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G-protein-coupled receptors (GPCRs) are key regulators of human physiology and are the targets of many small-molecule research compounds and therapeutic drugs. While most of these ligands bind to their target GPCR with high affinity, selectivity is often limited at the receptor, tissue and cellular levels. Antibodies have the potential to address these limitations but their properties as GPCR ligands remain poorly characterized. Here, using protein engineering, pharmacological assays and structural studies, we develop maternally selective heavy-chain-only antibody ('nanobody') antagonists against the angiotensin II type I receptor and uncover the unusual molecular basis of their receptor antagonism. We further show that our nanobodies can simultaneously bind to angiotensin II type I receptor with specific small-molecule antagonists and demonstrate that ligand selectivity can be readily tuned. Our work illustrates that antibody fragments can exhibit rich and evolvable pharmacology, attesting to their potential as next-generation GPCR modulators.
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BACKGROUND: Recently, tracheal narrowing has been recognized as a significant comorbid condition in patients with Morquio A, also known as mucopolysaccharidosis IVA. We studied a large cohort of patients with Morquio A to describe the extent of their tracheal narrowing and its relationship to airway management during anesthesia care. METHODS: This is an observational study, collecting data retrospectively, of a cohort of patients with Morquio A. Ninety-two patients with Morquio A syndrome were enrolled, among whom 44 patients had their airway evaluated by computed tomography angiography and had undergone an anesthetic within a year of the evaluation. Our hypothesis was that the tracheal narrowing as evaluated by computed tomography angiography increases with age in patients with Morquio A. The primary aim of the study was to examine the degree of tracheal narrowing in patients with Morquio A and describe the difficulties encountered during airway management, thus increasing awareness of both the tracheal narrowing and airway management difficulties in this patient population. In addition, the degree of tracheal narrowing was evaluated for its association with age or spirometry parameters using Spearman's rank correlation. Analysis of variance followed by the Bonferroni test was used to further examine the age-based differences in tracheal narrowing for the 3 age groups: 1 to 10 years, 11 to 20 years, and >21 years. RESULTS: Patient age showed a positive correlation with tracheal narrowing ( rs= 0.415; 95% confidence interval [95% CI], 0.138-0.691; P = .005) with older patients having greater narrowing of the trachea. Among spirometry parameters, FEF25%-75% showed an inverse correlation with tracheal narrowing as follows: FEF25%-75% versus tracheal narrowing: ( rs = -0.467; 95% CI, -0.877 to -0.057; P = .007). During anesthetic care, significant airway management difficulties were encountered, including cancelation of surgical procedures, awake intubation using flexible bronchoscope, and failed video laryngoscopy attempts. CONCLUSIONS: Clinically significant tracheal narrowing was present in patients with Morquio A, and the degree of such narrowing likely contributed to the difficulty with airway management during their anesthetic care. Tracheal narrowing worsens with age, but the progression appears to slow down after 20 years of age. In addition to tracheal narrowing, spirometry values of FEF25%-75% may be helpful in the overall evaluation of the airway in patients with Morquio A.
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Anestesia , Anestésicos , Mucopolisacaridosis IV , Humanos , Lactante , Preescolar , Niño , Adulto Joven , Adulto , Adolescente , Mucopolisacaridosis IV/cirugía , Estudios Retrospectivos , Anestesia/métodos , Intubación Intratraqueal/efectos adversos , Intubación Intratraqueal/métodos , Laringoscopía/métodosRESUMEN
Engineering protein biosensors that sensitively respond to specific biomolecules by triggering precise cellular responses is a major goal of diagnostics and synthetic cell biology. Previous biosensor designs have largely relied on binding structurally well-defined molecules. In contrast, approaches that couple the sensing of flexible compounds to intended cellular responses would greatly expand potential biosensor applications. Here, to address these challenges, we develop a computational strategy for designing signaling complexes between conformationally dynamic proteins and peptides. To demonstrate the power of the approach, we create ultrasensitive chemotactic receptor-peptide pairs capable of eliciting potent signaling responses and strong chemotaxis in primary human T cells. Unlike traditional approaches that engineer static binding complexes, our dynamic structure design strategy optimizes contacts with multiple binding and allosteric sites accessible through dynamic conformational ensembles to achieve strongly enhanced signaling efficacy and potency. Our study suggests that a conformationally adaptable binding interface coupled to a robust allosteric transmission region is a key evolutionary determinant of peptidergic GPCR signaling systems. The approach lays a foundation for designing peptide-sensing receptors and signaling peptide ligands for basic and therapeutic applications.
