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1.
Sci Rep ; 11(1): 2224, 2021 01 26.
Article in English | MEDLINE | ID: mdl-33500453

ABSTRACT

Phylogenetic analysis has demonstrated that the etiologic agent of the 2020 pandemic outbreak is a betacoronavirus named SARS-CoV-2. For public health interventions, a diagnostic test with high sensitivity and specificity is required. The gold standard protocol for diagnosis by the Word Health Organization (WHO) is RT-PCR. To detect low viral loads and perform large-scale screening, a low-cost diagnostic test is necessary. Here, we developed a cost-effective test capable of detecting SARS-CoV-2. We validated an auxiliary protocol for molecular diagnosis with the SYBR Green RT-PCR methodology to successfully screen negative cases of SARS-CoV-2. Our results revealed a set of primers with high specificity and no homology with other viruses from the Coronovideae family or human respiratory tract pathogenic viruses, presenting with complementarity only for rhinoviruses/enteroviruses and Legionella spp. Optimization of the annealing temperature and polymerization time led to a high specificity in the PCR products. We have developed a more affordable and swift methodology for negative SARS-CoV-2 screening. This methodology can be applied on a large scale to soften panic and economic burden through guidance for isolation strategies.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Organic Chemicals , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2 , Benzothiazoles , DNA, Single-Stranded , Diamines , Enterovirus , Genome, Viral , Humans , Phylogeny , Polymerase Chain Reaction , Quinolines , Rhinovirus , Sensitivity and Specificity , Temperature , Viral Load
2.
Int J Dev Neurosci ; 75: 13-18, 2019 Jun.
Article in English | MEDLINE | ID: mdl-30940500

ABSTRACT

Undernourishment is a global issue, especially in developing countries, affecting newborns and children in a vulnerable period of brain development. Previous studies of undernourishment models suggested a relationship between undernourishment and epilepsy. The exposure to both undernourishment and recurrent seizures early in life appears to have detrimental effects on the developing brain. This study aims to investigate the neurobiological consequences of undernourishment and recurrent seizures exposure early in life, investigating Long-Term Potentiation (LTP) induction and gene expression of NMDA receptor subunits in the hippocampus during adulthood (P60). Animals were exposed to maternal deprivation protocol from P2 to P15 to control food intake in rat pups and Flurothyl-induced seizures from P7 to P10. Electrophysiological records of hippocampal slices were recorded and gene expression of NR1A, NR2A, NR2B, NR2C, NR2D and BDNF were investigated. Animals exposed to undernourishment or recurrent seizures failed to promote LTP after stimulation. Furthermore, seizure exposure early in life led to increased expression of hippocampal NR1A, NR2A, NR2B, NR2C and NR2D when compared to controls. Interestingly, when animals were exposed to undernourishment paradigm early in life, this upregulation of NDMA subunits was absent. In conclusion, our study showed impaired LTP after undernourishment and recurrent seizures early in life, together with differential expression of NDMA expression in the hippocampus during adulthood.


Subject(s)
Hippocampus/metabolism , Long-Term Potentiation/physiology , Malnutrition/physiopathology , Receptors, AMPA/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Seizures/physiopathology , Animals , Brain-Derived Neurotrophic Factor/genetics , Brain-Derived Neurotrophic Factor/metabolism , Flurothyl , Gene Expression , Malnutrition/metabolism , Maternal Deprivation , Rats , Receptors, AMPA/genetics , Receptors, N-Methyl-D-Aspartate/genetics , Seizures/chemically induced , Seizures/metabolism
3.
Int J Dev Neurosci ; 69: 112-118, 2018 Oct.
Article in English | MEDLINE | ID: mdl-30048676

ABSTRACT

Focal Cortical Dysplasia (FCD), a common type of Malformations of cortical development, may result from an early disturbance in the migration and final arrangement of the cortical architecture of immature neurons. FCD type II is now known to be due to a post-zygotic somatic mutation that involves the mTOR and AKT pathways. The aim of the present study was to investigate the possible differences in neurogenesis and neurodifferentiation of iPSCs (induced pluripotent stem cells) from fibroblasts of individuals affected by FCD type II (2) and normal individuals (2). iPSCs were generated from skin fibroblasts of FCD individuals and healthy individuals. The reprogramming was done through the fibroblasts exposure to viral vectors containing the OCT4, KLF4, SOX2, and c-MYC genes and the clones were characterized by immunohistochemistry. iPSCs were neurodifferentiated and analyzed at the 14th, 22nd and 35th days. We also analyzed the cerebral cortex tissue, fibroblasts and iPSCs cells from the individuals. Through qRT-PCR, the expression of 4 genes involved in Notch signaling process were quantified. In general, individuals with dysplasia presented increase and decrease in the relative quantification in the most genes analyzed compared to control individuals in all processes and study groups. We suggest that, during embryonic neurogenesis, the neural precursor cells of FCD type II individuals present increase and decrease in gene expression in the Notch signaling pathway causing cortical formation disorders and can be seen as a candidate for the developmental changes observed in the cerebral cortex of individuals with FCD type II. This altered gene expression may be related to brain formation with dysplasia.


