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OBJECTIVE: To evaluate the agreement between nurse and dietician nutritional risk assessments when using the Nutritional Risk Screening 2002 (NRS2002) protocol, and to explore the relations of falsely labeling patients 'not at risk' for malnutrition and the screening time difference (STD) between nurse and dietician with the length of stay (LoS). METHODS: Included are all patients hospitalized in a tertiary care center between January 2017 and December 2019 and screened for malnutrition by both a nurse and a dietician. The inter-rater reliability is evaluated using Cohen's Kappa. The relation between STD and the patient classification (PCET) is assessed by a linear mixed effect model. The relation between the LoS and PCET is evaluated with the Kaplan-Meier method and multivariable Cox regression including STD with pathology group and severity of illness as random effect. RESULTS: 9085 patients are assessed by nurse and dietician. 72% of all assessments agree (Kappa = 0.44 [0.43-0.46]). The dietician is involved later for patients falsely labeled 'not at risk' (1.06 [0.92-1.20] days; p < 0.001). Compared to patients where the dietician is involved within 3 days, the LoS is 7.37 days (Hazard Ratio (HR): 0.51 [0.43-0.61]) longer for patients falsely labeled 'not at risk', while only 3.51 days (HR: 0.72 [0.64-0.80]) longer for patients correctly labeled 'at risk'. CONCLUSIONS: Agreement of screening for malnutrition between nurses and dieticians is weak. Avoiding falsely labeling patients 'not at risk' should be a main concern upon patient admission as later involvement of dieticians is correlated with a longer LoS.
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Desnutrição , Avaliação Nutricional , Humanos , Hospitalização , Tempo de Internação , Desnutrição/diagnóstico , Estado Nutricional , Nutricionistas , Reprodutibilidade dos Testes , Centros de Atenção TerciáriaRESUMO
INTRODUCTION: In patients with cystic fibrosis (CF), it is still unclear to which extent glucose abnormalities - preceding the diagnosis of cystic fibrosis related diabetes (CFRD) - are associated with pulmonary and nutritional outcome parameters. This study related circadian glycemic patterns to clinical outcomes in a group of CF patients not previously diagnosed with diabetes. METHODS: Continuous glucose monitoring (CGM) readings (7 days) of 47 CF patients (26 children, 21 adults) with an impaired oral glucose tolerance test (OGTT) (n = 25) and/or increased Hb1Ac (> 5.5%) were analyzed. Biometric, pulmonary function and clinical parameters were retrospectively collected over a period of 1 year before (T-1) and 1 year after (T + 1) CGM (T0). RESULTS: 96% (45/47) of CGM readings showed glucose values > 140 mg/dL ≥ 4.5% of the time and at least one ≥ 200 mg/dL. In the pediatric cohort, no significant associations were found between CGM parameters and pulmonary and nutritional outcome parameters. In the adult cohort, an area under the curve (AUC) > 140 mg/dL and%-time > 140 mg/dL during the night were associated with a lower forced expiratory volume in 1 s (FEV1)% predicted (pp) at time of evaluation but not with change in FEV1pp. CONCLUSION: This is the first study reporting the circadian glycemic pattern in children and adults at risk for CFRD. In the adult cohort an association between detection of abnormal glucose exposure and a lower FEV1pp was found. Our results support continued screening for glucose intolerance in patients with CF.