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Quimiotaxis , Péptidos , Humanos , Quimiotaxis/fisiología , Transducción de Señal , Proteínas , Sitio Alostérico , LigandosRESUMEN
Communication across membranes controls critical cellular processes and is achieved by receptors translating extracellular signals into selective cytoplasmic responses. While receptor tertiary structures can be readily characterized, receptor associations into quaternary structures are challenging to study and their implications in signal transduction remain poorly understood. Here, we report a computational approach for predicting receptor self-associations, and designing receptor oligomers with various quaternary structures and signaling properties. Using this approach, we designed chemokine receptor CXCR4 dimers with reprogrammed binding interactions, conformations, and abilities to activate distinct intracellular signaling proteins. In agreement with our predictions, the designed CXCR4s dimerized through distinct conformations and displayed different quaternary structural changes upon activation. Consistent with the active state models, all engineered CXCR4 oligomers activated the G protein Gi, but only specific dimer structures also recruited ß-arrestins. Overall, we demonstrate that quaternary structures represent an important unforeseen mechanism of receptor biased signaling and reveal the existence of a bias switch at the dimer interface of several G protein-coupled receptors including CXCR4, mu-Opioid and type-2 Vasopressin receptors that selectively control the activation of G proteins vs ß-arrestin-mediated pathways. The approach should prove useful for predicting and designing receptor associations to uncover and reprogram selective cellular signaling functions.
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Arrestinas , Transducción de Señal , Arrestinas/metabolismo , beta-Arrestinas/metabolismo , Transducción de Señal/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Unión al GTP/metabolismoRESUMEN
Following the hugely successful application of deep learning methods to protein structure prediction, an increasing number of design methods seek to leverage generative models to design proteins with improved functionality over native proteins or novel structure and function. The inherent flexibility of proteins, from side-chain motion to larger conformational reshuffling, poses a challenge to design methods, where the ideal approach must consider both the spatial and temporal evolution of proteins in the context of their functional capacity. In this review, we highlight existing methods for protein design before discussing how methods at the forefront of deep learning-based design accommodate flexibility and where the field could evolve in the future.
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Natural circular RNAs have been found to sequester microRNAs and suppress their function. We have used this principle as a molecular tool and produced artificial circular RNA sponges in a cell-free system by in vitro transcription and ligation. Formerly, we were able to inhibit hepatitis C virus propagation by applying a circular RNA decoy strategy against microRNA-122, which is essential for the viral life cycle. In another proof-of-principle study, we used circular RNAs to sequester microRNA-21, an oncogenic and pro-proliferative microRNA. This strategy slowed tumor growth in a 3D cell culture model system, as well as in xenograft mice upon systemic delivery. In the wake of the global use of an in vitro transcribed RNA in coronavirus disease 2019 (COVID-19) vaccines, the question arose whether therapeutic circular RNAs trigger cellular antiviral defense mechanisms when delivered systemically. In this study, we present data on the cellular innate immune response as a consequence of liposome-based transfection of the circular RNA sponges we previously used to inhibit microRNA function. We find that circular RNAs produced by the presented methodology do not trigger the antiviral response and do not activate innate immune-signaling pathways.
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The demonstration that spray-induced gene silencing (SIGS) can confer strong disease resistance, bypassing the laborious and time-consuming transgenic expression of double-stranded (ds)RNA to induce the gene silencing of pathogenic targets, was ground-breaking. However, future field applications will require fundamental mechanistic knowledge of dsRNA uptake, processing, and transfer. There is increasing evidence that extracellular vesicles (EVs) mediate the transfer of transgene-derived small interfering (si)RNAs in host-induced gene silencing (HIGS) applications. In this study, we establish a protocol for barley EV isolation and assess the possibilities for EVs regarding the translocation of sprayed dsRNA from barley (Hordeum vulgare) to its interacting fungal pathogens. We found barley EVs that were 156 nm in size, containing predominantly 21 and 19 nucleotide (nts) siRNAs, starting with a 5'-terminal Adenine. Although a direct comparison of the RNA cargo between HIGS and SIGS EV isolates is improper given their underlying mechanistic differences, we identified sequence-identical siRNAs in both systems. Overall, the number of siRNAs isolated from the EVs of dsRNA-sprayed barley plants with sequence complementarity to the sprayed dsRNA precursor was low. However, whether these few siRNAs are sufficient to induce the SIGS of pathogenic target genes requires further research. Taken together, our results raise the possibility that EVs may not be mandatory for the spray-delivered siRNA uptake and induction of SIGS.
