RESUMO
OBJECTIVE: Opening of voltage-gated sodium channels is crucial for neuronal depolarization. Proper channel opening and influx of Na+ through the ion pore, is dependent upon binding of Na+ ion to a specific amino-acid motif (DEKA) within the pore. In this study we used molecular dynamic simulations, an advanced bioinformatic tool, to research the dysfunction caused by pathogenic variants in SCN1a, SCN2a and SCN8a genes. METHOD: Molecular dynamic simulations were performed in six patients: three patients with Dravet syndrome (p.Gly177Ala,p.Ser259Arg and p.Met1267Ile, SCN1a), two patients with early onset drug resistant epilepsy(p.Ala263Val, SCN2a and p.Ile251Arg, SCN8a), and a patient with autism (p.Thr155Ala, SCN2a). After predicting the 3D-structure of mutated proteins by homology modeling, time dependent molecular dynamic simulations were performed, using the Schrödinger algorithm. The opening of the sodium channel, including the detachment of the sodium ion to the DEKA motif and pore diameter were assessed. Results were compared to the existent patch clamp analysis in four patients, and consistency with clinical phenotype was noted. RESULTS: The Na+ ion remained attached to DEKA filter longer when compared to wild type in the p.Gly177Ala, p.Ser259Arg,SCN1a, and p.Thr155Ala, SCN2a variants, consistent with loss-of-function. In contrast, it detached quicker from DEKA than wild type in the p.Ala263Val,SCN2a variant, consistent with gain-of-function. In the p.Met1267Ile,SCN1a variant, detachment from DEKA was quicker, but pore diameter decreased, suggesting partial loss-of-function. In the p.Leu251Arg,SCN8a variant, the pore remained opened longer when compared to wild type, consistent with a gain-of-function. The molecular dynamic simulation results were consistent with the existing patch-clamp analysis studies, as well as the clinical phenotype. SIGNIFICANCE: Molecular dynamic simulation can be useful in predicting pathogenicity of variants and the disease phenotype, and selecting targeted treatment based on channel dysfunction. Further development of these bioinformatic tools may lead to "virtual patch-clamp analysis".
Assuntos
Epilepsias Mioclônicas , Canal de Sódio Disparado por Voltagem NAV1.1 , Epilepsias Mioclônicas/genética , Humanos , Mutação/genética , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Fenótipo , Sódio/metabolismoRESUMO
Here we describe a DNA analog in which the mesyl (methanesulfonyl) phosphoramidate group is substituted for the natural phosphodiester group at each internucleotidic position. The oligomers show significant advantages over the often-used DNA phosphorothioates in RNA-binding affinity, nuclease stability, and specificity of their antisense action, which involves activation of cellular RNase H enzyme for hybridization-directed RNA cleavage. Biological activity of the oligonucleotide analog was demonstrated with respect to pro-oncogenic miR-21. A 22-nt anti-miR-21 mesyl phosphoramidate oligodeoxynucleotide specifically decreased the miR-21 level in melanoma B16 cells, induced apoptosis, reduced proliferation, and impeded migration of tumor cells, showing superiority over isosequential phosphorothioate oligodeoxynucleotide in the specificity of its biological effect. Lower overall toxicity compared with phosphorothioate and more efficient activation of RNase H are the key advantages of mesyl phosphoramidate oligonucleotides, which may represent a promising group of antisense therapeutic agents.
