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INTRODUCTION: Hospital readmissions are recognized as a prevalent, yet potentially preventable, personal and economic burden. Length of stay, Acuity of admission, Comorbidities, and number of Emergency Department visits in the preceding 6 months can be quantified into one score, the LACE score. LACE scores have previously been identified to correlate with hospital readmissions within 30 days of discharge, but research specific to the pediatric population is scant. The objective of the present study was to investigate if LACE scores, in addition to other factors, can be utilized to create a predictive pediatric hospital readmission model that may ultimately be used to decrease readmission rates. METHODS: This study included 25,616 hospitalizations of patients under the age of 18 years. Data were extracted from a hospital network electronic medical record. Demographics included LACE scores, age, gender, race/ethnicity, median household income, and medical centers. The primary exposure variable was LACE score. The main outcome measures were readmissions within 7, 14, and 30 days. The area under the curve (AUC) was used to assess the predictive capability of the regression model on patient 30-day admission. RESULTS: LACE scores, age, gender, race/ethnicity, median household income, and medical centers were examined in a multivariable model to assess patient risk of a 30-day readmission. Only age and LACE score were observed to be statistically significant. The AUC for the combined model was 0.69. DISCUSSION: As only age and LACE score were observed to be statistically significant and the AUC for the combined model was 0.69, this model is considered to have poor predictive capability. CONCLUSIONS: In this study, LACE scores, as well the other factors, had a poor predictive capability for pediatric readmissions.
Assuntos
Readmissão do Paciente , Humanos , Readmissão do Paciente/estatística & dados numéricos , Criança , Feminino , Masculino , Adolescente , Pré-Escolar , Lactente , Tempo de Internação/estatística & dados numéricos , Fatores de Risco , Recém-Nascido , Fatores Etários , Estudos Retrospectivos , Área Sob a Curva , Medição de Risco/métodos , Valor Preditivo dos Testes , Serviço Hospitalar de Emergência/estatística & dados numéricosRESUMO
Like multiblock copolymers, spider silk proteins are built of repetitive sequence motives. One prominent repetitive motif is based on the consensus sequence of spidroin 4 of the spider Araneus diadematus ADF4. The number x of the repeating sequence motives (C) determines the molecular weight of the recombinant ADF4-based, engineered spider silk protein denoted as eADF4(Cx). eADF4(Cx) can be used as a model for intrinsically disordered proteins (IDP) and to elucidate their folding. Herein, the influence of the variation of the sequence motive repeating number x (x = 1, 2, 4, 8, 16) on the protein folding within eADF4(Cx) films was investigated. eADF4(Cx) films were cast from 1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP) solutions onto planar silicon model substrates, revealing mainly helical or random coil structure. Upon treatment with methanol vapor (ptm), the formation of crystalline ß-sheets was triggered. Dichroic Fourier-transform infrared (FTIR) spectroscopy, circular dichroism, spectroscopic ellipsometry, atomic force microscopy, grazing-incidence small-angle X-ray scattering (GISAXS), grazing-incidence wide-angle X-ray scattering (GIWAXS), and electrokinetic and contact angle measurements were used to get information concerning the secondary structure and folding kinetics, orientation of ß-sheets, the ratio of parallel/antiparallel ß-sheets, domain sizes and distributions, surface topography, surface potential, hydrophobicity and the film integrity under water. Significant differences in the final ß-sheet content, the share of antiparallel ß-sheet structures, film integrity, surface potential, and isoelectric points between eADF4(Cx) with x = 1, 2 and eADF4(Cx) with x = 4, 8, 16 gave new insights in the molecular weight-dependent structure formation and film properties of IDP systems. GISAXS and kinetic measurements confirmed a relation between ß-sheet crystal growth rate and final ß-sheet crystal size. Further, competing effects of reduced diffusibility hindering accelerated crystal growth and enhanced backfolding promoting accelerated crystal growth with increasing molecular weight were discussed.