Subject(s)
Epilepsy/genetics , Epilepsy/metabolism , Malformations of Cortical Development, Group I/genetics , Malformations of Cortical Development, Group I/metabolism , Neural Stem Cells/metabolism , Pluripotent Stem Cells/metabolism , Receptors, Notch/metabolism , Signal Transduction/genetics , Cerebral Cortex/cytology , Child , Excitatory Postsynaptic Potentials/genetics , Female , Fibroblasts , Gene Expression Regulation , Healthy Volunteers , Humans , Immunohistochemistry , Kruppel-Like Factor 4 , Male , Middle Aged , Neurogenesis/genetics , Skin/cytology
4.
In Vitro Cell Dev Biol Anim ; 51(1): 26-35, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25134818

ABSTRACT

Human umbilical cord blood is an attractive source of stem cells; however, it has a heterogeneous cell population with few mesenchymal stem cells. Cell reprogramming induced by different methodologies can confer pluripotency to differentiated adult cells. The objective of this study was to evaluate the reprogramming of fibroblasts and their subsequent neural differentiation after co-culture with umbilical cord blood mononuclear cells. Cells were obtained from four human umbilical cords. The mononuclear cells were cultured for 7 d and subsequently co-cultured with mouse fibroblast NIH-3T3 cells for 6 d. The pluripotency of the cells was evaluated by RT-PCR using primers specific for pluripotency marker genes. The pluripotency was also confirmed by adipogenic and osteogenic differentiation. Neural differentiation of the reprogrammed cells was evaluated by immunofluorescence. All co-cultured cells showed adipogenic and osteogenic differentiation capacity. After co-cultivation, cells expressed the pluripotency gene KLF4. Statistically significant differences in cell area, diameter, optical density, and fractal dimension were observed by confocal microscopy in the neurally differentiated cells. Contact in the form of co-cultivation of fibroblasts with umbilical cord blood mononuclear fraction for 6 d promoted the reprogramming of these cells, allowing the later induction of neural differentiation.


Subject(s)
Cell Differentiation , Coculture Techniques/methods , Gene Expression Regulation , Leukocytes, Mononuclear/cytology , Neurons/cytology , Umbilical Cord/cytology , Animals , Biomarkers/metabolism , Cell Differentiation/genetics , Cell Proliferation , Fibroblasts/cytology , Fluorescent Antibody Technique , Humans , Imaging, Three-Dimensional , Interleukin-2/metabolism , Kruppel-Like Factor 4 , Leukocytes, Mononuclear/metabolism , Mesoderm/cytology , Mice , NIH 3T3 Cells , Neurons/metabolism , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism
5.
Int J Antimicrob Agents ; 42(1): 76-9, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23664677

ABSTRACT

The emergence and spread of resistance mechanisms in Gram-negative bacilli has complicated the treatment of serious nosocomial infections. Current automated systems for detection of Klebsiella pneumoniae carbapenemase (KPC)-producing isolates are unreliable. One possible straightforward alternative method is evaluation of ertapenem resistance. However, the accuracy of this method is affected by other resistance mechanisms such as AmpC gene expression or extended-spectrum ß-lactamase production associated with porin loss. This study included 128 samples of K. pneumoniae and Enterobacter spp. that were non-susceptible to ertapenem. The disk diffusion and Etest method were applied to determine susceptibility to imipenem, meropenem and ertapenem. Isolates exhibiting intermediate or complete resistance to ertapenem were evaluated for resistance mechanisms. bla(TEM), bla(SHV), bla(CTX-M), bla(CTX-M-2) and bla(KPC) genes were tested for by PCR, and the presence of outer membrane protein was investigated by dot-blot assay. bla(TEM) was detected in 52.9% and 10.3%, bla(SHV) in 29.4% and 0.94%, bla(CTX-M) in 41.4% and 1.9% and bla(CTX-M-2) in 23.5% and 1.9% of K. pneumoniae and Enterobacter cloacae isolates, respectively. The bla(KPC) gene was present in 12.6% of Enterobacter spp. isolates. OmpC and OmpF were present in 6.6% of E. cloacae isolates. These results indicate that several resistance mechanisms contribute to potential therapeutic failure of carbapenem therapy and point to the need for better detection methods and surveillance strategies.