Assuntos
Amidas/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos/metabolismo , Fosfatos/metabolismo , Ácidos Fosfóricos/metabolismo , Animais , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , DNA/metabolismo , Melanoma Experimental , Camundongos , MicroRNAs/metabolismo , RNA/metabolismo , Ribonuclease H/metabolismoRESUMO
The ability of a series of novel modified external guide sequences (EGS oligonucleotides) to induce the hydrolysis of target RNA with bacterial ribonuclease P has been studied; the most efficient modification variants have been selected. We have found patterns of the oligonucleotide sugar-phosphate backbone modi-fications that enhance oligonucleotide stability in the biological environment and do not violate the ability to interact with the enzyme and induce the RNA hydrolysis. It has been shown that analogues of EGS oligonucleotides selectively modified at 2'-position (2'-O-methyl and 2'-fluoro) or at internucleotide phosphates (phosphoryl guanidines) can be used for the addressed cleavage of a model RNA target by bacterial RNase P. The ability of new phosphoryl guanidine analogues of oligodeoxyribonucleotides that are stable in biological media to induce the hydrolysis of target RNA with bacterial ribonuclease P has been shown for the first time. The modified EGS oligonucleotides with an optimal balance between functional activity and stability in biological media can be considered as potential antibacterial agents.
Assuntos
Oligonucleotídeos/química , Clivagem do RNA , RNA/química , Ribonuclease P/química , BactériasRESUMO
Here we make a brief survey of the present state of antibiotic research and use. We also describe a novel antibiotic that contains a basic peptide covalently bound to a morpholino oligonucleotide.
Assuntos
Antibacterianos/uso terapêutico , Infecções Bacterianas/tratamento farmacológico , Pesquisa Biomédica/tendências , Previsões , Antibacterianos/química , Infecções Bacterianas/microbiologia , Pesquisa Biomédica/métodos , Humanos , Viabilidade Microbiana/efeitos dos fármacos , Morfolinos/químicaRESUMO
AIM: Our aim was to determine the frequency and economic impact of anastomotic leakage (AL) at local and national levels in England. METHOD: All patients who underwent AR in Oxford between 2007 and 2009 were evaluated for AL. Hospital Episode Statistics (HES) data were used to determine reoperation rates after elective AR (n = 23 388) in England between 2000 and 2008. Hospital episode remuneration costs were calculated by the local commissioning department and compared with Department of Health (DH) reference index costs. RESULTS: The frequency of AL following anterior resection was 10.9% (31 out of 285) in Oxford. Laparotomy for leakage was performed in 5.6% of cases. The 30-day hospital mortality rate for all ARs was 2.1%, compared with 3.2% after AL. The national relaparotomy rate (within 28 days) and 30-day hospital mortality in English National Health Service (NHS) trusts following AR were 5.9% and 2.9%, respectively. Institutional remunerated tariffs (£6233 (SD ± 965)) were similar to DH reference costs (£6319 (SD ± 1830)) after uncomplicated AR. However, there was a significant (P = 0.008) discrepancy between the remunerated tariff for AL (£9605 (SD ± 6908)) and the actual cost (£17 220 (SD ± 9642)). AL resulted in an additional annual cost of approximately £1.1 million to £3.5 million when extrapolated nationally. CONCLUSION: The estimated economic burden of anastomotic leakage following AR is approximately double that of the remunerated tariff.
Assuntos
Adenocarcinoma/cirurgia , Adenoma/cirurgia , Fístula Anastomótica/economia , Fístula Anastomótica/terapia , Custos Hospitalares/estatística & dados numéricos , Reembolso de Seguro de Saúde/economia , Neoplasias Retais/cirurgia , Medicina Estatal/economia , Idoso , Inglaterra , Enterostomia/economia , Feminino , Mortalidade Hospitalar , Humanos , Masculino , Pessoa de Meia-Idade , Reoperação/economiaRESUMO
AIMS: The hypothesis that surrogate planktonic pathogens (Bacillus cereus and polystyrene microspheres) could be integrated in biofilms and protected from decontamination was tested. METHODS AND RESULTS: Pseudomonas fluorescens biofilms were grown on polyvinyl chloride coupons in annular reactors under low nutrient conditions. After biofilm growth, B. cereus spores and polystyrene microspheres (an abiotic control) were introduced separately. Shear stress at the biofilm surface was varied between 0.15 and 1.5 N m(-2). The amount of surrogate pathogens introduced ranged from approximately 10(5) CFU ml(-1) to 10(10 )spheres ml(-1). The quantity of surrogate pathogens integrated in the biofilm was proportional to the amount introduced. In 14 of the 16 cases, 0.4-3.0% of the spores or spheres introduced were measured in the biofilms. The other two cases had 10% and 21% of the spores detected. Data suggested that the spores germinated in the system. The amount of surrogate pathogens detected in the biofilms was higher in the mid-shear range. Chlorine treatment reduced the quantity of both surrogate pathogens and biofilm organisms. In one experiment, the biofilms and B. cereus recovered when the chlorine treatment was terminated. CONCLUSIONS: Planktonic surrogate pathogens can be integrated in biofilms and protected from chlorination decontamination. SIGNIFICANCE AND IMPACT OF THE STUDY: This knowledge assists in understanding the impact of biofilms on harbouring potential pathogens in drinking-water systems and protecting the pathogens from decontamination.
