Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Criança , Humanos , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão Oncogênica/genéticaRESUMO
Glial cells have been implicated in temporal lobe epilepsy in humans and in its models. Astrocytes are lost in several brain regions after acute seizures induced by pilocarpine and may suffer hyperplasia at subsequent time points. This study investigated the effect of N-methyl-(2S,4R)-trans-4-hydroxy-L-proline (NMP) on astrocytes exposed to cytotoxic concentrations of pilocarpine. Astrocytes were incubated with pilocarpine (half maximal inhibitory concentration (IC50)=31.86 mM) for 24 h. Afterwards, they were treated with NMP at concentrations ranging from 3.12 to 100 µg/mL for 24 h. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cytoplasmic reactive oxygen species (ROS) and mitochondrial transmembrane potential (ΔΨm) were analyzed by flow cytometry using 2',7'-dichlorofluorescein diacetate (DCFH-DA) and rhodamine-123 (Rho123), respectively. Expression of glial fibrillary acidic protein (GFAP) and voltage-dependent anion channel-1 (VDAC-1) were measured by western blot. Pilocarpine significantly decreased cell viability and mitochondrial potential and increased ROS concentration significantly by 6.7 times compared to the control. NMP concentrations ≥25 µg/mL protected astrocytes against pilocarpine-induced injury in a concentration-dependent manner. Concomitantly, NMP reduced cytoplasmic ROS accumulation to 27.3, 24.8, and 12.3% in the groups treated with 25, 50, and 100 µg/mL NMP, respectively. NMP also protected mitochondria from pilocarpine-induced depolarization. These effects were associated with improvement of pilocarpine-induced GFAP and VDAC-1 overexpression, which are important biomarkers of astrocyte dysfunction. In conclusion, the improvement of ROS accumulation, VDAC-1 overexpression, and mitochondrial depolarization are possible mechanisms of the NMP protective action on reactive astrocytes.
Assuntos
Pilocarpina , Sapotaceae , Humanos , Pilocarpina/farmacologia , Astrócitos , Espécies Reativas de Oxigênio/metabolismo , Sapotaceae/metabolismoRESUMO
Doxorubicin (DOX) is a chemotherapeutic agent that has been used in the treatment of breast cancer. However, serious toxic effects have limited its use, mainly cardiotoxicity. To minimize the adverse effects, liposomal preparations containing DOX have been developed. These preparations can reach the target in the tumor region as well as bypass the resistance-related problems. An alternative to increased therapeutic efficacy may be the fusion of liposomes with exosomes released from tumor cells to facilitate membrane and fusion interactions, achieving greater cell uptake. Thus, the purpose of this study was the fusion of exosomes derived from breast tumor cells with long-circulating and pH-sensitive liposomes loading DOX (ExoSpHL-DOX) for the treatment of breast cancer. The mean diameter of ExoSpHL-DOX was 100.8 ± 7.8 nm, the polydispersity index was 0.122 ± 0.004, and the encapsulated DOX content was equal to 83.5 ± 2.5%. The fusion of exosomes with long-circulating and pH-sensitive liposomes was confirmed by Fourier transform infrared spectroscopy, Raman spectroscopy, and nano-flow cytometry. The physicochemical characteristics of ExoSpHL-DOX were maintained for 60 days, at 4 °C. The study of the release of DOX from ExoSpHL-DOX in dilution media with different pH values showed the pH sensitivity characteristic of the nanosystem, since 96.6 ± 0.2% of DOX was released from ExoSpHL-DOX at pH 5.0, while at pH 7.4, the release was 70.1 ± 1.7% in the medium. The cytotoxic study against the breast cancer cell line demonstrated that ExoSpHL-DOX treatment significantly reduced the cancer cell viability.
Assuntos
Antineoplásicos , Neoplasias da Mama , Exossomos , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Doxorrubicina/química , Doxorrubicina/farmacologia , Exossomos/patologia , Feminino , Humanos , Concentração de Íons de Hidrogênio , Lipossomos/químicaRESUMO
Glial cells have been implicated in temporal lobe epilepsy in humans and in its models. Astrocytes are lost in several brain regions after acute seizures induced by pilocarpine and may suffer hyperplasia at subsequent time points. This study investigated the effect of N-methyl-(2S,4R)-trans-4-hydroxy-L-proline (NMP) on astrocytes exposed to cytotoxic concentrations of pilocarpine. Astrocytes were incubated with pilocarpine (half maximal inhibitory concentration (IC50)=31.86 mM) for 24 h. Afterwards, they were treated with NMP at concentrations ranging from 3.12 to 100 μg/mL for 24 h. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cytoplasmic reactive oxygen species (ROS) and mitochondrial transmembrane potential (ΔΨm) were analyzed by flow cytometry using 2',7'-dichlorofluorescein diacetate (DCFH-DA) and rhodamine-123 (Rho123), respectively. Expression of glial fibrillary acidic protein (GFAP) and voltage-dependent anion channel-1 (VDAC-1) were measured by western blot. Pilocarpine significantly decreased cell viability and mitochondrial potential and increased ROS concentration significantly by 6.7 times compared to the control. NMP concentrations ≥25 µg/mL protected astrocytes against pilocarpine-induced injury in a concentration-dependent manner. Concomitantly, NMP reduced cytoplasmic ROS accumulation to 27.3, 24.8, and 12.3% in the groups treated with 25, 50, and 100 µg/mL NMP, respectively. NMP also protected mitochondria from pilocarpine-induced depolarization. These effects were associated with improvement of pilocarpine-induced GFAP and VDAC-1 overexpression, which are important biomarkers of astrocyte dysfunction. In conclusion, the improvement of ROS accumulation, VDAC-1 overexpression, and mitochondrial depolarization are possible mechanisms of the NMP protective action on reactive astrocytes.
