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An increase in plasma high glucose promotes endothelial dysfunction mainly through increasing mitochondrial ROS production. High glucose ROS-induced has been implicated in the fragmentation of the mitochondrial network, mainly by an unbalance expression of mitochondrial fusion and fission proteins. Mitochondrial dynamics alterations affect cellular bioenergetics. Here, we assessed the effect of PDGF-C on mitochondrial dynamics and glycolytic and mitochondrial metabolism in a model of endothelial dysfunction induced by high glucose. High glucose induced a fragmented mitochondrial phenotype associated with the reduced expression of OPA1 protein, high DRP1pSer616 levels and reduced basal respiration, maximal respiration, spare respiratory capacity, non-mitochondrial oxygen consumption and ATP production, regarding normal glucose. In these conditions, PDGF-C significantly increased the expression of OPA1 fusion protein, diminished DRP1pSer616 levels and restored the mitochondrial network. On mitochondrial function, PDGF-C increased the non-mitochondrial oxygen consumption diminished by high glucose conditions. These results suggest that PDGF-C modulates the damage induced by HG on the mitochondrial network and morphology of human aortic endothelial cells; additionally, it compensates for the alteration in the energetic phenotype induced by HG.
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Dinaminas , Doenças Vasculares , Humanos , Dinaminas/genética , Células Endoteliais/metabolismo , Glucose/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Doenças Vasculares/metabolismoRESUMO
Faithful DNA replication is essential for normal cell division and differentiation. In eukaryotic cells, DNA replication takes place on chromatin. This poses the critical question as to how DNA replication can progress through chromatin, which is inhibitory to all DNA-dependent processes. Here, we developed a novel genome-wide method to measure chromatin accessibility to micrococcal nuclease (MNase) that is normalized for nucleosome density, the NCAM (normalized chromatin accessibility to MNase) assay. This method enabled us to discover that chromatin accessibility increases specifically at and ahead of DNA replication forks in normal S phase and during replication stress. We further found that Mec1, a key regulatory ATR-like kinase in the S-phase checkpoint, is required for both normal chromatin accessibility around replication forks and replication fork rate during replication stress, revealing novel functions for the kinase in replication stress response. These results suggest a possibility that Mec1 may facilitate DNA replication fork progression during replication stress by increasing chromatin accessibility around replication forks.
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Cromatina/metabolismo , Replicação do DNA , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Cromatina/química , Mapeamento Cromossômico , Genoma/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Nuclease do Micrococo/metabolismo , Mutação/genética , Nucleossomos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Fase S/genética , Proteínas de Saccharomyces cerevisiae/genética , Estresse FisiológicoRESUMO
Eukaryotic DNA replication initiates from multiple discrete sites in the genome, termed origins of replication (origins). Prior to S phase, multiple origins are poised to initiate replication by recruitment of the pre-replicative complex (pre-RC). For proper replication to occur, origin activation must be tightly regulated. At the population level, each origin has a distinct firing time and frequency of activation within S phase. Many studies have shown that chromatin can strongly influence initiation of DNA replication. However, the chromatin parameters that affect properties of origins have not been thoroughly established. We found that nucleosome occupancy in G1 varies greatly around origins across the S. cerevisiae genome, and nucleosome occupancy around origins significantly correlates with the activation time and efficiency of origins, as well as pre-RC formation. We further demonstrate that nucleosome occupancy around origins in G1 is established during transition from G2/M to G1 in a pre-RC-dependent manner. Importantly, the diminished cell-cycle changes in nucleosome occupancy around origins in the orc1-161 mutant are associated with an abnormal global origin usage profile, suggesting that proper establishment of nucleosome occupancy around origins is a critical step for regulation of global origin activities. Our work thus establishes nucleosome occupancy as a novel and key chromatin parameter for proper origin regulation.
