RESUMEN
Mitochondria are versatile organelles that continuously change their morphology via fission and fusion. However, the detailed functions of mitochondrial dynamics-related genes in pluripotent stem cells remain largely unclear. Here, we aimed to determine the effects on energy metabolism and differentiation ability of mouse embryonic stem cells (ESCs) following deletion of the mitochondrial fission-related gene Dnml1. Resultant Dnm1l-/- ESCs maintained major pluripotency characteristics. However, Dnm1l-/- ESCs showed several phenotypic changes, including the inhibition of differentiation ability (dissolution of pluripotency). Notably, Dnm1l-/- ESCs maintained the expression of the pluripotency marker Oct4 and undifferentiated colony types upon differentiation induction. RNA sequencing analysis revealed that the most frequently differentially expressed genes were enriched in the glutathione metabolic pathway. Our data suggested that differentiation inhibition of Dnm1l-/- ESCs was primarily due to metabolic shift from glycolysis to OXPHOS, G2/M phase retardation, and high level of Nanog and 2-cell-specific gene expression.
Asunto(s)
Ciclo Celular , Dinaminas , Glucólisis , Células Madre Embrionarias de Ratones , Células Madre Pluripotentes , Animales , Ratones , Diferenciación Celular/genética , División Celular , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Pluripotentes/metabolismo , Dinaminas/genética , Dinaminas/fisiología , Eliminación de Gen , Glucólisis/genéticaRESUMEN
Gut microbes play diverse roles in modulating host fitness, including longevity; however, the molecular mechanisms underlying their mediation of longevity remain poorly understood. We performed genome-wide screens using 3,792 Escherichia coli mutants and identified 44 E. coli mutants that modulated Caenorhabditis elegans longevity. Three of these mutants modulated C. elegans longevity via the bacterial metabolite methylglyoxal (MG). Importantly, we found that low MG-producing E. coli mutants, Δhns E. coli, extended the lifespan of C. elegans through activation of the DAF-16/FOXO family transcription factor and the mitochondrial unfolded protein response (UPRmt). Interestingly, the lifespan modulation by Δhns did not require insulin/insulin-like growth factor 1 signaling (IIS) but did require TORC2/SGK-1 signaling. Transcriptome analysis revealed that Δhns E. coli activated novel class 3 DAF-16 target genes that were distinct from those regulated by IIS. Taken together, our data suggest that bacteria-derived MG modulates host longevity through regulation of the host signaling pathways rather than through nonspecific damage on biomolecules known as advanced glycation end products. Finally, we demonstrate that MG enhances the phosphorylation of hSGK1 and accelerates cellular senescence in human dermal fibroblasts, suggesting the conserved role of MG in controlling longevity across species. Together, our studies demonstrate that bacteria-derived MG is a novel therapeutic target for aging and aging-associated pathophysiology.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans , Factores de Transcripción Forkhead/metabolismo , Longevidad/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Piruvaldehído , Animales , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/microbiología , Escherichia coli/metabolismo , Microbioma Gastrointestinal/fisiología , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Modelos Biológicos , Piruvaldehído/metabolismo , Piruvaldehído/farmacología , Transducción de Señal/efectos de los fármacos , Transcriptoma/genéticaRESUMEN
Bovine prolactin (PRL) gene is essential for the initiation and maintenance of lactation and exerts multiple effects on mammary alveoli to promote the synthesis and secretion of major components of milk. The objectives of this study were to identify mutations in PRL gene and to evaluate the mutations as potential markers of milk performance traits in cattle populations of Ethiopia. For this purpose, genomic DNA from whole blood was extracted through salting out procedure from 87 animals of five cattle populations of Ethiopia. Accordingly, three single nucleotide polymorphisms (SNPs) were identified of which one SNP g.8323T > A showed missense mutation while the other two SNPs revealed silent mutations. FST values showed statistically significant genetic differentiation among the studied populations. Intermediate polymorphic information content was noted for most SNPs, which indicates the presence of sufficient genetic variation at this locus. Two SNPs showed heterozygote deficiency as a result of positive FIS values. Only g.8398A > G SNP have statistically significant (p < 0.05) effect on average daily milk yield, fat and solid not fat percentage in all studied cattle populations. Therefore, g.8398A > G SNP identified in this study influences cattle milk production and may be used as possible candidate SNP for marker-assisted selection programs in cattle populations of Ethiopia.
