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INTRODUCTION: The aim of this study was to investigate the features of imaging differences between Clarus and Optomap ultra-widefield imaging systems after implantable collamer lens (ICL) implantation. METHODS: This was a non-randomized controlled study. Ninety-two eyes of 46 consecutive patients were enrolled. Full-scale ophthalmological examinations were conducted preoperatively. All patients underwent Clarus (CLARUS 500; Carl Zeiss, Dublin, USA) and Optomap (Daytona; Optos, UK) ultra-wide imaging sequentially under the same circumstance preoperatively and 1 month after EVO-ICL implantation. A single image was acquired from each. Dx was defined as the distance between the upper furcation of the central retinal artery and the central fovea of macula. Pixels of the optic cup and disc and Dx as well as the optic cup/disc ratio were calculated and compared on each machine before and after surgery. RESULTS: All surgeries were uneventful without complications. Safety and efficacy indices were both 100% at 1 month. Values of both optic cup and disc areas were in decrease after surgery with statistically significant differences (p < 0.001), while the cup/disc ratio remained the same (Clarus mean of differences = -0.0028, p = 0.83; Optomap mean of differences = -0.0016, p = 0.76). Dx of images captured with either machine was statistically significantly decreased (p < 0.001). Differences of both optic cup (p = 0.057) and disc (p = 0.041) areas of Clarus were more obvious than that of Optomap, while only the latter was with statistical significance. Difference of Dx of Clarus was statistically significantly larger than Optomap. CONCLUSIONS: Display ranges tend to be broadened after EVO-ICL implantation in both Clarus and Optomap ultra-widefield imaging systems, while Clarus shows a wider display range of the two, which encourages the application of Clarus when it comes to the detection of more peripheral retinal lesions.
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Lentes Intraoculares , Miopía , Disco Óptico , Humanos , Implantación de Lentes Intraoculares/métodos , Miopía/diagnóstico , Miopía/cirugíaRESUMEN
Although large-scale and accurate identification of cis-regulatory elements on pig protein-coding and long non-coding genes has been reported, similar study on pig miRNAs is still lacking. Here, we systematically characterized the cis-regulatory elements of pig miRNAs in muscle and fat by adopting miRNAomes, ChIP-seq, ATAC-seq, RNA-seq and Hi-C data. In total, the cis-regulatory elements of 257 (85.95%) expressed miRNAs including 226 known and 31 novel miRNAs were identified. Especially, the miRNAs associated with super-enhancers, active promoters, and "A" compartment were significantly higher than those associated by typical enhancers, prompters without H3K27ac, and "B" compartment, respectively. The tissue specific transcription factors were the primary determination of core miRNA expression pattern in muscle and fat. Moreover, the miRNA promoters are more evolutionarily conserved than miRNA enhancers, like other type genes. Our study adds additional important information to existing pig epigenetic data and provides essential resource for future in-depth investigation of pig epigenetics.
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Epigenómica , MicroARNs , Animales , Secuenciación de Inmunoprecipitación de Cromatina , MicroARNs/genética , Músculos , Secuencias Reguladoras de Ácidos Nucleicos , Porcinos/genéticaRESUMEN
The Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes viral encephalitis in humans, pigs and other mammals across Asia and the Western Pacific. Genetic screening tools such as CRISPR screening, DNA sequencing and RNA interference have greatly improved our understanding of JEV replication and its potential antiviral approaches. However, information on exon and intron mutations associated with JEV replication is still scanty. CRISPR-Cas9-mediated cytosine base editing can efficiently generate C: G-to-T: A conversion in the genome of living cells. One intriguing application of base editing is to screen pivotal variants for gene function that is yet to be achieved in pigs. Here, we illustrate that CRISPR-Cas9-mediated cytosine base editor, known as AncBE4max, can be used for the functional analysis of calreticulin (CALR) variants. We conducted a CRISPR-Cas9-mediated cytosine base editing screen using 457 single guide RNAs (sgRNAs) against all exons and introns of CALR to identify loss-of-function variants involved in JEV replication. We unexpectedly uncovered that two enriched sgRNAs targeted the same site in intron-2 of the CALR gene. We found that mutating four consecutive G bases in the intron-2 of the CALR gene to four A bases significantly inhibited JEV replication. Thus, we established a CRISPR-Cas9-mediated cytosine-base-editing point mutation screening technique in pigs. Our results suggest that CRISPR-mediated base editing is a powerful tool for identifying the antiviral functions of variants in the coding and noncoding regions of the CALR gene.