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Protección de Cultivos/métodos , Hordeum/genética , Hordeum/microbiología , ARN Interferente Pequeño/administración & dosificación , Familia 3 del Citocromo P450/genética , Resistencia a la Enfermedad/genética , Vesículas Extracelulares/genética , Vesículas Extracelulares/microbiología , Silenciador del Gen , Interacciones Microbiota-Huesped/genética , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/prevención & control , Interferencia de ARN , ARN de Planta/genética , ARN de Planta/aislamiento & purificación , ARN Interferente Pequeño/aislamiento & purificaciónRESUMEN
Recent observations of the potentially habitable planets TRAPPIST-1 e, f, and g suggest that they possess large water mass fractions of possibly several tens of weight percent of water, even though the host star's activity should drive rapid atmospheric escape. These processes can photolyze water, generating free oxygen and possibly desiccating the planet. After the planets formed, their mantles were likely completely molten with volatiles dissolving and exsolving from the melt. To understand these planets and prepare for future observations, the magma ocean phase of these worlds must be understood. To simulate these planets, we have combined existing models of stellar evolution, atmospheric escape, tidal heating, radiogenic heating, magma-ocean cooling, planetary radiation, and water-oxygen-iron geochemistry. We present MagmOc, a versatile magma-ocean evolution model, validated against the rocky super-Earth GJ 1132b and early Earth. We simulate the coupled magma-ocean atmospheric evolution of TRAPPIST-1 e, f, and g for a range of tidal and radiogenic heating rates, as well as initial water contents between 1 and 100 Earth oceans. We also reanalyze the structures of these planets and find they have water mass fractions of 0-0.23, 0.01-0.21, and 0.11-0.24 for planets e, f, and g, respectively. Our model does not make a strong prediction about the water and oxygen content of the atmosphere of TRAPPIST-1 e at the time of mantle solidification. In contrast, the model predicts that TRAPPIST-1 f and g would have a thick steam atmosphere with a small amount of oxygen at that stage. For all planets that we investigated, we find that only 3-5% of the initial water will be locked in the mantle after the magma ocean solidified.
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Atmósfera , Planetas , Planeta Tierra , Evolución Planetaria , Exobiología , Medio Ambiente Extraterrestre , Océanos y Mares , AguaRESUMEN
OBJECTIVE: The objective of this study was to evaluate the feasibility of telehealth visits and compare office-based visits for pediatric patients undergoing evaluation of recurrent acute otitis media or sleep-disordered breathing. METHODS: A retrospective cohort study compared telehealth patients with matched controls seen in the office. The feasibility of a thorough patient evaluation in a single telehealth visit without a follow-up office visit was assessed. Both groups were also compared for completeness of physical exam, management, follow-up recommendations, and correlation of physical exam findings with intraoperative findings. RESULTS: 100 children [mean age (SD) = 20.7 (15.6) months] with a chief complaint of recurrent acute otitis media and 128 children [5.4 (3.2) years] with a chief complaint of sleep-disordered breathing were evaluated. Recommendations for surgery, additional studies, or routine follow-up were similar between telehealth and office-based groups. Physical exam feasibility was significantly different for the nasal cavity, oropharynx, and middle ear (P < .001). Patients who underwent office-based consultation were much more likely to have findings of middle ear fluid at the time of tympanostomy tube placement (79.3% vs 39.3%, P = .002). There was no significant difference between preoperative and intraoperative tonsil size discrepancies (P = .749). CONCLUSION: Telehealth can be used successfully for the evaluation of pediatric patients with sleep-disordered breathing; however, reliance on history alone may result in unnecessary tympanostomy tube placement in patients with recurrent acute otitis media. Physical examination of the oropharynx, nasal cavity, and middle ear via telehealth presents a unique challenge in pediatric otolaryngology.