Asunto(s)
Leche , Polimorfismo de Nucleótido Simple , Femenino , Bovinos/genética , Animales , Polimorfismo de Nucleótido Simple/genética , Prolactina/genética , Etiopía , Lactancia/genética , ExonesRESUMEN
Antimicrobial peptides (AMPs) are promising alternatives to existing treatments for multidrug-resistant bacteria-infected wounds. Therefore, the effect of protegrin-1 (PG1), a potent porcine AMP with broad-spectrum activity, on wound healing was evaluated. PG1-overexpressing transgenic mice were used as an in vivo model to evaluate its healing efficiency against Staphylococcus aureus-infected (106 colony forming units) wounds. We analyzed the wounds under four specific conditions in the presence or absence of antibiotic treatment. We observed the resolution of bacterial infection and formation of neo-epithelium in S. aureus-infected wounds of the mice, even without antibiotic treatment, whereas all wild-type mice with bacterial infection died within 8 to 10 days due to uncontrolled bacterial proliferation. Interestingly, the wound area on day 7 was smaller (p < 0.01) in PG1 transgenic mice than that in the other groups, including antibiotic-treated mice, suggesting that PG1 exerts biological effects other than bactericidal effect. Additionally, we observed that the treatment of primary epidermal keratinocytes with recombinant PG1 enhanced cell migration in in vitro scratch and cell migration assays. This study contributes to the understanding of broad-spectrum endogenous cathelicidins with potent antimicrobial activities, such as PG1, on wound healing. Furthermore, our findings suggest that PG1 is a potent therapeutic candidate for wound healing.
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Infecciones Estafilocócicas , Infección de Heridas , Porcinos , Ratones , Animales , Catelicidinas/genética , Catelicidinas/farmacología , Staphylococcus aureus , Ratones Transgénicos , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/uso terapéutico , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Infección de Heridas/tratamiento farmacológico , Infección de Heridas/microbiología , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiologíaRESUMEN
The effects of ΔPb-CATH4, a cathelicidin derived from Python bivittatus, were evaluated against Staphylococcus aureus-infected wounds in mice. These effects were comparable to those of classical antibiotics. ΔPb-CATH4 was resistant to bacterial protease but not to porcine trypsin. A reduction in the level of inflammatory cytokines and an increase in the migration of immune cells was observed in vitro. Thus, ΔPb-CATH4 can promote wound healing by controlling infections including those caused by multidrug-resistant bacteria via its immunomodulatory effects.
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Catelicidinas/administración & dosificación , Infecciones Estafilocócicas/tratamiento farmacológico , Infección de Heridas/tratamiento farmacológico , Animales , Boidae , Catelicidinas/química , Humanos , Ratones , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/fisiopatología , Staphylococcus aureus/fisiología , Cicatrización de Heridas/efectos de los fármacos , Infección de Heridas/microbiología , Infección de Heridas/fisiopatologíaRESUMEN
The efficiency of existing cell lysis methods to isolate nucleic acids from diverse bacteria varies depending on cell wall structures. This study tested a novel idea of using broad-spectrum antimicrobial peptides to improve the lytic efficiency of hard-to-lyse bacteria and characterized their differences. The lysis conditions of Staphylococcus aureus using recombinant porcine myeloid antimicrobial peptide 36 (PMAP-36), a broad-spectrum pig cathelicidin, was optimized, and RNA isolation was performed with cultured pellets of ten bacterial species using various membranolytic proteins. Additionally, three other antimicrobial peptides, protegrin-1 (PG-1), melittin, and nisin, were evaluated for their suitability as the membranolytic agents of bacteria. However, PMAP-36 use resulted in the most successful outcomes in RNA isolation from diverse bacterial species. The amount of total RNA obtained using PMAP-36 increased by ~2-fold compared to lysozyme in Salmonella typhimurium. Streptococci species were refractory to all lytic proteins tested, although the RNA yield from PMAP-36 treatment was slightly higher than that from other methods. PMAP-36 use produced high-quality RNA, and reverse transcription PCR showed the efficient amplification of the 16S rRNA gene from all tested strains. Additionally, the results of genomic DNA isolation were similar to those of RNA isolation. Thus, our findings present an additional option for high quality and unbiased nucleic acid isolation from microbiomes or challenging bacterial strains.
