RESUMEN
Proteins represent powerful biomacromolecules due to their unique functionality and broad utility both in the cell and in non-biological applications. The genetic encoding of non-canonical amino acids (ncAAs) facilitates functional diversification of these already powerful proteins. Specifically, ncAAs have been demonstrated to provide unique functional handles to bioorthogonally introduce novel functionality via conjugation reactions. Herein we examine the ability of a single ncAA to serve as a handle to generate multivalent bioconjugates to introduce two or more additional components to a protein, yielding a multivalent conjugate. To accomplish this aim, p-bromopropargyloxyphenyalanine (pBrPrF) was genetically encoded into both superfolder green fluorescent protein (sfGFP) and ubiquitin model proteins to serve as a conjugation handle. A sequential bioconjugation sequence involving a copper-assisted cycloaddition reaction coupled with a subsequent Sonogashira cross-coupling was then optimized. The linkage of two additional molecules to the model protein via these reactions yielded the desired multivalent bioconjugate. This domino approach using a single ncAA has a plethora of applications in both therapeutics and diagnostics as multiple unique moieties can be introduced into proteins in a highly controlled fashion.
Asunto(s)
Aminoácidos , Aminoácidos/química , Proteínas Fluorescentes Verdes/químicaRESUMEN
The efficient preparation of protein bioconjugates represents a route to novel materials, diagnostics, and therapeutics. We previously reported a novel bioorthogonal Glaser-Hay reaction for the preparation of covalent linkages between proteins and a reaction partner; however, deleterious protein degradation was observed under extended reaction conditions. Herein, we describe the systematic optimization of the reaction to increase coupling efficiency and decrease protein degradation. Two optimized conditions were identified varying either the pH of the reaction or the bidentate ligand employed, allowing for more rapid conjugations and/or less protein oxidation.
Asunto(s)
Alquinos/síntesis química , Técnicas de Química Sintética/métodos , Proteínas Fluorescentes Verdes/química , Línea Celular Tumoral , Cobre/química , Proteínas Fluorescentes Verdes/genética , Humanos , Yoduros/química , Ligandos , Fenilalanina/análogos & derivados , Fenilalanina/genética , Rodaminas/síntesis química , TemperaturaRESUMEN
BACKGROUND: Smell and taste loss are highly prevalent symptoms in coronavirus disease 2019 (COVID-19), although few studies have employed objective measures to quantify these symptoms, especially dysgeusia. Reports of unrecognized anosmia in COVID-19 patients suggests that self-reported measures are insufficient for capturing patients with chemosensory dysfunction. OBJECTIVES: The purpose of this study was to quantify the impact of recent COVID-19 infection on chemosensory function and demonstrate the use of at-home objective smell and taste testing in an at-risk population of healthcare workers. METHODS: Two hundred and fifty healthcare workers were screened for possible loss of smell and taste using online surveys. Self-administered smell and taste tests were mailed to respondents meeting criteria for elevated risk of infection, and one-month follow-up surveys were completed. RESULTS: Among subjects with prior SARS-CoV-2 infection, 73% reported symptoms of olfactory and/or gustatory dysfunction. Self-reported smell and taste loss were both strong predictors of COVID-19 positivity. Subjects with evidence of recent SARS-CoV-2 infection (<45 days) had significantly lower olfactory scores but equivalent gustatory scores compared to other subjects. There was a time-dependent increase in smell scores but not in taste scores among subjects with prior infection and chemosensory symptoms. The overall infection rate was 4.4%, with 2.5% reported by PCR swab. CONCLUSION: Healthcare workers with recent SARS-CoV-2 infection had reduced olfaction and normal gustation on self-administered objective testing compared to those without infection. Rates of infection and chemosensory symptoms in our cohort of healthcare workers reflect those of the general public.
RESUMEN
OBJECTIVE: To characterize differences in vestibular testing results among patients presenting with balance-related complaints; to stratify patterns of testing abnormalities by age. STUDY DESIGN: Retrospective chart review. SETTING: Academic Balance Center at a Tertiary Referral Center. PATIENT POPULATION: All patients who underwent vestibular testing in a 1-year period from 2017 to 2018. MAIN OUTCOME MEASURE: Balance function test results. RESULTS: We reviewed 1,116 patients with age ranging from 11 to 94âyears, including 521 patients ≥60âyears. Most patients had at least 1 abnormal result, with only 21% of patients ≥60âyears and 43% of patientsâ<60âyears yielding no test abnormalities (pâ<â0.001). Among 754 individuals with abnormal testing results, caloric testing did not show any significant difference between age groups. Patients ≥60âyears of age were more likely to demonstrate abnormalities on saccadic and horizontal tracking eye movements (pâ<â0.01; sacc 20.8% vs 6.3%; HT 9.2% vs 4.7%), as well as positional and Dix-Hallpike testing with videonystagmography (pâ<â0.001; pos 52.3% vs 37.5%, DH 14.4% vs 6.8%). On computerized dynamic posturography, there were significantly more abnormal composite scores in the older group for both sensory organization and motor control testing (pâ<â0.001; SOT 31.8% vs 8.8%, MCT 7.6% vs 1.8%). CONCLUSION: Among patients presenting balance-related complaints, a majority demonstrate at least one abnormality on vestibular testing. While caloric abnormalities occur across the life span, patients ≥60âyears of age are more likely to have abnormal results in random saccades, positional, Dix-Hallpike testing, and posturography, with greater effect sizes in tests of central function. While the causes of imbalance and vertigo remain multifactorial, vestibular dysfunction can be considered a major contributor to balance dysfunction in the elderly population and should be thoroughly evaluated.
Asunto(s)
Mareo , Equilibrio Postural , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Pruebas Calóricas , Niño , Mareo/diagnóstico , Humanos , Persona de Mediana Edad , Estudios Retrospectivos , Vértigo , Pruebas de Función Vestibular , Adulto JovenRESUMEN
The importance of bioconjugates within the field of chemistry drives the need for novel methodologies for their preparation. Well-defined and stable bioconjugates are easily accessible via the utilization of unnatural amino acids (UAAs). As such, we have synthesized and incorporated two new UAAs into green fluorescent protein, and optimized a novel Cadiot-Chodkiewicz bioconjugation, effectively expanding the toolbox of chemical reactions that can be employed in the preparation of bioconjugates.