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Atención Ambulatoria/estadística & datos numéricos , Visita a Consultorio Médico/estadística & datos numéricos , Otolaringología , Telemedicina/estadística & datos numéricos , Preescolar , Humanos , Lactante , Ventilación del Oído Medio , Estudios RetrospectivosRESUMEN
The global coronavirus disease-2019 (COVID-19) pandemic has changed the prevalence and management of many pediatric infectious diseases, including acute otitis media (AOM). Coronaviruses are a group of RNA viruses that cause respiratory tract infections in humans. Before the COVID-19 pandemic, coronavirus serotypes OC43, 229E, HKU1, and NL63 were infrequently detected in middle ear fluid (MEF) specimens and nasopharyngeal aspirates in children with AOM during the 1990s and 2000s and were associated with a mild course of the disease. At times when CoV was detected in OM cases, the overall viral load was relatively low. The new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative pathogen responsible for the eruption of the COVID-19 global pandemic. Following the pandemic declaration in many countries and by the World Health Organization in March 2020, preventive proactive measures were imposed to limit COVID-19. These included social distancing; lockdowns; closure of workplaces; kindergartens and schools; increased hygiene; use of antiseptics and alcohol-based gels; frequent temperature measurements and wearing masks. These measures were not the only ones taken, as hospitals and clinics tried to minimize treating non-urgent medical referrals such as OM, and elective surgical procedures were canceled, such as ventilating tube insertion (VTI). These changes and regulations altered the way OM is practiced during the COVID-19 pandemic. Advents in technology allowed a vast use of telemedicine technologies for OM, however, the accuracy of AOM diagnosis in those encounters was in doubt, and antibiotic prescription rates were still reported to be high. There was an overall decrease in AOM episodes and admissions rates and with high spontaneous resolution rates of MEF in children, and a reduction in VTI surgeries. Despite an initial fear regarding viral shedding during myringotomy, the procedure was shown to be safe. Special draping techniques for otologic surgery were suggested. Other aspects of OM practice included the presentation of adult patients with AOM who tested positive for SARS-2-CoV and its detection in MEF samples in living patients and in the mucosa of the middle ear and mastoid in post-mortem specimens.
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COVID-19 , Otitis Media , Niño , Control de Enfermedades Transmisibles , Humanos , Otitis Media/epidemiología , Otitis Media/prevención & control , Pandemias/prevención & control , SARS-CoV-2RESUMEN
OBJECTIVE: To determine the rates and primary causes of missed appointments (MAs) for telehealth visits and present remedies for improvement. METHODS: This cross-sectional survey was conducted at a tertiary care pediatric otolaryngology practice during expansion of telehealth-based visits. A review of questionnaire responses was performed for 103 consecutive patients with MAs over 50 business days from March 20, 2020, to May 29, 2020. Families were asked a brief survey regarding the cause of the MA and assisted with technical support and rescheduling. MA rates and causes were analyzed. RESULTS: The overall MA rate during the initiation of telehealth services was significantly increased at 12.4% as compared with clinic-based visits of a similar duration before COVID of 5.2% (P < .001). Technical issues were the most common causes of MAs (51.3%). Of the caregivers, 23.8% forgot or reported cancellation of the appointment. Five percent of patients were non-English speaking and scheduled without translator support. Minorities and patients with public insurance represented 53.6% and 61.9% of MAs, respectively. DISCUSSION: Technical difficulties were the most commonly reported cause of missed telehealth appointments. Optimization of applications by providing patient reminders, determining need for translator assistance, and reducing required upload/download speeds may significantly reduce rates of MAs and conversions to other communication. IMPLICATIONS FOR PRACTICE: Clear, concise education materials on the technical aspects of telehealth, platform optimization, and robust technical and administrative support may be necessary to reduced missed telehealth appointments and support large-scale telehealth operations. An assessment of institutional capacity is critical when considering telehealth expansion.