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Péptidos Catiónicos Antimicrobianos/química , ARN Bacteriano/química , Staphylococcus aureus/química , Péptidos Catiónicos Antimicrobianos/farmacología , Fraccionamiento Celular/métodos , Fraccionamiento Celular/normas , ADN Bacteriano/química , ADN Bacteriano/aislamiento & purificación , ARN Bacteriano/aislamiento & purificación , Staphylococcus aureus/efectos de los fármacosRESUMEN
Antimicrobial peptides (AMPs) are of interest as alternatives to antibiotics or immunomodulators. We generated and characterized the phenotypes of transgenic mice overexpressing protegrin 1 (PG1), a potent porcine cathelicidin. No obvious differences were observed between PG1 transgenic and wild-type mice in terms of growth, development, general behaviour, and the major immune cell population. However, PG1 transgenic mice intranasally infected with Staphylococcus aureus resulted in a reduction in microscopic pulmonary injury, improved clearance of bacteria, and lower proinflammatory cytokine secretion, compared to those of wild-type mice. On the other hand, approximately 25% of PG1 transgenic mice (n = 54/215) showed corneal opacity and developed inflammation in the eye, resulting ultimately in phthisis bulbi. Immunohistochemical analyses revealed that PG1 and its activator, neutrophil elastase, localized to the basal cells of the cornea and glands in eyelids, respectively. In addition, apoptosis indicated by a Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive signal was detected from flat cells of the cornea. Our study suggests that the expression regulation or localization of AMPs such as PG1 is important to prevent their adverse effects. However, our results also showed that the cytotoxic effects of PG1 on cells could be tolerated in animals, except for the eyes.
Asunto(s)
Antiinfecciosos/metabolismo , Péptidos Catiónicos Antimicrobianos/fisiología , Opacidad de la Córnea/patología , Oftalmopatías/patología , Inflamación/patología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/fisiología , Animales , Opacidad de la Córnea/etiología , Opacidad de la Córnea/metabolismo , Oftalmopatías/etiología , Oftalmopatías/metabolismo , Femenino , Inflamación/etiología , Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mucina-1/genética , Regiones Promotoras Genéticas , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/patología , PorcinosRESUMEN
The biorecognition ability of hybridized toll-like receptors (TLRs) 2 and 6 proteins on electrode surfaces has been studied. TLR biosensors have been designed to be non-specific to particular bacterial strains but rather to provide broad spectrum detection of cells and toxins containing relevant pathogen-associated molecular patterns (PAMPs). Our electrochemical TLR2/6 biosensors demonstrated selective detection towards Gram-positive bacterial whole-cells and a synthetic diacylated lipopeptide (Pam2CSK4), a PAMP. Responses towards Bacillus licheniformis (B. licheniformis) and Enterococcus hirae (E. hirae) were obtained. The biosensor was able to differentiate signals between B. licheniformis and a Gram-negative bacterial cell (control) as low as 100 CFU mL-1. One challenge in developing protein-based biosensors is to improve the shelf-life of the biosensor chips and preserve the detection activity of the protein molecules, therefore we did our first exploration into storage conditions. The activity of stored biosensors was found to be strongly dependent on storage medium, and that effective 'shelf-life' was obtained makes an important step towards creating robust sensors for real-life applications.
