RESUMEN
OBJECTIVE: For treatment of medication-related osteonecrosis of the jaw, one proposed approach is the use of a topical agent to block entry of these medications in oral soft tissues. We tested the ability of phosphonoformic acid (PFA), an inhibitor of bisphosphonate entry through certain sodium-dependent phosphate contransporters (SLC20A1, 20A2, 34A1-3) as well as Dynasore, a macropinocytosis inhibitor, for their abilities to prevent zoledronate-induced (ZOL) death in human gingival fibroblasts (HGFs). METHODOLOGY: MTT assay dose-response curves were performed to determine non-cytotoxic levels of both PFA and Dynasore. In the presence of 50 µM ZOL, optimized PFA and Dynasore doses were tested for their ability to restore HGF viability. To determine SLC expression in HGFs, total HGF RNA was subjected to quantitative real-time RT-PCR. Confocal fluorescence microscopy was employed to see if Dynasore inhibited macropinocytotic HGF entry of AF647-ZOL. Endosomal acidification in the presence of Dynasore was measured by live cell imaging utilizing LysoSensor Green DND-189. As a further test of Dynasore's ability to interfere with ZOL-containing endosomal maturation, perinuclear localization of mature endosomes containing AF647-ZOL or TRITC-dextran as a control were assessed via confocal fluorescence microscopy with CellProfiler™ software analysis of the resulting photomicrographs. RESULTS: 0.5 mM PFA did not rescue HGFs from ZOL-induced viability loss at 72 hours while 10 and 30 µM geranylgeraniol did partially rescue. HGFs did not express the SLC transporters as compared to the expression in positive control tissues. 10 µM Dynasore completely prevented ZOL-induced viability loss. In the presence of Dynasore, AF647-ZOL and FITC-dextran co-localized in endosomes. Endosomal acidification was inhibited by Dynasore and perinuclear localization of both TRITC-dextran- and AF647-ZOL-containing endosomes was inhibited by 30 µM Dynasore. CONCLUSION: Dynasore prevents ZOL-induced viability loss in HGFs by partially interfering with macropinocytosis and by inhibiting the endosomal maturation pathway thought to be needed for ZOL delivery to the cytoplasm.
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Supervivencia Celular , Difosfonatos , Endosomas , Fibroblastos , Encía , Hidrazonas , Imidazoles , Ácido Zoledrónico , Ácido Zoledrónico/farmacología , Humanos , Fibroblastos/efectos de los fármacos , Encía/efectos de los fármacos , Encía/citología , Difosfonatos/farmacología , Imidazoles/farmacología , Supervivencia Celular/efectos de los fármacos , Endosomas/efectos de los fármacos , Hidrazonas/farmacología , Células Cultivadas , Factores de Tiempo , Reacción en Cadena en Tiempo Real de la Polimerasa , Conservadores de la Densidad Ósea/farmacología , Reproducibilidad de los Resultados , Microscopía Confocal , Relación Dosis-Respuesta a Droga , Pinocitosis/efectos de los fármacosRESUMEN
PURPOSE: Napabucasin (NP) is a natural compound that can suppress cancer cell proliferation and cell cycle by inhibition of the signal transducer and activator of transcription 3 (STAT3) gene. We examined the effects of NP on the proliferation and invasion of neuroblastoma cells (SH-SY5Y). METHODS: Human neuroblastoma SH-SY5Y cell line was used in this study. NP was administered to groups at the doses of 0.3-1 µM. Cell viability was analyzed by MTT assay. Real-time quantitative reverse transcription polymerase chain reaction and western blot analysis assessed the expressions of interleukin (IL)-6 dependent Jak2/Stat3 signaling pathway. The MTT cell viability method was applied to determine the antagonistic-synergistic effects and inhibitory concentration (IC50) doses of doxorubicin (DX) and NP. RESULTS: It was determined that 0.3-1 µM doses of NP killed the cells almost completely after 48 hours, and also that Jak2/Stat3 expressions decreased dose-dependently via IL-6. At the protein level, NP and DX were found to reduce Jak2 and Stat3 levels. CONCLUSIONS: NP showed that it suppresses the proliferation of neuroblastoma cells. Due to its inhibitory effect on Jak2 and Stat3, it can be used to prevent invasion of SH-SY5Y cells. NP, which can inactivate Jak2/Stat3, can be used as a treatment agent by combining with DX in proliferation pathway in neuroblastoma.