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Calreticulina , Virus de la Encefalitis Japonesa (Especie) , Virus de la Encefalitis Japonesa (Subgrupo) , Animales , Humanos , Antivirales , Calreticulina/genética , Sistemas CRISPR-Cas/genética , Citosina , Virus de la Encefalitis Japonesa (Especie)/genética , Edición Génica , Intrones/genética , Mamíferos , Mutación , ARN Guía de Sistemas CRISPR-Cas , PorcinosRESUMEN
Managed pressure drilling (MPD) is the most effective means to ensure drilling safety, and MPD is able to avoid further deterioration of complex working conditions through precise control of the wellhead back pressure. The key to the success of MPD is the well control strategy, which currently relies heavily on manual experience, hindering the automation and intelligence process of well control. In response to this issue, an MPD knowledge graph is constructed in this paper that extracts knowledge from published papers and drilling reports to guide well control. In order to improve the performance of entity extraction in the knowledge graph, a few-shot Chinese entity recognition model CEntLM-KL is extended from the EntLM model, in which the KL entropy is built to improve the accuracy of entity recognition. Through experiments on benchmark datasets, it has been shown that the proposed model has a significant improvement compared to the state-of-the-art methods. On the few-shot drilling datasets, the F-1 score of entity recognition reaches 33%. Finally, the knowledge graph is stored in Neo4J and applied for knowledge inference.
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On-site quantitative analysis of pesticide is of significant importance for addressing serious public health issues in clinical, food, and environmental settings. Herein, we designed a novel smartphone-assisted sensing platform for on-site monitoring of 2,4-dichlorophenoxyacetic acid (2,4-D) based on carbon dots/cobalt oxyhydroxide nanosheet (CDs/CoOOH) composite. In this work, a red emissive CDs/CoOOH composite was proposed as a signal indicator for shielding background interference, enhancing anti-interference capability. 2,4-D as an inhibitor of alkaline phosphatase could specifically suppress the production of ascorbic acid, which restrained in situ etching of the CDs/CoOOH composite and further triggered the fluorescence response of the biosensor. By employing a lab-on-smartphone based device and self-designed application software, the fluorescence image was directly captured and analyzed with a sensitive detection limit of 100 µg L-1 for 2,4-D. Merging the CDs/CoOOH composite-based fluorometric system with the smartphone-assisted optical reader, such a cost-effective and portable platform provided a new sight for on-site monitoring of pesticide and expanded application prospect in the field of biological analysis.
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Ácido 2,4-Diclorofenoxiacético/análisis , Técnicas Biosensibles , Carbono/química , Inhibidores Enzimáticos/análisis , Puntos Cuánticos/química , Teléfono Inteligente , Ácido 2,4-Diclorofenoxiacético/farmacología , Fosfatasa Alcalina/antagonistas & inhibidores , Fosfatasa Alcalina/metabolismo , Cobalto/química , Inhibidores Enzimáticos/farmacología , Nanopartículas/química , Imagen Óptica , Óxidos/química , Programas Informáticos , Espectrofotometría UltravioletaRESUMEN
Atomically thin two-dimensional transition metal dichalcogenides (TMDCs) have been regarded as ideal and promising nanomaterials that bring broad application prospects in extensive fields due to their ultrathin layered structure, unique electronic band structure, and multiple spatial phase configurations. TMDCs with different phase structures exhibit great diversities in physical and chemical properties. By regulating the phase structure, their properties would be modified to broaden the application fields. In this mini review, focusing on the most widely concerned molybdenum dichalcogenides (MoX2: X = S, Se, Te), we summarized their phase structures and corresponding electronic properties. Particularly, the mechanisms of phase transformation are explained, and the common methods of phase regulation or phase stabilization strategies are systematically reviewed and discussed. We hope the review could provide guidance for the phase regulation of molybdenum dichalcogenides nanomaterials, and further promote their real industrial applications.
