RESUMEN
The aim of this study was to inhibit adipogenic differentiation by transfecting two growth factors, platelet-derived growth factor (PDGF-BB) and bone morphogenic protein 2 (BMP-2), into modified rat bone marrow mesenchymal stem cells (rBMSCs) and then compounded with platelet-rich plasma (PRP). To achieve rBMSCs, the osteoporosis model of rats was established, and then the rBMSCs from the rats were isolated and identified. Co-transfection of rBMSCs with PDGF-BB-GFP and BMP-2 and detection of PDGF-BB/BMP-2 expression in transfected BMSCs was assessed by qRT-PCR and western blot, respectively. Moreover, the effect of the two growth factors transfection of rBMSCs on adipogenic differentiation was evaluated by oil red O staining and western blot, respectively. Finally, construction of the two growth factors transfection of rBMSCs compounded with PRP and detection of adipogenic differentiation were assessed by oil red O staining, CCK-8, and western blot, respectively. In vitro studies revealed that the two growth factors transfection of rBMSCs compounded with PRP promoted cell viability and inhibited adipogenic differentiation and could be promising for inhibiting adipogenic differentiation.
Asunto(s)
Animales , Ratas , Diferenciación Celular , Tejido Adiposo/citología , Plasma Rico en Plaquetas , Proteína Morfogenética Ósea 2/genética , Células Madre Mesenquimatosas/citología , Becaplermina/genética , Transfección , Células CultivadasRESUMEN
The aim of this study was to inhibit adipogenic differentiation by transfecting two growth factors, platelet-derived growth factor (PDGF-BB) and bone morphogenic protein 2 (BMP-2), into modified rat bone marrow mesenchymal stem cells (rBMSCs) and then compounded with platelet-rich plasma (PRP). To achieve rBMSCs, the osteoporosis model of rats was established, and then the rBMSCs from the rats were isolated and identified. Co-transfection of rBMSCs with PDGF-BB-GFP and BMP-2 and detection of PDGF-BB/BMP-2 expression in transfected BMSCs was assessed by qRT-PCR and western blot, respectively. Moreover, the effect of the two growth factors transfection of rBMSCs on adipogenic differentiation was evaluated by oil red O staining and western blot, respectively. Finally, construction of the two growth factors transfection of rBMSCs compounded with PRP and detection of adipogenic differentiation were assessed by oil red O staining, CCK-8, and western blot, respectively. In vitro studies revealed that the two growth factors transfection of rBMSCs compounded with PRP promoted cell viability and inhibited adipogenic differentiation and could be promising for inhibiting adipogenic differentiation.
Asunto(s)
Tejido Adiposo/citología , Becaplermina/genética , Proteína Morfogenética Ósea 2/genética , Diferenciación Celular , Células Madre Mesenquimatosas/citología , Plasma Rico en Plaquetas , Animales , Células Cultivadas , Ratas , TransfecciónRESUMEN
The aim of this study was to evaluate the knowledge of and attitudes toward coronavirus disease 2019 (COVID-19) among the parents of child dental patients in Shenzhen during the outbreak. A structured questionnaire containing 10 questions was used, and each question had 2 or 3 possible answers. The parents of children (aged 0-14 years) who visited the dental department of our hospital last year were eligible to participate in this study. A total of 148 parents were interviewed by telephone in February 2020 by research staff. A total of 94.59% of the parents said they paid high attention to COVID-19 and explained it to their children; 66.22% thought the dental department environment was more dangerous than other public places; 91.89% believed the dental department had a higher risk of virus infection; and 83.78% said they would take their children to a dental department if the children had a severe toothache. Approximately 81.08% of the parents expressed confidence after we informed them about the preventive measures taken in the dental department to ensure safe treatment for their children. In conclusion, all parents were concerned about COVID-19, and most of them had talked about it with their children often. In addition, a considerable percentage of them would not take their children to the dental department even if they had severe dental pain and thought that the dental environment could be more dangerous than other environments. More information about this topic should be delivered to this population.