Asunto(s)
Técnicas Biosensibles , Receptores Toll-Like , Bacterias Gramnegativas , Bacterias GrampositivasRESUMEN
Nonylphenol (NP) is an alkylphenol that is widely used in chemical manufacturing. Exposure to this toxic environmental contaminant has been shown to negatively affect the reproductive system. Herein, we evaluated the toxicity of NP in mouse testes, while using in vitro organ culture. Mouse testicular fragments (MTFs), derived from five-day postpartum neonatal mouse testes, were exposed to different concentrations of NP (1-50 µM) for 30 days. The results showed that NP impaired germ cell development and maintenance. Furthermore, NP significantly downregulated the transcript levels of both undifferentiated and differentiated germ cell marker genes relative to those in controls. In particular, a high dose of NP (50 µM) led to complete germ cell depletion and resulted in spermatogenic failure, despite the presence of Sertoli and Leydig cells. In addition, the mRNA expression of steroidogenic enzymes, such as steroidogenic acute regulatory protein (STAR), Cytochrome P450 Family 11 Subfamily A Member 1 (Cyp11α1), Cytochrome P450 17A1 (Cyp17α1), and androgen receptor (AR), increased with increasing concentration of NP. Conversely, the expression of estrogen receptor alpha (ESR1) and Cytochrome P450 family 19 subfamily A member 1 (Cyp19α1) in NP-exposed MTFs decreased when compared to that of the control. Taken together, this study demonstrates that NP has a negative effect on prepubertal spermatogenesis and germ cell maintenance and it disrupts steroidogenesis and induces hormonal imbalance in MTFs.
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Técnicas de Cultivo de Órganos , Fenoles/toxicidad , Testículo/crecimiento & desarrollo , Animales , Animales Recién Nacidos , Biomarcadores/metabolismo , Femenino , Feto/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Células Germinativas/efectos de los fármacos , Células Germinativas/metabolismo , Células Intersticiales del Testículo/efectos de los fármacos , Células Intersticiales del Testículo/metabolismo , Masculino , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células de Sertoli/efectos de los fármacos , Células de Sertoli/metabolismo , Testículo/efectos de los fármacos , Testículo/embriologíaRESUMEN
The thermoplasmonic properties of platinum nanoparticles (PtNPs) render them desirable for use in diagnosis, detection, therapy, and surgery. However, their toxicological effects and impact at the molecular level remain obscure. Nanotoxicology is mainly focused on the interactions of nanostructures with biological systems, particularly with an emphasis on elucidating the relationship between the physical and chemical properties such as size and shape. Therefore, we hypothesized whether these unique anisotropic nanoparticles could induce cytotoxicity similar to that of spherical nanoparticles and the mechanism involved. Thus, we synthesized unique and distinct anisotropic PtNPs using lycopene as a biological template and investigated their biological activities in model human acute monocytic leukemia (THP-1) macrophages. Exposure to PtNPs for 24 h dose-dependently decreased cell viability and proliferation. Levels of the cytotoxic markers lactate dehydrogenase and intracellular protease significantly and dose-dependently increased with PtNP concentration. Furthermore, cells incubated with PtNPs dose-dependently produced oxidative stress markers including reactive oxygen species (ROS), malondialdehyde, nitric oxide, and carbonylated protein. An imbalance in pro-oxidants and antioxidants was confirmed by significant decreases in reduced glutathione, thioredoxin, superoxide dismutase, and catalase levels against oxidative stress. The cell death mechanism was confirmed by mitochondrial dysfunction and decreased ATP levels, mitochondrial copy numbers, and PGC-1α expression. To further substantiate the mechanism of cell death mediated by endoplasmic reticulum stress (ERS), we determined the expression of the inositol-requiring enzyme (IRE1), (PKR-like ER kinase) PERK, activating transcription factor 6 (ATF6), and activating transcription factor 4 ATF4, the apoptotic markers p53, Bax, and caspase 3, and the anti-apoptotic marker Bcl-2. PtNPs could activate ERS and apoptosis mediated by mitochondria. A proinflammatory response to PtNPs was confirmed by significant upregulation of interleukin-1-beta (IL-1ß), interferon γ (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin (IL-6). Transcriptomic and molecular pathway analyses of THP-1 cells incubated with the half maximal inhibitory concentration (IC50) of PtNPs revealed the altered expression of genes involved in protein misfolding, mitochondrial function, protein synthesis, inflammatory responses, and transcription regulation. We applied transcriptomic analyses to investigate anisotropic PtNP-induced toxicity for further mechanistic studies. Isotropic nanoparticles are specifically used to inhibit non-specific cellular uptake, leading to enhanced in vivo bio-distribution and increased targeting capabilities due to the higher radius of curvature. These characteristics of anisotropic nanoparticles could enable the technology as an attractive platform for nanomedicine in biomedical applications.