Assuntos
Bacillus cereus/crescimento & desenvolvimento , Biofilmes/crescimento & desenvolvimento , Cloro/farmacologia , Desinfetantes/farmacologia , Pseudomonas fluorescens/crescimento & desenvolvimento , Esporos Bacterianos/crescimento & desenvolvimento , Bacillus cereus/efeitos dos fármacos , Aderência Bacteriana/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Reatores Biológicos/microbiologia , Contagem de Colônia Microbiana , Descontaminação/métodos , Cloreto de Polivinila , Pseudomonas fluorescens/efeitos dos fármacos , Resistência ao Cisalhamento , Esporos Bacterianos/efeitos dos fármacosRESUMO
PURPOSE: Catheter-related blood stream infections pose a significant risk for patients living with vascular catheters. The cost to manage these infections is substantial. Although the etiology of these infections is multifactorial, tap water has been implicated as a significant causative factor. This retrospective review evaluates the effectiveness of a surgical dressing, the CD-1000, at protecting catheters and exit site wounds from fluid and debris when patients engage in high risk activities like showering. METHODS: All patients who received the CD-1000 from a single national medical supplier from September 2006 through to March 2007 were contacted to participate in this retrospective review; 209 patients, representing 34 states and 175 unique physicians, participated in this study. Effectiveness of the dressing along with prior and current history of catheter events was queried. RESULTS: The CD-1000 was 95% effective at keeping the catheter and exit site dry while patients engaged in high risk activities like showering. Prior to using the CD-1000, the 209 patients reported a historical catheter infection rate of 1.83 per 1000 catheter days. While using the CD-1000 the 209 patients reported a catheter infection rate of 0.47 per 1000 catheter days. CONCLUSION: The CD-1000 catheter specific composite dressing adequately protects vascular catheters and exit sites when patients engage in high risk activities like showering. In this geographically diverse retrospective review, use of the CD-1000 was associated with a 75% reduction in catheter associated infections.
Assuntos
Atividades Cotidianas , Bandagens , Infecções Relacionadas a Cateter/prevenção & controle , Cateteres de Demora/efeitos adversos , Falência Renal Crônica/terapia , Diálise Renal , Infecções Relacionadas a Cateter/microbiologia , Desenho de Equipamento , Feminino , Humanos , Higiene , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Inquéritos e Questionários , Microbiologia da ÁguaRESUMO
Sporothrix schenckii causes sporotrichosis, a disease that most commonly presents as a subacute or chronic skin infection. An unusually high incidence of clinical cases of sporotrichosis occurred in the southwest of Western Australia over the last 5 years. Anecdotal accounts from patients implicated contact with hay prior to infection. Isolates of S. schenckii from hay and clinical cases were investigated by traditional phenotypic methods and pulsed-field gel electrophoresis (PFGE). The phenotypic evaluation separated S. schenckii from Ophiostoma spp. A DNA macrorestriction method using SfiI and NotI macrorestriction digestion by PFGE was developed to investigate the epidemiological connections. BioNumerics software was used to analyze the results. DNA macrorestriction digestion patterns for the recent Western Australian clinical isolates and four hay isolates were indistinguishable. Eastern state clinical isolates, national Quality Assurance Program isolates, and other environmental isolates gave different macrorestriction patterns. Clinical isolates from the southwest of Western Australia collected in the 1980s and 1990s were also characterized using PFGE. The patterns generated were indistinguishable from those of the recent clinical isolates. PFGE showed that the dominant strain of S. schenckii causing sporotrichosis in Western Australia is present in hay, has caused sporotrichosis for at least 15 years, and is a different strain from the strains found in other parts of Australia.
