A novel PCR-based method for high throughput prokaryotic expression of antimicrobial peptide genes

<p>Abstract</p> <p>Background</p> <p>To facilitate the screening of large quantities of new antimicrobial peptides (AMPs), we describe a cost-effective method for high throughput prokaryotic expression of AMPs. EDDIE, an autoproteolytic mutant of the N-terminal autoprotease, Npro, from classical swine fever virus, was selected as a fusion protein partner. The expression system was used for high-level expression of six antimicrobial peptides with different sizes: Bombinin-like peptide 7, Temporin G, hexapeptide, Combi-1, human Histatin 9, and human Histatin 6. These expressed AMPs were purified and evaluated for antimicrobial activity.</p> <p>Results</p> <p>Two or four primers were used to synthesize each AMP gene in a single step PCR. Each synthetic gene was then cloned into the pET30a/His-EDDIE-GFP vector via an <it>in vivo </it>recombination strategy. Each AMP was then expressed as an Npro fusion protein in <it>Escherichia coli</it>. The expressed fusion proteins existed as inclusion bodies in the cytoplasm and the expression levels of the six AMPs reached up to 40% of the total cell protein content. On <it>in vitro </it>refolding, the fusion AMPs was released from the C-terminal end of the autoprotease by self-cleavage, leaving AMPs with an authentic N terminus. The released fusion partner was easily purified by Ni-NTA chromatography. All recombinant AMPs displayed expected antimicrobial activity against <it>E. coli</it>, <it>Micrococcus </it>luteus and <it>S. cerevisia</it>.</p> <p>Conclusions</p> <p>The method described in this report allows the fast synthesis of genes that are optimized for over-expression in <it>E. coli </it>and for the production of sufficiently large amounts of peptides for functional and structural characterization. The Npro partner system, without the need for chemical or enzymatic removal of the fusion tag, is a low-cost, efficient way of producing AMPs for characterization. The cloning method, combined with bioinformatic analyses from genome and EST sequence data, will also be useful for screening new AMPs. Plasmid pET30a/His-EDDIE-GFP also provides green/white colony selection for high-throughput recombinant AMP cloning.</p

Supplement with Cystoseira canariensis promotes an increase of resistance to the maximum load in the grastrocnemius muscle of female rats

Was evaluated the maximum load supported by the grastrocnemius muscle of female rats after the ministering of Cystoseira canariensis, either associated or not to swimming. Twenty-eight young Wistar female rats were used, divided into 4 groups: control (C, n=7); supplement (S, n=7); supplement and swimming (SSw, n=7); swimming (Sw, n=7). Each one pertaining to the groups S and SSw received 20 mg of myostatin inhibitor per day. The swimming consisted in an aerobe protocol, three times in a week, during eight weeks. The right grastrocnemius muscle of each animal was removed and a tension test was performed in an Emic testing machine. The results (Mean&#177;SEM) were evaluated through ANOVA and Tukey test (p<0.05). A significant difference for maximum load (in N) was verified among the groups C (35.41&#177;1.06) and S (39.98&#177;1.15); Sw (27.94&#177;2.19) and S (39.98&#177;1.15); Sw (27.94&#177;2.19) and SSw (37.78&#177;1.28). In relation to the stretching at the maximum limit (in x10-3m) at the maximum load, the group SSw obtained a value (20.68&#177;1.19) significantly greater than the groups C (17.15&#177;1.11), S and Sw (16.11&#177;1.60). There was a significant difference for body weight gain among the groups treated with supplement and supplement associated to the swimming, with smaller values for this last. The myostatin inhibitor either, associated or not to the swimming, promotes an increase of resistance to the maximum load in the tension test in grastrocnemius muscle of young female rats

Neuropsychiatric symptoms of the elderly with Alzheimer's disease and the family caregivers' distress