Asunto(s)
Betacoronavirus , Infecciones por Coronavirus , Atención Dental para Niños/estadística & datos numéricos , Conocimientos, Actitudes y Práctica en Salud , Pandemias , Padres , Neumonía Viral , Adolescente , Adulto , Distribución por Edad , COVID-19 , Niño , Preescolar , China , Infecciones por Coronavirus/prevención & control , Escolaridad , Femenino , Humanos , Lactante , Entrevistas como Asunto , Masculino , Persona de Mediana Edad , Pandemias/prevención & control , Neumonía Viral/prevención & control , Factores de Riesgo , SARS-CoV-2 , Distribución por Sexo , Encuestas y Cuestionarios , Adulto JovenRESUMEN
Structural knowledge of gastropod hemocyanins is scarce. To better understand their evolution and diversity we studied the hemocyanin of a caenogastropod, Pomacea canaliculata (PcH). Through a proteomic and genomic approach, we identified 4 PcH subunit isoforms, in contrast with other gastropods that usually have 2 or 3. Each isoform has the typical Keyhole limpet-type hemocyanin architecture, comprising a string of eight globular functional units (FUs). Correspondingly, genes are organized in eight FUs coding regions. All FUs in the 4 genes are encoded by more than one exon, a feature not found in non- caenogastropods. Transmission electron microscopy images of PcH showed a cylindrical structure organized in di, tri and tetra-decamers with an internal collar structure, being the di and tri-decameric cylinders the most abundant ones. PcH is N-glycosylated with high mannose and hybrid-type structures, and complex-type N-linked glycans, with absence of sialic acid. Terminal ß-N-GlcNAc residues and nonreducing terminal α-GalNAc are also present. The molecule lacks O-linked glycosylation but presents the T-antigen (Gal-ß1,3-GalNAc). Using an anti-PcH polyclonal antibody, no cross-immunoreactivity was observed against other gastropod hemocyanins, highlighting the presence of clade-specific structural differences among gastropod hemocyanins. This is, to the best of our knowledge, the first gene structure study of a Caenogastropoda hemocyanin.
Asunto(s)
Gastrópodos/genética , Gastrópodos/metabolismo , Hemocianinas/química , Hemocianinas/genética , Animales , Evolución Molecular , Gastrópodos/química , Perfilación de la Expresión Génica , Genómica , Hemocianinas/metabolismo , Espectrometría de Masas , Microscopía Electrónica de Transmisión , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , ProteómicaRESUMEN
Abstract: The aim of this study was to evaluate the knowledge of and attitudes toward coronavirus disease 2019 (COVID-19) among the parents of child dental patients in Shenzhen during the outbreak. A structured questionnaire containing 10 questions was used, and each question had 2 or 3 possible answers. The parents of children (aged 0-14 years) who visited the dental department of our hospital last year were eligible to participate in this study. A total of 148 parents were interviewed by telephone in February 2020 by research staff. A total of 94.59% of the parents said they paid high attention to COVID-19 and explained it to their children; 66.22% thought the dental department environment was more dangerous than other public places; 91.89% believed the dental department had a higher risk of virus infection; and 83.78% said they would take their children to a dental department if the children had a severe toothache. Approximately 81.08% of the parents expressed confidence after we informed them about the preventive measures taken in the dental department to ensure safe treatment for their children. In conclusion, all parents were concerned about COVID-19, and most of them had talked about it with their children often. In addition, a considerable percentage of them would not take their children to the dental department even if they had severe dental pain and thought that the dental environment could be more dangerous than other environments. More information about this topic should be delivered to this population.
Asunto(s)
Humanos , Masculino , Femenino , Lactante , Preescolar , Niño , Adolescente , Adulto , Adulto Joven , Padres , Neumonía Viral/prevención & control , Conocimientos, Actitudes y Práctica en Salud , Infecciones por Coronavirus/prevención & control , Atención Dental para Niños/estadística & datos numéricos , Pandemias/prevención & control , Betacoronavirus , China , Entrevistas como Asunto , Encuestas y Cuestionarios , Factores de Riesgo , Distribución por Sexo , Distribución por Edad , Escolaridad , SARS-CoV-2 , COVID-19 , Persona de Mediana EdadRESUMEN
Breast milk consumption reduces the incidence of necrotizing enterocolitis (NEC) in preterm infants compared to formula. Branched-chain fatty acids (BCFAs) are present in breast milk but not in most formulas intended for preterm infants. We aimed to determine the composition of BCFAs in the breast milk of mothers with preterm infants, and to understand the impact of gestational age at birth and stage of lactation on BCFA content. The main BCFAs in preterm breast milk were iso-14:0, iso-15:0, anteiso-15:0, iso-16:0, iso-17:0, and anteiso-17:0. Breast milk BCFAs as a percent of total fatty acids (g per 100 g, %) were significantly different across lactation stages, with the highest concentration in colostrum, followed by transitional and mature breast milk (median: 0.41, 0.31, and 0.28%, respectively, p < 0.05). Lower BCFAs in preterm breast milk compared to term breast milk may have been related to maternal intake, or the ability of the mammary gland to extract BCFA from plasma, or differences in mammary gland BCFA synthesis. BCFAs were mainly in the sn-2 position (52-65%), similar to palmitic acid. Overall, preterm and term breast milk BCFAs were similar and showed specific concentration patterns, resembling 16:0 with respect to sn-2 positional distribution. BCFAs were reduced with lactation stage, similar to highly unsaturated fatty acids.