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Benzofuranos , Proliferación Celular , Supervivencia Celular , Doxorrubicina , Janus Quinasa 2 , Neuroblastoma , Factor de Transcripción STAT3 , Transducción de Señal , Humanos , Janus Quinasa 2/metabolismo , Janus Quinasa 2/efectos de los fármacos , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Línea Celular Tumoral , Transducción de Señal/efectos de los fármacos , Doxorrubicina/farmacología , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/patología , Supervivencia Celular/efectos de los fármacos , Benzofuranos/farmacología , Interleucina-6/metabolismo , Western Blotting , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , NaftoquinonasRESUMEN
This study aimed to illustrate the biological behavior and changes in cell function during the progression of apical periodontitis in deciduous teeth and to explore the underlying molecular mechanism. Deciduous teeth periodontal ligament stem cells (DePDLSCs) were derived and their identity was confirmed. The viability, inflammation, and osteogenic ability of cells were tested by exposing them to various concentrations of lipopolysaccharide (LPS) (0-100 µg/mL) using the cell counting kit-8 (CCK-8) assay, reverse transcription polymerase chain reaction (real-time PCR), alkaline phosphatase (ALP) staining, and ALP activity assay. In addition, osteogenic-induced cells with and without 10 µg/mL LPS were harvested for high-throughput sequencing. Based on sequencing data, proinflammatory factors and ALP expression were measured after interference with the PI3K-AKT signaling pathway activator, 740Y-P. LPS biphasically affected the proliferation and osteogenesis of DePDLSCs. Low concentrations of LPS showed stimulatory effects, whereas inhibitory effects were observed at high concentrations. Sequencing analysis showed that the PI3K-AKT signaling pathway was significantly downregulated when DePDLSCs were treated with 10 µg/mL LPS. The LPS-induced inflammation and osteogenesis inhibition of DePDLSCs were partially rescued by 740Y-P treatment. In conclusion, LPS affected DePDLSCs proliferation and osteogenesis in a biphasic manner. Moderate activation of PI3K-AKT signaling pathway was beneficial for osteogenic differentiation and anti-inflammatory effect in DePDLSCs. This research may provide etiological probes for apical periodontitis and its treatment.
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Osteogénesis , Ligamento Periodontal , Células Madre , Diente Primario , Ligamento Periodontal/citología , Ligamento Periodontal/efectos de los fármacos , Humanos , Osteogénesis/efectos de los fármacos , Osteogénesis/fisiología , Diente Primario/citología , Células Madre/efectos de los fármacos , Lipopolisacáridos/farmacología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inflamación , Transducción de Señal/efectos de los fármacos , Células Cultivadas , Reacción en Cadena en Tiempo Real de la Polimerasa , Fosfatasa Alcalina/metabolismo , Fosfatasa Alcalina/análisisRESUMEN
Early postnatal administration of antibiotics has been linked to lasting effects on brain development and behavior. Research conducted on animals that are free from germs has demonstrated that the impact of microbiome colonization on the regulation of the hypothalamic-pituitary-adrenal (HPA) axis and neuroendocrine pathways is substantial, which play a crucial role in stress management. Nevertheless, it is still uncertain if the exposure to antibiotics in rat dams (F0-generation) before weaning is associated with neurobehavioral changes in rat offspring (F1-generation) during adulthood. In order to investigate the effects, we perturbed the intestinal microbiota of rat dams (F0 generation) by administering cefixime (CEF), an antibiotic commonly used for obstetric purposes, at clinically relevant doses (1 mg/kg, 2.5 mg/kg or 5 mg/kg). Anxiety-like behaviors in adult offspring was evaluated through the utilization of elevated plus maze (EPM) and open field paradigm (OFP) following a six-week interval from birth (PND42). Subsequent to behavioral assessments, the rats were euthanized, and their brains and blood was collected for biochemical analysis. Plasma corticosterone concentration was used to assess HPA activity, whereas the quantitative real-time polymerase chain reaction (PCR) was employed to determine the transcription levels of the glucocorticoid receptor (GR) Nr3c1. The offspring of F1 that were administered antibiotics before being weaned spent less time in the EPM open arm. The alterations were accompanied by increased levels of corticosterone in the bloodstream. The gene expression study revealed a decrease in the levels of mRNA transcription of Nr3c1. This research emphasizes the possible long-term effects of antibiotic exposure before weaning on the development of anxiety in offspring upon adulthood.
