RESUMEN
RNA modifications regulate multiple aspects of cellular function including RNA splicing, translation, export, decay, stability, and phase separation. One of the comprehensive ways to detect such modifications is by the recent advancement of direct RNA sequencing from Oxford Nanopore Technologies (ONT). However, this method obtains a large amount of data with high complexity in the form of raw current signal that poses a new informatics challenge to accurately detect those modifications. Here, we provide nanoDoc2, a software to detect multiple types of RNA modification from nanopore direct RNA sequencing data. The nanoDoc2 includes a novel signal segmentation algorithm based on the trace value-a base probability feature that is added by the Guppy basecalling program from ONT during processing of the raw signal. The core of nanoDoc2 includes a machine learning algorithm in which a 6-mer segmented raw current signal is analyzed by deep one-class classification using a WaveNet-based neural network. As an output, an RNA modification is detected by a statistical score in each candidate position. Herein, we describe the detailed instructions on how to use nanoDoc2 for signal segmentation, train/test the neural network, and finally predict RNA modifications present in nanopore direct RNA sequencing data.
Asunto(s)
Nanoporos , ARN , ARN/genética , Programas Informáticos , Algoritmos , Análisis de Secuencia de ARN/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodosRESUMEN
Transposons are known to participate in tissue aging, but their effects on aged stem cells remain unclear. Here, we report that in the Drosophila ovarian germline stem cell (GSC) niche, aging-related reductions in expression of Piwi (a transposon silencer) derepress retrotransposons and cause GSC loss. Suppression of Piwi expression in the young niche mimics the aged niche, causing retrotransposon depression and coincident activation of Toll-mediated signaling, which promotes Glycogen synthase kinase 3 activity to degrade ß-catenin. Disruption of ß-catenin-E-cadherin-mediated GSC anchorage then results in GSC loss. Knocking down gypsy (a highly active retrotransposon) or toll, or inhibiting reverse transcription in the piwi-deficient niche, suppresses GSK3 activity and ß-catenin degradation, restoring GSC-niche attachment. This retrotransposon-mediated impairment of aged stem cell maintenance may have relevance in many tissues, and could represent a viable therapeutic target for aging-related tissue degeneration.
Asunto(s)
Proteínas Argonautas/metabolismo , Senescencia Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Células Germinativas/metabolismo , Animales , Proteínas Argonautas/genética , Cadherinas/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Silenciador del Gen , Glucógeno Sintasa Quinasa 3/metabolismo , Ovario/citología , Ovario/metabolismo , Retroelementos/genética , Transducción de Señal , Nicho de Células Madre/fisiología , Células Madre/metabolismo , Receptores Toll-Like/metabolismo , beta Catenina/metabolismoRESUMEN
Surface coating of antifouling materials on the substrates offers convenient strategies and great opportunities to improve their biocompatibility and functions of host substrates for wide biomedical applications. In this work, we present a general surface zwitterionization strategy to improve surface biocompatibility and antifouling properties of titanium (Ti) by grafting zwitterionic poly(sulfobetaine methacrylate) (polySBMA). This method also demonstrates its general applicability to graft polySBMA onto Ti surface using different anchoring agents of dopamine and silane. The resulting polySBMA grafted from dopamine- (pTi-D-pSBMA) and silane-anchored titanium surfaces (pTi-Si-pSBMA) surfaces exhibit superlow fouling ability to highly resist the adhesions of plasma proteins, platelets, erythrocytes, leukocytes, human fibroblast (HT1080), E. coli, and S. epidermidis. The interfacial properties of the surface-modified Ti surfaces are analyzed and correlated with their antifouling properties. The new method and materials provide a more general, flexible, and robust way to produce an excellent nonfouling surface with adjustable interfacial structures of grafted polymers, which hopefully can be expanded to wider applications based on both the structure and surface superiorities.