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Cromatina/genética , Replicação do DNA/genética , Nucleossomos/genética , Complexo de Reconhecimento de Origem/genética , Origem de Replicação/genética , Proteínas de Saccharomyces cerevisiae/genética , Ciclo Celular/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Proteínas Mutantes/genética , Fase S/genética , Saccharomyces cerevisiae/genéticaRESUMO
In this study, we identified, at the single-cell level, naturally induced cytokine-producing circulating cells (CPCCs) in children with dengue virus (DENV) infection ranging clinically from mild to severe disease. Tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) CPCCs were detected in children with primary or secondary acute dengue virus (DENV) infection, and the pattern of these cytokines was similar to that seen in the supernatant of cultured peripheral blood mononuclear cells and partially comparable to that found in plasma. Monocytes, B cells, and myeloid dendritic cells (mDCs) were the primary CPCCs detected, and the frequency of mDCs was significantly higher in severe disease. B cells isolated from children with dengue spontaneously secreted TNF-α, IL-6, and interleukin 10, and supernatants from cultures of purified B cells induced activation of allogeneic T cells, supporting an antibody-independent function of these cells during DENV infection. Thus, CPCCs could be a new immune parameter with potential use to evaluate pathogenesis in this infection.
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Linfócitos B/metabolismo , Citocinas/metabolismo , Células Dendríticas/metabolismo , Dengue/imunologia , Monócitos/metabolismo , Criança , Dengue/metabolismo , Feminino , Regulação da Expressão Gênica/imunologia , Humanos , MasculinoRESUMO
Hereditary angioedema (HAE) is a heterogeneous genetic disease caused by a deficit in C1 inhibitor (C1-INH) and clinically characterized by sudden events of edema, swelling, and pruritus. Here, we describe the first SERPING1 genotyping in 22 subjects from 4 non-related families, all from southern Colombia. The previously reported heterozygous gene mutations, c.1081C>T (p.Gln361*), c.1396C>G (p.Arg466Gly), c.1029+84G>A, or c.106_107del (p.Ser36Phefs*21), were found in 12 patients. Of note, a single patient clinically characterized as severe HAE type 2 expressed mutations in exon 8 and intron 6, whereas all the others have type 1 HAE and expressed one pathogenic variant. One of the subjects, a 5-year-old girl was discovered to have a pathogenic variant, and she is still asymptomatic. This is the first report focused on HAE genetic analysis in a Colombian population.
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Angioedemas Hereditários/diagnóstico , Angioedemas Hereditários/genética , Proteína Inibidora do Complemento C1/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética , Genótipo , Adolescente , Adulto , Angioedemas Hereditários/sangue , Biomarcadores , Criança , Pré-Escolar , Colômbia , Complemento C4 , Feminino , Estudos de Associação Genética/métodos , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Linhagem , Adulto JovemRESUMO
Garlic oil (GO) and cinnamaldehyde (CIN) have shown potential to modify rumen fermentation. The aim of this study was to assess the effects of GO and CIN on rumen fermentation, microbial protein synthesis (MPS), and microbial populations in Rusitec fermenters fed a mixed diet (50:50 forage/concentrate), as well as whether these effects were maintained over time. Six fermenters were used in two 15-day incubation runs. Within each run, two fermenters received no additive, 180 mg/L of GO, or 180 mg/L of CIN. Rumen fermentation parameters were assessed in two periods (P1 and P2), and microbial populations were studied after each of these periods. Garlic oil reduced the acetate/propionate ratio and methane production (p < 0.001) in P1 and P2 and decreased protozoal DNA concentration and the relative abundance of fungi and archaea after P1 (p < 0.05). Cinnamaldehyde increased bacterial diversity (p < 0.01) and modified the structure of bacterial communities after P1, decreased bacterial DNA concentration after P2 (p < 0.05), and increased MPS (p < 0.001). The results of this study indicate that 180 mg/L of GO and CIN promoted a more efficient rumen fermentation and increased the protein supply to the animal, respectively, although an apparent adaptive response of microbial populations to GO was observed.