Subject(s)
Drug Resistance, Bacterial , Enterobacteriaceae Infections/microbiology , Enterobacteriaceae/drug effects , Enterobacteriaceae/enzymology , Porins/genetics , beta-Lactamases/genetics , Anti-Bacterial Agents/pharmacology , Enterobacteriaceae/genetics , Enterobacteriaceae/isolation & purification , Ertapenem , Humans , Microbial Sensitivity Tests , Polymerase Chain Reaction , beta-Lactams/pharmacology
6.
Food Chem Toxicol ; 53: 119-25, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23220610

ABSTRACT

The antioxidant effects of the hydro-alcoholic guaraná extract (Paullinia cupana var. sorbilis Mart.) on nitric oxide (NO) and other compounds generated from the degradation of sodium nitroprusside (SNP) in an embryonic fibroblast culture (NIH-3T3 cells) were evaluated. The guaraná bioactive compounds were initially determined by high-performance liquid chromatography: caffeine=12.240 mg/g, theobromine=6.733 mg/g and total catechins=4.336 mg/g. Cells were exposed to 10 µM SNP during a 6 h period because the cells exhibited >90% mortality at this concentration. Guaraná was added to the cultures in five concentrations (0.5, 1, 5, 10 and 20 mg/mL). The guaraná antioxidant effect was evaluated by viability assays, biochemical oxidation [lipid peroxidation, catalase and superoxide dismutase (SOD) activity] and genotoxicity (DNA Comet assay) analysis. Additionally, oxidative stress was evaluated by a 2,7-dihydrodichlorofluorescein diacetate fluorescence assay. Guaraná reverted the SNP toxicity mainly at lower concentrations (<5 mg), which decreased cell mortality, lipid peroxidation, DNA damage and cell oxidative stress as well as increased the SOD levels. These results demonstrate that guaraná has an antioxidant effect on NO metabolism in situations with higher cellular NO levels.


Subject(s)
Fibroblasts/drug effects , Nitroprusside/adverse effects , Paullinia/chemistry , Plant Extracts/pharmacology , Animals , Antioxidants/pharmacology , Caffeine/analysis , Caffeine/pharmacology , Catechin/analysis , Catechin/pharmacology , Chromatography, High Pressure Liquid , Comet Assay , DNA Damage/drug effects , Fibroblasts/cytology , Fluoresceins/analysis , Mice , NIH 3T3 Cells , Nitric Oxide/metabolism , Oxidative Stress/drug effects , Theobromine/analysis , Theobromine/pharmacology
7.
Neuroscience ; 210: 431-41, 2012 May 17.
Article in English | MEDLINE | ID: mdl-22441035

ABSTRACT

Despite the beneficial effects of cell-based therapies on brain repair shown in most studies, there has not been a consensus regarding the optimal dose of human umbilical cord blood cells (HUCBC) for neonatal hypoxia-ischemia (HI). In this study, we compared the long-term effects of intravenous administration of HUCBC at three different doses on spatial memory and brain morphological changes after HI in newborn Wistar rats. In addition, we tested whether the transplanted HUCBC migrate to the injured brain after transplantation. Seven-day-old animals underwent right carotid artery occlusion and were exposed to 8% O(2) inhalation for 2 h. After 24 h, randomly selected animals were assigned to four different experimental groups: HI rats administered with vehicle (HI+vehicle), HI rats treated with 1×10(6) (HI+low-dose), 1×10(7) (HI+medium-dose), and 1×10(8) (HI+high-dose) HUCBC into the jugular vein. A control group (sham-operated) was also included in this study. After 8 weeks of transplantation, spatial memory performance was assessed using the Morris water maze (MWM), and subsequently, the animals were euthanized for brain morphological analysis using stereological methods. In addition, we performed immunofluorescence and polymerase chain reaction (PCR) analyses to identify HUCBC in the rat brain 7 days after transplantation. The MWM test showed a significant spatial memory recovery at the highest HUCBC dose compared with HI+vehicle rats (P<0.05). Furthermore, the brain atrophy was also significantly lower in the HI+medium- and high-dose groups compared with the HI+vehicle animals (P<0.01; 0.001, respectively). In addition, HUCBC were demonstrated to be localized in host brains by immunohistochemistry and PCR analyses 7 days after intravenous administration. These results revealed that HUCBC transplantation has the dose-dependent potential to promote robust tissue repair and stable cognitive improvement after HI brain injury.


Subject(s)
Brain/pathology , Cord Blood Stem Cell Transplantation/methods , Hypoxia-Ischemia, Brain/surgery , Memory Disorders/prevention & control , Animals , Animals, Newborn , Disease Models, Animal , Fetal Blood/transplantation , Fluorescent Antibody Technique , Humans , Hypoxia-Ischemia, Brain/complications , Maze Learning , Memory , Memory Disorders/etiology , Microscopy, Confocal , Rats , Rats, Wistar
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