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Fibrose Cística , Diabetes Mellitus , Intolerância à Glucose , Humanos , Adulto , Criança , Glicemia , Índice Glicêmico , Automonitorização da Glicemia/métodos , Estudos Retrospectivos , Diabetes Mellitus/diagnóstico , Intolerância à Glucose/diagnóstico , GlucoseRESUMO
Advanced glycation end products (AGEs) are important in pathophysiology of type 2 diabetes mellitus (T2DM) and diabetic kidney disease (DKD). Dietary AGEs (dAGEs) contribute to the overall AGE pool in the body. Forty elderly T2DM patients with DKD were randomly allocated to a low-AGE (n = 20) or regular diabetic (n = 20) diet group. A three-day meal questionnaire was used to estimate average quantity of dAGEs. AGE accumulation was measured using skin autofluorescence and urine spectroscopy. sRAGE (soluble receptor AGE) was quantified using ELISA. After 8 weeks, the mean consumption of dAGEs was considerably reduced, both in the low-AGE diet (p = 0.004) and the control (p = 0.019) group. The expected urinary emission peak at 490 nm was shifted to 520 nm in some spectra. dAGEs did not correspond with urine AGE output. An AGE-limited diet for two months did not affect AGE content in skin and urine, or sRAGE concentration in the blood. The role of glycemia is likely to be greater than the impact of dAGE consumption. The unique observation of a fluorescence pattern at 520 nm warrants further examination, since it might point to genetic differences in AGE regulation, which could have clinical consequences, as AGE content depends on its formation and elimination.
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Diabetes Mellitus Tipo 2 , Nefropatias Diabéticas , Idoso , Glicemia , Dieta , Feminino , Produtos Finais de Glicação Avançada , Humanos , Lactente , Masculino , Receptor para Produtos Finais de Glicação AvançadaRESUMO
BACKGROUND & AIMS: Despite its negative impact on patients and health care expenditures, malnutrition remains an under-recognized problem in hospitals. The objectives were thus: 1) to study the prevalence of malnutrition risk, protein-calorie malnutrition and cachexia in a Belgian tertiary care hospital, 2) to evaluate the impact thereof on patient outcomes, and 3) to evaluate the impact of optimizing malnutrition screening, diagnosing, registration and coding on hospital reimbursement. METHODS: Data was included from all multi-day admissions between January 1, 2017 and December 31, 2019. The NRS2002 was used as screening tool. Patient outcomes were modeled using (generalized) linear mixed models, with pathology and severity of illness as random effects. The financial impact of the screening, diagnosing and registration process was evaluated comparing net revenues related to a malnutrition diagnosis in the year before (2017) and the year after (2019) the optimization process. RESULTS: 55,345 patients were evaluated for malnutrition risk at admission of whom 23.6% are considered malnourished or at risk for malnutrition, 0.6% have cachexia and 4.6% protein-calorie malnutrition. Overall length of stay is 2.2 days (p < 0.001) longer in the at-risk population, and 6.2 and 5.0 days longer in patients with cachexia and protein-calorie malnutrition as compared to patients not at risk. Odds ratio for in-hospital mortality is 2.9 (p < 0.001) for the at-risk patients and 3.0 (p < 0.001) for patients with cachexia. Optimization of dietetic workflow and registration, specifying malnutrition severity and facilitating malnutrition coding can increase hospital reimbursement, with approximately 0.4% of all justified beds. CONCLUSIONS: Malnutrition still affects both patients and health care finances. Patients at risk for, or having malnutrition at admission have worse outcomes than those without. Importantly, hospital reimbursement for these patients can effectively be increased by implementing an automated nutritional screening and diagnosing protocol with optimized dietetic registration and enhanced nutritional coding.
Assuntos
Desnutrição , Avaliação Nutricional , Hospitais de Ensino , Humanos , Desnutrição/diagnóstico , Desnutrição/epidemiologia , Estado Nutricional , Centros de Atenção TerciáriaRESUMO
Patients with cystic fibrosis (CF) have a two- to four-fold higher sodium chloride sweat content compared with healthy controls. This high sweat salt loss increases the risk for electrolyte disturbances, associated with subacute or chronic complications. Sodium status therefore needs to be adequately monitored and salt intake adjusted to individual needs. The lack of current evidence to formulate specific recommendations and assess sodium status is reflected in a variability of recommendations in international guidelines. This narrative review presents an overview of the current evidence. Infants with CF in particular are at risk for severe sodium deficiency, potentially leading to metabolic alkalosis due to low intake and high sweat losses. More research on the assessment of sodium status and efficacy of sodium chloride supplements in the population of patients with CF, especially given the changing era of CF transmembrane conductance regulator modulatory treatment, is warranted.