ABSTRACT Objective: to analyze the relationship between the distress of the family caregiver and the presence of neuropsychiatric symptoms in elderly patients with Alzheimer's disease or mixed dementia. Method: a descriptive, cross-sectional study conducted in the Geriatric and Dementias Clinic of a general tertiary hospital, with 96 elderly people with Alzheimer's disease or mixed dementia and their family caregivers. Questionnaires to characterize the elderly and caregivers, and the Neuropsychiatric Inventory were used. Descriptive statistics and Pearson correlation test were performed. Results: 68.7% of the elderly were women, average age 80.8 years, 56.2% had Alzheimer's disease and 43.7%, mixed dementia. Among caregivers, 90.6% were women, average age 56, 70.8% took care of parents and 64.6% lived with the elderly. There was a strong (r = 0.82) and significant (p <0.01) correlation between the total score on the Neuropsychiatric Inventory and the total score on the Neuropsychiatric Inventory-Distress and strong (r = 0.80) and significant (p <0 01) correlation between the total score on the Neuropsychiatric Inventory Distress and the number of neuropsychiatric symptoms, i.e., the higher the number, frequency and severity of these symptoms in the elderly, the more intense is the caregiver distress. Conclusion: the presence of neuropsychiatric symptoms in the elderly was related to increased distress in caregivers

Activation of the mitochondrial ATP-sensitive K+ channel reduces apoptosis of spleen mononuclear cells induced by hyperlipidemia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Background: We have previously demonstrated that increased rates of superoxide generation by extramitochondrial enzymes induce the activation of the mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) in the livers of hypertriglyceridemic (HTG) mice. The resulting mild uncoupling mediated by mitoK(ATP) protects mitochondria against oxidative damage. In this study, we investigate whether immune cells from HTG mice also present increased mitoK(ATP) activity and evaluate the influence of this trait on cell redox state and viability. Methods: Oxygen consumption (Clark-type electrode), reactive oxygen species production (dihydroethidium and H2-DCF-DA probes) and cell death (annexin V, cytocrome c release and Trypan blue exclusion) were determined in spleen mononuclear cells. Results: HTG mice mononuclear cells displayed increased mitoK(ATP) activity, as evidenced by higher resting respiration rates that were sensitive to mitoK(ATP) antagonists. Whole cell superoxide production and apoptosis rates were increased in HTG cells. Inhibition of mitoK(ATP) further increased the production of reactive oxygen species and apoptosis in these cells. Incubation with HTG serum induced apoptosis more strongly in WT cells than in HTG mononuclear cells. Cytochrome c release into the cytosol and caspase 8 activity were both increased in HTG cells, indicating that cell death signaling starts upstream of the mitochondria but does involve this organelle. Accordingly, a reduced number of blood circulating lymphocytes was found in HTG mice. Conclusions: These results demonstrate that spleen mononuclear cells from hyperlipidemic mice have more active mitoKATP channels, which downregulate mitochondrial superoxide generation. The increased apoptosis rate observed in these cells is exacerbated by closing the mitoK(ATP) channels. Thus, mitoK(ATP) opening acts as a protective mechanism that reduces cell death induced by hyperlipidemia.12Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2006/53705-8, 2006/59786-0, 2011/50400-0]CNPq [304532/2010-0

Cyclin-Dependent Kinase-Like 5 Deficiency Disorder: Clinical Review

CDKL5 deficiency disorder (CDD) is a developmental encephalopathy caused by pathogenic variants in the gene cyclin-dependent kinase-like 5 (CDKL5). This unique disorder includes early infantile onset refractory epilepsy, hypotonia, developmental intellectual and motor disabilities, and cortical visual impairment. We review the clinical presentations and genetic variations in CDD based on a systematic literature review and experience in the CDKL5 Centers of Excellence (COEs). We propose minimum diagnostic criteria. Pathogenic variants include deletions, truncations, splice variants, and missense variants. Pathogenic missense variants occur exclusively within the kinase domain or affect splice sites. The CDKL5 protein is widely expressed in the brain, predominantly in neurons, with roles in cell proliferation, neuronal migration, axonal outgrowth, dendritic morphogenesis and synapse development. The molecular biology of CDD is revealing opportunities in precision therapy, with phase 2 and 3 clinical trials underway or planned to assess disease specific and disease modifying treatments