Asunto(s)
Apoptosis/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Inflamación/genética , Leucemia Monocítica Aguda/patología , Nanopartículas del Metal/toxicidad , Platino (Metal)/toxicidad , Transducción de Señal/efectos de los fármacos , Transcriptoma/genética , Adenosina Trifosfato/metabolismo , Anisotropía , Antioxidantes/farmacología , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Humanos , Leucemia Monocítica Aguda/genética , Peroxidación de Lípido/efectos de los fármacos , Licopeno/química , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Nanopartículas del Metal/ultraestructura , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Óxido Nítrico/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Carbonilación Proteica/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Pluripotent stem cells can be established from parthenogenetic embryos, which only possess maternal alleles with maternal-specific imprinting patterns. Previously, we and others showed that parthenogenetic embryonic stem cells (pESCs) and parthenogenetic induced pluripotent stem cells (piPSCs) progressively lose the bimaternal imprinting patterns. As ESCs and iPSCs are naïve pluripotent stem cells, parthenogenetic primed pluripotent stem cells have not yet been established, and thus, their imprinting patterns have not been studied. Here, we first established parthenogenetic epiblast stem cells (pEpiSCs) from 7.5 dpc parthenogenetic implantation embryos and compared the expression patterns and DNA methylation status of the representative imprinted genes with biparental EpiSCs. We found that there were no striking differences between pEpiSCs and biparental EpiSCs with respect to morphology, pluripotency gene expression, and differentiation potential, but there were differences in the expression and DNA methylation status of imprinted genes (H19, Igf2, Peg1, and Peg3). Moreover, pEpiSCs displayed a different DNA methylation pattern compared with that of parthenogenetic neural stem cells (pNSCs), which showed a typical bimaternal imprinting pattern. These results suggest that both naïve pluripotent stem cells and primed pluripotent stem cells have an unstable imprinting status.
Asunto(s)
Células Madre Embrionarias/metabolismo , Impresión Genómica/genética , Estratos Germinativos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Partenogénesis/genética , Células Madre Pluripotentes/metabolismo , Animales , Diferenciación Celular/genética , Células Cultivadas , Metilación de ADN , Células Madre Embrionarias/citología , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Estratos Germinativos/citología , Células Madre Pluripotentes Inducidas/citología , Factor II del Crecimiento Similar a la Insulina/genética , Ratones , Células Madre Pluripotentes/citología , ARN Largo no Codificante/genéticaRESUMEN
OBJECTIVE: We tried to analyze allele-specific expression in the pig neocortex using bioinformatic analysis of high-throughput sequencing results from the parental genomes and offspring transcriptomes from reciprocal crosses between Korean Native and Landrace pigs. METHODS: We carried out sequencing of parental genomes and offspring transcriptomes using next generation sequencing. We subsequently carried out genome scale identification of SNPs in two different ways using either individual genome mapping or joint genome mapping of the same breed parents that were used for the reciprocal crosses. Using parent-specific SNPs, allele-specifically expressed genes were analyzed. RESULTS: Because of the low genome coverage (~4x) of the sequencing results, most SNPs were non-informative for parental lineage determination of the expressed alleles in the offspring and were thus excluded from our analysis. Consequently, 436 SNPs covering 336 genes were applicable to measure the imbalanced expression of paternal alleles in the offspring. By calculating the read ratios of parental alleles in the offspring, we identified seven genes showing allele-biased expression (P < 0.05) including three previously reported and four newly identified genes in this study. CONCLUSION: The newly identified allele-specifically expressing genes in the neocortex of pigs should contribute to improving our knowledge on genomic imprinting in pigs. To our knowledge, this is the first study of allelic imbalance using high throughput analysis of both parental genomes and offspring transcriptomes of the reciprocal cross in outbred animals. Our study also showed the effect of the number of informative animals on the genome level investigation of allele-specific expression using RNA-seq analysis in livestock species.