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Fibrose Cística/sangue , Suplementos Nutricionais/estatística & dados numéricos , Estado Nutricional , Cloreto de Sódio/administração & dosagem , Sódio/sangue , Adulto , Criança , Pré-Escolar , Fibrose Cística/complicações , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/sangue , Eletrólitos/sangue , Feminino , Humanos , Hiponatremia/etiologia , Lactente , Masculino , Sódio/deficiência , Cloreto de Sódio/análise , Suor/química , Equilíbrio HidroeletrolíticoRESUMO
BACKGROUND: Nutritional therapy is one of the cornerstones in cystic fibrosis (CF) therapy. There is a strong association between nutritional status and pulmonary function and thus longevity. Therefore nutritional therapy should be continuously adapted to preserve or improve the nutritional status. This narrative review was written to reconsider nutritional therapy in CF based on the latest evidence available since the publication of the ESPEN - ESPGHAN - ECFS guidelines on nutrition care for infants, children and adults with CF. METHODS: A literature search in Pubmed, Scopus and Web of Science was conducted to identify new research focusing on the use of growth charts, body composition, protein intake and pancreatic enzyme therapy (PERT) in CF between June 2014 and June 2017. RESULTS: The search strategy resulted in a total of 1810 hits across the databases. After reviewing title and abstract only 17 studies were included of which 2 animal studies. The use of growth charts was discussed in 3 studies, body composition in 6, protein intake and digestion in 4 and PERT in 4. CONCLUSION: According to the current guidelines and the available evidence of the discussed topics, it is important that the nutritional therapy in CF is redefined according to age, pancreatic function and disease stage. Macronutrients balances are of importance and change over lifetime. As a consequence an accurate PERT intake is required and thus further research on timing and dosage is necessary. To improve the nutritional assessment a proper use of the growth charts and a consensus on body composition measurements, references and thresholds is advised.
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Fibrose Cística/dietoterapia , Terapia Nutricional , Estado Nutricional , Composição Corporal , Índice de Massa Corporal , Pré-Escolar , Bases de Dados Factuais , Humanos , Lactente , Avaliação Nutricional , Apoio NutricionalRESUMO
Raw bovine milk is highly nutritious as well as pH-neutral, providing the ideal conditions for microbial growth. The microbiota of raw milk is diverse and originates from several sources of contamination including the external udder surface, milking equipment, air, water, feed, grass, feces, and soil. Many bacterial and fungal species can be found in raw milk. The autochthonous microbiota of raw milk immediately after milking generally comprises lactic acid bacteria such as Lactococcus, Lactobacillus, Streptococcus, and Leuconostoc species, which are technologically important for the dairy industry, although they do occasionally cause spoilage of dairy products. Differences in milking practices and storage conditions on each continent, country and region result in variable microbial population structures in raw milk. Raw milk is usually stored at cold temperatures, e.g., about 4°C before processing to reduce the growth of most bacteria. However, psychrotrophic bacteria can proliferate and contribute to spoilage of ultra-high temperature (UHT) treated and sterilized milk and other dairy products with a long shelf life due to their ability to produce extracellular heat resistant enzymes such as peptidases and lipases. Worldwide, species of Pseudomonas, with the ability to produce these spoilage enzymes, are the most common contaminants isolated from cold raw milk although other genera such as Serratia are also reported as important milk spoilers, while for others more research is needed on the heat resistance of the spoilage enzymes produced. The residual activity of extracellular enzymes after high heat treatment may lead to technological problems (off flavors, physico-chemical instability) during the shelf life of milk and dairy products. This review covers the contamination patterns of cold raw milk in several parts of the world, the growth potential of psychrotrophic bacteria, their ability to produce extracellular heat-resistant enzymes and the consequences for dairy products with a long shelf life. This problem is of increasing importance because of the large worldwide trade in fluid milk and milk powder.
