RESUMEN
OBJECTIVE: The study aimed to characterize the experiences of primary caregivers of children with medical complexity (CMC) in engaging with other members of the child's caregiving network, thereby informing the design of health information technology (IT) for the caregiving network. Caregiving networks include friends, family, community members, and other trusted individuals who provide resources, information, health, or childcare. MATERIALS AND METHODS: We performed a secondary analysis of two qualitative studies. Primary studies conducted semi-structured interviews (n = 50) with family caregivers of CMC. Interviews were held in the Midwest (n = 30) and the mid-Atlantic region (n = 20). Interviews were transcribed verbatim for thematic analysis. Emergent themes were mapped to implications for the design of future health IT. RESULTS: Thematic analysis identified 8 themes characterizing a wide range of primary caregivers' experiences in constructing, managing, and ensuring high-quality care delivery across the caregiving network. DISCUSSION: Findings evidence a critical need to create flexible and customizable tools designed to support hiring/training processes, coordinating daily care across the caregiving network, communicating changing needs and care updates across the caregiving network, and creating contingency plans for instances where caregivers are unavailable to provide care to the CMC. Informaticists should additionally design accessible platforms that allow primary caregivers to connect with and learn from other caregivers while minimizing exposure to sensitive or emotional content as indicated by the user. CONCLUSION: This article contributes to the design of health IT for CMC caregiving networks by uncovering previously underrecognized needs and experiences of CMC primary caregivers and drawing direct connections to design implications.
Asunto(s)
Cuidadores , Informática Médica , Niño , Humanos , Cuidadores/psicología , Investigación Cualitativa , Mid-Atlantic Region , EmocionesRESUMEN
N-demethylases have been reported to remove the methyl groups on primary or secondary amines, which could further affect the properties and functions of biomacromolecules or chemical compounds; however, the substrate scope and the robustness of N-demethylases have not been systematically investigated. Here we report the recreation of natural evolution in key microdomains of the Thermomicrobium roseum sarcosine oxidase (TrSOX), an N-demethylase with marked stability (melting temperature over 100 °C) and enantioselectivity, for enhanced substrate scope and catalytic efficiency on -C-N- bonds. We obtained the structure of TrSOX by crystallization and X-ray diffraction (XRD) for the initial framework. The natural evolution in the nonconserved residues of key microdomains-including the catalytic loop, coenzyme pocket, substrate pocket, and entrance site-was then identified using ancestral sequence reconstruction (ASR), and the substitutions that accrued during natural evolution were recreated by site-directed mutagenesis. The single and double substitution variants catalyzed the N-demethylation of N-methyl-L-amino acids up to 1800- and 6000-fold faster than the wild type, respectively. Additionally, these single substitution variants catalyzed the terminal N-demethylation of non-amino-acid compounds and the oxidation of the main chain -C-N- bond to a -C=N- bond in the nitrogen-containing heterocycle. Notably, these variants retained the enantioselectivity and stability of the initial framework. We conclude that the variants of TrSOX are of great potential use in N-methyl enantiomer resolution, main-chain Schiff base synthesis, and alkaloid modification or degradation.
Asunto(s)
Chloroflexi , Oxidorreductasas N-Desmetilantes , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/metabolismo , Catálisis , Chloroflexi/enzimología , Chloroflexi/genética , Mutagénesis Sitio-Dirigida , Oxidorreductasas N-Desmetilantes/genética , Oxidorreductasas N-Desmetilantes/metabolismo , Ingeniería de Proteínas , Especificidad por SustratoRESUMEN
Thermomicrobium roseum sarcosine oxidase (TrSOX) was a N-demethylase with specific substrate chiral selectivity, outstanding thermostability and environmental resistance. To promote the expression of TrSOX in Bacillus subtilis W600, the HpaII promoter of pMA5 plasmid was replaced by constitutive or inducible promoters. Through orthogonal experiment, the expression process was optimized, B. subtilis W600 cells containing pMA5-Pxyl-trSOX plasmid were cultivated until OD600nm reached 2.0 and were then induced with 1.6% xylose at 37 °C for 2 h, and the native environment of T. roseum was simulated by heating at 80 °C, with the productivity of TrSOX increased from ~8.3 to ~66.7 µg/g wet cells; and the simulated high temperature was the key switch for the final folding. To reduce the surface hydrophobicity, a S320R mutant was built to form a hydrophilic lid around the entrance of the substrate pocket, and the yield of TrSOX (S320R) was ~163.0 µg/g wet cells, approximately 20 folds as that in the initial expression system. This mutant revealed the similar secondary structure, stability, resistance, chiral substrate selectivity and optimal reaction environment with wild type TrSOX; however, the N-demethylation activities for amino acid derivative substrates were dramatically increased, while those for hydrophobic non-amino acid compounds were repressed.