Neuroimmunomodulatory and neuroprotective effects of the flavonoid apigenin in in vitro models of neuroinflammation associated with Alzheimer's disease

Neurodegenerative disorders (ND) are characterized by the progressive and irreversible loss of neurons. Alzheimer’s Disease (AD) is the most incident age-related ND, in which the presence of a chronic inflammatory compound seems to be related to its pathogenesis. Different stimuli in the central nervous system (CNS) can induce activation, proliferation, and changes in phenotype and glial function, which can be modulated by anti-inflammatory agents. Apigenin (4,5,7–trihydroxyflavone) is a flavonoid found in abundance in many fruits and vegetables, that has shown important effects upon controlling the inflammatory response. This study evaluated the neuroprotective and neuroimmunomodulatory potential of apigenin using in vitro models of neuroinflammation associated with AD. Co-cultures of neurons and glial cells were obtained from the cortex of newborn and embryonic Wistar rats. After 26 days in vitro, cultures were exposed to lipopolysaccharide (LPS; 1 μg/ml), or IL-1β (10 ng/ml) for 24 h, or to Aβ oligomers (500 nM) for 4 h, and then treated with apigenin (1 μM) for further 24 h. It was observed that the treatment with apigenin preserved neurons and astrocytes integrity, determined by Rosenfeld’s staining and immunocytochemistry for β-tubulin III and GFAP, respectively. Moreover, it was observed by Fluoro-Jade-B and caspase-3 immunostaining that apigenin was not neurotoxic and has a neuroprotective effect against inflammatory damage. Additionally, apigenin reduced microglial activation, characterized by inhibition of proliferation (BrdU+ cells) and modulation of microglia morphology (Iba-1 + cells), and decreased the expression of the M1 inflammatory marker CD68. Moreover, as determined by RT-qPCR, inflammatory stimuli induced by IL-1β increased the mRNA expression of IL-6, IL-1β, and CCL5, and decreased the mRNA expression of IL-10. Contrary, after treatment with apigenin in inflammatory stimuli (IL-1β or LPS) there was a modulation of the mRNA expression of inflammatory cytokines, and reduced expression of OX42, IL-6 and gp130. Moreover, apigenin alone and after an inflammatory stimulus with IL-1β also induced the increase in the expression of brain-derived neurotrophic factor (BDNF), an effect that may be associated with anti-inflammatory and neuroprotective effects. Together these data demonstrate that apigenin presents neuroprotective and anti-inflammatory effects in vitro and might represent an important neuroimmunomodulatory agent for the treatment of neurodegenerative conditions

Phytoestrogen agathisflavone ameliorates neuroinflammation-induced by LPS and IL-1β and protects neurons in cocultures of glia/neurons

Inflammation and oxidative stress are common aspects of most neurodegenerative diseases in the central nervous system. In this context, microglia and astrocytes are central to mediating the balance between neuroprotective and neurodestructive mechanisms. Flavonoids have potent anti-inflammatory and antioxidant properties. Here, we have examined the anti-inflammatory and neuroprotective potential of the flavonoid agathisflavone (FAB), which is derived from the Brazilian plant Poincianella pyramidalis, in in vitro models of neuroinflammation. Cocultures of neurons/glial cells were exposed to lipopolysaccharide (LPS, 1 µg/mL) or interleukin (IL)-1β (10 ng/mL) for 24 h and treated with FAB (0.1 and 1 µM, 24 h). FAB displayed a significant neuroprotective effect, as measured by nitric oxide (NO) production, Fluoro-Jade B (FJ-B) staining, and immunocytochemistry (ICC) for the neuronal marker β-tubulin and the cell death marker caspase-3, preserving neuronal soma and increasing neurite outgrowth. FAB significantly decreased the LPS-induced microglial proliferation, identified by ICC for Iba-1/bromodeoxyuridine (BrdU) and CD68 (microglia M1 profile marker). In contrast, FAB had no apparent effect on astrocytes, as determined by ICC for glial fibrillary acidic protein (GFAP). Furthermore, FAB protected against the cytodestructive and proinflammatory effects of IL-1β, a key cytokine that is released by activated microglia and astrocytes, and ICC showed that combined treatment of FAB with α and β estrogen receptor antagonists did not affect NF-κB expression. In addition, qPCR analysis demonstrated that FAB decreased the expression of proinflammatory molecules TNF-α, IL-1β, and connexins CCL5 and CCL2, as well as increased the expression of the regulatory molecule IL-10. Together, these findings indicate that FAB has a significant neuroprotective and anti-inflammatory effect in vitro, which may be considered as an adjuvant for the treatment of neurodegenerative diseases

