RESUMO
1,8-Napthalimides (NIs) have been widely used as fluorescent molecules in biological, chemical, and medical fields because NIs shows high stability and various fluorescence properties under different conditions. However, NIs typically display a fluorescence emission wavelength in the range of 350 - 550 nm which can be notably interfered with by autofluorescence in living cells, significantly limiting their bio-applications. Moreover, low solubility in aqueous media is another major limitation for NIs. In this project, four derivatives of NIs (1-4) have been synthesized via an aromatic nucleophilic substitution reaction and their photophysical properties have been investigated in various media (water, MeOH, MeCN, DMSO, EtOAc, and THF). All of these derivatives (1-4) show a long emission wavelength around 600 nm and high solubility in polar solvents. Particularly molecules (1-4) show the longest emission (624-629 nm) in water and the fluorescence intensity is not significantly varied in the range of pH 4-11. These unique features, long emission wavelength, high solubility, and high stability in difference pH media, will allow these derivative (1-4) to be used as excellent labeling reagents in the biological system.
RESUMO
A reaction-based sensor (NAS-1) showed a high affinity and sensitivity to HSO3- via a nucleophilic addition reaction in the aqueous media, giving dual signals from absorption and emission spectra. NAS-1 was successfully applied in RK13 epithelial cells to detect HSO3- in a cellular environment.
Assuntos
Corantes Fluorescentes/química , Naftalimidas/química , Sulfitos/análise , Animais , Células Cultivadas , Corantes Fluorescentes/síntese química , Estrutura Molecular , Naftalimidas/síntese química , Imagem Óptica , Coelhos , Espectrometria de FluorescênciaRESUMO
AIM: This study assessed the cost consciousness of nurses and physicians in German neonatal intensive care units (NICUs) and identified factors affecting cost consciousness. METHODS: This study on cost consciousness was part of the German Safety4NICU study, a cross-sectional survey conducted from 2015 to 2016. All 224 German NICUs were invited to take part in the survey, and written consent was obtained from the leading physicians and nurses. The various professions were addressed via specific questionnaires. The cost survey tool identified the participants' responsibility and their desired focus on cost consciousness. RESULTS: Of the 1406 nurses and 496 physicians from 84 NICUs, 64.4% of the nurses and 62.5% of the physicians agreed that they shared responsibility for controlling costs. The computed score to define the overall cost consciousness level was 4.47. We identified a significantly positive association between cost consciousness, longer total clinical work experience and a decreased number of NICU intensive care beds. Increased cost consciousness was found in both men and physicians. Other hospital characteristics did not have an effect. CONCLUSION: Neonatology is a medical speciality where the tension between economics and the benefit of patients is extremely high. We found a moderate level of cost consciousness among NICU physicians and nurses.
Assuntos
Atitude do Pessoal de Saúde , Custos de Cuidados de Saúde , Unidades de Terapia Intensiva Neonatal/economia , Enfermeiras e Enfermeiros/psicologia , Médicos/psicologia , Adulto , Idoso , Estudos Transversais , Feminino , Alemanha , Humanos , Masculino , Pessoa de Meia-Idade , Enfermeiras e Enfermeiros/estatística & dados numéricos , Médicos/estatística & dados numéricos , Adulto JovemRESUMO
Nucleosides play an essential role in the physiology of eukaryotes by acting as metabolic precursors in de novo nucleic acid synthesis and energy metabolism. Nucleosides also act as ligands for purinergic receptors. Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that aid in regulating plasmalemmal flux of purine and pyrimidine nucleosides and nucleobases. ENTs exhibit broad substrate selectivity across different isoforms and utilize diverse mechanisms to drive substrate flux across membranes. However, the molecular mechanisms and chemical determinants of ENT-mediated substrate recognition, binding, inhibition, and transport are poorly understood. To determine how ENT-mediated transport occurs at the molecular level, greater chemical insight and assays employing purified protein are essential. This article focuses on the expression and purification of human ENT1, human ENT2, and Saccharomyces cerevisiae ScENT1 using novel expression and purification strategies to isolate recombinant ENTs. ScENT1, hENT1, and hENT2 were expressed in W303 Saccharomyces cerevisiae cells and detergent solubilized from the membrane. After detergent extraction, these ENTs were further purified using immobilized metal affinity chromatography and size exclusion chromatography. This effort resulted in obtaining quantities of purified protein sufficient for future biophysical analysis.
