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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, TGFβ2, 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 TGFβ2 (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., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

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 (MoX 2 : 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.

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., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Han, Wei, Liang and Guan.)

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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (©2024PublishedbyElsevierB.V.)

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 (K 1 ), flattest meridian keratometry (K 2 ), steepest anterior keratometry (K max ), 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. K 2 , 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Han, Shen, Gu, Zhang, Sun, Chen and Zhou.)

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.

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.

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.

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.

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., (© 2023. The Author(s).)

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 &lt; 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 &lt; 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., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

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.

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., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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 NaErF 4 : 0.5% Tm 3+ @NaYF 4 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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

Oxalate is commonly employed as adjuvant of pesticide agent, causing renal injury of human even in trace residues. Despite the great achievements of the existing point-of-care testing (POCT) technology, accurate on-site screening of oxalate remains a tricky issue. To this aim, we proposed a "lab in a tube" platform which integrated portable hydrogel kit with smartphone for real-time monitoring of oxalate to achieve quantitatively precise analysis. In this work, a stimuli-responsive hydrogel-based kit was constructed via embedding manganese dioxide (MnO 2 ) nanosheets into sodium alginate hydrogel system. Based on the intrinsic oxidase-like activity, MnO 2 nanosheets-based nanozyme triggered color reaction by introducing a common sensing probe 3,3',5,5'-tetramethylbenzidine. Meanwhile, the presence of oxalate would decompose MnO 2 nanosheets, inducing the decrease of nanozyme activity, which resulted in the color response of portable kit. Coupling with ImageJ software, the image information of kit captured via smartphone could be transduced into the hue intensity, which provided a directly quantitative tool to detect oxalate with a detection limit of 8.0 μmol L -1 . This portable smartphone biosensor was successfully applied for screening urine sample within 10 min for high-throughput analysis (twelve samples) without the need for any advanced analytical instruments. Based on the merits of simple operation, cost-efficiency, and good selectivity, the availability of the miniaturized biosensor platform for POCT will achieve the requirements of routine screening and disease prevention., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus that causes encephalitis and reproductive disorders in mammalian species. However, the host factors critical for its entry, replication, and assembly are poorly understood. Here, we design a porcine genome-scale CRISPR/Cas9 knockout (PigGeCKO) library containing 85,674 single guide RNAs targeting 17,743 protein-coding genes, 11,053 long ncRNAs, and 551 microRNAs. Subsequently, we use the PigGeCKO library to identify key host factors facilitating JEV infection in porcine cells. Several previously unreported genes required for JEV infection are highly enriched post-JEV selection. We conduct follow-up studies to verify the dependency of JEV on these genes, and identify functional contributions for six of the many candidate JEV-related host genes, including EMC3 and CALR. Additionally, we identify that four genes associated with heparan sulfate proteoglycans (HSPGs) metabolism, specifically those responsible for HSPGs sulfurylation, facilitate JEV entry into porcine cells. Thus, beyond our development of the largest CRISPR-based functional genomic screening platform for pig research to date, this study identifies multiple potentially vulnerable targets for the development of medical and breeding technologies to treat and prevent diseases caused by JEV.

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.

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 Transwell TM 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 Transwell TM 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.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology is effective for genome editing and now widely used in life science research. However, the key factors determining its editing efficiency and off-target cleavage activity for single-guide RNA (sgRNA) are poorly documented. Here, we systematically evaluated the effects of sgRNA length on genome editing efficiency and specificity. Results showed that sgRNA 5'-end lengths can alter genome editing activity. Although the number of predicted off-target sites significantly increased after sgRNA length truncation, sgRNAs with different lengths were highly specific. Because only a few predicted off-targets had detectable cleavage activity as determined by Target capture sequencing (TargetSeq). Interestingly, > 20% of the predicted off-targets contained microsatellites for selected sgRNAs targeting the dystrophin gene, which can produce genomic instability and interfere with accurate assessment of off-target cleavage activity. We found that sgRNA activity and specificity can be sensitively detected by TargetSeq in combination with in silico prediction. Checking whether the on- and off-targets contain microsatellites is necessary to improve the accuracy of analyzing the efficiency of genome editing. Our research provides new features and novel strategies for the accurate assessment of CRISPR sgRNA activity and specificity., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The ectopic overexpression of foreign genes in animal genomes is an important strategy for gain-of-function study and establishment of transgenic animal models. Previous studies showed that two loci ( Rosa26 and pH11 ) were identified as safe harbor locus in pig genomes, which means foreign genes can be integrated into this locus for stable expression. Moreover, integration of a transgene may interfere with the endogenous gene expression of the target locus after the foreign fragments are inserted. Here, we provide a new strategy for efficient transgene knock-in in the endogenous GAPDH gene via CRISPR/Cas9 mediated homologous recombination. This strategy has no influence on the expression of the endogenous GAPDH gene. Thus, the GAPDH locus is a new alternative safe harbor locus in the pig genome for foreign gene knock-ins. This strategy is promising for agricultural breeding and biomedical model applications.