The flavonoid agathisflavone modulates the microglial neuroinflammatory response and enhances remyelination

Myelin loss is the hallmark of the demyelinating disease multiple sclerosis (MS) and plays a significant role in multiple neurodegenerative diseases. A common factor in all neuropathologies is the central role of microglia, the intrinsic immune cells of the central nervous system (CNS). Microglia are activated in pathology and can have both pro- and anti-inflammatory functions. Here, we examined the effects of the flavonoid agathisflavone on microglia and remyelination in the cerebellar slice model following lysolecithin induced demyelination. Notably, agathisflavone enhances remyelination and alters microglial activation state, as determined by their morphology and cytokine profile. Furthermore, these effects of agathisflavone on remyelination and microglial activation were inhibited by blockade of estrogen receptor α. Thus, our results identify agathisflavone as a novel compound that may act via ER to regulate microglial activation and enhance remyelination and repair

ALTERAÇÕES MORFOLÓGICAS DE LEUCÓCITOS E TROMBÓCITOS SANGUÍNEOS EM Piaractus mesopotamicus DURANTE A SEPSE

Sepsis is defined as organ dysfunction with high mortality. During this process, the hematological system plays a critical role. Thus, the work aimed to analyze the morphological changes in the leukogram and thrombogram by the indirect method in the course of sepsis in Piaractus mesopotamicus. For this, 98 pacus were used, divided into two groups, one received 0.5 ml of sterile 0.65% sodium chloride solution (control) and the other received the same volume containing the bacterial inoculum (1.8 x 108 CFU/ml – challenge). To assess such changes in the evolution of sepsis, blood samples were collected from fish 1, 3, 6 and 9 hours after inoculation and in the control group (n = 10). One aliquot of the blood was used for blood culture and another for determining the number of leukocytes and thrombotics by the indirect method and the evaluation of morphological changes in blood extensions. Aeromonosis was confirmed by positive blood culture in all samples from the challenged groups. The leukogram analysis showed a significant increase (p <0.05) of leukocytes in the later times, 9 and 6 HPI in relation to the control group. Differential analysis showed leukopenia (p <0.05; 3 HPI) and consumption of thrombocytes. It was found that with the increase of the infection the septicemic pacus develops a picture of granulocytosis and monocytosis (p <0.05). It was concluded that inoculation with A. hydrophila induced sepsis with positive blood culture with leukocytosis in the challenged pacus and caused an intense change in the leukocyte and thrombotic morphology.KEYWORDS: Hemoculture, Leukogram, Morphology, Aeromonas hydrophila, Teleosts.La sepsis se define como disfunción orgánica con alta mortalidad. Durante este proceso, el sistema hematológico juega un papel crítico. Por lo tanto, el trabajo tuvo como objetivo analizar los cambios morfológicos en el leucograma y el trombograma por el método indirecto en el curso de la sepsis en Piaractus mesopotamicus. Para esto, se utilizaron 98 pacus, divididos en dos grupos, uno recibió 0,5 ml de solución estéril de cloruro de sodio al 0,65% (control) y el otro recibió el mismo volumen que contenía el inóculo bacteriano (1,8 x 108 UFC / ml - desafío). Para evaluar dichos cambios en la evolución de la sepsis, se recogieron muestras de sangre de peces 1, 3, 6 y 9 horas después de la inoculación y en el grupo de control (n = 10). Una parte alícuota de la sangre se usó para el hemocultivo y otra para determinar el número de leucocitos y trombóticos mediante el método indirecto y la evaluación de los cambios morfológicos en las extensiones de sangre. La aeromonosis se confirmó por hemocultivo positivo en todas las muestras de los grupos desafiados. El análisis de leucogramas mostró un aumento significativo (p <0.05) de leucocitos en los últimos tiempos, 9 y 6 HPI en relación con el grupo de control. El análisis diferencial mostró leucopenia (p <0.05; 3 HPI) y consumo de trombocitos. Se encontró que con el aumento de la infección, el pacus septicémico desarrolla una imagen de granulocitosis y monocitosis (p <0.05). Se concluyó que la inoculación con A. hydrophila indujo sepsis con hemocultivo positivo con leucocitosis en pacus desafiado y causa un cambio intenso en los leucocitos y la morfología trombóticaPALABRAS CLAVES Hemocultivo, Leucograma, Morfología, Aeromonas hydrophila, Teleósteos.A sepse é definida como disfunção orgânica com alta mortalidade. Durante esse processo o sistema hematológico desempenha um papel crítico. Assim, o trabalho objetivou analisar as alterações morfológicas no leucograma e no trombograma pelo método indireto no curso da sepse em Piaractus mesopotamicus. Para tanto, foram utilizados 98 pacus, divididos em dois grupos, um deles recebeu 0,5 ml de solução de cloreto de sódio esterilizada a 0,65% (controle) e outro recebeu o mesmo volume contendo o inoculo bacteriano (18 x 108 CFU/ml – desafio). Para avaliar tais alterações na evolução da sepse foram realizadas coletas de sangue dos peixes 1, 3, 6 e 9 horas após inoculação e no grupo controle (n=10). Uma alíquota do sangue foi destina a hemocultura e outra na determinação do número de leucócitos e de trombóticos pelo método indireto e a avaliação das alterações morfológicas nas extensões sanguíneas. A aeromonose foi confirmada pela hemocultura positiva em todas as amostras dos grupos desafiados. A análise do leucograma demonstrou aumento significativa (p<0,05) dos leucócitos nos tempos mais tardios, 9 e 6 HPI em relação ao grupo controle. Da análise diferencial observou-se leucopenia (p<0,05; 3 HPI) e consumo dos trombócitos. Verificou-se que com o aumento da infecção os pacus septicêmicos desenvolvem um quadro de granulocitose e monocitose (p<0,05). Concluiu-se que a inoculação com A. hydrophila induziu a sepse com hemocultura positiva com leucocitose nos pacus desafiados e causa intensa alteração na morfologia dos leucócitos e trombóticos.PALAVRAS-CHAVE: Hemocultura, Leucograma, Morfologia, Aeromonas hydrophila, Teleósteos