Assuntos
Fibroínas , Aranhas , Animais , Seda/química , Fibroínas/química , Proteínas de Artrópodes , Proteínas Recombinantes/química , Dobramento de Proteína , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Biomaterials are an indispensable part of biomedical research. However, although many materials display suitable application-specific properties, they provide only poor biocompatibility when implanted into a human/animal body leading to inflammation and rejection reactions. Coatings made of spider silk proteins are promising alternatives for various applications since they are biocompatible, non-toxic and anti-inflammatory. Nevertheless, the biological response toward a spider silk coating cannot be generalized. The properties of spider silk coatings are influenced by many factors, including silk source, solvent, the substrate to be coated, pre- and post-treatments and the processing technique. All these factors consequently affect the biological response of the environment and the putative application of the appropriate silk coating. Here, we summarize recently identified factors to be considered before spider silk processing as well as physicochemical characterization methods. Furthermore, we highlight important results of biological evaluations to emphasize the importance of adjustability and adaption to a specific application. Finally, we provide an experimental matrix of parameters to be considered for a specific application and a guided biological response as exemplarily tested with two different fibroblast cell lines.
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Intrinsically disordered proteins (IDPs) play an important role in molecular biology and medicine because their induced folding can lead to so-called conformational diseases, where ß-amyloids play an important role. Still, the molecular folding process into the different substructures, such as parallel/antiparallel or extended ß-sheet/crossed ß-sheet is not fully understood. The recombinant spider silk protein eADF4(Cx) consisting of repeating modules C, which are composed of a crystalline (pep-c) and an amorphous peptide sequence (pep-a), can be used as a model system for IDP since it can assemble into similar structures. In this work, blend films of the pep-c and pep-a sequences were investigated to modulate the ß-sheet formation by varying the molar fraction of pep-c and pep-a. Dichroic Fourier-transform infrared spectroscopy (FTIR), circular dichroism, spectroscopic ellipsometry, atomic force microscopy, and IR nanospectroscopy were used to examine the secondary structure, the formation of parallel and antiparallel ß-sheets, their orientation, and the microscopic roughness and phase formation within peptide blend films upon methanol post-treatment. New insights into the formation of filament-like structures in these silk blend films were obtained. Filament-like structures could be locally assigned to ß-sheet-rich structures. Further, the antiparallel or parallel character and the orientation of the formed ß-sheets could be clearly determined. Finally, the ideal ratio of pep-a and pep-c sequences found in the fibroin 4 of the major ampullate silk of spiders could also be rationalized by comparing the blend and spider silk protein systems.
Assuntos
Fibroínas , Aranhas , Animais , Seda/química , Conformação Proteica em Folha beta , Peptídeos/química , Fibroínas/química , Estrutura Secundária de Proteína , Proteínas RecombinantesAssuntos
Hematologia , Farmácia , Humanos , Pandemias , Drogas em Investigação/uso terapêutico , Opinião PúblicaRESUMO
Improving biomaterials by engineering application-specific and adjustable properties is of increasing interest. Most of the commonly available materials fulfill the mechanical and physical requirements of relevant biomedical applications, but they lack biological functionality, including biocompatibility and prevention of microbial infestation. Thus, research has focused on customizable, application-specific, and modifiable surface coatings to cope with the limitations of existing biomaterials. In the case of adjustable degradation and configurable interaction with body fluids and cells, these coatings enlarge the applicability of the underlying biomaterials. Silks are interesting coating materials, e.g., for implants, since they exhibit excellent biocompatibility and mechanical properties. Herein, we present putative implant coatings made of five engineered recombinant spider silk proteins derived from the European garden spider Araneus diadematus fibroins (ADF), differing in amino acid sequence and charge. We analyzed the influence of the underlying amino acid composition on wetting behavior, blood compatibility, biodegradability, serum protein adsorption, and cell adhesion. The outcome of the comparison indicates that spider silk coatings can be engineered for explicit biomedical applications.
