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In monocrystalline Si (c-Si) solar cells, identification and mitigation of bulk defects are crucial to achieving a high photoconversion efficiency. To spectroscopically detect defects in the c-Si bulk, it is desirable to passivate the surface defects. Passivation of the c-Si surface with dielectrics such as Al2O3 and SiNx requires deposition at elevated temperatures, which can influence defects in the bulk. Herein, we report on the passivation of different Czochralski (Cz) Si wafer surfaces by an organic copolymer, Nafion. We test the efficacy of the surface passivation at temperatures ranging from 6 to 473 K to detect bulk defects using electron paramagnetic resonance (EPR) spectroscopy. By comparing with state-of-the-art passivation layers, including Al2O3 and liquid HF/HCl, we found that at room temperature, Nafion can provide comparable passivation of n-type Cz Si with an implied open-circuit voltage (iVoc) of 713 mV and a recombination current prefactor J0 of 5 fA/cm2. For p-type Cz Si, we obtained an iVoc of 682 mV with a J0 of 22.4 fA/cm2. Scanning electron microscopy and photoluminescence reveal that Nafion can also be used to passivate the surface of c-Si solar cell fragments scribed from a solar cell module by using a laser. Consistent with previous studies, analysis of the EPR spectroscopy data confirms that the H-terminated surface is necessary, and fixed negative charge in Nafion is responsible for the field-effect passivation. While the surface passivation quality was maintained for almost 24 h, which is sufficient for spectroscopic measurements, the passivation degraded over longer durations, which can be attributed to surface SiOx growth. These results show that Nafion is a promising room-temperature surface passivation technique to study bulk defects in c-Si.
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The high cost of substrates for III-V growth can be cost limiting for technologies that require large semiconductor areas. Thus, being able to separate device layers and reuse the original substrate is highly desirable, but existing techniques to lift a film from a substrate have substantial drawbacks. This work discusses some of the complexities with the growth of a water-soluble, alkali halide salt thin film between a III-V substrate and overlayer. Much of the difficulty stems from the growth of GaAs on an actively decomposing NaCl surface at elevated temperatures. Interestingly, the presence of an in situ electron beam incident on the NaCl surface, prior to and during GaAs deposition, affects the crystallinity and morphology of the III-V overlayer. Here, we investigate a wide range of growth temperatures and the timing of the impinging flux of both elemental sources and high energy electrons at different points during the growth. We show that an assortment of morphologies (discrete islands, porous material, and fully dense layers with sharp interfaces) and crystallinity (amorphous, crystalline, and highly textured) occur depending on the specific growth conditions, driven largely by changes in GaAs nucleation which is greatly affected by the presence of the reflection high energy electron diffraction beam.
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Renewable forms of electricity generation like solar and wind require low-cost energy storage solutions to meet climate change deployment goals. Here, we explore the use of depleted hydraulically fractured ("fracked") oil and gas wells to store electrical energy in the form of compressed natural gas to be released to spin an expander/generator when electrical demand is high. Our reservoir model indicates that the same dual-porosity geological environment of fracked wells used to liberate hydrocarbons is also suitable for storing and releasing gas in a diurnal or seasonal cycle. Round-trip storage efficiency is calculated to be 40%-70% depending on the natural reservoir temperature. Levelized cost of storage is estimated to be $70-270/MWh, on par with pumped hydro storage. This study indicates that repurposed "fracked" wells could provide a much-needed low-cost seasonal energy storage solution at the TWh scale.
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High-efficiency silicon solar cells rely on some form of passivating contact structure to reduce recombination losses at the crystalline silicon surface and losses at the metal/Si contact interface. One such structure is polycrystalline silicon (poly-Si) on oxide, where heavily doped poly-Si is deposited on a SiOx layer grown directly on the crystalline silicon (c-Si) wafer. Depending on the thickness of the SiOx layer, the charge carriers can cross this layer by tunneling (<2 nm SiOx thickness) or by direct conduction through disruptions in the SiOx, often referred to as pinholes, in thicker SiOx layers (>2 nm). In this work, we study structures with tunneling- or pinhole-like SiOx contacts grown on pyramidally textured c-Si wafers and expose variations in the SiOx layer properties related to surface morphology using electron-beam-induced current (EBIC) imaging. Using EBIC, we identify and mark regions with potential pinholes in the SiOx layer. We further perform high-resolution transmission electron microscopy on the same areas, thus allowing us to directly correlate locally enhanced carrier collection with variations in the structure of the SiOx layer. Our results show that the pinholes in the SiOx layer preferentially form in different locations based on the annealing conditions used to form the device. With greater understanding of these processes and by controlling the surface texture geometry, there is potential to control the size and spatial distribution of oxide disruptions in silicon solar cells with poly-Si on oxide-type contacts; usually, this is a random phenomenon on polished or planar surfaces. Such control will enable us to consistently produce high-efficiency devices with low recombination currents and low junction resistances using this contact structure.