Low-bias RNA sequencing of the HIV-2 genome from blood plasma

Accurate determination of the genetic diversity present in the HIV quasispecies is critical for the development of a preventative vaccine: in particular, little is known about viral genetic diversity for the second type of HIV, HIV-2. A better understanding of HIV-2 biology is relevant to the HIV vaccine field because a substantial proportion of infected people experience long-term viral control, and prior HIV-2 infection has been associated with slower HIV-1 disease progression in coinfected subjects. The majority of traditional and next-generation sequencing methods have relied on target amplification prior to sequencing, introducing biases that may obscure the true signals of diversity in the viral population. Additionally, target enrichment through PCR requires a priori sequence knowledge, which is lacking for HIV-2. Therefore, a target enrichment free method of library preparation would be valuable for the field. We applied an RNA shotgun sequencing (RNA-Seq) method without PCR amplification to cultured viral stocks and patient plasma samples from HIV-2-infected individuals. Libraries generated from total plasma RNA were analyzed with a two-step pipeline: (i) de novo genome assembly, followed by (ii) read remapping. By this approach, whole-genome sequences were generated with a 28× to 67× mean depth of coverage. Assembled reads showed a low level of GC bias, and comparison of the genome diversities at the intrahost level showed low diversity in the accessory gene vpx in all patients. Our study demonstrates that RNA-Seq is a feasible full-genome de novo sequencing method for blood plasma samples collected from HIV-2-infected individuals.IMPORTANCE An accurate picture of viral genetic diversity is critical for the development of a globally effective HIV vaccine. However, sequencing strategies are often complicated by target enrichment prior to sequencing, introducing biases that can distort variant frequencies, which are not easily corrected for in downstream analyses. Additionally, detailed a priori sequence knowledge is needed to inform robust primer design when employing PCR amplification, a factor that is often lacking when working with tropical diseases localized in developing countries. Previous work has demonstrated that direct RNA shotgun sequencing (RNA-Seq) can be used to circumvent these issues for hepatitis C virus (HCV) and norovirus. We applied RNA-Seq to total RNA extracted from HIV-2 blood plasma samples, demonstrating the applicability of this technique to HIV-2 and allowing us to generate a dynamic picture of genetic diversity over the whole genome of HIV-2 in the context of low-bias sequencing