Assuntos
Impressões Digitais de DNA , Técnicas de Tipagem Micológica , Poaceae/microbiologia , Polimorfismo de Fragmento de Restrição , Sporothrix/classificação , Sporothrix/genética , Esporotricose/microbiologia , Austrália/epidemiologia , Análise por Conglomerados , DNA Fúngico/genética , Desoxirribonucleases de Sítio Específico do Tipo II , Eletroforese em Gel de Campo Pulsado , Humanos , Epidemiologia Molecular , Sporothrix/isolamento & purificação , Esporotricose/epidemiologiaRESUMO
OBJECTIVE: Factor analytic studies of the Positive and Negative Syndrome Scale (PANSS) have consistently isolated a factor that is frequently labeled as 'cognitive'. The present study sought to further explore the factor by examining the relationships between 4 versions of the cognitive factor and a set of neuropsychological tests. METHOD: Thirty-seven inpatients diagnosed with schizophrenia or schizoaffective disorder were assessed with the PANSS and neuropsychological measures. RESULTS: Verbal intelligence and verbal memory were found to be most closely associated with cognitive factor scores. A global rating of illness severity showed greater relationships to cognitive variables than any cognitive factor. CONCLUSIONS: The PANSS cognitive factor may reflect verbal ability and memory, but is not sufficiently comprehensive to be considered as a replacement for direct assessment of cognitive functioning.
Assuntos
Cognição , Escalas de Graduação Psiquiátrica , Transtornos Psicóticos/classificação , Esquizofrenia/classificação , Adolescente , Adulto , Análise Fatorial , Feminino , Humanos , Inteligência , Masculino , Memória , Pessoa de Meia-IdadeRESUMO
The assessment of outcomes after treatment with antipsychotic medication is fundamental to clinical care and research. The Routine Assessment of Patient Progress (RAPP) is a reliable multidimensional scale that employs nurses' ratings of symptoms and functioning in psychiatric inpatients. The present study sought to extend validity evidence for the RAPP by examining its ability to reflect changes associated with treatment by antipsychotic medications. The use of a different sample in this study also provided the opportunity to replicate earlier validity data collected on the original set of patients. Ninety-seven separate trials were conducted, involving 65 consecutive admissions to a unit that specializes in the assessment and treatment of patients with long standing severe psychiatric disorders. The RAPP, along with the Positive and Negative Syndrome Scale and global measures of severity, were administered at baseline and at the end of each trial. Both factor scores and clinically-derived subscales were analysed for sensitivity to change. Patients were globally rated as improved, unchanged or worsened at the end of the medication trial. Results indicated that the RAPP factor, clinical scale and total scores compared favourably to other outcome measures in patients rated as improved or worse. In patients rated as unchanged, RAPP scores displayed significantly less change than did the PANSS scores. These findings support the validity of the RAPP as an outcome measure in treatment trials.
Assuntos
Antipsicóticos/uso terapêutico , Transtornos Psicóticos/tratamento farmacológico , Adulto , Ensaios Clínicos como Assunto , Coleta de Dados/métodos , Feminino , Humanos , Masculino , Escalas de Graduação Psiquiátrica , Reprodutibilidade dos Testes , Resultado do TratamentoRESUMO
This study sought to: a) ascertain the effect on rates of violence by varying its operational definition and b) compare characteristics of violent and nonviolent patients. Aggressive behavior was recorded daily for every patient (N = 78) during a 2-year period. Standardized rating scales were used to rate psychopathology and functioning. Almost two thirds of patients were aggressive to others, and 26% violently assaulted another person. Official incident reports underestimated rates of violence to others, self- harm, and property damage. Multivariate predictive models that greatly improved accuracy over base rates showed that violent patients tended to be female, schizophrenic (nonparanoid type), and abusive of alcohol before admission. Violence is more common in treatment resistant psychotic inpatients than suggested by incident reports. Standardized definitions of violence are urged in order to accurately study its prevalence and correlates. Models combining both historical/demographic and clinical data may enhance prediction of violence.