RESUMEN
Primary porcine alveolar macrophages (PAM) are useful for studying viral infections and immune response in pigs; however, long-term use of these cells is limited by the cells' short lifespan. We immortalized primary PAMs by transfecting them with both hTERT and SV40LT and established two immortalized cell lines (iPAMs) actively proliferating even after 35 passages. These cells possessed the characteristics of primary PAMs, including strong expression of swine leukocyte antigen (SLA) class II genes and the inability to grow anchorage-independently. We characterized their SLA genes and subsequently performed peptide-SLA binding assays using a peptide from porcine circovirus type 2 open reading frame 2 to experimentally measure the binding affinity of the peptide to SLA class II. The number of peptides bound to cells measured by fluorescence was very low for PK15 cells (7.0% ± 1.5), which are not antigen-presenting cells, unlike iPAM61 (33.7% ± 3.4; SLA-DQA*0201/0303, DQB1*0201/0901, DRB1*0201/1301) and iPAM303 (73.3% ± 5.4; SLA DQA*0106/0201, DQB1*0202/0701, DRB1*0402/0602). The difference in peptide binding between the two iPAMs was likely due to the allelic differences between the SLA class II molecules that were expressed. The development of an immortal PAM cell panel harboring diverse SLA haplotypes and the use of an established method in this study can become a valuable tool for evaluating the interaction between antigenic peptides and SLA molecules and is important for many applications in veterinary medicine including vaccine development.
Asunto(s)
Genes MHC Clase II/fisiología , Antígenos de Histocompatibilidad Clase I/metabolismo , Macrófagos Alveolares/metabolismo , Porcinos/metabolismo , Animales , Línea Celular , Péptidos/metabolismo , Unión ProteicaRESUMEN
For 10,000 years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars â¼1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model.
Asunto(s)
Genoma/genética , Filogenia , Sus scrofa/clasificación , Sus scrofa/genética , Animales , Demografía , Modelos Animales , Datos de Secuencia Molecular , Dinámica PoblacionalRESUMEN
The rapid development of nanotechnology has led to the use of silver nanoparticles (AgNPs) in biomedical applications, including antibacterial, antiviral, anti-inflammatory, and anticancer therapies. The molecular mechanism of AgNPs-induced cytotoxicity has not been studied thoroughly using a combination of cellular assays and RNA sequencing (RNA-Seq) analysis. In this study, we prepared AgNPs using myricetin, an anti-oxidant polyphenol, and studied their effects on NIH3T3 mouse embryonic fibroblasts as an in vitro model system to explore the potential biomedical applications of AgNPs. AgNPs induced loss of cell viability and cell proliferation in a dose-dependent manner, as evident by increased leakage of lactate dehydrogenase (LDH) from cells. Reactive oxygen species (ROS) were a potential source of cytotoxicity. AgNPs also incrementally increased oxidative stress and the level of malondialdehyde, depleted glutathione and superoxide dismutase, reduced mitochondrial membrane potential and adenosine triphosphate (ATP), and caused DNA damage by increasing the level of 8-hydroxy-2'-deoxyguanosine and the expressions of the p53 and p21 genes in NIH3T3 cells. Thus, activation of oxidative stress may be crucial for NIH3T3 cytotoxicity. Interestingly, gene ontology (GO) term analysis revealed alterations in epigenetics-related biological processes including nucleosome assembly and DNA methylation due to AgNPs exposure. This study is the first demonstration that AgNPs can alter bulk histone gene expression. Therefore, our genome-scale study suggests that the apoptosis observed in NIH3T3 cells treated with AgNPs is mediated by the repression of genes required for cell survival and the aberrant enhancement of nucleosome assembly components to induce apoptosis.