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High-efficiency crystalline silicon (Si) solar cells require textured surfaces for efficient light trapping. However, passivation of a textured surface to reduce carrier recombination is difficult. Here, we relate the electrical properties of cells fabricated on a KOH-etched, random pyramidal-textured Si surface to the nanostructure of the passivated contact and the textured surface morphology. The effects of both microscopic pyramidal morphology and nanoscale surface roughness on passivated contacts consisting of polycrystalline Si (poly-Si) deposited on top of an ultrathin, 1.5-2.2 nm, SiOx layer are investigated. Using atomic force microscopy, we show a pyramid face, which is predominantly a Si(111) plane to be significantly rougher than a polished Si(111) surface. This roughness results in a nonuniform SiOx layer as determined by transmission electron microscopy of a poly-Si/SiOx contact. Our device measurements also show an overall more resistive and hence a thicker SiOx layer over the pyramidal surface as compared to a polished Si(111) surface, which we relate to increased surface roughness. Using electron-beam-induced current measurements of poly-Si/SiOx contacts, we further show that the SiOx layer near the pyramid valleys is preferentially more conducting and hence likely thinner than over pyramid tips, edges, and faces. Hence, both the microscopic pyramidal morphology and nanoscale roughness lead to a nonuniform SiOx layer, thus leading to poor poly-Si/SiOx contact passivation. Finally, we report >21% efficient and ≥80% fill-factor front/back poly-Si/SiOx solar cells on both single-side and double-side textured wafers without the use of transparent conductive oxide layers, and show that the poorer contact passivation on a textured surface is limited to boron-doped poly-Si/SiOx contacts.
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Therapeutic botulinum toxin injections are commonly performed in pediatric otolaryngology. Aerodigestive complications from botulinum toxin injections, although rare, may be serious. Oral pyridostigmine is effective in the symptomatic treatment of these complications. We report 2 cases of aerodigestive complications arising from injection of botulinum toxin that were successfully treated with pyridostigmine.
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Toxinas Botulínicas Tipo A/efeitos adversos , Inibidores da Colinesterase/uso terapêutico , Fármacos Neuromusculares/efeitos adversos , Brometo de Piridostigmina/uso terapêutico , Criança , Transtornos de Deglutição/induzido quimicamente , Transtornos de Deglutição/tratamento farmacológico , Feminino , Humanos , LactenteRESUMO
Microorganisms have universally adapted their RNAs and proteins to survive at a broad range of temperatures and growth conditions. However, for RNAs, there is little quantitative understanding of the effects of mutations on function at high temperatures. To understand how variant tRNA function is affected by temperature change, we used the tRNA nonsense suppressor SUP4oc of the yeast Saccharomyces cerevisiae to perform a high-throughput quantitative screen of tRNA function at two different growth temperatures. This screen yielded comparative values for 9243 single and double variants. Surprisingly, despite the ability of S. cerevisiae to grow at temperatures as low as 15°C and as high as 39°C, the vast majority of variants that could be scored lost half or more of their function when evaluated at 37°C relative to 28°C. Moreover, temperature sensitivity of a tRNA variant was highly associated with its susceptibility to the rapid tRNA decay (RTD) pathway, implying that RTD is responsible for most of the loss of function of variants at higher temperature. Furthermore, RTD may also operate in a met22Δ strain, which was previously thought to fully inhibit RTD. Consistent with RTD acting to degrade destabilized tRNAs, the stability of a tRNA molecule can be used to predict temperature sensitivity with high confidence. These findings offer a new perspective on the stability of tRNA molecules and their quality control at high temperature.