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Antibacterianos , Ansiedad , Regulación hacia Abajo , Sistema Hipotálamo-Hipofisario , Sistema Hipófiso-Suprarrenal , Ratas Wistar , Receptores de Glucocorticoides , Animales , Receptores de Glucocorticoides/metabolismo , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Sistema Hipófiso-Suprarrenal/efectos de los fármacos , Femenino , Antibacterianos/farmacología , Masculino , Regulación hacia Abajo/efectos de los fármacos , Ratas , Corticosterona/sangre , Embarazo , Efectos Tardíos de la Exposición Prenatal , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Prueba de Campo Abierto/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa , Animales Recién NacidosRESUMEN
Bartonelloses represent a group of potentially fatal diseases associated with various clinical manifestations including endocarditis. Caused by bacteria belonging to the genus Bartonella, these microorganisms have a remarkable ability to infect mammals, and their transmission is commonly associated with hematophagous vectors such as fleas, lice, mosquitoes, and ticks. The aim of this study was to evaluate the occurrence of Bartonella sp. DNA in 81 triatomines of the species Triatoma sordida collected in the field in peridomiciliary areas of the Brazilian city of Seabra, located in the state of Bahia. Nested PCR was conducted targeting the ftsZ gene and real-time PCR targeting the gltA gene, both representing specific reactions for Bartonella henselae. Additionally, conventional PCR targeting kDNA was employed to evaluate the presence of Trypanosoma cruzi. Of the samples tested, 23/81 (28.39 %) bugs showed positive PCR for B. henselae. No sample showed positive PCR for T. cruzi. The high prevalence of triatomines with a positive PCR for B. henselae emphasizes the close relationship between these insects and the bacteria, indicating the need for further studies to investigate the vectorial potential of these kissing bugs.
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Bartonella henselae , ADN Bacteriano , Insectos Vectores , Triatoma , Animales , Triatoma/microbiología , Triatoma/parasitología , Bartonella henselae/genética , Bartonella henselae/aislamiento & purificación , Insectos Vectores/microbiología , ADN Bacteriano/análisis , Brasil , Reacción en Cadena de la Polimerasa , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Python regius or ball pythons are the famous exotic pets because of their beautiful color and pattern. The albino ball python is one type of ball python, but it is very difficult to determine the difference of phenotype between wildtype and heterozygous genotype of albino (het albino). In this study, PCR and qPCR can distinguish between wildtype and het albino. The PCR product size of wildtype and het albino was 415 bp, but the intensity of PCR product of wildtype was more intense than that of het albinos. No PCR amplicon was found in albinos and the Ct value of wildtype was lower than Ct of het albinos. The molecular detection technique, especially PCR and qPCR, can determine the difference between wildtype and het albinos of ball pythons.
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Boidae , Genotipo , Reacción en Cadena de la Polimerasa , Reacción en Cadena en Tiempo Real de la Polimerasa , Animales , Boidae/genética , Boidae/clasificación , Heterocigoto , Fenotipo , Pigmentación/genéticaRESUMEN
Peripheral regulation emerges as a promising intervention in the early stages of Alzheimer's disease (AD). The hub genes in the peripheral blood of MCI patients from GEO database (GSE63060, GSE63061) were screened using weighted gene co-expression analysis (WGCNA). Meanwhile, behavioral tests, HE staining and Nissl staining were used to detect the memory impairment and histopathological changes in 24-week-old male 3×Tg-AD mice. Thioflavin-S and immunohistochemical staining were used to determine the Aß deposition in both intracellular and extracellular neurons. Subsequently, the MCI-hub genes were verified by quantitative real-time PCR (qRT-PCR) in the peripheral blood of 3×Tg-AD mice. The research revealed ten hub genes associated with MCI were identified WGCNA. Short-term memory loss, intracellular Aß deposition and limited of extracellular amyloid plaques in 3×Tg-AD mice. The qRT-PCR analysis of peripheral blood from these mice revealed significantly down-regulation in the expression levels of ATP5C1, ITGB2, EFTUD2 and RPS27A genes; whereas the expression level of VCP gene was significantly up-regulated. These findings confirmed that 24-week-old male 3×Tg-AD mice were a valuable animal model for simulating the early symptomatic stages of AD. Additionally, the peripheral blood MCI-hub genes related to immune response, energy metabolism and ribosomal coding efficiency provide potential biomarkers for this stage.