Assuntos
Fibroínas , Seda , Aminoácidos , Proteínas de Artrópodes , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Fibroínas/química , Proteínas Recombinantes/química , Seda/químicaRESUMO
Polypeptide coatings are a cornerstone in the field of surface modification due to their widespread biological potential. As their properties are dictated by their structural features, subsequent control thereof using unique fabrication strategies is important. Herein, we report a facile method of precisely creating densely crosslinked polypeptide films with unusually high random coil content through continuous assembly polymerization via reversible addition-fragmentation chain transfer (CAP-RAFT). CAP-RAFT was fundamentally investigated using methacrylated poly-l-lysine (PLLMA) and methacrylated poly-l-glutamic acid (PLGMA). Careful technique refinement resulted in films up to 36.1±1.1â nm thick which could be increased to 94.9±8.2â nm after using this strategy multiple times. PLLMA and PLGMA films were found to have 30-50 % random coil conformations. Degradation by enzymes present during wound healing reveals potential for applications in drug delivery and tissue engineering.
RESUMO
Orientation analysis of the ß-sheet structure within films of the established recombinant spider silk protein eADF4(C16) was performed using a concept based on dichroic transmission- and attenuated total reflection-Fourier transform infrared spectroscopy, lineshape analysis, assignment of amide I components to specific vibration modes, and transition dipole moment directions of ß-sheet structures. Based on the experimental dichroic ratio R, the order parameter S of ß-sheet structures was calculated with respect to uniaxial orientation. Films of eADF4(C16) were deposited on untexturized (Si) and unidirectionally scratched silicon substrates (Si-sc) and post-treated with MeOH vapor. Freshly cast thin and thick eADF4(C16) films out of hexafluoroisopropanol featured ß-sheet contents of ≈6%, which increased to >30% after MeOH post-treatment in dependence of time. Pseudo-first order folding kinetics were obtained, suggesting a transition from an unfolded to a folded state. In MeOH post-treated thin films with diameters in the nanometer range, a significant orientation of ß-sheets was obtained regardless of the texturization of the silicon substrate (Si, Si-sc). This was rationalized by dichroic ratios of the amide I component at 1696 cm-1 assigned to the (0, π) mode of antiparallel ß-sheet structures, whose transition dipole moment M is located in parallel to both ß-sheet plane and chain direction. The calculated high molecular order parameter S ≈ 0.40 suggested vertically (out-of-plane) oriented antiparallel ß-sheet stacks with tilt angles of γ ≈ 39° to the surface normal. Microscale (thick) films, in contrast, revealed low order parameters S ≈ 0. Scanning force microscopy on thin eADF4 films at silicon substrates showed dewetted polymer film structures rather at the micro-scale. These findings give new insights in the role of the ß-sheet crystallite orientation for the mechanical properties of spider silk materials.
Assuntos
Silício , Seda , Conformação Proteica em Folha beta , Proteínas Recombinantes , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
In recent years, spider silk-based materials have attracted attention because of their biocompatibility, processability, and biodegradability. For their potential use in biomaterial applications, i.e., as drug delivery systems and implant coatings for tissue regeneration, it is vital to understand the interactions between the silk biomaterial surface and the biological environment. Like most polymeric carrier systems, spider silk material surfaces can adsorb proteins when in contact with blood, resulting in the formation of a biomolecular corona. Here, we assessed the effect of surface net charge of materials made of recombinant spider silk on the biomolecular corona composition. In-depth proteomic analysis of the biomolecular corona revealed that positively charged spider silk materials surfaces interacted predominantly with fibrinogen-based proteins. This fibrinogen enrichment correlated with blood clotting observed for both positively charged spider silk films and particles. In contrast, negative surface charges prevented blood clotting. Genetic engineering allows the fine-tuning of surface properties of the spider silk particles providing a whole set of recombinant spider silk proteins with different charges or peptide tags to be used for, for example, drug delivery or cell docking, and several of these were analyzed concerning the composition of their biomolecular corona. Taken together this study demonstrates how the surface net charge of recombinant spider silk surfaces affects the composition of the biomolecular corona, which in turn affects macroscopic effects such as fibrin formation and blood clotting.