Assuntos
Transtornos Psicóticos/psicologia , Violência/psicologia , Adolescente , Adulto , Idoso , Agressão/psicologia , Colúmbia Britânica , Estudos Transversais , Feminino , Hospitais Psiquiátricos , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Transtornos Psicóticos/diagnóstico , Transtornos Psicóticos/epidemiologia , Recidiva , Violência/estatística & dados numéricosAssuntos
Endorribonucleases/metabolismo , RNA Catalítico/metabolismo , Resinas de Troca Aniônica , Resinas de Troca de Cátion , Cromatografia em Gel , Cromatografia por Troca Iônica , Endorribonucleases/isolamento & purificação , Células HeLa , Humanos , RNA Catalítico/isolamento & purificação , Ribonuclease PRESUMO
Rpp21, a protein subunit of human nuclear ribonuclease P (RNase P) was cloned by virtue of its homology with Rpr2p, an essential subunit of Saccharomyces cerevisiae nuclear RNase P. Rpp21 is encoded by a gene that resides in the class I gene cluster of the major histocompatibility complex, is associated with highly purified RNase P, and binds precursor tRNA. Rpp21 is predominantly localized in the nucleoplasm but is also observed in nucleoli and Cajal bodies when expressed at high levels. Intron retention and splice-site selection in Rpp21 precursor mRNA regulate the intranuclear distribution of the protein products and their association with the RNase P holoenzyme. Our study reveals that dynamic nuclear structures that include nucleoli, the perinucleolar compartment and Cajal bodies are all involved in the production and assembly of human RNase P.
Assuntos
Endorribonucleases/química , RNA Catalítico/química , Células 3T3 , Processamento Alternativo , Sequência de Aminoácidos , Animais , Western Blotting , Núcleo Celular/química , Núcleo Celular/metabolismo , Células Cultivadas , Clonagem Molecular , DNA Complementar/metabolismo , Endorribonucleases/metabolismo , Fibroblastos/metabolismo , Células HeLa , Humanos , Íntrons , Complexo Principal de Histocompatibilidade , Camundongos , Microscopia de Fluorescência , Modelos Genéticos , Dados de Sequência Molecular , Testes de Precipitina , Ligação Proteica , Splicing de RNA , RNA Catalítico/metabolismo , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ribonuclease P , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
A yeast three-hybrid system was employed to analyze interactions in vivo between H1 RNA, the RNA subunit of human nuclear RNase P, and eight of the protein subunits of the enzyme. The genetic analysis indicates that subunits Rpp21, Rpp29, Rpp30, and Rpp38 interact directly with H1 RNA. The results of direct UV crosslinking studies of the purified RNase P holoenzyme confirm the results of the three-hybrid assay.
Assuntos
Núcleo Celular/enzimologia , Endorribonucleases/metabolismo , RNA Catalítico/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Western Blotting , Endorribonucleases/química , Células HeLa , Humanos , RNA Catalítico/química , Ribonuclease PRESUMO
Narrow spectrum antimicrobial activity has been designed to reduce the expression of two essential genes, one coding for the protein subunit of RNase P (C5 protein) and one for gyrase (gyrase A). In both cases, external guide sequences (EGS) have been designed to complex with either mRNA. Using the EGS technology, the level of microbial viability is reduced to less than 10% of the wild-type strain. The EGSs are additive when used together and depend on the number of nucleotides paired when attacking gyrase A mRNA. In the case of gyrase A, three nucleotides unpaired out of a 15-mer EGS still favor complete inhibition by the EGS but five unpaired nucleotides do not.