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Apoptosis/efectos de los fármacos , Embrión de Mamíferos/citología , Fibroblastos/citología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Nanopartículas del Metal/toxicidad , Plata/toxicidad , Adenosina Trifosfato/metabolismo , Animales , Antioxidantes/farmacología , Apoptosis/genética , Autofagosomas/efectos de los fármacos , Autofagosomas/metabolismo , Autofagosomas/ultraestructura , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Daño del ADN , Endocitosis/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Flavonoides/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , L-Lactato Deshidrogenasa/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Lisosomas/ultraestructura , Malondialdehído/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Nanopartículas del Metal/ultraestructura , Ratones , Células 3T3 NIH , Nucleosomas/efectos de los fármacos , Nucleosomas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Electricidad Estática , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
In this study, we sought to identify novel antimicrobial peptides (AMPs) in Python bivittatus through bioinformatic analyses of publicly available genome information and experimental validation. In our analysis of the python genome, we identified 29 AMP-related candidate sequences. Of these, we selected five cathelicidin-like sequences and subjected them to further in silico analyses. The results showed that these sequences likely have antimicrobial activity. The sequences were named Pb-CATH1 to Pb-CATH5 according to their sequence similarity to previously reported snake cathelicidins. We predicted their molecular structure and then chemically synthesized the mature peptide for three putative cathelicidins and subjected them to biological activity tests. Interestingly, all three peptides showed potent antimicrobial effects against Gram-negative bacteria but very weak activity against Gram-positive bacteria. Remarkably, ΔPb-CATH4 showed potent activity against antibiotic-resistant clinical isolates and also was observed to possess very low hemolytic activity and cytotoxicity. ΔPb-CATH4 also showed considerable serum stability. Electron microscopic analysis indicated that ΔPb-CATH4 exerts its effects via toroidal pore preformation. Structural comparison of the cathelicidins identified in this study to previously reported ones revealed that these Pb-CATHs are representatives of a new group of reptilian cathelicidins lacking the acidic connecting domain. Furthermore, Pb-CATH4 possesses a completely different mature peptide sequence from those of previously described reptilian cathelicidins. These new AMPs may be candidates for the development of alternatives to or complements of antibiotics to control multidrug-resistant pathogens.
Asunto(s)
Antibacterianos/farmacología , Boidae/genética , Catelicidinas/genética , Catelicidinas/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Antibacterianos/sangre , Antibacterianos/química , Catelicidinas/sangre , Línea Celular Tumoral , Pollos , Eritrocitos/efectos de los fármacos , Genoma/genética , Células HEK293 , Hemólisis/efectos de los fármacos , Humanos , Células MCF-7 , Estructura Secundaria de ProteínaRESUMEN
Glyoxalase 1 (Glo1) is the first enzyme involved in glutathione-dependent detoxification of methylglyoxal, eventually generating d-lactate by the second enzyme glyoxalase 2 (Glo2). An accumulation of intracellular glyoxal and methylglyoxal leads to protein malfunction and mutation via formation of the advanced glycation end products (AGEs). Studies on mouse behavior suggest that methylglyoxal has anxiolytic properties. In this report, we generated and characterized a mouse knockout for Glo1. The knockout mice were viable without a pronounced phenotypic defect. Increased level of AGEs in Glo1 knockout mice was detected by immunoblotting with anti-MGH1 in liver homogenate, but not in brain. Alterations in behavior were observed in open field, light-dark transition, and tail suspension test. Open field data indicate increased exploration for novel environment and entry/stay in center zone in Glo1 knockout mice. In addition, increased light-dark transition and immobility was observed in the knockout mice. These data indicate that Glo1 knockout reduces anxiety-like behavior, but increases depression-like behavior.