RESUMO
BACKGROUND AND AIMS: Introduction: The familial hypercholesterolemia (FH) is one of the main causes of cardiovascular diseases, and it is mainly caused by genetic variants at the low-density lipoprotein receptor (LDLR). Although ultrasequencing technology has allowed the identification of several genetic variants, few of them was functional analyzed. The CRISPR/ Cas9 tool promotes precise genetic editing and allows the creation of experimental models, therefore contributing to the functional validation process. Aim: To use the CRISPR/Cas9 tool to perform in vitro functional analysis of LDLR variants identified in FH patients. METHODS: Two missense LDLR variants were selected within a group of variants identified in FH patients, based on in silico data, the affected protein domain and MAF. Three sgRNAs were designed for each of the variants c.551G>A and c.1118G>A, to analyze the accuracy of the sgRNAs. The sgRNAs were inserted on PX458 plasmid, cloned, purified in E. coli DH5a, and then co-transfected with the DNA template at HepG2 cells. The DNAs templates were designed to contain the selected variants. RESULTS: HepG2 cells co-transfected with PX458 constructs and DNA templates showed considerably transfection rate, being possible to visualize it at fluorescence microscopy. However, it was noted that single transfection of sgRNAs showed a higher transfection efficiency than cotransfection. CONCLUSIONS: We designed sgRNA for c.551G>A and c.1118G>A variants, being able to analyze the transfection efficiency. In further steps, we will select new sgRNAs for LDLR variants that have not been described yet, and functional analysis will be performed to determine the clinical relevance of these variants.
Assuntos
Hiperlipoproteinemia Tipo II , Lipídeos , Lipoproteínas HDL , GenéticaRESUMO
The determination of necrophagous fly specie's development time is considered an accurate method for estimating postmortem interval (PMI). However, pesticides and other chemicals can alter the flies' life cycle, inducing errors in PMI estimation. Thus, this work aimed to evaluate the effect of different doses of Terbufos (Organophosphates) on the temporal dispersion pattern and development of immature dipterans associated with decaying rat carcasses. For this, 150 g female Wistar rats received, via gavage, 200 µl of Terbufos (5 or 10 mg/kg) or distilled water (control) and, after 30 min of administration, the animals were euthanized and distributed in suspended traps to decompose under environmental conditions. The dispersing immatures were collected daily, and their development time was monitored until the emergence of adult flies. After data analysis, it was observed that Terbufos altered 1) the temporal pattern of larval dispersion; 2) the composition and structure of the colonizing assemblage (emerged adults); 3) species' development time, accelerating or delaying their cycle, depending on the dose used; and 4) the califorids and sarcophagids emergence rate, increasing the mortality of pupae from intoxicated carcasses. Thus, this work demonstrates experimentally that Terbufos directly influences the development of flies with forensic potential and discusses the implications for PMI estimation, which can assist in future investigative processes with suspected poisoning by this organophosphate.
Assuntos
Calliphoridae/efeitos dos fármacos , Entomologia Forense , Inseticidas/toxicidade , Muscidae/efeitos dos fármacos , Compostos Organotiofosforados/toxicidade , Sarcofagídeos/efeitos dos fármacos , Animais , Cadáver , Calliphoridae/crescimento & desenvolvimento , Relação Dose-Resposta a Droga , Feminino , Muscidae/crescimento & desenvolvimento , Ratos , Ratos Wistar , Sarcofagídeos/crescimento & desenvolvimentoRESUMO
PURPOSE: To investigate the relationship between aesthetic perception and molar incisor hypomineralization (MIH) in schoolchildren aged 8-12 years. METHODS: The study included 467 pupils enrolled in elementary schools. Once authorization was received, the students were examined for DMFT and dmft indexes (WHO criteria), and for MIH (EAPD criteria). The aesthetic perceptions were verified by the Child Perceptions Questionnaire About Tooth Appearance (CQATA), a questionnaire divided into several items, including physical, psychological and social domains; perception about tooth pleasantness, tooth alignment, tooth discoloration and tooth health; and reported pleasant colour. Data analysis was carried out using descriptive statistics, the Chi-square, the Mann-Whitney and the Kruskal-Wallis tests, and linear regression at a significance level of 0.05. RESULTS: The tooth health perception was worse when the child had MIH, and the tooth alignment and tooth discoloration perceptions, when MIH affected the incisors. The number of teeth affected by MIH was associated with higher averages in the tooth discoloration perception. CONCLUSIONS: Children with MIH showed more negative perceptions toward tooth health, tooth alignment and tooth discoloration. However, none of the clinically investigated variables negatively impacted the reported pleasant colour question.