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BACKGROUND: Spastic paraplegia 11 (SPG11) is the most prevalent form of autosomal recessive hereditary spastic paraplegia, resulting from biallelic pathogenic variants in the SPG11 gene (MIM *610844). METHODS: The proband is a 36-year-old female referred for genetic evaluation due to cognitive dysfunction, gait impairment, and corpus callosum atrophy (brain MRI was normal at 25-years-old). Diagnostic approaches included CGH array, next-generation sequencing, and whole transcriptome sequencing. RESULTS: CGH array revealed a 180 kb deletion located upstream of SPG11. Sequencing of SPG11 uncovered two rare single nucleotide variants: the novel variant c.3143C>T in exon 17 (in cis with the deletion), and the previously reported pathogenic variant c.6409C>T in exon 34 (in trans). Whole transcriptome sequencing revealed that the variant c.3143C>T caused exon 17 skipping. CONCLUSION: We report a novel sequence variant in the SPG11 gene resulting in exon 17 skipping, which, along with a nonsense variant, causes Spastic Paraplegia 11 in our proband. In addition, a deletion upstream of SPG11 was identified in the patient, whose implication in the phenotype remains uncertain. Nonetheless, the deletion apparently affects cis-regulatory elements of the gene, suggesting a potential new pathogenic mechanism underlying the disease in a subset of undiagnosed patients. Our findings further support the hypothesis that the origin of thin corpus callosum in patients with SPG11 is of progressive nature.
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Paraplegia Espástica Hereditária , Humanos , Feminino , Adulto , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/diagnóstico , Paraplegia Espástica Hereditária/patologia , Éxons , Proteínas/genética , Códon sem Sentido , Corpo Caloso/patologia , Corpo Caloso/diagnóstico por imagem , Deleção de Sequência , FenótipoRESUMO
Identification of early determinants of dengue disease progression, which could potentially enable individualized patient care are needed at present times. Soluble ST2 (sST2) has been recently reported to be elevated in the serum of children older than 2 years old and adults with dengue infection and it was correlated with secondary infections as well as with severe presentations of the disease. The mechanism by which secreted ST2 is linked to severe dengue and plasma leakage remains unclear. One possibility is that IL-33 ligand may be elevated, contributing to membrane bound ST2 as part of the immune activation in dengue infection. We determined plasma levels of sST2 and the ligand IL-33 in 66 children with acute secondary dengue infections clinically classified using the guidelines of the World Health Organization, 2009. Dengue infection showed significant increases in cytokines IL-12p70, IL-10, IL-8, IL-6, IL-1ß and TNFα measured by flow cytometry based assay compared to uninfected individuals. In contrast, IL-33 levels remained unchanged between infected and uninfected individuals. The levels of sST2 positively correlated with values of IL-6 and IL-8 and inversely correlated with number of median value of platelet levels. In addition to circulating cytokine positive correlations we found that sST2 and isoenzyme creatine kinase-MB (CK-MB), a marker of myocardial muscle damage present in severe dengue cases were associated. Our pediatric study concluded that in dengue infections sST2 elevation does not involve concomitant changes of IL-33 ligand. We propose a study to assess its value as a predictor factor of disease severity.
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Dengue/sangue , Dengue/imunologia , Interleucinas/sangue , Receptores de Superfície Celular/sangue , Adulto , Criança , Pré-Escolar , Estudos de Coortes , Demografia , Dengue/patologia , Feminino , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33 , Interleucina-6/sangue , Interleucina-8/sangue , Ligantes , Masculino , Índice de Gravidade de Doença , SolubilidadeRESUMO
BACKGROUND: Pediatric dengue and sepsis share clinical and pathophysiologic aspects. Multiple inflammatory and regulatory cytokines, decoy receptors and vascular permeability factors have been implicated in the pathogenesis of both diseases. The differential pattern and dynamic of these soluble factors, and the relationship with clinical severity between pediatric dengue and sepsis could offer new diagnosis and therapeutic strategies. METHODS: We evaluated the concentration levels of 11 soluble factors with proinflammatory, regulatory and vascular permeability involvement, in plasma from children with dengue or sepsis, both clinically ranging from mild to severe, in the early, late and convalescence phases of the disease. RESULTS: During early acute infection, children with sepsis exhibited specific higher concentration levels of IL-6, vascular endothelial growth factor (VEGF), and its soluble decoy receptor II (sVEGFR2) and lower concentration levels of IL-10 and the soluble tumor necrosis factor receptor 2 (sTNFR2), in comparison with children with severe dengue. In addition, the circulating amounts of soluble ST2, and VEGF/sVEGFR2 were widely associated with clinical and laboratory indicators of dengue severity, whereas secondary dengue virus infections were characterized by an enhanced cytokine response, relative to primary infections. In severe forms of dengue, or sepsis, the kinetics and the cytokines response during the late and convalescence phases of the disease also differentiate. CONCLUSIONS: Dengue virus infection and septic processes in children are characterized by cytokine responses of a specific magnitude, pattern and kinetics, which are implicated in the pathophysiology and clinical outcome of these diseases.