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Enfermedad de Alzheimer , Modelos Animales de Enfermedad , Ratones Transgénicos , Proteínas tau , Animales , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/sangre , Masculino , Ratones , Proteínas tau/genética , Precursor de Proteína beta-Amiloide/genética , Presenilina-1/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Redes Reguladoras de GenesRESUMEN
Several reptile species have been described as hosts of Trypanosoma cruzi, the causative agent of Chagas disease, and therefore, they have become vertebrates of epidemiological interest. In recent decades, there has been a growing interest in animal welfare, especially in populations with small numbers where lethal sampling could have catastrophic consequences, and non-lethal methodologies have been developed for detecting zoonotic parasites. In this study, we compared three non-lethal sampling methodologies for detecting T. cruzi DNA in 21 captured specimens of the native lizard Liolaemus monticola, collected from the semiarid Mediterranean ecosystem of Chile. Specimens were subjected to xenodiagnosis (XD), tail clipping, and living syringe sampling procedures to evaluate whether lizards could serve as sentinel species for T. cruzi in endemic regions. To detect the protozoan, real-time PCR (qPCR) was performed on the DNA extracted from the samples (intestinal contents, tail tissues, and blood from living syringes). Trypanosoma cruzi DNA was detected in 12 of 21 lizards, considering all three methodologies. By XD, 12 specimens showed infection (57.1 %), and both living syringe and tail sampling methodologies detected only one infected lizard (4.8 %). Therefore, T. cruzi can be detected in lizards by qPCR using the three methodologies but XD is by far the most effective non-lethal detection methodology. The use of tail and living syringe methodologies showed a large underestimation; however, they might be options for monitoring the presence of T. cruzi in lizard populations when large sample sizes are available.
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Enfermedad de Chagas , ADN Protozoario , Reservorios de Enfermedades , Lagartos , Trypanosoma cruzi , Animales , Lagartos/parasitología , Trypanosoma cruzi/aislamiento & purificación , Trypanosoma cruzi/genética , Chile/epidemiología , ADN Protozoario/análisis , ADN Protozoario/aislamiento & purificación , Enfermedad de Chagas/veterinaria , Enfermedad de Chagas/diagnóstico , Enfermedad de Chagas/parasitología , Enfermedad de Chagas/epidemiología , Reservorios de Enfermedades/parasitología , Reservorios de Enfermedades/veterinaria , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Zoonosis/parasitologíaRESUMEN
BACKGROUND: Circular RNAs (circRNAs) hold critical importance due to their notable function in developing Gastric Cancer (GC), which is a malignancy with the third most frequent occurrence worldwide. The aim of this study was to see if circRNA_0044516 would control GC cell proliferation and establish more effective therapeutic strategies. METHODS: In GC tissues or cells, quantitative RealTime Polymerase Chain Reaction (qRT-PCR) was employed for the detection of the expression of circRNA_100349, Insulin-like Growth Factor II (IGF2), and miR-218-5p. CCK-8 assays were employed to gauge the proliferation of cells. A luciferase reporter was employed to establish the relationship of circRNA_100349 or IGF2 with miR-218-5p. RESULTS: CircRNA_100349 was observed to undergo upregulation in GC cell lines along with tissues. GC cell proliferation was prevented by downregulating circRNA_100349. MiR-149 was targeted by CircRNA_100349, and its downregulation increased the amount of miR-218-5p in GC cells. Simultaneously silencing circRNA_100349 decreased IGF2 expression via miR-218-5p, and thus suppressed GC cell proliferation. Furthermore, in nude mice, circRNA_100349 knockdown prevented the tumor development of GC cells. CONCLUSIONS: The findings furnished evidence of the critical involvement of circRNA_100349 in GC and that its downregulation impedes GC cell proliferation via the miR-218-5p/IGF2 axis.
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Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Factor II del Crecimiento Similar a la Insulina , MicroARNs , ARN Circular , Neoplasias Gástricas , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Neoplasias Gástricas/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , ARN Circular/genética , Proliferación Celular/genética , Humanos , Factor II del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/genética , Animales , Regulación hacia Abajo , Regulación hacia Arriba , Ratones Desnudos , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , MasculinoRESUMEN
BACKGROUND: B chromosomes are extra non-essential elements present in several eukaryotes. Unlike A chromosomes which are essential and present in all individuals of a species, B chromosomes are not necessary for normal functioning of an organism. Formerly regarded as genetically inactive, B chromosomes have been discovered to not only express their own genes, but also to exert influence on gene expression in A chromosomes. Recent studies have shown that, in some Psalidodon (Characiformes, Characidae) species, B chromosomes might be associated with phenotypic effects, such as changes in the reproductive cycle and gene expression. METHODS AND RESULTS: In this study, we aimed to establish stable reference genes for RT-qPCR experiments conducted on gonads of three fish species within Psalidodon genus, both in the presence and absence of B chromosomes. The stability of five selected reference genes was assessed using NormFinder, geNorm, BestKeeper, and RefFinder algorithms. We determined ppiaa and pgk1 as the most stable genes in P. fasciatus, whereas ppiaa and hmbsa showed the highest stability in P. bockmanni. For P. paranae, tbp and hprt1 were the most stable genes in females, and ppiaa and hprt1 were the most stable in males. CONCLUSIONS: We determined the most stable reference genes in gonads of three Psalidodon species considering the presence of B chromosomes. This is the first report of reference gene stability in the genus and provides valuable tools to better understand the effects of B chromosomes at gene expression level.