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Ansiedad/genética , Depresión/genética , Lactoilglutatión Liasa/genética , Animales , Ansiedad/metabolismo , Depresión/metabolismo , Eliminación de Gen , Glutatión/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Glioxal/metabolismo , Lactoilglutatión Liasa/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Piruvaldehído/metabolismoRESUMEN
The overuse of antibiotics plays a major role in the emergence and spread of multidrug-resistant bacteria. A molecularly targeted, specific treatment method for bacterial pathogens can prevent this problem by reducing the selective pressure during microbial growth. Herein, we introduce a nonviral treatment strategy delivering genome editing material for targeting antibacterial resistance. We apply the CRISPR-Cas9 system, which has been recognized as an innovative tool for highly specific and efficient genome engineering in different organisms, as the delivery cargo. We utilize polymer-derivatized Cas9, by direct covalent modification of the protein with cationic polymer, for subsequent complexation with single-guide RNA targeting antibiotic resistance. We show that nanosized CRISPR complexes (= Cr-Nanocomplex) were successfully formed, while maintaining the functional activity of Cas9 endonuclease to induce double-strand DNA cleavage. We also demonstrate that the Cr-Nanocomplex designed to target mecA-the major gene involved in methicillin resistance-can be efficiently delivered into Methicillin-resistant Staphylococcus aureus (MRSA), and allow the editing of the bacterial genome with much higher efficiency compared to using native Cas9 complexes or conventional lipid-based formulations. The present study shows for the first time that a covalently modified CRISPR system allows nonviral, therapeutic genome editing, and can be potentially applied as a target specific antimicrobial.
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Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica/métodos , Genoma Bacteriano , Staphylococcus aureus Resistente a Meticilina/genética , ARN Guía de Kinetoplastida/administración & dosificación , Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR , Endonucleasas/metabolismo , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Polímeros/metabolismo , ARN Guía de Kinetoplastida/genética , Streptococcus pyogenes/enzimologíaRESUMEN
Pigs with severe combined immunodeficiency (SCID) may provide useful models for regenerative medicine, xenotransplantation, and tumor development and will aid in developing therapies for human SCID patients. Using a reporter-guided transcription activator-like effector nuclease (TALEN) system, we generated targeted modifications of recombination activating gene (RAG) 2 in somatic cells at high efficiency, including some that affected both alleles. Somatic-cell nuclear transfer performed with the mutated cells produced pigs with RAG2 mutations without integrated exogenous DNA. Biallelically modified pigs either lacked a thymus or had one that was underdeveloped. Their splenic white pulp lacked B and T cells. Under a conventional housing environment, the biallelic RAG2 mutants manifested a "failure to thrive" phenotype, with signs of inflammation and apoptosis in the spleen compared with age-matched wild-type animals by the time they were 4 wk of age. Pigs raised in a clean environment were healthier and, following injection of human induced pluripotent stem cells (iPSCs), quickly developed mature teratomas representing all three germ layers. The pigs also tolerated grafts of allogeneic porcine trophoblast stem cells. These SCID pigs should have a variety of uses in transplantation biology.
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Proteínas de Unión al ADN/genética , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/trasplante , Proteínas Nucleares/genética , Inmunodeficiencia Combinada Grave/metabolismo , Trasplante Heterólogo , Alelos , Animales , Secuencia de Bases , Fibroblastos/metabolismo , Genotipo , Humanos , Datos de Secuencia Molecular , Mutación , Fenotipo , Regeneración , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/terapia , Porcinos , Porcinos Enanos , Timo/metabolismo , Cordón Umbilical/citologíaRESUMEN
Here we report a comprehensive analysis of the vomeronasal receptor repertoire in pigs. We identified a total of 25 V1R sequences consisting of 10 functional genes, 3 pseudogenes, and 12 partial genes, while functional V2R and FPR genes were not present in the pig genome. Pig V1Rs were classified into three subfamilies, D, F, and J. Using direct high resolution sequencing-based typing of all functional V1Rs from 10 individuals of 5 different breeds, a total of 24 SNPs were identified, indicating that the allelic diversity of V1Rs is much lower than that of the olfactory receptors. A high expression level of V1Rs was detected in the vomeronasal organ (VNO) and testes, while a low expression level of V1Rs was observed in all other tissues examined. Our results showed that pigs could serve as an interesting large animal model system to study pheromone-related neurobiology because of their genetic simplicity.