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Pacientes no Presentados/estadística & datos numéricos , Otolaringología , Pediatría , Telemedicina , Niño , Preescolar , Estudios Transversales , Humanos , Lactante , Otolaringología/organización & administración , Pediatría/organización & administración , Telemedicina/organización & administraciónRESUMEN
CONTEXT: To describe this new clinical entity, diagnosis, and potential management of pediatric intratonsillar/peritonsillar abscesses in children affected by infectious mononucleosis. METHODS: After institutional review board approval, a retrospective chart review of patients who underwent testing for infectious mononucleosis and also had a computed tomography scan of the head and neck was completed. Those who did not have imaging showing the palatine tonsils and those with insufficient testing to diagnose infectious mononucleosis were excluded. MAIN FINDINGS: One hundred patients were included in the study; 15 had a peritonsillar abscess and 29 had an intratonsillar abscess. Four of the patients with a peritonsillar abscess (26.7%) had a positive Monospot or Epstein-Barr virus IgM result, and two of 15 (13.3%) had positive rapid strep or culture results. Of the 29 patients with an intratonsillar abscess, eight (27.6%) had a positive Monospot or Epstein-Barr virus IgM result while two (6.9%) had a positive rapid strep or culture result. Of those with bilateral intratonsillar abscess, five of 12 (41.7%) patients showed laboratory markers for infectious mononucleosis compared with three of 17 (17.6%) with unilateral intratonsillar abscess. This difference was not statistically significant (Fischer's, p = 0.218). CONCLUSION: In our cohort of patients undergoing computed tomography scan and acute infectious mononucleosis testing, patients with intratonsillar and peritonsillar abscess tested positive for mononucleosis markers more commonly than for streptococcus markers. Recognizing uncomplicated intratonsillar and peritonsillar abscess in the setting of infectious mononucleosis in these pediatric patients may help tailor management in this population.
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Mononucleosis Infecciosa/virología , Tonsila Palatina/virología , Absceso Peritonsilar/virología , Biomarcadores , Niño , Femenino , Herpesvirus Humano 4/inmunología , Humanos , Inmunoglobulina M/análisis , Mononucleosis Infecciosa/complicaciones , Mononucleosis Infecciosa/diagnóstico , Masculino , Tonsila Palatina/diagnóstico por imagen , Absceso Peritonsilar/diagnóstico , Absceso Peritonsilar/etiología , Proyectos Piloto , Estudios Retrospectivos , Tomografía Computarizada por Rayos XRESUMEN
The D2 dopamine receptor (DRD2) is a therapeutic target for Parkinson's disease1 and antipsychotic drugs2. DRD2 is activated by the endogenous neurotransmitter dopamine and synthetic agonist drugs such as bromocriptine3, leading to stimulation of Gi and inhibition of adenylyl cyclase. Here we used cryo-electron microscopy to elucidate the structure of an agonist-bound activated DRD2-Gi complex reconstituted into a phospholipid membrane. The extracellular ligand-binding site of DRD2 is remodelled in response to agonist binding, with conformational changes in extracellular loop 2, transmembrane domain 5 (TM5), TM6 and TM7, propagating to opening of the intracellular Gi-binding site. The DRD2-Gi structure represents, to our knowledge, the first experimental model of a G-protein-coupled receptor-G-protein complex embedded in a phospholipid bilayer, which serves as a benchmark to validate the interactions seen in previous detergent-bound structures. The structure also reveals interactions that are unique to the membrane-embedded complex, including helix 8 burial in the inner leaflet, ordered lysine and arginine side chains in the membrane interfacial regions, and lipid anchoring of the G protein in the membrane. Our model of the activated DRD2 will help to inform the design of subtype-selective DRD2 ligands for multiple human central nervous system disorders.
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Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/ultraestructura , Lípidos de la Membrana/metabolismo , Membranas Artificiales , Receptores de Dopamina D2/química , Receptores de Dopamina D2/ultraestructura , Bromocriptina/química , Bromocriptina/metabolismo , Dopamina/química , Dopamina/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Humanos , Lípidos de la Membrana/química , Modelos Moleculares , Conformación Proteica , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D2/metabolismo , Transducción de SeñalRESUMEN
Plasmids are extrachromosomal genetic elements that replicate independently of the chromosome and play a vital role in the environmental adaptation of bacteria. Due to potential mobilization or conjugation capabilities, plasmids are important genetic vehicles for antimicrobial resistance genes and virulence factors with huge and increasing clinical implications. They are therefore subject to large genomic studies within the scientific community worldwide. As a result of rapidly improving next-generation sequencing methods, the quantity of sequenced bacterial genomes is constantly increasing, in turn raising the need for specialized tools to (i) extract plasmid sequences from draft assemblies, (ii) derive their origin and distribution, and (iii) further investigate their genetic repertoire. Recently, several bioinformatic methods and tools have emerged to tackle this issue; however, a combination of high sensitivity and specificity in plasmid sequence identification is rarely achieved in a taxon-independent manner. In addition, many software tools are not appropriate for large high-throughput analyses or cannot be included in existing software pipelines due to their technical design or software implementation. In this study, we investigated differences in the replicon distributions of protein-coding genes on a large scale as a new approach to distinguish plasmid-borne from chromosome-borne contigs. We defined and computed statistical discrimination thresholds for a new metric: the replicon distribution score (RDS), which achieved an accuracy of 96.6â%. The final performance was further improved by the combination of the RDS metric with heuristics exploiting several plasmid-specific higher-level contig characterizations. We implemented this workflow in a new high-throughput taxon-independent bioinformatics software tool called Platon for the recruitment and characterization of plasmid-borne contigs from short-read draft assemblies. Compared to PlasFlow, Platon achieved a higher accuracy (97.5â%) and more balanced predictions (F1=82.6â%) tested on a broad range of bacterial taxa and better or equal performance against the targeted tools PlasmidFinder and PlaScope on sequenced Escherichia coli isolates. Platon is available at: http://platon.computational.bio/.