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Dengue , Sepse , Dengue Grave , Humanos , Criança , Dengue Grave/diagnóstico , Dengue Grave/complicações , Fator A de Crescimento do Endotélio Vascular , Dengue/diagnóstico , Dengue/complicações , Convalescença , Citocinas , Sepse/diagnóstico , Sepse/complicações , BiomarcadoresRESUMO
This study presents an analysis of the impacts of the changes in bottom depth along the Guadalquivir Estuary on tidal dynamics. A realistic non-linear 1D numerical model, incorporating changes in both breadth and bottom depth, was employed to investigate the involved effects. The findings reveal a significant amplification of the M2 tidal wave towards the upper region of the Estuary, resulting from the gradual deepening caused by multiple dredging operations. The Estuary exhibits a pronounced tendency towards resonance, which is further enhanced by its deepening, resulting in reduced bottom friction and a smaller decrease in tidal wave amplitude as it propagates through the Estuary. The alterations in depth, particularly in breadth, along the Estuary play a crucial role in determining the magnitude of the resonant response of the M2 tidal wave.
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Endothelial dysfunction is an early marker for cardiovascular diseases. Hyperglycemia induces endothelial dysfunction, increasing the production of reactive oxygen species. Platelet-derived growth factor C stimulates angiogenesis and revascularization in ischemic tissues of diabetic mice and promotes the migration of progenitors and mature ECs to injury sites; however, the molecular mechanisms of its actions are not described yet. Here, we evaluated the effect of PDGF-C on oxidative stress induced by HG. Human aortic endothelial cells were grown in glucose concentrations ranging from 5 mmol/L to 35 mmol/L for 1 to 24 h. Treatment with 50 ng/mL PDGF-C was done for 1 to 3 h. Cytosolic and mitochondrial ROS were measured by fluorometry, and the expression of antioxidant enzymes was evaluated by Western blot. Nrf2 and Keap1 expression was assessed by real-time PCR. High glucose induced mitochondrial ROS production. PDGF-C diminished the oxidative stress induced by high glucose, increasing SOD2 expression and SOD activity, and modulating the Keap1 expression gene. These results give new evidence about the mitochondrial antioxidant effect that PDGF-C could exert on endothelial cells exposed to high glucose and its considerable role as a therapeutic target in diabetes.
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Silencing of multiple cancer-related genes is associated with de novo methylation of linked CpG islands. Additionally, bivalent histone modification profiles characterized by the juxtaposition of active and inactive histone marks have been observed in genes that become hypermethylated in cancer. It is unknown how these ambiguous epigenetic states are maintained and how they interrelate with adjacent genomic regions with different epigenetic landscapes. Here, we present the analysis of a set of neighboring genes, including many frequently silenced in colon cancer cells, in a chromosomal region at 5q35.2 spanning 1.25 Mb. Promoter DNA methylation occurs only at genes maintained at a low transcriptional state and is characterized by the presence of bivalent histone marks, namely trimethylation of lysines 4 and 27 in histone 3. Chemically induced hyperacetylation and DNA demethylation lead to up-regulation of silenced genes in this locus yet do not resolve bivalent domains into a domain-wide active chromatin conformation. In contrast, active genes in the region become down-regulated after drug treatment, accompanied by a partial loss of chromatin domain boundaries and spreading of the inactive histone mark trimethylated lysine 27 in histone 3. Our results demonstrate that bivalent domains mark the promoters of genes that will become DNA methylated in adult tumor cells to enforce transcriptional silence. These bivalent domains not only remain upon drug induced gene reactivation, but also spread over adjacent CpG islands. These results may have important implications in understanding and managing epigenetic therapies of cancer.