Assuntos
Coroa de Proteína/metabolismo , Seda/química , Aranhas/química , Adsorção , Sequência de Aminoácidos , Animais , Fibrinogênio/metabolismo , Humanos , Ligação Proteica , Engenharia de Proteínas , Seda/genética , Seda/metabolismo , Eletricidade Estática , Propriedades de SuperfícieRESUMO
Fluoroquinolones are a class of antibacterial agents used clinically to treat a wide array of bacterial infections and target bacterial type-II topoisomerases (DNA gyrase and topoisomerase IV). Fluoroquinolones, however potent, are susceptible to bacterial resistance with prolonged use, which limits their use in the clinic. Quinazoline-2,4-diones also target bacterial type-II topoisomerases and are not susceptible to bacterial resistance similar to fluoroquinolones, however, their potency pales in comparison to fluoroquinolones. To meet the increasing demand for antibacterial development, nine modified quinazoline-2,4-diones were developed to probe quinazoline-2,4-dione structure modification for possible new binding contacts with the bacterial type-II topoisomerase, DNA gyrase. Evaluation of compounds for inhibition of the supercoiling activity of DNA gyrase revealed a novel ethyl 5,6-dihydropyrazolo[1,5-c]quinazoline-1-carboxylate derivative as a modest inhibitor of DNA gyrase, having an IC50 of 3.5 µM. However, this ethyl 5,6-dihydropyrazolo[1,5-c]quinazoline-1-carboxylate does not trap the catalytic intermediate like fluoroquinolones or typical quinazoline-2,4-diones do. Thus, the ethyl 5,6-dihydropyrazolo[1,5-c]quinazoline-1-carboxylate derivative discovered in this work acts as a catalytic inhibitor of DNA gyrase and therefore represents a new structural type of catalytic inhibitor of DNA gyrase.
Assuntos
DNA Girase/metabolismo , Inibidores da Topoisomerase II/farmacologia , Biocatálise , Relação Dose-Resposta a Droga , Escherichia coli/enzimologia , Estrutura Molecular , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/síntese química , Inibidores da Topoisomerase II/químicaRESUMO
Fluoroquinolones substituted with N-1 biphenyl and napthyl groups were discovered to act as catalytically inhibitors of human topoisomerases I and II, and to possess anti-proliferative activity in vivo. Structural requirements for these novel quinolones to inhibit catalytic activity of human topoisomerase I have not been explored. In this work novel derivatives of the N-1 biphenyl fluoroquinolone were designed, synthesized and evaluated to understand structural requirements of the C-3 carboxylic acid, C-6 fluorine, C-7 aminomethylpyrrolidine, C-8 methoxy, and the N-1 biphenyl functional groups for hTopoI inhibition. Characterization of each analog for inhibition of hTopoI catalytic inhibition reveals critical insight into structural requirements of these novel quinolones for activity. Additionally, results of DNA binding and modeling studies suggest that N-1 biphenyl fluoroquinolones intercalate between the DNA base pairs with the N-1 biphenyl functional group, rather than the quinolone core, and that this mode of DNA intercalation contributes to inhibition of hTopoI by these novel structures. The results presented here support further development and evaluation of N-1 biphenyl fluoroquinolone analogs as a novel class of anti-cancer agents that act through catalytic inhibition of hTopoI.