O envelhecimento ativo sob o olhar de idosos funcionalmente independentes El envejecimiento activo bajo la visión de ancianos funcionalmente independientes Active aging from the perspective of aged individuals who are functionally independent

Esta pesquisa teve como objetivo apreender as representações sociais de idosos sobre o envelhecimento ativo. Foi realizada na cidade de João Pessoa, Paraíba, com 100 idosos funcionalmente independentes. Como instrumentos, utilizaram-se entrevistas semi-estruturadas. Os dados foram organizados e analisados pelo software Alceste. Os resultados demonstraram que os discursos dos idosos sobre o envelhecimento ativo são permeados por conteúdos positivos. No entanto, quando não está associado à palavra ativo, o envelhecimento ainda é representado como perdas e incapacidades. Mesmo com a existência de perdas durante o processo, o envelhecimento de maneira ativa deve ser estimulado entre os idosos, uma vez que ele é sinônimo de vida plena e com qualidade. Manter os idosos funcionalmente independentes é o primeiro passo para se atingir um envelhecimento ativo e com melhor qualidade de vida.<br>Esta investigación tuvo como objetivo captar las representaciones sociales de ancianos sobre el envejecimiento activo. Fue realizada en la ciudad de João Pessoa - PB, con 100 ancianos funcionalmente independientes. Como instrumento, fue utilizada la entrevista semiestructurada. Los datos se organizaron y procesaron a través del software Alceste. Los resultados demostraron que los testimonios de los ancianos acerca del envejecimiento activo están impregnados por contenidos positivos. Sin embargo, cuando no está asociado a la palabra activo, el envejecimiento aún es representado como pérdidas e incapacidades. Incluso con la existencia de pérdidas durante el proceso, el envejecimiento activo debe ser estimulado entre los ancianos, toda vez que es sinónimo de vida plena y con calidad. Mantener a los ancianos funcionalmente independientes es el primer paso para alcanzar un envejecimiento activo y con mejor calidad de vida.<br>The objective of this study was to identify the social representations of the elderly regarding active aging. Semi-structured interviews were performed with 100 functionally independent aged individuals from João Pessoa, Paraíba, Brazil. The data was organized and analyzed using Alceste software. Results showed that the aged individuals' statements about active aging are permeated with positive contents. However, when aging is not associated with the word active, it is still represented as losses and disabilities. Despite the existence of losses during the process, active aging should be encouraged among the elderly, as it means living a quality, plentiful life. Maintaining the elderly functionally independent is the first step to achieving active aging and thus improving their quality of life

Insuficiência cardíaca: estratégias usadas por idosos na busca por qualidade de vida

Estudo descritivo com abordagem qualitativa cujos objetivos foram identificar as repercussões da insuficiência cardíaca na qualidade de vida dos idosos; descrever as estratégias utilizadas no enfrentamento destas repercussões e conhecer as percepções acerca dos cuidados de enfermagem. Foram realizadas entrevistas semiestruturadas com dez idosos com insuficiência cardíaca em um hospital militar do Rio de Janeiro-RJ. Identificou-se que a insuficiência cardíaca gera repercussões emocionais, físicas e financeiras na vida dos idosos, ocasionando impacto na qualidade de vida. Dentre as estratégias criadas pelos idosos, verificou-se o enfrentamento paliativo e o enfrentamento emocional. Os cuidados de enfermagem evidenciados foram orientação, carinho, zelo e dedicação. Cuidar do idoso com insuficiência cardíaca implica em avaliar as repercussões na capacidade funcional e autonomia dos anciãos, exigindo habilidades e competências do enfermeiro para identificar suas especificidades na perspectiva de um cuidado integral que contribua para a qualidade de vida dos mesmos