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Introduction: Acupuncture and tuina, acknowledged as ancient and highly efficacious therapeutic modalities within the domain of Traditional Chinese Medicine (TCM), have provided pragmatic treatment pathways for numerous patients. To address the problems of ambiguity in the concept of Traditional Chinese Medicine (TCM) acupuncture and tuina treatment protocols, the lack of accurate quantitative assessment of treatment protocols, and the diversity of TCM systems, we have established a map-filling technique for modern literature to achieve personalized medical recommendations. Methods: (1) Extensive acupuncture and tuina data were collected, analyzed, and processed to establish a concise TCM domain knowledge base. (2)A template-free Chinese text NER joint training method (TemplateFC) was proposed, which enhances the EntLM model with BiLSTM and CRF layers. Appropriate rules were set for ERE. (3) A comprehensive knowledge graph comprising 10,346 entities and 40,919 relationships was constructed based on modern literature. Results: A robust TCM KG with a wide range of entities and relationships was created. The template-free joint training approach significantly improved NER accuracy, especially in Chinese text, addressing issues related to entity identification and tokenization differences. The KG provided valuable insights into acupuncture and tuina, facilitating efficient information retrieval and personalized treatment recommendations. Discussion: The integration of KGs in TCM research is essential for advancing diagnostics and interventions. Challenges in NER and ERE were effectively tackled using hybrid approaches and innovative techniques. The comprehensive TCM KG our built contributes to bridging the gap in TCM knowledge and serves as a valuable resource for specialists and non-specialists alike.
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Objectives: Osteoarthritis (OA) is characterized by a high prevalence, poor prognosis, and a propensity to lead to disability. Despite the availability of standard therapies, they are associated with potential side effects and don't provide a complete cure for patients. Consequently, there is an urgent demand for the development of novel drugs. Method: The gene expression profiles (GSE64394, GSE178557 and GSE215039) of normal and OA chondrocytes samples were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified by the "LIMMA" R package. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were conducted using the R package clusterProfiler. A protein-protein (PPI) interaction network was performed to identify hub genes by using the Search Tool for the Retrieval of Interacting Genes (STRING) and Cytoscape. Small molecule compounds linked to OA were predicted through the NetworkAnalyst platform. Finally, molecular docking was conducted using AutoDock and Pymol software. Results: We identified 98 DEGs primarily implicated in endochondral ossification, extracellular matrix degradation, and Wnt signaling pathways. 23 DEGs were closely associated with OA, and 10 hub genes were found to be potential drug targets for OA. Two new targeted compounds, tetrachlorodibenzodioxin (TCDD) and valproic acid (VPA), were screened. And they both exhibited strong binding affinity to their respective targets. Conclusions: Reducing exposure to TCDD could be a crucial strategy in preventing OA, and VPA has gained recognition as a novel drug candidate for OA treatment.