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Fatores de Terminação de Peptídeos/genética , Estabilidade de RNA/genética , RNA de Transferência/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Biblioteca Gênica , Genes Reporter , Mutação , RNA de Transferência/química , Saccharomyces cerevisiae/fisiologia , Análise de Sequência de DNA , TemperaturaRESUMO
Although synonymous mutations can affect gene expression, they have generally not been considered in genomic studies that focus on mutations that increase the risk of cancer. However, mounting evidence implicates some synonymous mutations as driver mutations in cancer. Here, a massively parallel assay, based on cell sorting of a reporter containing a segment of p53 fused to GFP, was used to measure the effects of nearly all synonymous mutations in exon 6 of TP53 In this reporter context, several mutations within the exon caused strong expression changes including mutations that may cause potential gain or loss of function. Further analysis indicates that these effects are largely attributed to errors in splicing, including exon skipping, intron inclusion, and exon truncation, resulting from mutations both at exon-intron junctions and within the body of the exon. These mutations are found at extremely low frequencies in healthy populations and are enriched a few-fold in cancer genomes, suggesting that some of them may be driver mutations in TP53 This assay provides a general framework to identify previously unknown detrimental synonymous mutations in cancer genes.Implications: Using a massively parallel assay, this study demonstrates that synonymous mutations in the TP53 gene affect protein expression, largely through their impact on splicing.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/15/10/1301/F1.large.jpg Mol Cancer Res; 15(10); 1301-7. ©2017 AACR.
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Separação Celular/métodos , Citometria de Fluxo/métodos , Análise de Sequência/métodos , Mutação Silenciosa , Proteína Supressora de Tumor p53/genética , Linhagem Celular , Éxons , Proteínas de Fluorescência Verde/metabolismo , Humanos , Análise de Sequência de DNA , Análise de Sequência de RNARESUMO
Subepithelial changes to the vocal fold mucosa, such as fibrosis, are difficult to identify using visual assessment of the tissue surface. Moreover, without suspicion of neoplasm, mucosal biopsy is not a viable clinical option, as it carries its own risk of iatrogenic injury and scar formation. Given these challenges, we assessed the ability of high- (4.7â T) and ultrahigh-field (9.4â T) magnetic resonance imaging to resolve key vocal fold subepithelial tissue structures in the rat, an important and widely used preclinical model in vocal fold biology. We conducted serial in vivo and ex vivo imaging, evaluated an array of acquisition sequences and contrast agents, and successfully resolved key anatomic features of naïve, acutely injured, and chronically scarred vocal fold mucosae on the ex vivo scans. Naïve lamina propria was hyperintense on T1-weighted imaging with gadobenate dimeglumine contrast enhancement, whereas chronic scar was characterized by reduced lamina propria T1 signal intensity and mucosal volume. Acutely injured mucosa was hypointense on T2-weighted imaging; lesion volume steadily increased, peaked at 5â days post-injury, and then decreased - consistent with the physiology of acute, followed by subacute, hemorrhage and associated changes in the magnetic state of hemoglobin and its degradation products. Intravenous administration of superparamagnetic iron oxide conferred no T2 contrast enhancement during the acute injury period. These findings confirm that magnetic resonance imaging can resolve anatomic substructures within naïve vocal fold mucosa, qualitative and quantitative features of acute injury, and the presence of chronic scar.
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Cicatriz/diagnóstico por imagem , Cicatriz/patologia , Imageamento por Ressonância Magnética , Mucosa/diagnóstico por imagem , Mucosa/patologia , Prega Vocal/diagnóstico por imagem , Prega Vocal/patologia , Animais , Injeções Intravenosas , Laringe/diagnóstico por imagem , Laringe/patologia , Nanopartículas de Magnetita/química , Masculino , Ratos Endogâmicos F344 , Fatores de TempoRESUMO
Progress in DNA-sequencing technologies has provided a catalogue of millions of DNA variants in the human population, but characterization of the functional effects of these variants has lagged far behind. For example, sequencing of tumor samples is driving an urgent need to classify whether or not mutations seen in cancers affect disease progression or treatment effectiveness or instead are benign. Furthermore, mutations can interact with genetic background and with environmental effects. A new approach, termed deep mutational scanning, has enabled the quantitative assessment of the effects of thousands of mutations in a protein. However, this type of experiment is carried out in model organisms, tissue culture, or in vitro; typically addresses only a single biochemical function of a protein; and is generally performed under a single condition. The current challenge lies in using these functional data to generate useful models for the phenotypic consequences of genetic variation in humans.