Asunto(s)
Proteínas Sanguíneas/química , Titanio/química , Células Sanguíneas/química , Escherichia coli/química , Fibroblastos , Humanos , Propiedades de SuperficieRESUMEN
The influence of the topographic morphology of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) films on human mesenchymal stem cells (hMSCs) was investigated in this study. PHBHHx films with various surface characteristics were prepared by compression-molding, solvent-casting and electrospinning. The adhesion, proliferation and differentiation behaviors of hMSCs were significantly modulated by the surface characteristics of these films. HMSCs could aggregate and form cellular clusters on the cast PHBHHx films, and the time to form cellular aggregates increased as the surface roughness increased. The aggregated hMSCs on the cast films kept their original surface markers and presented much higher viability during the regular culture and lower differentiation ability upon osteogenic induction than the spread cells on the compression-molded films and TCPS. HMSCs spread well and showed a specific orientation on the surface of the random electrospun fibrous films, they were not able to migrate into the interior of electrospun fibrous films, and they revealed the highest viability during the regular culture but a lower differentiation activity upon osteogenic induction. The electrospun fibrous PHBHHx films could serve as a suitable substrate for large quantity culturing of hMSCs when undifferentiated hMSCs are desired.
Asunto(s)
Ácido 3-Hidroxibutírico/química , Ácido 3-Hidroxibutírico/farmacología , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Caproatos/química , Caproatos/farmacología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Adulto , Agregación Celular/efectos de los fármacos , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Células Madre Mesenquimatosas/ultraestructura , Osteogénesis/efectos de los fármacos , Solventes/química , Propiedades de Superficie , Factores de Tiempo , Ingeniería de TejidosRESUMEN
Polyhydroxyalkanoates (PHAs) are a newer family of biomaterials for tissue-engineering applications. The objective of this study is to investigate the behavior of human mesenchymal stem cells (hMSCs) grown on various PHA films. The surface characteristics of PHA co-polymer films were varied by the content of 3-hydroxyvalerate (HV) or 3-hydroxyhexanoate (HHx) and by the film preparation methods such as compression-molding and solvent-casting. Hyaluronic acid (HA) was further applied to modify the surface properties of PHA membranes. As HV content increased, the crystallinity and the hydrophobicity of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) membranes decreased and the metabolic activity of hMSCs raised, although the distribution and morphology of hMSCs did not show significant variation. Hyaluronic acid (HA) coating on PHA membranes could improve the metabolic activity and reduce the death rate of hMSCs. Aggregates and spheroidal clusters of hMSCs were found on the surface of cast poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) membranes. The growth of hMSCs was remarkably influenced by various surface characteristics of the PHA films.
Asunto(s)
Materiales Biocompatibles/química , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Polihidroxialcanoatos/química , Adulto , Caproatos/química , Proliferación Celular , Células Cultivadas , Humanos , Ácido Hialurónico/química , Ácidos Pentanoicos/química , Propiedades de SuperficieRESUMEN
The new biodegradable polyester poly(3-hydroxybutyrate-co-3-hydroxyhexnoate) (PHBHHx) has a potential application in tissue engineering. The aim of this study was to present a deeper picture of the relationship between the cellular behavior and the surface characteristics of PHBHHx films. The pristine PHBHHx film was prepared by adopting the compression-molding method, and then the acrylic acid molecules were grafted on PHBHHx membrane surface by UV irradiation. The hydrophilic nature and surface roughness of various PHBHHx films were controlled by adjusting the acrylic acid concentration and the UV irradiation time. Although the surface characteristics of various PHBHHx films could not affect the metabolic activity of hMSCs, the performance of morphology of hMSCs was deeply affected by the hydrophilic nature and the orientation of surface scars. The hydrophilic nature would effectively improve the spread of hMSCs, and the orientation of surface scars would guide the growth direction of cytoskeleton (actin) inside hMSCs. In contrast, the behaviors of C3A/HepG2 hepatoma cells presented an opposite outcomes. Those surface characteristics were obviously associated with the performance of metabolic activity of C3A cells, but not with the morphology of C3A cells. Both hMSCs and C3A cells have unique cellular characteristics; therefore, their responses to environmental stimulations are significantly different.