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Cromossomos Humanos Par 5/genética , Neoplasias do Colo/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Genes Neoplásicos , Sequência de Bases , Transformação Celular Neoplásica/genética , Cromatina/metabolismo , Neoplasias do Colo/patologia , Neoplasias do Colo/terapia , Metilação de DNA/efeitos dos fármacos , Terapia Genética , Humanos , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Transcrição GênicaRESUMO
Cornelia de Lange syndrome (CdLS) is a rare disease affecting multiple organs and systems during development. Mutations in the cohesin loader, NIPBL/Scc2, were first described and are the most frequent in clinically diagnosed CdLS patients. The molecular mechanisms driving CdLS phenotypes are not understood. In addition to its canonical role in sister chromatid cohesion, cohesin is implicated in the spatial organization of the genome. Here, we investigate the transcriptome of CdLS patient-derived primary fibroblasts and observe the downregulation of genes involved in development and system skeletal organization, providing a link to the developmental alterations and limb abnormalities characteristic of CdLS patients. Genome-wide distribution studies demonstrate a global reduction of NIPBL at the NIPBL-associated high GC content regions in CdLS-derived cells. In addition, cohesin accumulates at NIPBL-occupied sites at CpG islands potentially due to reduced cohesin translocation along chromosomes, and fewer cohesin peaks colocalize with CTCF.
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Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Síndrome de Cornélia de Lange/genética , Genoma Humano , Transcriptoma/genética , Diferenciação Celular/genética , Cromatina/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Estabilidade Proteica , CoesinasRESUMO
The interleukin-1 receptor-like-1 protein (IL1RL1), also known as ST2, has been shown previously to regulate T-cell function and is produced by T cells and endothelial cells. It was reported recently to be elevated in mild dengue patients during acute disease. The ST2 gene encodes several splice products: L (long), V (short) and s (soluble). A cohort of 38 patients with dengue haemorrhagic fever (DHF) and mild dengue fever (DF) were evaluated using a secreted soluble ST2 (sST2) ELISA. The RNA expression of ST2 was evaluated by real-time quantitative RT-PCR using patients' peripheral blood mononuclear cells (PBMCs) and in vitro using human umbilical vein endothelial cells (HUVECs) exposed to sera from dengue patients. DHF patients had higher levels of serum sST2, tumour necrosis factor alpha (TNF-alpha), interleukin (IL)-8 and IL-10 compared with DF patients and normal healthy control individuals. However, viraemia was indistinguishable between mild and severe cases. No changes in ST2 mRNA expression were found in PBMCs from these two groups of dengue patients. In vitro, sST2 was elevated in HUVECs treated with patient sera. Neutralization of TNF-alpha in patient sera by pre-treatment with a TNF-alpha antibody inhibited the upregulation of sST2 expression in HUVECs. These results implicate serum TNF-alpha in the modulation of expression of sST2 in an in vitro system, and indicate that sST2 could be associated with the severity of disease. Further studies to determine whether sST2 levels are predictive of the severe form of the disease and the role of sST2 in immune regulation are warranted.