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PURPOSE: To evaluate changes of hydroxyproline concentration and its influencing factors of small incision lenticule extraction (SMILE)-derived corneal stromal lenticules with different preservation methods. METHODS: A total of 390 corneal stromal lenticules of 195 patients were derived from SMILE surgeries. Thirty of the lenticules were classified as the fresh (control) group, and the rest were randomly and evenly divided and stored in anhydrous glycerol, silicone oil, Optisol, and cryopreservation for 1 day, 1 week, or 1 month. A hydroxyproline assay kit (ab222941, Abcam) was used to measure the hydroxyproline concentration in each preservation method. Concentrations of MMP-2, TIMP-2, TNFα, TGFß2, and reactive oxygen species were also evaluated. RESULTS: In the anhydrous glycerol group, the concentration of hydroxyproline decreased within 1 week (fresh: 1 dΔ = 0.229, P < 0.001*; 1 d - 1 wΔ = 0.055, P < 0.001*) while that in the silicone oil group remained stable in 1 week (1 d - 1 wΔ = -0.005, P = 0.929) and decreased significantly in 1 m (1 m - 1 wΔ = -0.041, P = 0.003*). The sequence of hydroxyproline concentration in the Optisol group was 1 m > 1 day > 1 week. Hydroxyproline concentration in the cryopreservation group decreased within 1 m. Hydroxyproline concentration was highest in the Optisol group and lowest in the anhydrous glycerol group under the same preservation time. Hydroxyproline concentration was negatively correlated with MMP-2 (r = -0.16, P = 0.421) and TIMP-2 (r = -0.56, P = 0.002*) while MMP-2 and TNFα (r = 0.17, P = 0.242), TIMP-2 and TGFß2 (r = 0.21, P = 0.207), and TNFα and reactive oxygen species (r = 0.52, P = 0.007*) were positively correlated. CONCLUSIONS: More collagen was retained in SMILE lenticules preserved in Optisol under the same preservation time. The mechanism of the changes of collagen in preserved SMILE-derived lenticules and oxidative stress requires additional investigation.
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AIM: To investigate the long-term changes of corneal densitometry (CD) and its contributing elements after small incision lenticule extraction (SMILE). METHODS: Totally 31 eyes of 31 patients with mean spherical equivalent of -6.46±1.50 D and mean age 28.23±7.38y were enrolled. Full-scale examinations were conducted on all patients preoperatively and during follow-up. Visual acuity, manifest refraction, axial length, corneal thickness, corneal higher-order aberrations, and CD were evaluated. RESULTS: All surgeries were completed successfully without complications or adverse events. Ten-year safety index was 1.17±0.20 and efficacy 1.04±0.28. CD value of 0-6 mm zones in central layer was statistically significantly lower 10y postoperatively, compared with preoperative values (0-2 mmΔ=-1.62, 2-6 mmΔ=-1.24, P<0.01). There were no correlations between CD values and factors evaluated. CONCLUSION: SMILE is a safe and efficient procedure for myopia on a long-term basis. CD values get lower 10y postoperatively, whose mechanism is to be further discussed.
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PURPOSE: The aim of this study was to evaluate the safety, efficacy, and predictability of lenticule intrastromal keratoplasty (LIKE) for the correction of iatrogenic high hyperopia. METHODS: Three patients (4 eyes) were referred to our department because of overcorrection of myopia induced by femtosecond laser-assisted in situ keratomileusis. All eyes exhibited hyperopia (between +4.00 and +8.00 D) and thin corneas ranging from 307 to 378 µm. Because of the regression of the use of laser ablation to correct high hyperopia and thin corneas, we initially adopted LIKE to correct iatrogenic high hyperopia in all 4 eyes. The uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), manifest refraction, corneal topography, and anterior segment optical coherence tomography findings were recorded during each follow-up evaluation. RESULTS: No postoperative complications, such as interface haze and opacification, were observed in the 4 eyes during each follow-up evaluation, with an average follow-up of 9.50 months. All eyes had significantly improved UDVA postoperatively. A total of 3 eyes achieved an UDVA of 20/25 or better while the other 1 eye had an UDVA of 20/40 postoperatively. In addition, 2 of the 4 eyes had a postoperative UDVA equal to or better than preoperative CDVA. No eyes lost any CDVA lines. All 4 eyes were within ±0.50 D of the spherical power (intended target of 0). The central corneal thickness and curvature of the anterior corneal surface in all 4 eyes increased postoperatively. Anterior segment optical coherence tomography revealed that the lenticule was transparent, with no wrinkles or offsets, during each follow-up evaluation. CONCLUSIONS: LIKE for the correction of iatrogenic hyperopia has good efficacy and safety. Although an extremely thin cornea after overcorrection may not be suitable for hyperopic laser enhancement, LIKE is a good choice because of its good predictability and ability to restore the normal corneal structure.
