RESUMO
Lactate is shuttled between and inside cells, playing metabolic and signaling roles in healthy tissues. Lactate is also a harbinger of altered metabolism and participates in the pathogenesis of inflammation, hypoxia/ischemia, neurodegeneration and cancer. Many tumor cells show high rates of lactate production in the presence of oxygen, a phenomenon known as the Warburg effect, which has diagnostic and possibly therapeutic implications. In this article we introduce Laconic, a genetically-encoded Forster Resonance Energy Transfer (FRET)-based lactate sensor designed on the bacterial transcription factor LldR. Laconic quantified lactate from 1 µM to 10 mM and was not affected by glucose, pyruvate, acetate, betahydroxybutyrate, glutamate, citrate, α-ketoglutarate, succinate, malate or oxalacetate at concentrations found in mammalian cytosol. Expressed in astrocytes, HEK cells and T98G glioma cells, the sensor allowed dynamic estimation of lactate levels in single cells. Used in combination with a blocker of the monocarboxylate transporter MCT, the sensor was capable of discriminating whether a cell is a net lactate producer or a net lactate consumer. Application of the MCT-block protocol showed that the basal rate of lactate production is 3-5 fold higher in T98G glioma cells than in normal astrocytes. In contrast, the rate of lactate accumulation in response to mitochondrial inhibition with sodium azide was 10 times lower in glioma than in astrocytes, consistent with defective tumor metabolism. A ratio between the rate of lactate production and the rate of azide-induced lactate accumulation, which can be estimated reversibly and in single cells, was identified as a highly sensitive parameter of the Warburg effect, with values of 4.1 ± 0.5 for T98G glioma cells and 0.07 ± 0.007 for astrocytes. In summary, this article describes a genetically-encoded sensor for lactate and its use to measure lactate concentration, lactate flux, and the Warburg effect in single mammalian cells.
Assuntos
Técnicas Biossensoriais/métodos , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli/genética , Transferência Ressonante de Energia de Fluorescência , Glioma/patologia , Ácido Láctico/metabolismo , Análise de Célula Única/métodos , Fatores de Transcrição/genética , Animais , Transporte Biológico , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Células HEK293 , Humanos , Ácido Láctico/biossíntese , Masculino , Camundongos , Modelos Moleculares , Conformação Proteica , Análise Espaço-Temporal , Fatores de Transcrição/químicaRESUMO
The glycolytic rate is sensitive to physiological activity, hormones, stress, aging, and malignant transformation. Standard techniques to measure the glycolytic rate are based on radioactive isotopes, are not able to resolve single cells and have poor temporal resolution, limitations that hamper the study of energy metabolism in the brain and other organs. A new method is described in this article, which makes use of a recently developed FRET glucose nanosensor to measure the rate of glycolysis in single cells with high temporal resolution. Used in cultured astrocytes, the method showed for the first time that glycolysis can be activated within seconds by a combination of glutamate and K(+), supporting a role for astrocytes in neurometabolic and neurovascular coupling in the brain. It was also possible to make a direct comparison of metabolism in neurons and astrocytes lying in close proximity, paving the way to a high-resolution characterization of brain energy metabolism. Single-cell glycolytic rates were also measured in fibroblasts, adipocytes, myoblasts, and tumor cells, showing higher rates for undifferentiated cells and significant metabolic heterogeneity within cell types. This method should facilitate the investigation of tissue metabolism at the single-cell level and is readily adaptable for high-throughput analysis.
RESUMO
Biotinidase deficiency is an inherited metabolic disorder characterized by neurological and cutaneous symptoms. Fortunately, it can be treated and the symptoms prevented by oral administration of the vitamin biotin. Using dried blood-soaked filter paper cards, biotinidase activity was determined in the sera of 225,136 newborns in Brazil. Mutation analysis performed on DNA from 21 babies with low serum biotinidase activity confirmed that 3 had profound biotinidase deficiency (less than 10% of mean normal sera biotinidase activity), 10 had partial biotinidase deficiency (10 to 30% of mean normal serum activity), 1 was homozygous for partial biotinidase deficiency, 4 were heterozygous for either profound or partial deficiency, and 3 were normal. Variability in serum enzyme activities and discrepancies with mutation analyses were probably due to inappropriate handling and storage of samples sent to the laboratory. Obtaining an appropriate control serum at the same time as that of the suspected child will undoubtedly decrease the false-positive rate (0.09%). Mutation analysis can be used to confirm the genotype of these children. The estimated incidence of biotinidase deficiency in Brazil is about 1 in 9,000, higher than in most other countries. Screening and treatment of biotinidase deficiency are effective and warranted. These results strongly suggest that biotinidase deficiency should be included in the newborn mass screening program of Brazil.