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Proteínas Bacterianas/genética , Biología Computacional/métodos , Escherichia coli/genética , Genoma Bacteriano/genética , Plásmidos/genética , Cromosomas Bacterianos/genética , Mapeo Contig/métodos , Farmacorresistencia Bacteriana Múltiple/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Programas Informáticos , Secuenciación Completa del GenomaRESUMEN
The Rosetta software for macromolecular modeling, docking and design is extensively used in laboratories worldwide. During two decades of development by a community of laboratories at more than 60 institutions, Rosetta has been continuously refactored and extended. Its advantages are its performance and interoperability between broad modeling capabilities. Here we review tools developed in the last 5 years, including over 80 methods. We discuss improvements to the score function, user interfaces and usability. Rosetta is available at http://www.rosettacommons.org.
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Sustancias Macromoleculares/química , Modelos Moleculares , Proteínas/química , Programas Informáticos , Simulación del Acoplamiento Molecular , Peptidomiméticos/química , Conformación ProteicaRESUMEN
Membrane receptors sense and transduce extracellular stimuli into intracellular signaling responses but the molecular underpinnings remain poorly understood. We report a computational approach for designing protein allosteric signaling functions. By combining molecular dynamics simulations and design calculations, the method engineers amino-acid 'microswitches' at allosteric sites that modulate receptor stability or long-range coupling, to reprogram specific signaling properties. We designed 36 dopamine D2 receptor variants, whose constitutive and ligand-induced signaling agreed well with our predictions, repurposed the D2 receptor into a serotonin biosensor and predicted the signaling effects of more than 100 known G-protein-coupled receptor (GPCR) mutations. Our results reveal the existence of distinct classes of allosteric microswitches and pathways that define an unforeseen molecular mechanism of regulation and evolution of GPCR signaling. Our approach enables the rational design of allosteric receptors with enhanced stability and function to facilitate structural characterization, and reprogram cellular signaling in synthetic biology and cell engineering applications.
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Ingeniería de Proteínas , Receptores de Dopamina D2/química , Receptores de Dopamina D2/genética , Transducción de Señal , Regulación Alostérica/efectos de los fármacos , Sitio Alostérico , Secuencias de Aminoácidos , Técnicas Biosensibles , Simulación por Computador , Análisis Mutacional de ADN , Células HEK293 , Humanos , Concentración 50 Inhibidora , Cinética , Ligandos , Simulación de Dinámica Molecular , Mutagénesis , Serotonina/química , Programas InformáticosRESUMEN
Genome sequencing has revealed an increasing number of genetic variations that are associated with neuropsychiatric disorders. Frequently, studies limit their focus to likely gene-disrupting mutations because they are relatively easy to interpret. Missense variants, instead, have often been undervalued. However, some missense variants can be informative for developing a more profound understanding of disease pathogenesis and ultimately targeted therapies. Here we present an example of this by studying a missense variant in a well-known autism spectrum disorder (ASD) causing gene SHANK3. We analyzed Shank3's in vivo phosphorylation profile and identified S685 as one phosphorylation site where one ASD-linked variant has been reported. Detailed analysis of this variant revealed a novel function of Shank3 in recruiting Abelson interactor 1 (ABI1) and the WAVE complex to the post-synaptic density (PSD), which is critical for synapse and dendritic spine development. This function was found to be independent of Shank3's other functions such as binding to GKAP and Homer. Introduction of this human ASD mutation into mice resulted in a small subset of phenotypes seen previously in constitutive Shank3 knockout mice, including increased allogrooming, increased social dominance, and reduced pup USV. Together, these findings demonstrate the modularity of Shank3 function in vivo. This modularity further indicates that there is more than one independent pathogenic pathway downstream of Shank3 and correcting a single downstream pathway is unlikely to be sufficient for clear clinical improvement. In addition, this study illustrates the value of deep biological analysis of select missense mutations in elucidating the pathogenesis of neuropsychiatric phenotypes.