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Cromosomas , Animales , Masculino , Femenino , Cromosomas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Estándares de Referencia , Perfilación de la Expresión Génica/métodos , Perfilación de la Expresión Génica/normas , Gónadas/metabolismo , Characidae/genética , Characiformes/genéticaRESUMEN
OBJECTIVE: Ghrelin is an adipokine the placenta generates to control the maternal metabolic adaptation to pregnancy. It causes different pregnancy complications like preeclampsia (PE). Therefore, the aim of this study was to assess the association between ghrelin mRNA expression and rs26311 and rs27647 polymorphisms and PE development. METHODS: In total, 156 PE women (including 97 patients with mild PE and 59 patients with severe PE) and 152 healthy controls were recruited in this case-control study during 2019-2020. All participants with other diseases have been excluded from both groups. The ghrelin expression was analyzed with real-time PCR, and ghrelin variants were examined using the RFLP-PCR method. RESULTS: The maternal and placental ghrelin rs27647 and rs26311 variants were unrelated to PE susceptibility. Haplotype analyses showed no significant difference between the four haplotypes and PE. No relationship was revealed between rs27647 polymorphism and severe PE. However, the results indicated a relationship between rs27647 and severe PE compared to mild PE and controls. Therefore, the rs27647 variant was associated with severe PE compared to mild PE in codominant, recessive, and log-additive models and controls in codominant, dominant, recessive, and log-additive models. The placental ghrelin mRNA expression declined in PE women compared to controls (0.67-fold), but the difference was insignificant (p=0.263). No significant difference was found between various genotypes of rs27647 and rs26311 polymorphisms concerning ghrelin mRNA expression. CONCLUSION: The maternal and placental ghrelin polymorphisms, rs27647 and rs26311, showed no effect on PE. However, the rs27647 variant was associated with severe PE.
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Predisposición Genética a la Enfermedad , Ghrelina , Polimorfismo de Nucleótido Simple , Preeclampsia , ARN Mensajero , Índice de Severidad de la Enfermedad , Humanos , Femenino , Preeclampsia/genética , Ghrelina/genética , Embarazo , Estudios de Casos y Controles , Adulto , Predisposición Genética a la Enfermedad/genética , ARN Mensajero/genética , Haplotipos , Genotipo , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto JovenRESUMEN
OBJECTIVE: Osteoporosis, defined as a systemic skeletal disease, is characterized by increased bone fragility and fracture risk. Studies have shown that dysregulation of the functions of miRNAs or the mechanisms they mediate may be an important pathological factor in bone degeneration. Therefore, the aim of the study was to determine the role of miRNAs, which are thought to play a role in bone metabolism, in osteoporosis. METHODS: The study included 48 patients who were diagnosed with osteoporosis according to the results of a bone mineral density assessment by quantitative computed tomography and 36 healthy individuals. MiRNAs from plasma samples obtained from blood samples taken into ethylenediaminetetraacetic acid (EDTA) tubes were isolated with the miRNA isolation kit and converted to cDNA. Expression analysis of miR-21-5p, miR-34a-5p, miR-210, miR-122-5p, miR-125b-5p, miR-133a, miR-143-3p, miR-146a, miR-155-5p, and miR-223 was performed on the real-time PCR (RT-PCR) device. RESULTS: When miRNA expression levels in the patient group were compared with the control group, all miRNAs were found to be downregulated in the patients. When fold changes in expression levels in the patient group were examined, significant differences were found in miR-21-5p, miR-133a, mir143-3p, miR-210, and miR-223. In the receiver operating curve analysis, area under the curve=0.882 for the combination of miR-34, miR-125, miR-133, and miR-210. CONCLUSION: In this study, it was determined that the combined effects of miRNAs, as well as their single effects, were effective in the development of osteoporosis. Therefore, a miRNA panel to be created can make a significant contribution to the development of novel diagnostic and treatment approaches for this disease.