Asunto(s)
Ácidos Grasos/metabolismo , Leche Humana/química , Adulto , Calostro/química , Calostro/metabolismo , Ácidos Grasos/química , Femenino , Edad Gestacional , Humanos , Lactante , Recién Nacido/metabolismo , Lactancia , Masculino , Leche Humana/metabolismo , Estructura Molecular , EmbarazoRESUMEN
Intrafibrillar silicified collagen scaffold (SCS) is a promising biomaterial for bone regeneration because it promotes cell homing and angiogenesis in bone defects via monocyte modulation. In the present study, a rat femoral defect model was used to examine the contribution of monocyte signaling pathways to SCS modulation. Activation of the monocyte p38 signaling pathway by SCS resulted in monocyte differentiation into TRAP-positive mononuclear cells. These cells demonstrated increased secretion of SDF-1α, VEGFa and PDGF-BB, which, in turn, promoted homing of bone marrow stromal cells (BMSCs) and endothelial progenitor cells (EPCs), as well as local vascularization. Monocyte differentiation and secretion were blocked after inhibition of the p38 pathway, which resulted in reduction in cell homing and angiogenesis. Taken together, these novel findings indicate that the p38 signaling pathway is crucial in SCS-modulated monocyte differentiation and secretion, which has a direct impact on SCS-induced bone regeneration. STATEMENT OF SIGNIFICANCE: Intrafibrillar silicified collagen scaffold (SCS) is a promising biomaterial for bone regeneration. The present work demonstrates that SCS possesses favorable bone regeneration potential in a rat femoral defect model. The degrading scaffold modulates monocyte differentiation and release of certain cytokines to recruit MSCs and EPCs, as well as enhances local vascularization by activating the p38 MAPK signaling pathway. These findings indicate that SCS contributes to bone defect regeneration by stimulating host cell homing and promoting local angiogenesis and osteogenesis without the need for loading cytokines or xenogenous stem cells.
Asunto(s)
Regeneración Ósea/fisiología , Colágenos Fibrilares/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Monocitos/enzimología , Dióxido de Silicio/química , Andamios del Tejido/química , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Regeneración Ósea/efectos de los fármacos , Citocinas/metabolismo , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fémur/patología , Masculino , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Doxycycline (DC) has been shown to possess non-antibiotic properties including Fas/Fas Ligand (FasL)-mediated apoptosis against several tumor types in the concentration range of 10-40 µg/mL. However, the effect of DC in apoptotic signaling at much low concentrations was not studied. METHODS: The present study investigated the attenuation effect of low dose of DC on FasL-induced apoptosis in HeLa cell by the methods of MTT assay, fluorescence microscopy, DNA fragmentation, flow cytometry analysis, and western blotting. RESULTS AND CONCLUSION: In the present findings we showed that low concentration of DC (<2.0 µg/mL) exhibited protective effects against FasL-induced apoptosis in HeLa cells. FasL treatment to HeLa cells resulted in a concentration-dependent induction of cell death, and treatment with low concentrations of DC (0.1-2 µg/mL) significantly (p < 0.001) attenuated the FasL-induced cell death as measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Further, the FasL-induced apoptotic features in HeLa cells, such as morphological changes, DNA fragmentation and cell cycle arrest was also inhibited by DC (0.5 µg/mL). Tetracycline and minocycline also showed similar anti-apoptotic effects but were not significant when compared to DC, tested at same concentrations. Further, DC (0.01-16 µg/mL) did not influence the hydrogen peroxide- or cisplatin-induced intrinsic apoptotic pathway in HeLa cells. Protein analysis using Western blotting confirmed that FasL-induced cleavage/activation of caspase-8 and caspase-3, were inhibited by DC treatment at low concentration (0.5 µg/mL). Considering the overall data, we report for the first time that DC exhibited anti-apoptotic effects at low concentrations in HeLa cells by inhibition of caspase activation via FasL-induced extrinsic pathway.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasas/efectos de los fármacos , Doxiciclina/administración & dosificación , Proteína Ligando Fas/efectos de los fármacos , Animales , Western Blotting , Relación Dosis-Respuesta a Droga , Doxiciclina/farmacología , Activación Enzimática , Citometría de Flujo , Células HeLa , Humanos , Ratones , Células 3T3 NIHRESUMEN