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More and more people are under high pressure in modern society, leading to growing mental disorders, such as antenatal depression for pregnant women. Antenatal depression can affect pregnant woman's physical and psychological health and child outcomes, and cause postpartum depression. Therefore, it is essential to detect the antenatal depression of pregnant women early. This study aims to predict pregnant women's antenatal depression and identify factors that may lead to antenatal depression. First, a questionnaire was designed, based on the daily life of pregnant women. The survey was conducted on pregnant women in a hospital, where 5666 pregnant women participated. As the collected data is unbalanced and has high dimensions, we developed a one-class classifier named Stacked Auto Encoder Support Vector Data Description (SAE-SVDD) to distinguish depressed pregnant women from normal ones. To validate the method, SAE-SVDD was firstly applied on three benchmark datasets. The results showed that SAE-SVDD was effective, with its F-scores better than other popular classifiers. For the antenatal depression problem, the F-score of SAE- SVDD was higher than 0.87, demonstrating that the questionnaire is informative and the classification method is successful. Then, by an improved Term Frequency-Inverse Document Frequency (TF-IDF) analysis, the critical factors of antenatal depression were identified as work stress, marital status, husband support, passive smoking, and alcohol consumption. With its generalizability, SAE-SVDD can be applied to analyze other questionnaires.
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Complicaciones del Embarazo , Mujeres Embarazadas , Femenino , Humanos , Embarazo , Consumo de Bebidas Alcohólicas , Estado Civil , Complicaciones del Embarazo/diagnóstico , Mujeres Embarazadas/psicología , Encuestas y CuestionariosRESUMEN
Purpose: This study aimed to investigate the features of progressive keratoconus by means of machine learning. Methods: In total, 163 eyes from 127 patients with at least 3 examination records were enrolled in this study. Pentacam HR was used to measure corneal topography. Steepest meridian keratometry (K1), flattest meridian keratometry (K2), steepest anterior keratometry (Kmax), central corneal thickness (CCT), thinnest corneal thickness (TCT), anterior radius of cornea (ARC), posterior elevation (PE), index of surface variation (ISV), and index of height deviation (IHD) were input for analysis. Support vector machine (SVM) and logistic regression analysis were applied to construct prediction models. Results: Age, PE, and IHD showed statistically significant differences as the follow-up period extended. K2, PE, and ARC were selected for model construction. Logistic regression analysis presented a mean area under the curve (AUC) score of 0.780, while SVM presented a mean AUC of 0.659. The prediction sensitivity of SVM was 52.9%, and specificity was 79.0%. Conclusion: It is feasible to use machine learning to predict the progression and prognosis of keratoconus. Posterior elevation exhibits a sensitive prediction effect.
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The Rapid Visual CRISPR (RAVI-CRISPR) assay employs Cas12a and Cas13a enzymes for precise gene detection in a sample. However, RAVI-CRISPR is limited in single-tube multiplex detection applications due to the lack of specific single-strand (ss) DNA-fluorescently quenched (ssDNA-FQ) and RNA-fluorescently quenched (ssRNA-FQ) reporter cleavage mechanisms. We report the development of a sensitive and specific dual-gene Cas12a and Cas13a diagnostic system. To optimize the application for field testing, we designed a portable multiplex fluorescence imaging assay that could distinguish test results with the naked eye. Herein, dual gene amplified products from multiplex recombinase polymerase amplification (RPA) were simultaneously detected in a single tube using Cas12a and Cas13a enzymes. The resulting orthogonal DNA and RNA collateral cleavage specifically distinguishes individual and mixed ssDNA-FQ and ssRNA-FQ reporters using the green-red-yellow, fluorescent signal conversion reaction system, detectable with portable blue and ultraviolet (UV) light transilluminators. As a proof-of-concept, reliable multiplex RAVI-CRISPR detection of genome-edited pigs was demonstrated, exhibiting 100% sensitivity and specificity for the analysis of CD163 knockout, lactoferrin (LF) knock-in, and wild-type pig samples. This portable naked-eye multiplex RAVI-CRISPR detection platform can provide accurate point-of-care screening of genetically modified animals and infectious diseases in resource-limited settings.