Assuntos
Proteínas de Bactérias/genética , DNA Topoisomerases Tipo II/genética , Endorribonucleases/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Genes Bacterianos/fisiologia , RNA Catalítico/genética , Sequência de Bases , DNA Girase , Escherichia coli/fisiologia , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Ribonuclease PRESUMO
In the general population, low birthweight (LBW) is associated with neurological and psychological problems during childhood and adolescence. LBW may result from premature birth or poor fetal growth, and the independent effects of these two events on childhood development are not fully understood. The rate of low weight births is increased in schizophrenia and is associated with social withdrawal during childhood and an early onset of illness. However, it is unclear whether this LBW reflects poor fetal growth or premature birth, or whether these two risk factors have distinct implications for childhood functioning and age at onset of schizophrenia. Subjects included 270 patients with schizophrenia for whom a detailed history of obstetric events could be obtained. The rate of low weight births was high and was associated with poorer premorbid functioning and an earlier age at illness onset. The rate of both premature births and poor fetal growth was high relative to the normal population. Prematurity, but not poor fetal growth, was associated with premorbid social withdrawal and an early age at illness onset. Poor fetal growth, but not prematurity, was associated with low educational achievement. These results suggest that poor fetal growth and prematurity are associated with distinct patterns of childhood maladjustment in individuals who develop schizophrenia.
Assuntos
Desenvolvimento Embrionário e Fetal/fisiologia , Recém-Nascido de Baixo Peso , Esquizofrenia/epidemiologia , Adolescente , Adulto , Idade de Início , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Masculino , Pessoa de Meia-Idade , Alienação Social/psicologiaRESUMO
The protein subunit of RNase P from a thermophilic bacterium, Thermotoga maritima, was overexpressed in and purified from Escherichia coli. The cloned protein was reconstituted with the RNA subunit transcribed in vitro. The temperature optimum of the holoenzyme is near 50 degrees C, with no enzymatic activity at 65 degrees C or above. This finding is in sharp contrast to the optimal growth temperature of T.maritima, which is near 80 degrees C. However, in heterologous reconstitution experiments in vitro with RNase P subunits from other species, we found that the protein subunit from T.maritima was responsible for the comparative thermal stability of such complexes.
Assuntos
Endorribonucleases/genética , Endorribonucleases/metabolismo , Proteínas de Escherichia coli , RNA Catalítico/genética , RNA Catalítico/metabolismo , Thermotoga maritima/enzimologia , Thermotoga maritima/genética , Sequência de Aminoácidos , Aminoácidos/análise , Endorribonucleases/química , Endorribonucleases/isolamento & purificação , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/isolamento & purificação , Holoenzimas/metabolismo , Cinética , Dados de Sequência Molecular , Subunidades Proteicas , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , RNA Bacteriano/metabolismo , RNA Catalítico/química , RNA Catalítico/isolamento & purificação , RNA de Transferência de Tirosina/genética , RNA de Transferência de Tirosina/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Ribonuclease P , Alinhamento de Sequência , Temperatura , Transcrição GênicaRESUMO
A yeast two-hybrid system was used to analyze interactions among the protein subunits of human nuclear RNase P themselves and with other interacting partners encoded in a HeLa cell cDNA library. Subunits hpop1, Rpp21, Rpp29, Rpp30, Rpp38, and Rpp40 are involved in extensive, but weak, protein-protein interactions in the holoenzyme complex. Rpp14, Rpp20, and Rpp30 were found to have strong interactions with proteins encoded in the cDNA library. The small heat shock protein 27, which interacts with Rpp20 in the two-hybrid assay, binds to Rpp20 during affinity chromatography and can be found to be associated with, and enhances the activity of, highly purified RNase P. RNase P activity in HeLa cell nuclei also increases under the stress of heat shock.