Assuntos
Deficiência de Biotinidase/diagnóstico , Mutação/genética , Triagem Neonatal , Deficiência de Biotinidase/epidemiologia , Deficiência de Biotinidase/genética , Brasil/epidemiologia , Feminino , Genótipo , Humanos , Incidência , Recém-Nascido , MasculinoRESUMO
Biotinidase deficiency is an inherited metabolic disorder characterized by neurological and cutaneous symptoms. Fortunately, it can be treated and the symptoms prevented by oral administration of the vitamin biotin. Using dried blood-soaked filter paper cards, biotinidase activity was determined in the sera of 225,136 newborns in Brazil. Mutation analysis performed on DNA from 21 babies with low serum biotinidase activity confirmed that 3 had profound biotinidase deficiency (less than 10 percent of mean normal sera biotinidase activity), 10 had partial biotinidase deficiency (10 to 30 percent of mean normal serum activity), 1 was homozygous for partial biotinidase deficiency, 4 were heterozygous for either profound or partial deficiency, and 3 were normal. Variability in serum enzyme activities and discrepancies with mutation analyses were probably due to inappropriate handling and storage of samples sent to the laboratory. Obtaining an appropriate control serum at the same time as that of the suspected child will undoubtedly decrease the false-positive rate (0.09 percent). Mutation analysis can be used to confirm the genotype of these children. The estimated incidence of biotinidase deficiency in Brazil is about 1 in 9,000, higher than in most other countries. Screening and treatment of biotinidase deficiency are effective and warranted. These results strongly suggest that biotinidase deficiency should be included in the newborn mass screening program of Brazil.
Assuntos
Humanos , Masculino , Feminino , Recém-Nascido , Deficiência de Biotinidase/diagnóstico , Mutação , Triagem Neonatal , Brasil , Deficiência de Biotinidase/epidemiologia , Deficiência de Biotinidase/genética , Genótipo , IncidênciaRESUMO
BACKGROUND: Gram-negative bacilli are not infrequently encountered as etiologic organisms of pneumonia in children in warm-climate countries. OBJECTIVES: To investigate the nasopharyngeal carriage rate and antimicrobial susceptibility patterns of gram-negative bacilli colonizing children with community-acquired pneumonia in Fortaleza, Brazil. METHODS: A single nasopharyngeal specimen was collected from children 2 months to 5 years of age presenting at one of the three children's hospitals in Fortaleza and fulfilling the World Health Organization criteria for pneumonia. Randomly recruited healthy children from public daycare centers and immunization clinics served as controls. RESULTS: The study included 912 children, 482 (53%) with pneumonia and 430 (47%) controls. Aerobic gram-negative bacilli were seen in 79 (16%) of the 482 children with pneumonia and 51 (12%) of the 430 healthy controls. Nonfermentative gram-negative bacilli were seen in 85 (18%) of children with pneumonia and 54 (13%) of healthy controls. Neither gender, nutritional status, season, previous hospital admission nor antibiotic use was associated with carriage with gram-negative bacilli. However, pneumonia was associated with increased carriage, whereas concomitant colonization with Streptococcus pneumoniae or Haemophilus influenzae was associated with decreased carriage with gram-negative bacilli. Only 36% of all Escherichia species and 76% of all Klebsiella isolates were susceptible to cotrimoxazole; 90% of all Acinetobacter species were susceptible to gentamicin. CONCLUSION: Nasopharyngeal carriage with gram-negative bacilli, in particular with Acinetobacter species, is common and associated with a clinical diagnosis of community-acquired pneumonia in children in Fortaleza, Brazil.
Assuntos
Infecções Comunitárias Adquiridas/microbiologia , Bactérias Gram-Negativas/isolamento & purificação , Nasofaringe/microbiologia , Pneumonia Bacteriana/microbiologia , Brasil , Pré-Escolar , Infecções Comunitárias Adquiridas/tratamento farmacológico , Farmacorresistência Bacteriana , Feminino , Humanos , Lactente , Masculino , Pneumonia Bacteriana/tratamento farmacológico , Combinação Trimetoprima e Sulfametoxazol/uso terapêuticoRESUMO
The present study describes the carriage patterns and genetic variability of Moraxella catarrhalis strains isolated from children living in different countries. Moraxella catarrhalis is genetically heterogeneous, but little is known about its geographic distribution and phenotypic and genetic diversity in warm-climate countries. A collection of 99 isolates from 30 Brazilian, 19 Angolan and 50 Dutch healthy children, all less than 5 years of age, was investigated for phenotypic and genotypic relatedness. The isolates from the three countries were similar where biochemical reactivity was concerned: 89 strains were beta-lactamase-producing and 87 were complement-resistant as determined by phenotype. There was no geographical difference in the prevalence of beta-lactamase-producing isolates, but the carriage rate of complement-resistant strains was significantly higher in Dutch than in Angolan children (P=0.004). Complement resistance of 66 randomly selected strains was genetically confirmed in a Southern hybridization assay by a novel DNA probe that is specific for complement-resistant strains and that demonstrated a sensitivity of 97% and a specificity of 100%. PCR amplification based on the probe sequence had a sensitivity of 98% and a specificity of 57% when compared to the outcome of a conventional culture spot test. PCR restriction fragment length polymorphism analysis of the MU 46 locus and pulsed-field gel electrophoresis of SpeI DNA macrorestriction fragments revealed genetic heterogeneity of strains from within and between the three countries, and no geographical clustering could be established. In conclusion, similar phenotypic characteristics but genotypic heterogeneity was found among Moraxella catarrhalis strains colonizing children in three different continents.