BACKGROUND: Doxycycline (DC) has been shown to possess non-antibiotic properties including Fas/Fas Ligand (FasL)-mediated apoptosis against several tumor types in the concentration range of 10-40 µg/mL. However, the effect of DC in apoptotic signaling at much low concentrations was not studied. METHODS: The present study investigated the attenuation effect of low dose of DC on FasL-induced apoptosis in HeLa cell by the methods of MTT assay, fluorescence microscopy, DNA fragmentation, flow cytometry analysis, and western blotting. RESULTS AND CONCLUSION: In the present findings we showed that low concentration of DC (<2.0 µg/mL) exhibited protective effects against FasL-induced apoptosis in HeLa cells. FasL treatment to HeLa cells resulted in a concentration-dependent induction of cell death, and treatment with low concentrations of DC (0.1-2 µg/mL) significantly (p < 0.001) attenuated the FasL-induced cell death as measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide (MTT) assay. Further, the FasL-induced apoptotic features in HeLa cells, such as morphological changes, DNA fragmentation and cell cycle arrest was also inhibited by DC (0.5 µg/mL). Tetracycline and minocycline also showed similar anti-apoptotic effects but were not significant when compared to DC, tested at same concentrations. Further, DC (0.01-16 µg/mL) did not influence the hydrogen peroxide- or cisplatin-induced intrinsic apoptotic pathway in HeLa cells. Protein analysis using Western blotting confirmed that FasL-induced cleavage/activation of cas-pase-8 and caspase-3, were inhibited by DC treatment at low concentration (0.5 µg/mL). Considering the overall data, we report for the first time that DC exhibited anti-apoptotic effects at low concentrations in HeLa cells by inhibition of caspase activation via FasL-induced extrinsic pathway.
Asunto(s)
Humanos , Animales , Ratones , Apoptosis/efectos de los fármacos , Doxiciclina/administración & dosificación , Caspasas/efectos de los fármacos , Proteína Ligando Fas/efectos de los fármacos , Células HeLa , Western Blotting , Doxiciclina/farmacología , Células 3T3 NIH , Relación Dosis-Respuesta a Droga , Activación Enzimática , Citometría de FlujoRESUMEN
Although most eggs are intensely predated, the aerial egg clutches from the aquatic snail Pomacea canaliculata have only one reported predator due to unparalleled biochemical defenses. These include two storage-proteins: ovorubin that provides a conspicuous (presumably warning) coloration and has antinutritive and antidigestive properties, and PcPV2 a neurotoxin with lethal effect on rodents. We sequenced PcPV2 and studied whether it was able to withstand the gastrointestinal environment and reach circulation of a potential predator. Capacity to resist digestion was assayed using small-angle X-ray scattering (SAXS), fluorescence spectroscopy and simulated gastrointestinal proteolysis. PcPV2 oligomer is antinutritive, withstanding proteinase digestion and displaying structural stability between pH 4.0-10.0. cDNA sequencing and protein domain search showed that its two subunits share homology with membrane attack complex/perforin (MACPF)-like toxins and tachylectin-like lectins, a previously unknown structure that resembles plant Type-2 ribosome-inactivating proteins and bacterial botulinum toxins. The protomer has therefore a novel AB toxin combination of a MACPF-like chain linked by disulfide bonds to a lectin-like chain, indicating a delivery system for the former. This was further supported by observing PcPV2 binding to glycocalix of enterocytes in vivo and in culture, and by its hemaggutinating, but not hemolytic activity, which suggested an interaction with surface oligosaccharides. PcPV2 is able to get into predator's body as evidenced in rats and mice by the presence of circulating antibodies in response to sublethal oral doses. To our knowledge, a lectin-pore-forming toxin has not been reported before, providing the first evidence of a neurotoxic lectin in animals, and a novel function for ancient and widely distributed proteins. The acquisition of this unique neurotoxic/antinutritive/storage protein may confer the eggs a survival advantage, opening new perspectives in the study of the evolution of animal defensive strategies.