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Interpretação Estatística de Dados , Análise de Sequência de DNA/métodos , Variação Genética , Humanos , MutaçãoRESUMO
PURPOSE: To understand factors associated with poor attendance of follow-up appointments for care of glaucoma (GL), AMD, and diabetic retinopathy (DR) in a tertiary referral center, and to identify strategies to improve adherence. METHODS: Cross-sectional study of 240 adults attending follow-up appointments for GL, AMD, or DR. Cases (N = 102) were patients with poor follow-up who missed and failed to reschedule an appointment within 1 month of the recommended follow-up date during the preceding year. Controls (N = 138) were patients who completed the assigned follow-up. Data regarding the factors impacting adherence to appointments were collected via an orally administered questionnaire. Multivariate logistic regression was performed to determine factors associated with poor follow-up. RESULTS: In a multivariate logistic regression model, independent factors significantly associated with poor follow-up included incorrectly answering more than 50% of questions about eye disease (adjusted odds ratio [OR] = 3.24, P = 0.001), legal blindness (adjusted OR 2.64, P = 0.013), the presence of glaucomatous versus retinal disease (adjusted OR 2.06, P = 0.013), and difficulty for the study subject and/or escort taking time away from work for the appointments (adjusted OR 1.80, P = 0.049). Subjects identified the following strategies to improve follow-up: contact with others having the same eye condition (41.3%), greater education regarding eye disease (40.8%), and improved transportation services to the clinic (44.6%). CONCLUSIONS: Low disease knowledge scores, legal blindness, and difficulty getting time away from work for appointments adversely impacted follow-up independent of eye disease diagnosis. Improvements in patient education, transportation services, and clinic efficiency may increase adherence to recommended appointment intervals.
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Agendamento de Consultas , Atenção à Saúde/normas , Oftalmopatias/terapia , Cooperação do Paciente/estatística & dados numéricos , Guias de Prática Clínica como Assunto , Adulto , Doença Crônica , Estudos Transversais , Atenção à Saúde/tendências , Seguimentos , Humanos , Estudos Retrospectivos , Inquéritos e Questionários , Estados UnidosRESUMO
Interpreting variants of uncertain significance (VUS) is a central challenge in medical genetics. One approach is to experimentally measure the functional consequences of VUS, but to date this approach has been post hoc and low throughput. Here we use massively parallel assays to measure the effects of nearly 2000 missense substitutions in the RING domain of BRCA1 on its E3 ubiquitin ligase activity and its binding to the BARD1 RING domain. From the resulting scores, we generate a model to predict the capacities of full-length BRCA1 variants to support homology-directed DNA repair, the essential role of BRCA1 in tumor suppression, and show that it outperforms widely used biological-effect prediction algorithms. We envision that massively parallel functional assays may facilitate the prospective interpretation of variants observed in clinical sequencing.
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Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Domínios e Motivos de Interação entre Proteínas , Proteína BRCA1/química , Reparo do DNA , Genômica/métodos , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Síndrome Hereditária de Câncer de Mama e Ovário/metabolismo , Humanos , Mutação , Domínios e Motivos de Interação entre Proteínas/genética , Multimerização Proteica , Ubiquitina-Proteína LigasesRESUMO
Many protein interactions are conserved among organisms despite changes in the amino acid sequences that comprise their contact sites, a property that has been used to infer the location of these sites from protein homology. In an inter-species complementation experiment, a sequence present in a homologue is substituted into a protein and tested for its ability to support function. Therefore, substitutions that inhibit function can identify interaction sites that changed over evolution. However, most of the sequence differences within a protein family remain unexplored because of the small-scale nature of these complementation approaches. Here we use existing high throughput mutational data on the in vivo function of the RRM2 domain of the Saccharomyces cerevisiae poly(A)-binding protein, Pab1, to analyze its sites of interaction. Of 197 single amino acid differences in 52 Pab1 homologues, 17 reduce the function of Pab1 when substituted into the yeast protein. The majority of these deleterious mutations interfere with the binding of the RRM2 domain to eIF4G1 and eIF4G2, isoforms of a translation initiation factor. A large-scale mutational analysis of the RRM2 domain in a two-hybrid assay for eIF4G1 binding supports these findings and identifies peripheral residues that make a smaller contribution to eIF4G1 binding. Three single amino acid substitutions in yeast Pab1 corresponding to residues from the human orthologue are deleterious and eliminate binding to the yeast eIF4G isoforms. We create a triple mutant that carries these substitutions and other humanizing substitutions that collectively support a switch in binding specificity of RRM2 from the yeast eIF4G1 to its human orthologue. Finally, we map other deleterious substitutions in Pab1 to inter-domain (RRM2-RRM1) or protein-RNA (RRM2-poly(A)) interaction sites. Thus, the combined approach of large-scale mutational data and evolutionary conservation can be used to characterize interaction sites at single amino acid resolution.