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In the last years many populations of anurans have declined and extinctions have been recorded. They were related to environmental pollution, changes of land use and emerging diseases. The main objective of this study was to determine copper sensitivity of the anuran of the Amazon Rhinella granulosa and Scinax ruber tadpoles at stage 25 and Scinax ruber eggs exposed for 96 h to copper concentrations ranging from 15 µg Cu L-1 to 94 µg Cu L-1. LC50 at 96 h of Rhinella granulosa Gosner 25, Scinax ruber Gosner 25 and Scinax ruber eggs in black water of the Amazon were 23.48, 36.37 and 50.02 µg Cu L-1, respectively. The Biotic Ligand Model was used to predict the LC50 values for these species and it can be considered a promising tool for these tropical species and water conditions. Copper toxicity depends on water physical-chemical composition and on the larval stage of the tadpoles. The Gosner stage 19-21 (related to the appearance of external gills) is the most vulnerable and the egg stage is the most resistant. In case of contamination by copper, the natural streams must have special attention, since copper is more bioavailable.Nos últimos anos foram registrados muitas extinções e declínios de populações de anuros. Eles estavam relacionados com a poluição do ambiente, a mudanças no uso da terra e ao surgimento de doenças. O principal objetivo deste estudo foi determinar a sensibilidade dos anuros amazônicos ao cobre. Os girinos de Scinax ruber e Rhinella granulosa no estadio 25 e os ovos de Scinax ruber foram expostos por 96 horas a concentrações de cobre entre 15 µg Cu L-1 a 94 µg Cu L-1. A CL50 -96 h dos girinos de Rhinella granulosa, dos girinos de Scinax ruber e dos ovos de Scinax ruber em águas pretas da Amazônia foram 23,48; 36,37 e 50,02 µg Cu L-1, respectivamente. O modelo do ligante biótico foi usado para prever os valores de CL50 para essas duas espécies e pode ser considerado uma ferramenta promissora para essas espécies tropicais e para essas condições de água. A Toxicidade de cobre depende da composição físico-química da água e do estagio larval dos girinos. O estadio 19-21 de Gosner (relacionados ao aparecimento das brânquias externas) são os mais vulnerável e o estagio de ovo é o mais resistente. Em caso de contaminação por cobre, os igarapés naturais devem ter uma atenção especial, uma vez que o cobre é mais biodisponível nesse ambiente

Building The Sugarcane Genome For Biotechnology And Identifying Evolutionary Trends

Background: Sugarcane is the source of sugar in all tropical and subtropical countries and is becoming increasingly important for bio-based fuels. However, its large (10 Gb), polyploid, complex genome has hindered genome based breeding efforts. Here we release the largest and most diverse set of sugarcane genome sequences to date, as part of an on-going initiative to provide a sugarcane genomic information resource, with the ultimate goal of producing a gold standard genome.Results: Three hundred and seventeen chiefly euchromatic BACs were sequenced. A reference set of one thousand four hundred manually-annotated protein-coding genes was generated. A small RNA collection and a RNA-seq library were used to explore expression patterns and the sRNA landscape. In the sucrose and starch metabolism pathway, 16 non-redundant enzyme-encoding genes were identified. One of the sucrose pathway genes, sucrose-6-phosphate phosphohydrolase, is duplicated in sugarcane and sorghum, but not in rice and maize. A diversity analysis of the s6pp duplication region revealed haplotype-structured sequence composition. Examination of hom(e)ologous loci indicate both sequence structural and sRNA landscape variation. A synteny analysis shows that the sugarcane genome has expanded relative to the sorghum genome, largely due to the presence of transposable elements and uncharacterized intergenic and intronic sequences.Conclusion: This release of sugarcane genomic sequences will advance our understanding of sugarcane genetics and contribute to the development of molecular tools for breeding purposes and gene discovery. © 2014 de Setta et al.; licensee BioMed Central Ltd.151European Commission: Agriculture and Rural Development: Sugar http://ec.europa.eu/agriculture/sugar/index_en.htmKellogg, E.A., Evolutionary history of the grasses (2001) Plant Physiol, 125, pp. 1198-1205Grivet, L., Arruda, P., Sugarcane genomics: depicting the complex genome of an important tropical crop (2001) Curr Opin Plant Biol, 5, pp. 122-127Piperidis, G., Piperidis, N., D'Hont, A., Molecular cytogenetic investigation of chromosome composition and transmission in sugarcane (2010) Mol Genet Genomics, 284, pp. 65-73D'Hont, A., 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