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Dengue/imunologia , Dengue/patologia , Receptores de Superfície Celular/sangue , Fator de Necrose Tumoral alfa/imunologia , Adolescente , Adulto , Biomarcadores , Linhagem Celular , Células Cultivadas , Criança , Pré-Escolar , Dengue/diagnóstico , Células Endoteliais/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Perfilação da Expressão Gênica , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1 , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Soro/química , Adulto JovemRESUMO
Methylation of the cytosine is the most frequent epigenetic modification of DNA in mammalian cells. In humans, most of the methylated cytosines are found in CpG-rich sequences within tandem and interspersed repeats that make up to 45% of the human genome, being Alu repeats the most common family. Demethylation of Alu elements occurs in aging and cancer processes and has been associated with gene reactivation and genomic instability. By targeting the unmethylated SmaI site within the Alu sequence as a surrogate marker, we have quantified and identified unmethylated Alu elements on the genomic scale. Normal colon epithelial cells contain in average 25 486 +/- 10 157 unmethylated Alu's per haploid genome, while in tumor cells this figure is 41 995 +/- 17 187 (P = 0.004). There is an inverse relationship in Alu families with respect to their age and methylation status: the youngest elements exhibit the highest prevalence of the SmaI site (AluY: 42%; AluS: 18%, AluJ: 5%) but the lower rates of unmethylation (AluY: 1.65%; AluS: 3.1%, AluJ: 12%). Data are consistent with a stronger silencing pressure on the youngest repetitive elements, which are closer to genes. Further insights into the functional implications of atypical unmethylation states in Alu elements will surely contribute to decipher genomic organization and gene regulation in complex organisms.
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Elementos Alu , Carcinoma/genética , Neoplasias Colorretais/genética , Metilação de DNA , Genômica/métodos , Linhagem Celular Tumoral , Cromossomos Humanos , Colo/citologia , Biologia Computacional , Ilhas de CpG , Epigênese Genética , Genoma Humano , Humanos , Mucosa Intestinal/química , Reação em Cadeia da PolimeraseRESUMO
OBJECTIVE: To evaluate the clinical, laboratory, and immune characteristics of Zika virus (ZIKV)-associated encephalitis in pediatric patients after the epidemic in Huila, southern Colombia. METHODS: A pediatric neuro-surveillance hospital study was conducted in a referral health center in southern Colombia, from October 2016 to October 2017. Cases of encephalitis were confirmed by nucleic acid amplification tests and serological methods in cerebrospinal fluid (CSF), plasma, and/or urine. Levels of six cytokines were evaluated by flow cytometry. Patients underwent daily clinical and laboratory follow-up. RESULTS: Twenty children with probable encephalitis were included for further studies and 16 of them were confirmed. Four cases of bacterial meningoencephalitis (Streptococcus pneumoniae, group B Streptococcus, Staphylococcus epidermidis, and Escherichia coli) and 12 cases of viral encephalitis were identified, six of them associated with ZIKV infection. Other viral encephalitis cases were caused by herpes viruses (n=3), enterovirus (n=2), and dengue virus type 2 (DENV-2; n=1) infections. ZIKV-associated encephalitis symptoms subsided faster than those of patients with encephalitis caused by other agents. CSF analysis revealed lymphocytic pleocytosis. Compared to healthy controls, children with ZIKV-associated encephalitis presented modest plasma interleukin (IL)-10 but not IL-2, IL-4, IL-6, interferon gamma (IFN-γ), or tumor necrosis factor alpha (TNF-α). Cytokine expression was differentially regulated, as dramatically elevated IL-6, IL-10, and IFN-γ levels were observed in CSF but not in paired plasma samples in one of the patients with ZIKV detectable in CSF. CONCLUSIONS: This study provides evidence that ZIKV is responsible for pediatric encephalitis in endemic areas, and the local presence of the virus may induce cephalic but not systemic expression of cytokines.
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Encefalite Viral/virologia , Infecção por Zika virus/virologia , Adolescente , Criança , Pré-Escolar , Colômbia , Citocinas/sangue , Citocinas/líquido cefalorraquidiano , Encefalite Viral/diagnóstico , Encefalite Viral/imunologia , Feminino , Humanos , Lactente , Interferon gama/sangue , Interferon gama/líquido cefalorraquidiano , Masculino , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/líquido cefalorraquidiano , Zika virus/isolamento & purificação , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/imunologiaRESUMO
Olive oil extraction generates large amounts of a highly pollutant by-product called olive cake (OC), and its use in ruminant feeding could be an alternative. This study was designed to evaluate the effects of partially replacing forage by crude OC (COC) in a mixed dairy diet on rumen fermentation and microbial populations in Rusitec fermenters. The COC replaced 33% of the forage (66% maize silage and 33% barley straw) and was included at 16.6% of the total diet. Four fermenters were used in a cross-over design with two 13-day incubation periods. Experimental diets had a 50:50 forage-to-concentrate ratio and were formulated to contain the same protein (16.0%) and neutral detergent fiber (32.5%) levels. Compared with control fermenters, those fed the COC diet showed greater (p ≤ 0.02) pH (6.07 vs. 6.22), diet disappearance (0.709 vs. 0.748), and butyrate proportions (18.0 vs. 19.4), but there were no differences in volatile fatty acids and ammonia production. Microbial growth, bacterial diversity, protozoal abundance, and relative abundance of fungi and archaea were unaffected by diet, although the solid phase of COC-fed fermenters showed greater (p = 0.01) bacterial abundance than control ones. Results indicate that COC could replace 33% of the forage in a mixed dairy diet.