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Compostos de Bifenilo/farmacologia , DNA Topoisomerases Tipo I/metabolismo , Fluoroquinolonas/farmacologia , Inibidores da Topoisomerase I/farmacologia , Compostos de Bifenilo/síntese química , Compostos de Bifenilo/química , Relação Dose-Resposta a Droga , Fluoroquinolonas/síntese química , Fluoroquinolonas/química , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Inibidores da Topoisomerase I/síntese química , Inibidores da Topoisomerase I/químicaRESUMO
A Mg2+-water bridge between the C-3, C-4 diketo moiety of fluoroquinolones and the conserved amino acid residues in the GyrA/ParC subunit is critical for the binding of a fluoroquinolone to a topoisomerase-DNA covalent complex. The fluoroquinolone UING-5-249 (249) can bind to the GyrB subunit through its C7-aminomethylpyrrolidine group. This interaction is responsible for enhanced activities of 249 against the wild type and quinolone-resistant mutant topoisomerases. To further evaluate the effects of the 249-GyrB interaction on fluoroquinolone activity, we examined the activities of decarboxy- and thio-249 against DNA gyrase and conducted docking studies using the structure of a gyrase-ciprofloxacin-DNA ternary complex. We found that the 249-GyrB interaction rescued the activity of thio-249 but not that of decarboxy-249. A C7-group that binds more strongly to the GyrB subunit may allow for modifications at the C-4 position, leading to a novel compound that is active against the wild type and quinolone-resistant pathogens.
Assuntos
Ciprofloxacina/metabolismo , DNA Girase/metabolismo , DNA Bacteriano/metabolismo , Fluoroquinolonas/metabolismo , Pirrolidinas/química , Staphylococcus aureus/enzimologia , Compostos de Sulfidrila/química , Antibacterianos/química , Antibacterianos/metabolismo , Ciprofloxacina/química , DNA Girase/química , DNA Girase/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Descarboxilação , Escherichia coli/metabolismo , Fluoroquinolonas/química , Testes de Sensibilidade Microbiana , Subunidades ProteicasRESUMO
Fluoroquinolone-class agents selectively target the bacterial type IIA topoisomerases DNA gyrase and topoisomerase IV, with a few exceptions that target eukaryotic type IIA topoisomerases. Fluoroquinolones bind and stabilize type IIA topoisomerase-DNA covalent complexes that contain a double-strand break. This unique mode of action is referred to as 'topoisomerase poisoning'. We discovered that two novel fluoroquinolones having aryl functionality at the N-1 position, UITT-3-217 (217) and UITT-3-227 (227), could inhibit the catalytic activity of human topoisomerase II without stabilizing topoisomerase-DNA complexes, i.e., without poisoning it. Surprisingly, these compounds are more effective in inhibiting the catalytic activities of human and bacterial topoisomerase I. The National Cancer Institute's 60 human tumor cell lines screen revealed significant anti-proliferative activities with 217 and 227 against the majority of 60 cancer cell lines. A proof of concept in vivo efficacy study using an HT-29 xenograft model of human colorectal cancer showed that 217 could inhibit the proliferation of human colorectal cancer cells to a degree comparable to fluorouracil in mice. Although 227 also exhibited anti-proliferative activity, it was not as effective as 217 in this xenograft model. These novel fluoroquinolones may serve as promising lead compounds for the development of new anticancer drugs.
Assuntos
Antineoplásicos/farmacologia , Neoplasias do Colo/tratamento farmacológico , DNA Topoisomerases Tipo I/química , Fluoroquinolonas/farmacologia , Inibidores da Topoisomerase I/farmacologia , Animais , Antineoplásicos/química , Apoptose , Proliferação de Células , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Feminino , Fluoroquinolonas/química , Humanos , Camundongos , Camundongos Nus , Inibidores da Topoisomerase I/química , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Discrete gold nanoparticle crystals with tunable size and morphology are fabricated via a fast and inexpensive template-assisted method. The highly precise hierarchical organization of the plasmonic building blocks yields superstructures with outstanding behaviour for surface-enhanced Raman scattering analysis.