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Sistemas CRISPR-Cas , Sistemas de Atención de Punto , Animales , Porcinos , Sistemas CRISPR-Cas/genética , Bioensayo , ADN de Cadena Simple/genética , ARN , Técnicas de Amplificación de Ácido NucleicoRESUMEN
The development of a contamination-free and on-site nucleic acid detection platform with high sensitivity and specificity but low-cost for the detection of pathogenic nucleic acids is critical for infectious disease diagnosis and surveillance. In this study, we combined the recombinase-aided amplification (RAA) with the exonuclease III (Exo III)-assisted signal amplification into a platform for sensitive and specific detection of nucleic acids of African swine fever virus (ASFV). We found that this platform enabled a naked eye visual detection of ASFV at a detection limit as low as 2 copies/µL in 30 min. As expected, no cross-reactivity was observed with other porcine viruses. In addition, to avoid aerosol contamination, a one-tube RAA-Exo III colorimetric assay was also established for the accurate detection of ASFV in clinical samples. Taken together, we developed a rapid, instrument-free, and low-cost Exo III-assisted RAA colorimetric-assay-based nucleic acid detection platform.
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Virus de la Fiebre Porcina Africana , Ácidos Nucleicos , Animales , Porcinos , Sensibilidad y Especificidad , Colorimetría , Ácidos Nucleicos/genética , Recombinasas , Técnicas de Amplificación de Ácido NucleicoRESUMEN
Genetically modified pigs have shown considerable application potential in the fields of life science research and livestock breeding. Nevertheless, a barrier impedes the production of genetically modified pigs. There are too few safe harbor loci for the insertion of foreign genes into the pig genome. Only a few loci (pRosa26, pH11 and Pifs501) have been successfully identified to achieve the ectopic expression of foreign genes and produce gene-edited pigs. Here, we use CRISPR/Cas9-mediated homologous directed repair (HDR) to accurately knock the exogenous gene-of-interest fragments into an endogenous CKM gene in the porcine satellite cells. After porcine satellite cells are induced to differentiate, the CKM gene promoter simultaneously initiates the expression of the CKM gene and the exogenous gene. We infer preliminarily that the CKM gene can be identified as a potential muscle-specific safe harbor locus in pigs for the integration of exogenous gene-of-interest fragments.
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Sistemas CRISPR-Cas , Genoma , Animales , Genoma/genética , Ganado/genética , Músculos , Regiones Promotoras Genéticas , Porcinos/genéticaRESUMEN
On-site monitoring of carbamate pesticide in complex matrix remians as a challenge in terms of the real-time control of food safety and supervision of environmental quality. Herein, we fabricated robust upconversion nanoparticles (UCNPS)/polydopamine (PDA)-based hydrogel portable suit that precisely quantified carbaryl in complex tea samples with smartphone detector. UCNPS/PDA nanoprobe was developed by polymerization of dopamine monomers on the surface of NaErF4: 0.5% Tm3+@NaYF4 through electrostatic interaction, leading to efficient red luminescence quenching of UCNPS under near-infrared excitation, which circumvented autofluorescence and background interference in complicated environment. Such a luminescence quenching could be suppressed by thiocholine that was produced by acetylcholinesterase-mediated catalytic reaction, thus enabling carbaryl bioassay by inhibiting the activity of enzyme. Bestowed with the feasibility analysis of fluorescent output, portable platform was designed by integrating UCNPS-embedded sodium alginate hydrogel with 3D-printed smartphone device for quantitatively on-site monitoring of carbaryl in the range of 0.5-200 ng mL-1 in tea sample, accompanied by a detection limit of 0.5 ng mL-1. Owing to specific UCNPS signatures and hydrogel immobilization, this modular platform displayed sensitive response, portability and anti-interference capability in complex matrix analysis, thus holding great potential in point-of-care application.