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MicroARN Circulante , MicroARNs , Osteoporosis , Reacción en Cadena en Tiempo Real de la Polimerasa , Humanos , Osteoporosis/genética , Osteoporosis/sangre , Femenino , Persona de Mediana Edad , Masculino , Estudios de Casos y Controles , MicroARN Circulante/sangre , MicroARN Circulante/genética , MicroARNs/sangre , Anciano , Densidad Ósea/genética , Biomarcadores/sangre , Regulación hacia Abajo , AdultoRESUMEN
Rodents are commonly used as animal models in studies investigating various experimental conditions, often requiring gene expression analysis. Quantitative real-time reverse transcription PCR (RT-qPCR) is the most widely used tool to quantify target gene expression levels under different experimental conditions in various biological samples. Relative normalization with reference genes is a crucial step in RT-qPCR to obtain reliable quantification results. In this work, the main reference genes used in gene expression studies among the three rodents commonly employed in scientific research-hamster, rat, and mouse-are analyzed and described. An individual literature search for each rodent was conducted using specific search terms in three databases: PubMed, Scopus, and Web of Science. A total of 157 articles were selected (rats = 73, mice = 79, and hamsters = 5), identifying various reference genes. The most commonly used reference genes were analyzed according to each rodent, sample type, and experimental condition evaluated, revealing a great variability in the stability of each gene across different samples and conditions. Classic genes, which are expected to be stably expressed in both samples and conditions analyzed, demonstrated greater variability, corroborating existing concerns about the use of these genes. Therefore, this review provides important insights for researchers seeking to identify suitable reference genes for their validation studies in rodents.
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Perfilación de la Expresión Génica , Estándares de Referencia , Roedores , Animales , Ratones , Ratas , Expresión Génica/genética , Perfilación de la Expresión Génica/métodos , Perfilación de la Expresión Génica/normas , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Roedores/genéticaRESUMEN
Gene expression through RT-qPCR can be performed by the relative quantification method, which requires the expression normalization through reference genes. Therefore, it is essential to validate, experimentally, the candidate reference genes. Thus, although there are several studies that are performed to identify the most stable reference genes, most them validate genes for very specific conditions, not exploring the whole potential of the research since not all possible combinations of treatments and/or conditions of the study are explored. For this reason, new experiments must be conducted by researchers that have interest in analyzing gene expression of treatments and/or conditions present, but not explored, in these studies. Here, we present the RGeasy tool, which aims to facilitate the selection of reference genes, allowing the user to choose genes for a greater number of combinations of treatments/conditions, compared to the ones present in the original articles, through just a few clicks. RGeasy was validated with RT-qPCR data from gene expression studies performed in two coffee species, Coffea arabica and Coffea canephora, and it can be used for any animal, plant or microorganism species. In addition to displaying a rank of the most stable reference genes for each condition or treatment, the user also has access to the primer pairs for the selected reference genes.
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Perfilación de la Expresión Génica , Reacción en Cadena en Tiempo Real de la Polimerasa , Estándares de Referencia , Programas Informáticos , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Perfilación de la Expresión Génica/métodos , Perfilación de la Expresión Génica/normas , Genes de Plantas , Coffea/genética , Regulación de la Expresión Génica de las PlantasRESUMEN
Aquatic systems have traditionally played a key role in the development of human life, providing multiple ecosystem services to society and being a reservoir for a wide biodiversity of organisms. Among them, bacteria belonging to Legionella stand out, mainly because they are of great interest both in the field of microbial ecology and public health, since some of them turn out to be pathogenic for humans. The aim of this work was to study the monthly temporal dynamics of Legionella spp. and its relationship with the environmental variables measured in two Pampean shallow lakes (Gómez and Carpincho, Buenos Aires Province, Argentina). The analysis was carried out using a quantitative approach by real-time polymerase chain reaction (qPCR) and a non-quantitative approach using bacterial diversity data obtained by next-generation sequencing (NGS), using the Illumina MiSeq platform. Our results showed that the overall Legionella abundance was very high in the studied Pampean shallow lakes. Notably, fluctuations in dissolved organic carbon and temperature influenced the dynamics shifts in Legionella abundances. Correlation analyses between Legionella reads from NGS and copy numbers obtained through qPCR revealed positive relationships, unveiling distinctions attributable to the diverse sequence processing algorithms employed in the analysis of NGS data.