RESUMO
Two Pseudomonas strains, identified as closely related to Pseudomonas tolaasii, were isolated from milk of a farm with frequent false-positive Delvotest results for screening putative antibiotic residues in raw milk executed as part of the regulatory quality programme. Growth at 5 to 7°C of these isolates in milk resulted in high lipolysis and the production of bacterial inhibitors. The two main bacterial inhibitors have a molecular weight of 1168.7 and 1140.7 Da respectively, are heat-tolerant and inhibit Geobacillus stearothermophilus var. calidolactis, the test strain of most of the commercially available microbiological inhibitor tests for screening of antibiotic residues in milk. Furthermore, these bacterial inhibitors show antimicrobial activity against Staphylococcus aureus, Bacillus cereus and B. subtilis and also interfere negatively with yoghurt production. Following their isolation and purification with RP-HPLC, the inhibitors were identified by NMR analysis as cyclic lipodepsipeptides of the viscosin group. Our findings bring to light a new challenge for quality control in the dairy industry. By prolonging the refrigerated storage of raw milk, the keeping quality of milk is influenced by growth and metabolic activities of psychrotrophic bacteria such as pseudomonads. Besides an increased risk of possible spoilage of long shelf-life milk, the production at low temperature of natural bacterial inhibitors may also result in false-positive results for antibiotic residue screening tests based on microbial inhibitor assays thus leading to undue production loss.
Assuntos
Resíduos de Drogas/análise , Leite/microbiologia , Peptídeos Cíclicos/farmacologia , Pseudomonas/metabolismo , Animais , Cromatografia Líquida , Contagem de Colônia Microbiana , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/isolamento & purificaçãoRESUMO
OBJECTIVE: To evaluate parental stress after a false-positive result at the time of the cystic fibrosis (CF) newborn screening (NBS), attributable to heterozygotism or persistent hypertrypsinemia. STUDY DESIGN: A prospective study was conducted in 86 French families at 3, 12, and 24 months after NBS. A psychologist conducted interviews with a questionnaire, the Perceived Stress Scale, and the Vulnerable Child Scale. RESULTS: Overall, 96.5% of parents said they had been anxious at the time of the sweat test. However, 86% felt entirely reassured 3 months after the test. The mean Perceived Stress Scale score did not differ from that observed in the French population. Mean Vulnerable Child Scale scores were high, associated with a low Parental Perception of Child Vulnerability. These results did not differ significantly at 1 and 2 years. In total, 86% to 100% of families no longer worried about CF. All parents stated that they would have the test performed again for another child. CONCLUSIONS: CF NBS can lead to false-positive results, causing parental anxiety, which quickly decreases after a sweat test performed soon after the phone call.
Assuntos
Fibrose Cística/diagnóstico , Triagem Neonatal/psicologia , Pais/psicologia , Ansiedade/etiologia , Fibrose Cística/psicologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Reações Falso-Positivas , Feminino , Heterozigoto , Humanos , Recém-Nascido , Masculino , Mutação , Suor/química , Tripsina/sangueRESUMO
Pseudomonas fragi, Pseudomonas lundensis and members of the Pseudomonas fluorescens group may spoil Ultra High Temperature (UHT) treated milk and dairy products, due to the production of heat-stable proteases in the cold chain of raw milk. Since the aprX gene codes for a heat-resistant protease in P. fluorescens, the presence of this gene has also been investigated in other members of the genus. For this purpose an aprX-screening PCR test has been developed. Twenty-nine representatives of important milk Pseudomonas species and thirty-five reference strains were screened. In 42 out of 55 investigated Pseudomonas strains, the aprX gene was detected, which proves the potential of the aprX-PCR test as a screening tool for potentially proteolytic Pseudomonas strains in milk samples. An extensive study of the obtained aprX-sequences on the DNA and the amino acid level, however, revealed a large heterogeneity within the investigated milk isolates. Although this heterogeneity sets limitations to a general detection method for all proteolytic Pseudomonas strains in milk, it offers a great potential for the development of a multiplex PCR screening test targeting individual aprX-genes. Furthermore, our data illustrated the potential use of the aprX gene as a taxonomic marker, which may help in resolving the current taxonomic deadlock in the P. fluorescens group.