Assuntos
Hipoplasia do Esmalte Dentário , Dente Molar , Criança , Hipoplasia do Esmalte Dentário/epidemiologia , Estética , Humanos , Incisivo , Percepção , PrevalênciaRESUMO
OBJECTIVE: Expectant parents who live through perinatal loss experience intense grief, which is not always acknowledged or accepted. A screening tool to detect bereaved parents' grief reactions can guide professionals, including perinatal palliative care teams, to provide follow-up for those in need. This review's goal is to identify and synthesize the international published literature on existent instruments specifically measuring the grieving process after any perinatal loss and to identify factors that could moderate grief reactions. METHOD: Systematic review (PROSPERO # CRD42018092555) with critical synthesis. PUBMED, Cochrane, and PsycINFO databases were searched in English language articles using the keywords "perinatal" AND ("grief" OR "bereavement" OR mourning) AND ("scale" OR "questionnaire" OR "measure" OR "assessment") up to May 2018. Eligibility criteria included every study using a measure to assess perinatal grief after all kinds of perinatal losses, including validations and translations to other languages and interventions designed to alleviate grief symptoms. RESULTS: A total of 67 papers met inclusion criteria. Seven instruments measuring perinatal grief published between 1984 and 2002 are described. The Perinatal Grief Scale (PGS) was used in 53 of the selected studies. Of those, 39 analyzed factors associated with grief reactions. Six articles used PGS scores to evaluate pre- and post-bereavement interventions. Studies in English language only might have limited the number of articles. SIGNIFICANCE OF RESULTS: The PGS is the most used standardized measures to assess grief after perinatal loss. All parents living through any kind of perinatal loss should be screened.
Assuntos
Luto , Morte Fetal , Pesar , Feminino , Humanos , Pais , GravidezRESUMO
The aim of this study was to develop and characterize a double layer biomembrane for dual drug delivery to be used for the treatment of wounds. The membrane was composed of chitosan, hydroxypropyl methylcellulose and lidocaine chloride (anesthetic drug) in the first layer, and of sodium alginate-polymyxin B sulphate (antibiotic) nanoparticles as the second layer. A product with excellent thickness (0.01-0.02 mm), adequate mechanical properties with respect to elasticity, stiffness, tension, and compatible pH for lesion application has been successfully obtained. The incorporation of the drugs was confirmed analysing the membrane cross-sections by scanning electron microscopy. A strong interaction between the drugs and the functional groups of respective polymers was confirmed by Fourier-Transform Infrared Spectroscopy, thermal analysis and X-ray diffraction. Microbiological assays showed a high antimicrobial activity when polymyxin B was present to act against the Staphylococcus aureus and Pseudomonas aeruginosa strains. Low cytotoxicity observed in a cell viability colorimetric assay and SEM analysis suggest biocompatibility between the developed biomembrane and the cell culture. The in vivo assay allowed visualizing the healing potential by calculating the wound retraction index and by histological analysis. Our results confirm the effectiveness of the developed innovative biomaterial for tissue repair and regeneration in an animal model.
Assuntos
Quitosana , Nanopartículas , Alginatos , Animais , Bandagens , Lidocaína , Polimixinas , Espectroscopia de Infravermelho com Transformada de Fourier , CicatrizaçãoRESUMO
In Brazil, many studies on Forensic Entomology analyze the activity and succession of flies in animal models. Data on human corpses are always collected and evaluated in isolated cases. This study aimed to list the insect species associated with crime scenes investigated by the Technical-Scientific Institute of criminal expertise of the State of Rio Grande do Norte (ITEP-RN), in the Northeast of Brazil, a region exposed to high homicide rates. In total, 10 cases were investigated, of which 50% were in the initial stage of decomposition. The examined bodies were colonized by species of three orders of insects, Diptera, Coleoptera and Hymenoptera. The order Diptera represented 96% of the total insects, being represented by the following species: Chrysomya albiceps (Wiedemann) (Diptera: Calliphoridae), Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae), Chrysomya putoria (Wiedemann) (Diptera: Calliphoridae), and Cochliomyia macellaria, (Fabricius) (Diptera: Calliphoridae); Musca domestica (Linnaeus) (Diptera: Muscidae); and unidentified females of the Sarcophagidae family. Among beetles, the occurrence of Dermestes maculatus (DeGeer) (Coleoptera: Dermestidae), Necrobia rufipes (De Geer) (Coleoptera: Cleridae), and Onthophagus sp. (Scarabaeidae), as well as unidentified specimens of the families Tenebrionidae and Histeridae were recorded. In addition, specimens of Ectatomma sp. (Formicidae) were also recorded. Considering that the Rio Grande do Norte state presents a high homicide rate and the last cadaver study was conducted over a decade ago, these records update the list of species associated with cadaveric decomposition and contribute to consolidate forensic entomology in the Northeast region.