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Lagos , Legionella , Argentina , Lagos/microbiología , Legionella/genética , Microbiología del Agua , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Abdominal angiostrongyliasis (AA) is a zoonotic and severe parasitic infection caused by Angiostrongylus costaricensis. AA is currently diagnosed by the observation of A. costaricensis-compatible structures in biopsies or the detection of antibodies in serological tests. However, molecular methods targeting homologous sequences of A. costaricensis have not been designed before, and therefore, an HRM-coupled qPCR was developed to detect the internal transcribed spacer 1 (ITS1) of the parasite. The present assay successfully amplified DNA of A. costaricensis obtained from different hosts and identified slight sequence differences through the HRM analysis. The detection limit of the HRM-qPCR was 0.00036 ng/µL, 1.0 ng/µL, and 0.1 ng/µL when A. costaricensis DNA was diluted in nuclease-free water, whole blood, and sera, respectively, which highlights its potential use for cell-free DNA detection. Moreover, the reaction did not cross-amplify DNA of Angiostrongylus cantonensis, Strongyloides stercoralis, and other nematodes, thus emphasizing its specificity. Additionally, the assay tested positive in formalin-fixed paraffin embedded biopsies with visible A. costaricensis adults or eggs, but not in samples without evident parasites or a low number of larvae, which suggests that the reaction is useful for confirming the presence of the nematode in clinical samples. Finally, DNA of sera from patients with AA was evaluated with the HRM-qPCR but none tested positive, possibly due to long storage periods of the samples which could have led to cfDNA degradation. These results indicate that this assay may be useful in the confirmation of AA and its prospection for cell-free DNA detection protocols.
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Angiostrongylus , ADN de Helmintos , ADN Espaciador Ribosómico , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y Especificidad , Infecciones por Strongylida , Animales , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Angiostrongylus/genética , Angiostrongylus/aislamiento & purificación , Infecciones por Strongylida/diagnóstico , Infecciones por Strongylida/parasitología , ADN Espaciador Ribosómico/genética , ADN de Helmintos/genética , Humanos , Temperatura de Transición , Técnicas de Diagnóstico Molecular/métodosRESUMEN
Emerging evidence demonstrates that curcumin has an inhibitory effect on non-small cell lung cancer (NSCLC), and its targets and mechanism of action need further exploration. The goal of this study was to explore the potential targets and mechanism of curcumin against NSCLC by network pharmacology, bioinformatics, and experimental validation, thereby providing more insight into combination treatment with curcumin for NSCLC in preclinical and clinical research. Curcumin targets against NSCLC were predicted based on HIT2.0, STD, CTD, and DisGeNET, and the core targets were analyzed via protein-protein interaction network construction (PPI), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and molecular docking. The gene expression levels of samples in A549 cells, NCI-H460, and curcumin treated groups were detected by real-time quantitative PCR. A total of 67 common targets between curcumin and NSCLC were collected by screening public databases. GO and KEGG analysis suggested that curcumin treatment of NSCLC mainly involves cancer-related pathways, such as PI3K-AKT signaling pathway, Foxo signaling pathway, microRNAs, MAPK signaling pathway, HIF-1 signaling pathway, etc. The targets with the highest degree were identified through the PPI network, namely CASP3, CTNNB1, JUN, IL6, MAPK3, HIF1A, STAT3, AKT1, TP53, CCND1, VEGFA, and EGFR. The results of the in vitro experiments showed that curcumin treatment of NSCLC down-regulated the gene expressions of CCND1, CASP3, HIF1A, IL-6, MAPK3, STAT3, AKT1, and TP53. Our findings revealed that curcumin functions as a potential therapeutic candidate for NSCLC by suppressing multiple signaling pathways and interacting with multiple gene targets.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Biología Computacional , Curcumina , Neoplasias Pulmonares , Simulación del Acoplamiento Molecular , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Humanos , Curcumina/farmacología , Curcumina/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Mapas de Interacción de Proteínas/efectos de los fármacos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Transducción de Señal/efectos de los fármacos , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
OBJECTIVES: This study aimed to investigate the osseointegration of titanium (Ti) implants with micro-nano textured surfaces functionalized with strontium additions (Sr) in a pre-clinical rat tibia model. METHODOLOGY: Ti commercially pure (cp-Ti) implants were installed bilaterally in the tibia of 64 Holtzman rats, divided into four experimental groups (n=16/group): (1) Machined surface - control (C); (2) Micro-nano textured surface treatment (MN); (3) Micro-nano textured surface with Sr2+ addition (MNSr); and (4) Micro-nano textured surface with a higher complementary addition of Sr2+ (MNSr+). In total, two experimental euthanasia periods were assessed at 15 and 45 days (n=8/period). The tibia was subjected to micro-computed tomography (µ-CT), histomorphometry with the EXAKT system, removal torque (TR) testing, and gene expression analysis by PCR-Array of 84 osteogenic markers. Gene expression and protein production of bone markers were performed in an in vitro model with MC3T3-E1 cells. The surface characteristics of the implants were evaluated by scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS), and laser scanning confocal microscopy. RESULTS: SEM, confocal, and EDS analyses demonstrated the formation of uniform micro-nano textured surfaces in the MN group and Sr addition in the MNSr and MNSr+ groups. TR test indicated greater osseointegration in the 45-day period for treated surfaces. Histological analysis highlighted the benefits of the treatments, especially in cortical bone, in which an increase in bone-implant contact was found in groups MN (15 days) and MNSr (45 days) compared to the control group. Gene expression analysis of osteogenic activity markers showed modulation of various osteogenesis-related genes. According to the in vitro model, RT-qPCR and ELISA demonstrated that the treatments favored gene expression and production of osteoblastic differentiation markers. CONCLUSIONS: Micro-nano textured surface and Sr addition can effectively improve and accelerate implant osseointegration and is, therefore, an attractive approach to modifying titanium implant surfaces with significant potential in clinical practice.