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Trastorno del Espectro Autista/genética , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Mutación Missense/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Trastorno Autístico/genética , Proteínas del Citoesqueleto/metabolismo , Femenino , Humanos , Masculino , Ratones , Densidad Postsináptica/metabolismo , RatasRESUMEN
Laryngeal post-transplant lymphoproliferative disease (PTLD) is rare. Here, we describe two pediatric cases. The first, a 15-month-old who underwent liver transplantation at 5 weeks, presented with airway distress. Airway evaluation identified epiglottic and arytenoid infiltrate, and biopsy was consistent with polymorphic PTLD. The second, a 23-month-old who underwent liver transplantation at 13 months, presented with progressive stridor. Airway evaluation revealed sub-mucosal infiltrate of the epiglottis, arytenoids, post-cricoid region, and uvula. Biopsy was consistent with monomorphic PTLD. Airway findings and symptoms resolved for both after immunosuppression reduction. PTLD diagnosis requires a high index of suspicion in post-transplant patients with airway obstruction.
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INTRODUCTION: An increasing number of tracheostomies are performed in infants with complex comorbidities including bronchopulmonary dysplasia (BPD) and congenital heart disease (CHD). With this shift in indications, there is an urgent need to characterize outcomes in this population. METHODS: This 5-year retrospective chart review assessed rates of 12-month mortality in infants who were ≤12 months of age at the time of tracheostomy at a tertiary care pediatric hospital and risk factors associated with death. Patient characteristics evaluated included chronologic age and post-menstrual age at tracheostomy placement, gestational age and weight, sex, ethnicity, indication for tracheostomy, and comorbidities including BPD, CHD, subglottic stenosis (SGS), craniofacial syndromes, and chromosomal trisomy syndromes. Subgroup analysis was performed in infants with CHD. RESULTS: One hundred thirty-two tracheostomies were performed during the study period with an overall 12-month mortality of 14.4% (19/132). Mortality was increased in patients with CHD (35%) and decreased in patients with SGS (3.7%). No other patient characteristics were associated with differences in mortality. There was a trend towards improved mortality outcomes among patients born at earlier gestational ages. CONCLUSIONS: Among infants with tracheostomy in this cohort, overall mortality rates were relatively low but not insignificant. CHD was associated with increased mortality; however, children with SGS showed more favorable outcomes. Other patient characteristics were not associated with differences in mortality. These data clarify outcomes in a group of infants with tracheostomy.
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Traqueostomía/mortalidad , Delaware/epidemiología , Femenino , Cardiopatías Congénitas/mortalidad , Insuficiencia Cardíaca/mortalidad , Humanos , Lactante , Recién Nacido , Masculino , Insuficiencia Respiratoria/mortalidad , Estudios Retrospectivos , Factores de Riesgo , Sepsis/mortalidadRESUMEN
Solvent molecules interact intimately with proteins and can profoundly regulate their structure and function. However, accurately and efficiently modeling protein solvation effects at the molecular level has been challenging. Here, we present a method that improves the atomic-level modeling of soluble and membrane protein structures and binding by efficiently predicting de novo protein-solvent molecule interactions. The method predicted with unprecedented accuracy buried water molecule positions, solvated protein conformations, and challenging mutational effects on protein binding. When applied to homology modeling, solvent-bound membrane protein structures, pockets, and cavities were recapitulated with near-atomic precision even from distant homologs. Blindly refined atomic-level structures of evolutionary distant G protein-coupled receptors imply strikingly different functional roles of buried solvent between receptor classes. The method should prove useful for refining low-resolution protein structures, accurately modeling drug-binding sites in structurally uncharacterized receptors, and designing solvent-mediated protein catalysis, recognition, ligand binding, and membrane protein signaling.