Assuntos
Proteínas de Bactérias/genética , Microbiologia de Alimentos , Variação Genética , Temperatura Alta , Leite/microbiologia , Pseudomonas/genética , Serina Endopeptidases/genética , Adaptação Fisiológica/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Temperatura Baixa , DNA Bacteriano , Genes Bacterianos , Humanos , Filogenia , Reação em Cadeia da Polimerase , Pseudomonas/isolamento & purificação , RefrigeraçãoRESUMO
Psychrotolerant bacteria and their heat-resistant proteases play a major role in the spoilage of UHT-processed dairy products. Summer and winter raw milk samples were screened for the presence of such bacteria. One hundred and three proteolytic psychrotolerant bacteria were isolated, characterized by API tests, rep-PCR fingerprint analysis and evaluated for heat-resistant protease production. Twenty-nine strains (representing 79% of the complete collection) were further identified by 16S rRNA gene sequencing, rpoB gene sequencing and DNA-DNA hybridizations. A seasonal inter- and intra-species influence on milk spoilage capacity (e.g. growth rate and/or protease production) was demonstrated. Moreover, this polyphasic approach led to the identification of Pseudomonas fragi and Pseudomonas lundensis (representing 53% of all isolates) as predominant producers of heat-resistant proteases in raw milk. The role of Pseudomonas fluorescens, historically reported as important milk spoiler, could not unequivocally be established. The use of more reliable identification techniques and further revision of the taxonomy of P. fluorescens will probably result in a different perspective on its role in the milk spoilage issue.
Assuntos
Proteínas de Bactérias/metabolismo , Leite/microbiologia , Peptídeo Hidrolases/metabolismo , Pseudomonas/isolamento & purificação , Pseudomonas/metabolismo , Animais , Bélgica , Análise por Conglomerados , Impressões Digitais de DNA , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , RNA Polimerases Dirigidas por DNA , Genes de RNAr , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Filogenia , Pseudomonas/classificação , Pseudomonas/genética , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Estações do Ano , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
OBJECTIVES: The aims of this study were (1) to collect data on the stability of antibiotics in portable pumps for the treatment of bronchial superinfection in patients with cystic fibrosis and (2) to provide guidelines for prescribers. METHODS: The stability over 72 hours, in portable pumps stored at 35 degrees C, of piperacillin plus tazobactam, ticarcillin plus clavulanic acid, cefsulodin, cefepime, and aztreonam was checked at 3 different concentrations. Stability was assessed through visual examination, pH measurements, and direct measurements of drug concentrations by using high-performance liquid chromatography. All parameters were measured at time 0, time 0 plus 24 hours, and time 0 plus 72 hours. RESULTS: Degradation rates for penicillin plus beta-lactamase inhibitor combinations remained <10% at time 0 plus 24 hours for all drugs, but the rate for piperacillin reached 12% for the highest concentration tested. The cephalosporins cefepime and cefsulodin had significant respective degradation rates of 18% and 28% at time 0 plus 24 hours and 60% and 68.5% at time 0 plus 72 hours, which were linked to the storage temperature. Aztreonam seemed to be stable over 72 hours. CONCLUSIONS: This work provides data on drug stability that were lacking, allowing recommendations for physicians to optimize the safety and efficacy of antibiotic treatment of patients with cystic fibrosis. Piperacillin plus tazobactam and ticarcillin plus clavulanic acid infusions must be limited to 24 hours, and patients receiving cefepime or cefsulodin must wear a cold pack close to the ambulatory drug-delivery device during the infusion.