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Citrus pulp is a highly abundant by-product of the citrus industry. The aim of this study was to assess the effects of replacing extruded maize (EM; 20% of total diet) by dried citrus pulp (DCP; 20%) in a mixed diet on rumen fermentation and microbial populations in Rusitec fermenters. The two diets contained 50% alfalfa hay and 50% concentrate, and the same protein level. Four Rusitec fermenters were used in a cross-over design with two 13-d incubation runs. After 7-d of diet adaptation, diet disappearance, fermentation parameters, microbial growth, and microbial populations were assessed. Fermenters receiving the DCP showed greater pH values and fiber disappearance (p < 0.001) and lower methane production (p = 0.03) than those fed EM. Replacing EM by DCP caused an increase in the proportions of propionate and butyrate (p < 0.001) and a decrease in acetate (p = 0.04). Microbial growth, bacterial diversity, and the quantity of bacteria and protozoa DNA were not affected by the diet, but the relative abundances of fungi and archaea were greater (p < 0.03) in solid and liquid phases of DCP fermenters, respectively. Results indicate that DCP can substitute EM, promoting a more efficient ruminal fermentation.
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DNA methylation is an epigenetic modification that plays a crucial role in the control of gene expression and chromosome structure in plants and mammalian cells. Multiple types of DNA fingerprinting techniques have been developed and applied to investigate DNA methylation profiles in different experimental settings. One of these techniques, the amplification of intermethylated sites (AIMS) is a simple approach appropriate for genome-wide estimates of DNA methylation and the discovery of specific methylated sequences. AIMS is based on the differential enzymatic digestion of genomic DNA with methylation-sensitive and methylation-insensitive isoschizomers followed by restrained PCR amplification of methylated sequences. This method is appropriate to compare large series of samples and the simultaneous identification of hypo- and hypermethylation events. Applications of AIMS include the study of DNA methylation changes in cancer and aging, and the discovery of DNA methylation in a social insect.
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Impressões Digitais de DNA/métodos , Metilação de DNA , Animais , Sequência de Bases , Ilhas de CpG , DNA/química , DNA/genética , Primers do DNA/genética , Genômica , Humanos , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da PolimeraseRESUMO
The nutritive value of 26 agro-industrial by-products was assessed from their chemical composition, in vitro digestibility and rumen fermentation kinetics. By-products from sugar beet, grape, olive tree, almond, broccoli, lettuce, asparagus, green bean, artichoke, peas, broad beans, tomato, pepper, apple pomace and citrus were evaluated. Chemical composition, in vitro digestibility and fermentation kinetics varied largely across the by-products. Data were subjected to multivariate and principal component analyses (PCA). According to a multivariate cluster analysis chart, samples formed four distinctive groups (A-D). Less degradable by-products were olive tree leaves, pepper skins and grape seeds (group A); whereas the more degradable ones were sugar beet, orange, lemon and clementine pulps (group D). In the PCA plot, component 1 segregated samples of groups A and B from those of groups C and D. Considering the large variability among by-products, most of them can be regarded as potential ingredients in ruminant rations. Depending on the characteristic nutritive value of each by-product, these feedstuffs can provide alternative sources of energy (e.g., citrus pulps), protein (e.g., asparagus rinds), soluble fibre (e.g., sugar beet pulp) or less digestible roughage (e.g., grape seeds or pepper skin).