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Sequência de Aminoácidos/genética , Evolução Molecular , Mutação/genética , Proteínas de Ligação a Poli(A)/metabolismo , Mapas de Interação de Proteínas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Substituição de Aminoácidos/genética , Sítios de Ligação , Análise Mutacional de DNA , Fator de Iniciação Eucariótico 4G/genética , Fator de Iniciação Eucariótico 4G/metabolismo , Variação Genética , Humanos , Proteínas de Ligação a Poli(A)/genética , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de SequênciaRESUMO
OBJECTIVE: To examine the effect of the quadrivalent human papillomavirus vaccine, Gardasil, on the disease course of patients with recurrent respiratory papillomatosis (RRP). METHODS: A retrospective chart review of patients with RRP was conducted and 20 patients were selected who had received the Gardasil vaccine as part of their treatment. Efficacy was assessed by calculating the intersurgical interval (ISI) before and after receiving the vaccine, as well as number of complete and partial remissions. RESULTS: Analysis of all patients found a significant increase in the ISI of 3.1 months (95% confidence interval [CI]: 1.02-5.19, P=0.0061). Male patients experienced an increase in the ISI of 4.2 months (95% CI: 1.6-6.7, P=0.0048). Female patients had a nonsignificant increase in ISI of 1.2 months (95% CI: 3.1-5.4, P=0.51). Eight patients (40%; six male and two female) experienced complete remission. Five patients (25%) overall (three male and two female) experienced partial remission. In total, complete or partial remission was achieved in a total of 13 (65%) patients (nine male and four female). CONCLUSIONS: The Gardasil vaccine can modulate the severity of RRP and induce remission in some patients. The effect was much greater in males and in females in low estrogen states.
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Vacina Quadrivalente Recombinante contra HPV tipos 6, 11, 16, 18/uso terapêutico , Papillomaviridae/imunologia , Infecções por Papillomavirus/tratamento farmacológico , Infecções Respiratórias/tratamento farmacológico , Adolescente , Adulto , Idoso , Criança , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Infecções por Papillomavirus/fisiopatologia , Infecções por Papillomavirus/virologia , Indução de Remissão , Infecções Respiratórias/fisiopatologia , Infecções Respiratórias/virologia , Estudos Retrospectivos , Resultado do Tratamento , Adulto JovemRESUMO
Sequence variation in tRNA genes influences the structure, modification, and stability of tRNA; affects translation fidelity; impacts the activity of numerous isodecoders in metazoans; and leads to human diseases. To comprehensively define the effects of sequence variation on tRNA function, we developed a high-throughput in vivo screen to quantify the activity of a model tRNA, the nonsense suppressor SUP4oc of Saccharomyces cerevisiae. Using a highly sensitive fluorescent reporter gene with an ochre mutation, fluorescence-activated cell sorting of a library of SUP4oc mutant yeast strains, and deep sequencing, we scored 25,491 variants. Unexpectedly, SUP4oc tolerates numerous sequence variations, accommodates slippage in tertiary and secondary interactions, and exhibits genetic interactions that suggest an alternative functional tRNA conformation. Furthermore, we used this methodology to define tRNA variants subject to rapid tRNA decay (RTD). Even though RTD normally degrades tRNAs with exposed 5' ends, mutations that sensitize SUP4oc to RTD were found to be located throughout the sequence, including the anti-codon stem. Thus, the integrity of the entire tRNA molecule is under surveillance by cellular quality control machinery. This approach to assess activity at high throughput is widely applicable to many problems in tRNA biology.