Assuntos
Transportador Equilibrativo 1 de Nucleosídeo/genética , Transportador Equilibrativo 2 de Nucleosídeo/genética , Plasmídeos/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genética , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Cromatografia de Afinidade , Cromatografia em Gel , Clonagem Molecular , Detergentes/química , Transportador Equilibrativo 1 de Nucleosídeo/biossíntese , Transportador Equilibrativo 1 de Nucleosídeo/isolamento & purificação , Transportador Equilibrativo 2 de Nucleosídeo/biossíntese , Transportador Equilibrativo 2 de Nucleosídeo/isolamento & purificação , Expressão Gênica , Humanos , Plasmídeos/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Transporte Vesicular/biossíntese , Proteínas de Transporte Vesicular/isolamento & purificaçãoRESUMO
Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that transport nucleosides and, to a lesser extent, nucleobases across cell membranes. ENTs modulate efficacy for a range of human therapeutics and function in a diffusion-controlled bidirectional manner. A detailed understanding of ENT function at the molecular level has remained elusive. FUN26 (function unknown now 26) is a putative ENT homolog from S. cerevisiae that is expressed in vacuole membranes. In the present system, proteoliposome studies of purified FUN26 demonstrate robust nucleoside and nucleobase uptake into the luminal volume for a broad range of substrates. This transport activity is sensitive to nucleoside modifications in the C(2')- and C(5')-positions on the ribose sugar and is not stimulated by a membrane pH differential. [(3)H]Adenine nucleobase transport efficiency is increased â¼4-fold relative to nucleosides tested with no observed [(3)H]adenosine or [(3)H]UTP transport. FUN26 mutational studies identified residues that disrupt (G463A or G216A) or modulate (F249I or L390A) transporter function. These results demonstrate that FUN26 has a unique substrate transport profile relative to known ENT family members and that a purified ENT can be reconstituted in proteoliposomes for functional characterization in a defined system.
Assuntos
Proteínas de Transporte de Nucleosídeo Equilibrativas/metabolismo , Proteínas de Membrana Transportadoras/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Primers do DNA , Cinética , Proteínas de Membrana Transportadoras/metabolismo , Reação em Cadeia da Polimerase , Proteolipídeos , Proteínas de Saccharomyces cerevisiae/metabolismo , SolubilidadeRESUMO
Biofilms are complex biomaterials comprising a well-organized network of microbial cells encased in self-produced extracellular polymeric substances (EPS). This paper presents a detailed account of the implementation of optical coherence elastography (OCE) measurements tailored for the elastic characterization of biofilms. OCE is a non-destructive optical technique that enables the local mapping of the microstructure, morphology, and viscoelastic properties of partially transparent soft materials with high spatial and temporal resolution. We provide a comprehensive guide detailing the essential procedures for the correct implementation of this technique, along with a methodology to estimate the bulk Young's modulus of granular biofilms from the collected measurements. These consist of the system setup, data acquisition, and postprocessing. In the discussion, we delve into the underlying physics of the sensors used in OCE and explore the fundamental limitations regarding the spatial and temporal scales of OCE measurements. We conclude with potential future directions for advancing the OCE technique to facilitate elastic measurements of environmental biofilms.
