are tested against existing mutagenesis data and our own experimental measurements of the dissociation and association rates. The full kinetic model reveals an overall downhill but rugged binding funnel with multiple pathways. The overall strong binding arises from a variety of conformations with different hydrophobic contact surfaces that interconvert on the milliseconds timescale.
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Péptidos/química , Proteínas/química , Cinética , Simulación de Dinámica MolecularRESUMEN
Many intrinsically disordered proteins (IDPs) participate in coupled folding and binding reactions and form alpha helical structures in their bound complexes. Alanine, glycine, or proline scanning mutagenesis approaches are often used to dissect the contributions of intrinsic helicities to coupled folding and binding. These experiments can yield confounding results because the mutagenesis strategy changes the amino acid compositions of IDPs. Therefore, an important next step in mutagenesis-based approaches to mechanistic studies of coupled folding and binding is the design of sequences that satisfy three major constraints. These are (i) achieving a target intrinsic alpha helicity profile; (ii) fixing the positions of residues corresponding to the binding interface; and (iii) maintaining the native amino acid composition. Here, we report the development of a G: enetic A: lgorithm for D: esign of I: ntrinsic secondary S: tructure (GADIS) for designing sequences that satisfy the specified constraints. We describe the algorithm and present results to demonstrate the applicability of GADIS by designing sequence variants of the intrinsically disordered PUMA system that undergoes coupled folding and binding to Mcl-1. Our sequence designs span a range of intrinsic helicity profiles. The predicted variations in sequence-encoded mean helicities are tested against experimental measurements.
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Algoritmos , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Ingeniería de Proteínas/métodos , Secuencia de Aminoácidos , Modelos Moleculares , Mutagénesis , Estructura Secundaria de ProteínaRESUMEN
The site of the 'first hit' in the APC tumour suppressor gene determines the type of the 'second hit', both in familial adenomatous polyposis (FAP) and sporadic colorectal tumours. Mutations near codon 1300 are associated with loss of heterozygosity (LOH) of the wild-type allele; other tumours tend to have two protein-truncating mutations. In this study, we have confirmed and refined the LOH-associated region in colorectal FAP: allelic loss in adenomatous polyps tended to occur when the germline mutation lay in the region of the APC gene between the first and second beta-catenin degradation repeats (codons 1285-1378). LOH generally occurred by mitotic recombination, leaving two identical alleles, each encoding a protein with one remaining beta-catenin degradation repeat. For patients with germline mutations that truncated the protein before the first repeat (codon 1264), LOH was very rare and tumours generally acquired a somatic mutation which left two, or less often one, repeats remaining in the protein. In our sample set, patients with germline mutations after the second beta-catenin degradation repeat tended to have undetectable, presumably cryptic, somatic mutations in their polyps. Exceptions to these rules were, however, not uncommon. Although the site of the germline mutation was the strongest determinant of the somatic mutation in FAP tumours and most patients showed no clear tendency to acquire specific types of truncating 'second hit', a minority of patients did have unusual somatic mutation spectra in their polyps. Thus, some individuals may be predisposed to particular types of 'second hit' (for example, frameshift rather than nonsense changes). Overall, disease severity (polyp number) did not vary with individuals' spectrum of somatic APC mutations, providing no clear evidence for modifier genes that influence disease severity in this fashion. Our data are consistent with the hypothesis that there exists an optimal level of beta-catenin signalling in colorectal tumours and that the APC mutation spectrum principally reflects this fact. The association between 'first hits' and 'second hits' at APC is not, however, so strong as to suggest that tumorigenesis only occurs if the genotype is optimum; we suggest 'relaxed' terminology, the 'loose fit' model, to describe this situation.
Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/fisiología , Mutación , Poliposis Adenomatosa del Colon/genética , Alelos , Codón , Neoplasias Colorrectales/genética , Proteínas del Citoesqueleto/metabolismo , Exones , Eliminación de Gen , Mutación de Línea Germinal , Humanos , Pérdida de Heterocigocidad , Repeticiones de Microsatélite , Modelos Genéticos , Reacción en Cadena de la Polimerasa , Transducción de Señal , Transactivadores/metabolismo , beta CateninaRESUMEN
UNLABELLED: The muscarinic M1 receptor (M1R) is highly involved in cognition, and selective M1 agonists have procognitive properties. Loss of M1R has been found in postmortem brain tissue for several neuropsychiatric disorders and may be related to symptoms of cognitive dysfunction. (123)I-iododexetimide is used for imaging muscarinic acetylcholine receptors (mAchRs). Considering its high brain uptake and intense binding in M1R-rich brain areas, (123)I-iododexetimide may be an attractive radiopharmaceutical to image M1R. To date, the binding affinity and selectivity of (123)I-iododexetimide for the mAchR subtypes has not been characterized, nor has its brain distribution been studied intensively. Therefore, this study aimed to address these topics. METHODS: The in vitro affinity and selectivity of (127)I-iododexetimide (cold-labeled iododexetimide), as well as its functional antagonist properties (guanosine 5'-[γ-(35)S-thio]triphosphate [GTPγ(35)S] assay), were assessed on recombinant human M1R-M5R. Distributions of (127)I-iododexetimide and (123)I-iododexetimide in the brain were evaluated using liquid chromatography-mass spectrometry and storage phosphor imaging, respectively, ex vivo in rats, wild-type mice, and M1-M5 knock-out (KO) mice. Inhibition of (127)I-iododexetimide and (123)I-iododexetimide binding in M1R-rich brain areas by the M1R/M4R agonist xanomeline, or the antipsychotics olanzapine (M1R antagonist) and haloperidol (low M1R affinity), was assessed in rats ex vivo. RESULTS: In vitro, (127)I-iododexetimide displayed high affinity for M1R (pM range), with modest selectivity over other mAchRs. In biodistribution studies on rats, ex vivo (127)I-iododexetimide binding was much higher in M1R-rich brain areas, such as the cortex and striatum, than in cerebellum (devoid of M1Rs). In M1 KO mice, but not M2-M5 KO mice, (127)I-iododexetimide binding was strongly reduced in the frontal cortex compared with wild-type mice. Finally, acute administration of both an M1R/M4R agonist xanomeline and the M1R antagonist olanzapine was able to inhibit (123)I-iododexetimide ex vivo, and (123)I-iododexetimide binding in M1-rich brain areas in rats, whereas administration of haloperidol had no effect. CONCLUSION: The current results suggest that (123)I-iododexetimide preferentially binds to M1R in vivo and can be displaced by M1R ligands. (123)I-iododexetimide may therefore be a useful imaging tool as a way to further evaluate M1R changes in neuropsychiatric disorders, as a potential stratifying biomarker, or as a clinical target engagement biomarker to assess M1R.