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Técnicas Biosensibles , Nanopartículas , Plaguicidas , Acetilcolinesterasa , Carbamatos , HidrogelesRESUMEN
Objective To study the process and significance of intercellular mRNA transfer by using a Cre-loxP system. Methods Cre-expressing donor cells and loxP-DsRed-loxP-eGFP-expressing recipient cells were established. Co-culture and TranswellTM co-culture of both donor and recipient cells were analyzed with a separate culture of recipient cells only as controls. After 72 hours, the eGFP+ recipient cells in each group were observed and analyzed. Immunofluorescence staining was used to detect Cre expression in eGFP+ cells. The effects of donor cell death, cell membrane nanotubes, and ROCK signaling pathway on Cre mRNA transfer were explored. Results After co-culture for 72 hours, the eGFP+ cells were only observed in co-culture group, but not in the recipient cells alone group and the TranswellTM co-culture group, indicating that cell-cell contact could mediate Cre mRNA transfer. Immunofluorescence staining showed that Cre mRNA could be translated into proteins and transported to the nucleus in eGFP+ recipient cells. Moreover, the transport of Cre mRNA between living cells could be partially blocked by membrane nanotubes or ROCK signaling pathway inhibitors. Conclusion The mRNA can be exchanged among living cells through intercellular contact, thus leading to the changes of biological functions in the recipient cells.
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ARN Mensajero/genética , Membrana Celular , Núcleo Celular , IntegrasasRESUMEN
Colostrum quality is a vital factor in mortality and growth performance for piglets. Lactoferrin is an immuno-active milk protein that contributes to the formation of a protective layer above intestinal mucosa, possesses the antibacterial and antiviral activities that are favorable for piglet development. However, there is a notable reduction in lactoferrin in sow milk during lactation after the first few days, which causes many piglets to fail to ingest enough colostrum thereby leading to an increase in piglet mortality. In this study, we successfully constructed genome-edited Large-White pigs with marker-free site-specific knock-in of lactoferrin gene in the 3'-end of Casein alpha-s1 via CRISPR/Cas9 mediated homologous recombination. Thus, the lactoferrin protein can be expressed in the mammary gland in the control of Casein alpha-s1 promoter. As expected, the lactoferrin protein in genetically modified pigs sustained high expression in both colostrum and milk when compared with wild-type pigs. Moreover, the bacterial plate assay indicated that the milk from genetically modified pigs showed bacteriostatic effects when compared with control pigs. Taken together, our study demonstrated that the milk from genetically modified pigs had antibacterial activity which may reduce the costs of veterinary drug and improve the surviving rate of piglets, which is promising for pig breeding.
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Transgenic pigs play an important role in biomedicine and agriculture. The "safe harbor" locus maintains consistent foreign gene expression in cells and is important for transgenic pig generation. However, as only several safe harbor loci(Rosa26, pH11 and Pifs501) have been identified in pigs, meeting the needs of the insertion of various foreign genes is difficult. In this study, we develop a novel strategy for the efficient knock-in of gene-of-interest fragments into endogenous beta-actin(ACTB) gene via CRISPR/Cas9 mediated homologous recombination with normal expression of ACTB. Thus, we provide an alternative strategy to integrate exogenous genes into the pig genome that can be applied to agricultural breeding and biomedical models.