Assuntos
Técnicas de Imagem por Elasticidade , Biofilmes , Materiais Biocompatíveis , Módulo de ElasticidadeRESUMO
Replication forks must respond to changes in nutrient conditions, especially in bacterial cells. By investigating the single-molecule dynamics of replicative helicase DnaC, DNA primase DnaG, and lagging-strand polymerase DnaE in the model bacterium Bacillus subtilis, we show that proteins react differently to stress conditions in response to transient replication blocks due to DNA damage, to inhibition of the replicative polymerase, or to downshift of serine availability. DnaG appears to be recruited to the forks by a diffusion and capture mechanism, becomes more statically associated after the arrest of polymerase, but binds less frequently after fork blocks due to DNA damage or to nutritional downshift. These results indicate that binding of the alarmone (p)ppGpp due to stringent response prevents DnaG from binding to forks rather than blocking bound primase. Dissimilar behavior of DnaG and DnaE suggests that both proteins are recruited independently to the forks rather than jointly. Turnover of all three proteins was increased during replication block after nutritional downshift, different from the situation due to DNA damage or polymerase inhibition, showing high plasticity of forks in response to different stress conditions. Forks persisted during all stress conditions, apparently ensuring rapid return to replication extension.IMPORTANCE All cells need to adjust DNA replication, which is achieved by a well-orchestrated multiprotein complex, in response to changes in physiological and environmental conditions. For replication forks, it is extremely challenging to meet with conditions where amino acids are rapidly depleted from cells, called the stringent response, to deal with the inhibition of one of the centrally involved proteins or with DNA modifications that arrest the progression of forks. By tracking helicase (DnaC), primase (DnaG), and polymerase (DnaE), central proteins of Bacillus subtilis replication forks, at a single molecule level in real time, we found that interactions of the three proteins with replication forks change in different manners under different stress conditions, revealing an intriguing plasticity of replication forks in dealing with replication obstacles. We have devised a new tool to determine rates of exchange between static movement (binding to a much larger complex) and free diffusion, showing that during stringent response, all proteins have highly increased exchange rates, slowing down overall replication, while inactivation of polymerase or replication roadblocks leaves forks largely intact, allowing rapid restart once obstacles are removed.
Assuntos
Bacillus subtilis/genética , Replicação do DNA , Imagem Individual de Molécula/métodos , DNA Helicases/metabolismo , DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Microscopia de FluorescênciaRESUMO
Objective: This systematic review aims to analyze current capabilities, challenges, and impact of self-directed mobile health (mHealth) research applications such as those based on the ResearchKit platform. Materials and Methods: A systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. English publications were included if: 1) mobile applications were used in the context of large-scale collection of data for biomedical research, and not as medical or behavioral intervention of any kind, and 2) all activities related to participating in research and data collection methods were executed remotely without any face-to-face interaction between researchers and study participants. Results: Thirty-six unique ResearchKit apps were identified. The majority of the apps were used to conduct observational studies on general citizens and generate large datasets for secondary research. Nearly half of the apps were focused on chronic conditions in adults. Discussion: The ability to generate large biomedical datasets on diverse populations that can be broadly shared and re-used was identified as a promising feature of mHealth research apps. Common challenges were low participation retention, uncertainty regarding how use patterns influence data quality, need for data validation, and privacy concerns. Conclusion: ResearchKit and other mHealth-based studies are well positioned to enhance development and validation of novel digital biomarkers as well as generate new biomedical knowledge through retrospective studies. However, in order to capitalize on these benefits, mHealth research studies must strive to improve retention rates, implement rigorous data validation strategies, and address emerging privacy and security challenges.
Assuntos
Pesquisa Biomédica , Participação da Comunidade , Aplicativos Móveis , Confiabilidade dos Dados , Conjuntos de Dados como Assunto , Doença , Humanos , Seleção de Pacientes , Privacidade , SmartphoneRESUMO
This review describes emerging techniques within the last 5 years that employ ultrasound for detecting and staging malignancy, tracking metastasis, and guiding treatment. Ultrasound elastography quantifies soft tissue elastic properties that change as a tumor grows and proliferates. Hybrid imaging modalities that combine ultrasound with light or microwave energy provide novel contrast for mapping blood oxygen saturation, transport of particles through lymphatic vessels and nodes, and real-time feedback for guiding needle biopsies. Combining these methods with smart nanoparticles and contrast agents further promotes new paradigms for cancer imaging and therapy.