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Estabilidade de RNA/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Citometria de Fluxo , Variação Genética , Ensaios de Triagem em Larga Escala , Mutação/genética , Conformação de Ácido Nucleico , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
OBJECTIVE: Botulinum toxin is used to treat a wide range of dystonias in the head and neck. Occasionally, patients receiving laryngeal botulinum toxin experience severe dysphagia, dyspnea, or even distant and autonomic symptoms. Rarely, these patients may require hospitalization with possible intubation and placement of nasogastric tubes. Botulinum antitoxin is not readily available and ineffective once symptoms have progressed, so patients must wait until the toxin wears off over weeks to months. Pyridostigmine prevents the breakdown of acetylcholine at the neuromuscular junction, thus making more neurotransmitter available for the muscles. STUDY DESIGN: A retrospective case study of patients receiving botulinum toxin for dystonia in the head and neck from 1998 to 2012 who experienced adverse effects that were successfully treated with pyridostigmine. METHODS: Twenty cases were selected and reviewed to demonstrate how pyridostigmine was used to modulate severe dysphagia, breathiness, dyspnea, and some distant/autonomic symptoms. RESULTS: Pyridostigmine was well tolerated and resulted in significant symptom improvement. Only one significant adverse effect, bradycardia, occurred in a patient with severe cardiac disease. CONCLUSIONS: Given the safety and efficacy of this medication, pyridostigmine should be considered to modulate severe sequelae of botulinum toxin in select patients when conservative management is deemed insufficient. Also, physicians should be aware that patient complaints of symptoms at distant sites and temporally delayed from the injection may be a result of the botulinum toxin and relieved with pyridostigmine.
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Antídotos/uso terapêutico , Toxinas Botulínicas/efeitos adversos , Inibidores da Colinesterase/uso terapêutico , Transtornos de Deglutição/tratamento farmacológico , Dispneia/tratamento farmacológico , Distonia/tratamento farmacológico , Fármacos Neuromusculares/efeitos adversos , Brometo de Piridostigmina/uso terapêutico , Adulto , Idoso , Antídotos/efeitos adversos , Toxinas Botulínicas/administração & dosagem , Inibidores da Colinesterase/efeitos adversos , Transtornos de Deglutição/induzido quimicamente , Transtornos de Deglutição/diagnóstico , Transtornos de Deglutição/fisiopatologia , Dispneia/induzido quimicamente , Dispneia/diagnóstico , Dispneia/fisiopatologia , Distonia/diagnóstico , Distonia/fisiopatologia , Feminino , Humanos , Injeções , Masculino , Pessoa de Meia-Idade , Fármacos Neuromusculares/administração & dosagem , Brometo de Piridostigmina/efeitos adversos , Estudos Retrospectivos , Resultado do TratamentoRESUMO
OBJECTIVE: Chemical denervation with botulinum toxin A is the current standard of treatment for spasmodic dysphonia, but dosage is determined individually after a titration period that can take months to years. The objective of this study was to determine if age, body mass index (BMI), overall health, and socioeconomic factors were associated with a patient's optimal dose of botulinum toxin. STUDY DESIGN AND METHODS: This retrospective chart review looked at 32 patients with stabilized doses of botulinum toxin. Age and BMI were obtained from patient charts, and overall health was assessed by the Short-Form 36 survey. RESULTS: Analysis showed that BMI was positively correlated with botulinum toxin dose (r = 0.42, P = 0.02). Overall health showed a positive but nonsignificant association with dose, but subgroup analysis found that adductor spasmodic dysphonia (ADSD) patients without tremor had a significant positive correlation between overall health and dose (r = 0.50, P = 0.04), whereas tremor-only and mixed dystonia showed a negative nonsignificant correlation. Age was found to have no significant association with dose. Although socioeconomic factors were found to impact the number and frequency of injections, they had no significant impact on the ultimate dosage. CONCLUSION: BMI and overall health are positively correlated with higher effective dose and may be useful in guiding clinicians during the titration period.