Designing efficient and specific CRISPR single-guide RNAs (sgRNAs) is vital for the successful application of CRISPR technology. Currently, a growing number of new RNA-guided endonucleases with a different protospacer adjacent motif (PAM) have been discovered, suggesting the necessity to develop a versatile tool for designing sgRNA to meet the requirement of different RNA-guided DNA endonucleases. Here, we report the development of a flexible sgRNA design program named "CRISPR-offinder". Support for user-defined PAM and sgRNA length was provided to increase the targeting range and specificity. Additionally, evaluation of on- and off-target scoring algorithms was integrated into the CRISPR-offinder. The CRISPR-offinder has provided the bench biologist a rapid and efficient tool for identification of high quality target sites, and it is freely available at https://sourceforge.net/projects/crispr-offinder-v1-2/ or http://www.biootools.com., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

ADP-ribosylhydrolases (ARH1, ARH2 and ARH3) are a family of enzymes to catalyze ADP-ribosylation, a reversible and covalent post-translational modification (PTM). There are four phosphorylated sites (Tyr-4, Tyr-19, Tyr-20, and Tyr-205) in ARH1. To explore the structural changes and functional impact induced by phosphorylation, molecular dynamics (MD) simulations and steered molecular dynamics (SMD) simulations were performed for the phosphorylated and non-phosphorylated ARH1 with the ligands. MD simulations results indicate that: (1) Glu-25 is more frequently in the α helix group in the phosphorylated state with the adenosine-5-diphosphate-ribosylarginine (ADP-RA) complex (51.56%) than that of the non-phosphorylated state(2.12%); (2) Ser-124 and Ser-264 become less flexible in the phosphorylated state with ADP-RA complex, which helps two residues form hydrogen bonds with ADP-RA; and (3) Tyr-211 is also less flexible in the phosphorylated state with ADP-RA complex, which helps stabilize the cation-π interaction of Y211-R119. All these changes facilitate ADP-RA to bind ARH1. In addition, according to the crystal structure of adenosine-5-diphosphate-ribose (ADP-ribose) in complex with non-phosphorylated and phosphorylated ARH1, the possible unbinding pathways of ADP-ribose from non-phosphorylated and phosphorylated ARH1 were explored respectively using SMD simulations. Our results show that phosphorylated ARH1 has more ordered structures than the non-phosphorylated type.

Clenbuterol is a β-adrenergic agonist used as additive to increase the muscle mass of meat-producing animals. Previous studies were limited to evaluations of animal growth performance and determination of the residues. Several studies have focused on urine samples. Little information about the underlying molecular mechanisms that can explain Clenbuterol metabolism and promote energy repartition in animal muscle and fatty tissue is available. Therefore, this research aims to detect the metabolite variations in muscle and fatty tissue acquired from Chinese pigs fed with Clenbuterol using gas chromatography/mass spectrometry (GC/MS). Ten two-month old Enshi black pigs were fed under the same condition; five of which were fed with basic ration containing Clenbuterol for one month, whereas the other five pigs were fed only with basic ration. Muscle and fatty tissue were subjected to metabolomics analysis using GC/MS. Differences in metabolomic profiles between the two groups were characterized by multivariate statistical analysis. The muscle samples showed that 15 metabolites were significantly different in the Clenbuterol-treated group compared with the control group; 13 potential biomarkers were found in the fatty tissue. Most of the metabolites were associated with fatty acid metabolism and amino acid metabolism. Glycerol, phenylalanine, and leucine were the common metabolites between the muscle and fatty tissue. These metabolites may provide a new clue that contributes to the understanding of the energy reassignment induced by Clenbuterol., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In this work, we develop a novel and sensitive sensor for the detection of organophosphorus pesticides based on the inner-filter effect (IFE) between gold nanoparticles (AuNPs) and ratiometric fluorescent quantum dots (RF-QDs). The RF-QDs has been designed by hybridizing two differently colored CdTe QDs, in which the red emissive QDs entrapped in the silica sphere acting as the reference signal, and the green emissive QDs covalently attached on the silica surface serving as the response signal.The fluorescence of RF-QDs could be quenched by AuNPs based on IFE. Protamine could effectively turn on the fluorescence due to the electrostatic attraction between protamine and AuNPs. Trypsin can easily hydrolyze protamine, leading to the quench of the fluorescence. Then, the fluorescence could be recovered again by the addition of parathion-methyl (PM) which could inhibit the activity of trypsin. By measuring the fluorescence of RF-QDs, the inhibition efficiency of PM to trypsin activity was evaluated. Under the optimized conditions, the inhibition efficiency was proportional to the logarithm of PM concentration in the range of 0.04-400 ng mL(-1), with a detection limit of 0.018 ng mL(-1). Furthermore, the simple and convenient method had been used for PM detection in environmental and agricultural samples with satisfactory results., (Copyright © 2015 Elsevier B.V. All rights reserved.)