Asunto(s)
Toxinas Botulínicas/química , Lectinas/química , Neurotoxinas/metabolismo , Óvulo/metabolismo , Plantas/química , Proteínas Citotóxicas Formadoras de Poros/química , Caracoles/metabolismo , Secuencia de Aminoácidos , Animales , Células CACO-2 , Hemaglutinación/efectos de los fármacos , Hemólisis/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Inmunización , Mucosa Intestinal/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Neurotoxinas/química , Neurotoxinas/toxicidad , Filogenia , Conducta Predatoria , Estabilidad Proteica , Conejos , Ratas , Caracoles/inmunología , Caracoles/fisiologíaRESUMEN
This study was conducted to examine the in vitro activity of antimicrobials against Campylobacter spp. isolates from chicken and human sources and the genetic interrelation among them. During 2004-2008, a total of 173 Campylobacter spp. isolated from chicken meats (60 domestic and 62 imported chicken meats) and humans (n = 51) were tested for susceptibility to nine antimicrobials. Of 173 isolates, 140 (80.9%) showed multidrug resistance (MDR) against three to eight antimicrobials. The most frequent pattern type was MDR to four antimicrobials: ciprofloxacin, nalidixic acid, ampicillin, and tetracycline. Over 52.6% (91/173) of the isolates tested were resistant to these four antibiotics simultaneously. Especially, two and five isolates originated from Korea and Brazil showed resistance against all antibiotics tested, except for florfenicol. Further, 95% (57/60) of the isolates originated from domestic chicken showed resistance to ciprofloxacin, the antimicrobial agent of choice for treatment of human campylobacteriosis. Genotypic characterization of all Campylobacter isolates performed by pulsed-field gel electrophoresis yielded 74 types among the 173 isolates. Isolates sharing the same or similar genetic clusters were detected in different countries at different times. The pulsed-field gel electrophoresis patterns of chicken-related isolates were closely related to those of isolates from humans with gastroenteritidis. The results of this study suggest that MDR Campylobacter spp. are widespread and that Campylobacter with similar genotypes are circulating both in humans and in chicken meat in Korea.
Asunto(s)
Antibacterianos/farmacología , Infecciones por Campylobacter/microbiología , Campylobacter/efectos de los fármacos , Campylobacter/genética , Carne/microbiología , Animales , Brasil , Campylobacter/clasificación , Campylobacter/aislamiento & purificación , Pollos , ADN Bacteriano/química , Farmacorresistencia Bacteriana Múltiple , Electroforesis en Gel de Campo Pulsado , Europa (Continente) , Contaminación de Alimentos/análisis , Genotipo , Humanos , Pruebas de Sensibilidad Microbiana , República de Corea , Tailandia , Estados UnidosRESUMEN
Microbial biodiversity provides an increasingly important source of medically and industrially useful compounds. We have isolated 14 actinomycete species from a collection of approximately 300 plant stem samples from the upper Amazonian rainforest in Peru. All of the cultured isolates produce substances with inhibitory activity directed at a range of potential fungal and bacterial pathogens. For some organisms, this activity is very broad in spectrum while other organisms show specific activity against a limited number of organisms. Two of these organisms preferentially inhibit bacterial test organisms over eukaryotic organisms. rDNA sequence analysis indicates that these organisms are not equivalent to any other cultured deposits in GenBank. Our results provide evidence of the untapped biodiversity in the form of biologically active microbes present within the tissues of higher plants.
Asunto(s)
Actinobacteria/genética , Actinobacteria/aislamiento & purificación , Biodiversidad , Filogenia , Árboles/microbiología , Actinobacteria/clasificación , Actinobacteria/ultraestructura , Antibiosis , ADN Ribosómico/genética , Datos de Secuencia Molecular , Perú , ARN Bacteriano/genética , Análisis de Secuencia de ADN , Clima TropicalRESUMEN
BACKGROUND: A key argument in favor of conserving biodiversity is that as yet undiscovered biodiversity will yield products of great use to humans. However, the link between undiscovered biodiversity and useful products is largely conjectural. Here we provide direct evidence from bioassays of endophytes isolated from tropical plants and bioinformatic analyses that novel biology will indeed yield novel chemistry of potential value. METHODOLOGY/PRINCIPAL FINDINGS: We isolated and cultured 135 endophytic fungi and bacteria from plants collected in Peru. nrDNAs were compared to samples deposited in GenBank to ascertain the genetic novelty of cultured specimens. Ten endophytes were found to be as much as 15-30% different than any sequence in GenBank. Phylogenetic trees, using the most similar sequences in GenBank, were constructed for each endophyte to measure phylogenetic distance. Assays were also conducted on each cultured endophyte to record bioactivity, of which 65 were found to be bioactive. CONCLUSIONS/SIGNIFICANCE: The novelty of our contribution is that we have combined bioinformatic analyses that document the diversity found in environmental samples with culturing and bioassays. These results highlight the hidden hyperdiversity of endophytic fungi and the urgent need to explore and conserve hidden microbial diversity. This study also showcases how undergraduate students can obtain data of great scientific significance.