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Toxinas Botulínicas Tipo A/administração & dosagem , Disfonia/tratamento farmacológico , Músculos Laríngeos/fisiopatologia , Qualidade da Voz/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Disfonia/fisiopatologia , Disfonia/psicologia , Eletromiografia , Feminino , Seguimentos , Humanos , Injeções Intramusculares , Músculos Laríngeos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Fármacos Neuromusculares/administração & dosagem , Estudos Retrospectivos , Índice de Gravidade de Doença , Fatores Socioeconômicos , Inquéritos e Questionários , Resultado do TratamentoRESUMO
The RNA recognition motif (RRM) is the most common RNA-binding domain in eukaryotes. Differences in RRM sequences dictate, in part, both RNA and protein-binding specificities and affinities. We used a deep mutational scanning approach to study the sequence-function relationship of the RRM2 domain of the Saccharomyces cerevisiae poly(A)-binding protein (Pab1). By scoring the activity of more than 100,000 unique Pab1 variants, including 1246 with single amino acid substitutions, we delineated the mutational constraints on each residue. Clustering of residues with similar mutational patterns reveals three major classes, composed principally of RNA-binding residues, of hydrophobic core residues, and of the remaining residues. The first class also includes a highly conserved residue not involved in RNA binding, G150, which can be mutated to destabilize Pab1. A comparison of the mutational sensitivity of yeast Pab1 residues to their evolutionary conservation reveals that most residues tolerate more substitutions than are present in the natural sequences, although other residues that tolerate fewer substitutions may point to specialized functions in yeast. An analysis of â¼40,000 double mutants indicates a preference for a short distance between two mutations that display an epistatic interaction. As examples of interactions, the mutations N139T, N139S, and I157L suppress other mutations that interfere with RNA binding and protein stability. Overall, this study demonstrates that living cells can be subjected to a single assay to analyze hundreds of thousands of protein variants in parallel.
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Motivos de Aminoácidos , Proteína I de Ligação a Poli(A)/química , Proteína I de Ligação a Poli(A)/genética , Saccharomyces cerevisiae/genética , Substituição de Aminoácidos , Sequência de Bases , Sítios de Ligação , Técnicas de Inativação de Genes , Variação Genética , Mutação , Proteína I de Ligação a Poli(A)/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Sequência de RNA , Relação Estrutura-AtividadeRESUMO
Determining the half-life of proteins is critical for an understanding of virtually all cellular processes. Current methods for measuring in vivo protein stability, including large-scale approaches, are limited in their throughput or in their ability to discriminate among small differences in stability. We developed a new method, Stable-seq, which uses a simple genetic selection combined with high-throughput DNA sequencing to assess the in vivo stability of a large number of variants of a protein. The variants are fused to a metabolic enzyme, which here is the yeast Leu2 protein. Plasmids encoding these Leu2 fusion proteins are transformed into yeast, with the resultant fusion proteins accumulating to different levels based on their stability and leading to different doubling times when the yeast are grown in the absence of leucine. Sequencing of an input population of variants of a protein and the population of variants after leucine selection allows the stability of tens of thousands of variants to be scored in parallel. By applying the Stable-seq method to variants of the protein degradation signal Deg1 from the yeast Matα2 protein, we generated a high-resolution map that reveals the effect of â¼30,000 mutations on protein stability. We identified mutations that likely affect stability by changing the activity of the degron, by leading to translation from new start codons, or by affecting N-terminal processing. Stable-seq should be applicable to other organisms via the use of suitable reporter proteins, as well as to the analysis of complex mixtures of fusion proteins.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/métodos , Estabilidade Proteica , Proteômica/métodos , 3-Isopropilmalato Desidrogenase/química , 3-Isopropilmalato Desidrogenase/genética , 3-Isopropilmalato Desidrogenase/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Genes Reporter , Variação Genética , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Transferases Intramoleculares/química , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Dados de Sequência Molecular , Plasmídeos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Spermatogenesis is a complex process through which male germ line stem cells undergo a multi-step differentiation program and sequentially become spermatogonia, spermatocytes, spermatids, and eventually spermatozoa. In this process, transcription factors act as switches that precisely regulate the expression of genes that in turn control the developmental program of male germ cells. Transcription factors identified to be essential for normal haploid gene expression all display transcription-activating effects and thus serve as the "on" switch for haploid gene expression. Here, we report that ZMYND15 acts as a histone deacetylase-dependent transcriptional repressor and controls normal temporal expression of haploid cell genes during spermiogenesis. Inactivation of Zmynd15 results in early activation of transcription of numerous important haploid genes including Prm1, Tnp1, Spem1, and Catpser3; depletion of late spermatids; and male infertility. ZMYND15 represents the first transcriptional repressor identified to be essential for sperm production and male fertility.