Asunto(s)
Ácido 3-Hidroxibutírico/química , Caproatos/química , Hepatocitos/citología , Células Madre Mesenquimatosas/citología , Adulto , Materiales Biocompatibles/química , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Dióxido de Carbono/química , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Proliferación Celular , Células Cultivadas , Hepatoblastoma/metabolismo , Hepatocitos/metabolismo , Humanos , L-Lactato Deshidrogenasa/metabolismo , Neoplasias Hepáticas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Propiedades de SuperficieRESUMEN
The aim of this study was to evaluate the behaviors of mesenchymal stem cells (MSCs) on Poly(L-Lactic acid) (PLLA) membranes with different surface topographies. The double-sided micropatterns, island-patterned, and sunken-patterned PLLA membranes with diameters of 60 and 100 microm, were fabricated by the soft lithography method. The cell viability of MSCs on the island- and sunken-patterned PLLA membranes were characterized by scanning electron microscopy (SEM), MTT assay, and flow cytometric analysis. Cell adhesion and proliferation capability were superior for the MSCs seeding on the island-patterned PLLA membranes than those on the sunken-patterned PLLA membranes. Especially, we observed the best biocompatibility for MSCs on the island-patterned surface with diameter of 100 microm. In addition, the improvement of cell attachment and augmenting subsequent cellular response are investigated after the island-patterned membranes precoating with collagen and fibronectin. Furthermore, the flow cytometric analysis reveals the MSCs can expand and maintain the phenotype on these PLLA membranes without losing its potential for differentiation. Since scale-up of cell production and optimization of culture conditions are important for stem cell engineering, to control the stem cell proliferation and differentiation is necessary. Therefore, besides topographical properties play a crucial role on the stem cells attachment and proliferative activity, it is suggested that the "relative scale" between cell and pattern also affects the cell adhesion morphologies and cell behaviors. Based on the overall cellular response, this study provides a valuable guidance to prepare appropriate topographic surface for tissue engineering application.
Asunto(s)
Materiales Biocompatibles/química , Ácido Láctico/química , Células Madre Mesenquimatosas/citología , Polímeros/química , Adulto , Células de la Médula Ósea/citología , Adhesión Celular , Supervivencia Celular , Dimetilpolisiloxanos/química , Elastómeros , Citometría de Flujo/métodos , Humanos , Persona de Mediana Edad , Poliésteres , Siliconas/química , Propiedades de Superficie , Sales de Tetrazolio/farmacología , Tiazoles/farmacologíaRESUMEN
The influence of the properties and surface micropatterning of chitosan-collagen-gelatin (CCG) blended membranes on C3A cell's activities has been investigated. It is aimed to guide the cell growth and improve the growth rate in vitro for the application in tissue engineering. Masters with micropatterns are prepared on stainless steel plates by photolithography. The CCG membranes with surface micropatterns are then fabricated by soft lithography and dry-wet phase inversion techniques. The morphology and metabolic activity of cultured C3A cells on the membranes are recorded. When the C3A cells are seeded on the membranes with micropattern spacing of 200 microm width and 80 microm depth, they adhere and aggregate in the groove of the membranes in a few minutes. The aggregated cells migrate up to the surface of the ridge later. This phenomenon, however, is not found on membranes with a micropattern spacing of 500 microm width. In addition, it is demonstrated that the cells on the CCG membranes with micropatterns have higher metabolism and growth rates than those on the flat CCG membranes and on T-flask discs. Micropatterning on the membrane surface can affect the distribution of cells and the communication among cells, and results in a difference in cell adhesion, morphology, mobility, and growth activity.