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Antibacterianos , Broncopatias/tratamento farmacológico , Broncopatias/etiologia , Broncopatias/microbiologia , Criança , Fibrose Cística/complicações , Prescrições de Medicamentos , Estabilidade de Medicamentos , Guias como Assunto , Humanos , Bombas de Infusão , Superinfecção/tratamento farmacológico , Superinfecção/etiologiaRESUMO
Cholesterol-sphingolipid microdomains, or lipid rafts, are major regulators of molecular interactions in membrane organization. Because lipid rafts can move laterally and cluster into larger patches, they have been proposed to play a role in the redistribution of specific molecules to specialized cellular structures. Rafts have been shown to favor formation and maintenance of synaptic receptor clusters in neurons of the central nervous system. However, little is known about their role in formation of the neuromuscular junction (NMJ). To determine whether lipid rafts are involved in acetylcholine receptor (AChR) cluster formation and stabilization in myogenic cells, two standard tools were employed: (1) Perturbation of lipid rafts by drugs that deplete membrane cholesterol was carried out to verify that cholesterol is required for AChR clustering in agrin-treated C2C12 myotubes; and (2) detergent resistance of lipid-ordered domains was also used to demonstrate that AChRs, as well as key components of the postsynaptic membrane of the NMJ, are associated with rafts.
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Microdomínios da Membrana/fisiologia , Receptores Colinérgicos/fisiologia , Animais , Encéfalo/fisiologia , Linhagem Celular , Camundongos , Músculo Esquelético/fisiologia , RatosRESUMO
INTRODUCTION: Given many data about the stability of antibiotics in portable pump (elastomer) are lacking, this study was designed to make a point about available data and to evaluate the stability of antibiotics when exposed to temperature within 35 degrees C (average temperature measured in real conditions of use). MATERIAL AND METHODS: First, to collect information about the stability of antibiotics in portable pump and to confront them with the local antibiotics protocols dedicated to the treatment of bronchial superinfection in patients with cystic fibrosis; second, to evaluate the stability of piperacillin associated with tazobactam at 35 degrees C. RESULTS: While measured concentrations in tazobactam did not show significant variation during the study, piperacillin measurements showed a major reduction of concentration (up to 33%), both time and concentration related to. DISCUSSION: Such information must be pointed out to prescribers and patients to ensure a cold accumulator is placed in the pump carrying-bag and to limit the duration of infusion to 24 h with a single pump. CONCLUSION: This experimental program will keep on going with the stability study of both ticarcillin and cefsulodin in portable pump.
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Antibacterianos/uso terapêutico , Broncopatias/tratamento farmacológico , Infecções Respiratórias/tratamento farmacológico , Superinfecção/tratamento farmacológico , Antibacterianos/administração & dosagem , HumanosRESUMO
The chlorhexidine method was compared to the N-acetyl-l-cysteine-NaOH-oxalic acid decontamination method currently recommended for the recovery of nontuberculous mycobacteria (NTM) from patients with cystic fibrosis. Sputum samples (n = 827) treated with chlorhexidine yielded twice as many NTM-positive cultures as those treated by the reference method (54 [6.50%] versus 27 [3.25%]; P < 0.0001) despite a higher contamination rate (20% versus 14.2%; P = 0.0017).