Assuntos
Moraxella catarrhalis/genética , Nasofaringite/microbiologia , Nasofaringe/microbiologia , Infecções por Neisseriaceae/microbiologia , Angola , Brasil , Pré-Escolar , DNA Bacteriano/análise , Eletroforese em Gel de Campo Pulsado , Variação Genética , Genótipo , Humanos , Lactente , Moraxella catarrhalis/classificação , Países Baixos , Fenótipo , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de RestriçãoRESUMO
To study clonal diversity of penicillin-resistant Streptococcus pneumoniae, 161 randomly selected isolates with reduced susceptibility to penicillin, collected from the nasopharynx of children under 5 years of age with community-acquired pneumonia and healthy controls from public day-care and immunization centres in Fortaleza, Brazil, were characterized by microbiological and serological techniques and automated ribotyping. Also included were 44 randomly selected penicillin-susceptible strains and three international reference strains. With automated ribotyping 75 ribopatterns were observed: 50 ribogroups were unique and 25 ribogroups were represented by two or more isolates. Genetic diversity was extensive but some degree of genetic homogeneity was found in strains from children with pneumonia, strains from children in day-care centres, isolates with reduced susceptibility to penicillin and isolates expressing 'paediatric' serogroups. Fourteen (56%) clusters contained both isolates with reduced penicillin susceptibility and penicillin-susceptible isolates, suggesting emergence of penicillin resistance. In general, there was a good correlation between ribogroups and serogroups, but 12 (48%) clusters contained isolates with alternative serogroups. Isolates with such alternative serogroups were more often encountered in penicillin-susceptible strains (41%) than in strains with reduced susceptibility to penicillin (7%). Thirty-eight (19%) isolates (including seven penicillin-susceptible strains) showed ribotypes indistinguishable from those of two international epidemic clones of S. pneumoniae: ribogroup 54-S-1 (15 isolates) with a ribopattern characteristic of the 23F multiresistant 'Spanish/USA' clone and ribogroup 74-S-3 (23 isolates) with a pattern similar to that of the 6B multiresistant 'Spanish' clone.
Assuntos
Resistência às Penicilinas/genética , Pneumonia Pneumocócica/microbiologia , Streptococcus pneumoniae/genética , Análise de Variância , Brasil/epidemiologia , Distribuição de Qui-Quadrado , Creches/estatística & dados numéricos , Pré-Escolar , Infecções Comunitárias Adquiridas/epidemiologia , Infecções Comunitárias Adquiridas/microbiologia , Humanos , Lactente , Nasofaringe/microbiologia , Pneumonia Pneumocócica/epidemiologia , Sorotipagem , Streptococcus pneumoniae/efeitos dos fármacosRESUMO
Community-acquired pneumonia from enteric gram-negative bacilli is more common in developing than in industrialized countries. We investigated the nasopharyngeal flora in healthy children from Angola, Brazil and The Netherlands to see whether enteric gram-negative bacilli are more often part of the commensal flora in developing countries. Nasopharyngeal specimens were collected from children aged between 4 months and 5 years in day-care centres and immunization clinics. Children who had received antibiotics or were malnourished were excluded. Brazilian and Angolan children had a higher number of household members than Dutch children (5.5 and 7 vs 3.9 mean number of household members, respectively) (p < 0.0001). Enteric and non-fermentative gram-negative bacilli were much more prevalent in Brazilian (50%) and Angolan (57%) children than in Dutch (4%) children (p < 0.0001). By univariate analysis, carriage of enteric gram-negative bacilli was associated with the number of household members (r = 0.26; p < 0.001). The high carriage rate of enteric gram-negative bacilli in children from Angola and Brazil may explain why enteric gram-negative bacilli are a common cause of pneumonia in developing countries.
Assuntos
Países em Desenvolvimento/estatística & dados numéricos , Infecções por Enterobacteriaceae/epidemiologia , Enterobacteriaceae/classificação , Nasofaringe/microbiologia , Angola/epidemiologia , Brasil/epidemiologia , Pré-Escolar , Infecções por Enterobacteriaceae/microbiologia , Características da Família , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Países Baixos/epidemiologiaRESUMO
Children with biotinidase deficiency usually exhibit symptoms at several months to years of age. We describe four children who had symptoms later in childhood or during adolescence; they had motor limb weakness, spastic paresis, and eye problems, such as loss of visual acuity and scotomata, rather than the more characteristic symptoms observed in young untreated children with the disorder. These older children each have different mutations, but they are the same as those of children who have exhibited symptoms at an early age. Biotinidase deficiency should be considered in older children who suddenly experience limb weakness and/or spastic paresis and eye symptoms.