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Implantes Dentales , Oseointegración , Estroncio , Propiedades de Superficie , Tibia , Titanio , Microtomografía por Rayos X , Titanio/química , Oseointegración/efectos de los fármacos , Animales , Estroncio/farmacología , Estroncio/química , Factores de Tiempo , Tibia/efectos de los fármacos , Tibia/cirugía , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Ensayo de Materiales , Masculino , Osteogénesis/efectos de los fármacos , Microscopía Electrónica de Rastreo , Ratones , Torque , Expresión Génica/efectos de los fármacos , Análisis de Varianza , Reacción en Cadena en Tiempo Real de la Polimerasa , Ratas , Nanoestructuras , Valores de ReferenciaRESUMEN
OBJECTIVES: This study sought to determine effects of Thai propolis extract mixed in mineral trioxide aggregate (MTA) on matrix metalloproteinase-2 (MMP-2) expression and its activity in inflamed human dental pulp cells (HDPCs). MATERIALS AND METHODS: Interleukin-1ß-primed HDPCs were treated with either the eluate of MTA mixed with distilled water, of MTA mixed with 0.75 mg/ml of the propolis extract, or of Dycal®, 0.75 mg/ml of the propolis extract, or 0.2% (v/v) of chlorhexidine for 24 or 72 h. The viability of HDPCs was determined by the PrestoBlue® cytotoxic assay. HDPCs' lysates were analyzed for MMP-2 mRNA expression by RT-qPCR, while their supernatants were measured for MMP-2 activity by gelatin zymography. RESULTS: At 24 and 72 h, a non-toxic dose of the propolis extract at 0.75 mg/ml by itself or mixed in MTA tended to reduce MMP-2 expression upregulated by MTA, while it further decreased the MMP-2 activity as compared to that of MTA mixed with distilled water. The MMP-2 activity of interleukin-1ß-primed HDPCs treated with the eluate of the propolis extract mixed in MTA was significantly lower than that of interleukin-1ß-primed HDPCs at 24 h (p=0.012). As a control, treatment with chlorhexidine significantly inhibited MMP-2 expression induced by MTA and MMP-2 activity enhanced by interleukin-1ß (p<0.05). Treatment with Dycal® caused a significant increase in HDPC's death, resulting in a significant decrease in MMP-2 expression and activity (p<0.05). CONCLUSIONS: MTA mixed with Thai propolis extract can reduce MMP-2 mRNA expression and activity when compared to MTA mixed with distilled water in inflamed HDPCs.
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Compuestos de Aluminio , Compuestos de Calcio , Pulpa Dental , Metaloproteinasa 2 de la Matriz , Óxidos , Própolis , Silicatos , Compuestos de Aluminio/farmacología , Compuestos de Calcio/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Clorhexidina/farmacología , Pulpa Dental/efectos de los fármacos , Pulpa Dental/citología , Combinación de Medicamentos , Interleucina-1beta , Ensayo de Materiales , Metaloproteinasa 2 de la Matriz/efectos de los fármacos , Óxidos/farmacología , Própolis/farmacología , Própolis/química , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , ARN Mensajero/efectos de los fármacos , Silicatos/farmacología , Tailandia , Factores de Tiempo , HumanosRESUMEN
The aim of this work was to develop a real-time PCR assay with a TaqMan® probe that detects a species-specific part of the 16S rDNA gene of Ehrlichia canis. Canine blood samples (n = 207), collected and tested by a conventional PCR assay within a study conducted by De Salvo et al., were simultaneously analyzed with the novel designed real-time PCR, and the results of both assays were compared. The agreement between the two methods was 97.6 % with a kappa value of 0.92186. Hereby, the standard error was 0.034416 and the 95 % confidence interval from 0.8544 to 0.98931. While the conventional PCR assay showed false negative results (2.42 %; 5/207), the real-time PCR assays showed a specificity of 100 %. The results of the current study showed that the developed assay presents sensitivity and specificity for the detection of E. canis in blood samples, adding a new tool for the diagnosis of this pathogen.