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Clorexidina/farmacologia , Fibrose Cística/microbiologia , Descontaminação/métodos , Mycobacterium/isolamento & purificação , Escarro/microbiologia , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Humanos , Infecções por Mycobacterium/microbiologia , Ácido Oxálico/farmacologia , Hidróxido de Sódio/farmacologiaRESUMO
UNLABELLED: Treatment of Bronchial Superinfections: Data Related to Stability of Antibiotics in Portable Pumps. INTRODUCTION: Given many data about the stability of antibiotics in portable pump (elastomer) are lacking, this study was designed to make a point about available data and to evaluate the stability of antibiotics when exposed to temperature within 35°C (average temperature measured in real conditions of use). MATERIAL AND METHODS: First, to collect information about the stability of antibiotics in portable pump and to confront them with the local antibiotics protocols dedicated to the treatment of bronchial superinfection in patients with cystic fibrosis; second, to evaluate the stability of piperacillin associated with tazobactam at 35°C. RESULTS: While measured concentrations in tazobactam did not show significant variation during the study, piperacillin measurements showed a major reduction of concentration (up to 33%), both time and concentration related to. DISCUSSION: Such information must be pointed out to prescribers and patients to ensure a cold accumulator is placed in the pump can'ying-bag and to limit the duration of infusion to 24h with a single pump. CONCLUSION: This experimental program will keep on going with the stability study of both ticarcillin and cefsulodin in portable pump.
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Emerging data are sheding light on the critical task for synapses to locally control the production of neurotransmitter receptors ultimately leading to receptor accumulation and modulation at postsynaptic sites. By analogy with the epithelial-cell paradigm, the postsynaptic compartment may be regarded as a polarized domain favoring the selective recruitment and retention of newly delivered receptors at synaptic sites. Targeted delivery of receptors to synaptic sites is facilitated by a local organization of the exocytic pathway, likely resulting from spatial cues triggered by the nerve. This review focuses on the various mechanisms responsible for regulation of receptor assembly and trafficking. A particular emphasis is given to the role of synaptic anchoring and scaffolding proteins in the sorting and routing of their receptor companion along the exocytic pathway. Other cellular components such as lipidic microdomains, the docking and fusion machinery, and the cytoskeleton also contribute to the dynamics of receptor trafficking at the synapse.
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Proteínas do Tecido Nervoso/metabolismo , Transporte Proteico , Receptores de Neurotransmissores/metabolismo , Sinapses/metabolismo , Animais , Linhagem Celular , Polaridade Celular , Citoesqueleto/fisiologia , Cães , Exocitose/fisiologia , Humanos , Fusão de Membrana , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Modelos Neurológicos , Proteínas Musculares/metabolismo , Junção Neuromuscular/metabolismo , Mapeamento de Interação de Proteínas , Sinais Direcionadores de Proteínas/fisiologia , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismoRESUMO
The 43 kDa receptor-associated protein rapsyn is a myristoylated peripheral protein that plays a central role in nicotinic acetylcholine receptor (AChR) clustering at the neuromuscular junction. In a previous study, we demonstrated that rapsyn is specifically cotransported with AChR via post-Golgi vesicles targeted to the innervated surface of the Torpedo electrocyte (Marchand et al., 2000). In the present study, to further elucidate the mechanisms for sorting and assembly of postsynaptic proteins, we analyzed the dynamics of the intracellular trafficking of fluorescently labeled rapsyn in the transient-expressing COS-7 cell system. Our approach was based on fluorescence, time-lapse imaging, and immunoelectron microscopies, as well as biochemical analyses. We report that newly synthesized rapsyn associates with the trans-Golgi network compartment and traffics via vesiculotubular organelles toward the cell surface of COS-7 cells. The targeting of rapsyn organelles appeared to be mediated by a microtubule-dependent transport. Using cotransfection experiments of rapsyn and AChR, we observed that these two molecules codistribute within distal exocytic routes and at the plasma membrane. Triton X-100 extraction on ice and flotation gradient centrifugation demonstrated that rapsyn and AChR are recovered in low-density fractions enriched in two rafts markers: caveolin-1 and flotillin-1. We propose that sorting and targeting of these two companion molecules are mediated by association with cholesterol-sphingolipid-enriched raft microdomains. Collectively, these data highlight rapsyn as an itinerant vesicular protein that may play a dynamic role in the sorting and targeting of its companion receptor to the postsynaptic membrane. These data also raise the interesting hypothesis of the participation of the raft machinery in the targeting of signaling molecules to synaptic sites.