The signatures and crosstalk of gut microbiome, mycobiome, and metabolites in decompensated cirrhotic patients.

Background: Numerous studies have confirmed that gut microbiota plays a crucial role in the progression of cirrhosis. However, the contribution of gut fungi in cirrhosis is often overlooked due to the relatively low abundance. Methods: We employed 16S ribosomal RNA sequencing, internal transcribed spacer sequencing, and untargeted metabolomics techniques to investigate the composition and interaction of gut bacteria, fungi, and metabolites in cirrhotic patients. Results: Cirrhotic patients exhibited significant differences in the diversity and composition of gut microbiota and their metabolites in cirrhotic patients compared to healthy individuals. Increase in pathogenic microbial genera and a decrease in beneficial microbial genera including bacteria and fungi were observed. Various clinical indexes were closely connected with these increased metabolites, bacteria, fungi. Additionally, endoscopic treatment was found to impact the gut microbiota and metabolites in cirrhotic patients, although it did not significantly alter the gut ecology. Finally, we constructed a cirrhosis diagnostic model based on different features (bacteria, fungi, metabolites, clinical indexes) with an AUC of 0.938. Conclusion: Our findings revealed the characteristics of gut microbial composition and their intricate internal crosstalk in cirrhotic patients, providing cutting-edge explorations of potential roles of gut microbes in cirrhosis.

Insights into oral lentinan immunomodulation: Dectin-1-mediated lymphatic transport from Peyer's patch M cells to mononuclear phagocytes.

Lentinan (LNT), a natural polysaccharide, has been reported to exhibit immunomodulatory effects in the intestine after oral administration. Herein, we aimed to investigate the lymphatic transport of LNT in Peyer's patches (PPs) by traceable fluorescent labeling and to explore whether/how LNT contacts related immune cells. Near-infrared imaging confirmed the absorption of LNT in the small intestinal segment and its accumulation within PPs after oral administration. Subsequently, tissue imaging confirmed that M cells are the main cells responsible for transporting LNT to PPs, and an M cell model was established to explore the involvement of Dectin-1 in the absorption process. Systematic in vitro and in vivo studies revealed that the Dectin-1 further mediates the uptake of LNT by mononuclear phagocytes in PPs. Moreover, LNT can promote the proliferation and differentiation of mononuclear phagocytes, thereby activating immune responses. In summary, this study elucidates the pharmacokinetic mechanisms by which LNT exerts oral immunomodulatory effects, providing a theoretical basis for the development and application of other polysaccharides.

Accumulation and growth toxicity mechanisms of fluxapyroxad revealed by physiological, hepatopancreas transcriptome, and gut microbiome analysis in Pacific white shrimp (Litopenaeus vannamei).

Fluxapyroxad (FX), a typical succinate dehydrogenase inhibitor fungicide, is causing increased global concerns due to its fungicide effects. However, the accumulation and grow toxicity of FX to Litopenaeus vannamei (L. vannamei) is poorly understand. Therefore, the accumulation pattern of FX in L. vannamei was investigated for the first time in environmental concentrations. FX accumulated rapidly in shrimp muscle. Meanwhile, growth inhibition was observed and the mechanism derived by primarily accelerated glycolipid metabolism and reduced glycolipid content. Moreover, exposure to environmental concentrations of FX induced significant growth inhibition and oxidative stress and inhibited oxidative phosphorylation and TCA cycle in L. vannamei. The endocytosis signaling pathway genes were activated, thereby driving growth toxicity. Oxidative phosphorylation and cytosolic gene expression were further rescued in elimination experiments, demonstrating the mechanism of growth toxicity by FX exposure. The results revealed that FX persistently altered the gut microbiome of L. vannamei using gut microbiome sequencing, particularly with increased Garcinia Purple Pseudoalteromonas luteoviolacea for organic pollutant degradation. This study provided new insights into the potential toxicity of FX to marine organisms, emphasizing the need for further investigation and potential regulatory considerations.

Rapid determination and risk evaluation of multi-class antibiotics in aquatic products by one-step purification process coupled with ultra-high performance liquid chromatography-tandem mass spectrometry.

A sensitive and robust multiclass analytical method was established to simultaneously determine 55 antibiotics in aquatic products through liquid chromatography-tandem mass spectrometry. A simple one-step purification process was successfully developed, which combined post-acidic acetonitrile extraction directly by an enhanced matrix removal cartridge. This approach eliminated the need for solvent transition. The established method for 55 antibiotics achieved an excellent linear relationship with R2 values ≥ 0.9921 in the range of 0.05-200 µg/L. The quantitation limits ranged within 0.04-5.0 µg/kg. Satisfactory recoveries (76.2%-99.7 %) were achieved with the relative standard deviations below 13.9 %. Furthermore, the antibiotic residues in aquatic products were analyzed, and the health and antibiotic resistance risk assessments were conducted. Although the health risks of target antibiotics were acceptable, a resistance risk was observed. Therefore, monitoring antibiotic residue levels in aquatic products requires considerable attention and further research to ensure the quality of marine products and consumer safety.

Radiomic analysis based on magnetic resonance imaging for the prediction of VEGF expression in hepatocellular carcinoma patients.

OBJECTIVE: The purpose of this study was to investigate the ability of radiomic characteristics of magnetic resonance images to predict vascular endothelial growth factor (VEGF) expression in hepatocellular carcinoma (HCC) patients. METHODS: One hundred and twenty-four patients with HCC who underwent fat-suppressed T2-weighted imaging (FS-T2WI) and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) one week before surgical resection were enrolled in this retrospective study. Immunohistochemical analysis was used to evaluate the expression level of VEGF. Radiomic features were extracted from the axial FS-T2WI, DCE-MRI (arterial phase and portal venous phase) images of axial MRI. Least absolute shrinkage and selection operator (LASSO) and stepwise regression analyses were performed to select the best radiomic features. Multivariate logistic regression models were constructed and validated using tenfold cross-validation. Receiver operating characteristic (ROC) curve analysis, calibration curve analysis and decision curve analysis (DCA) were employed to evaluate these models. RESULTS: Our results show that there were 94 patients with high VEGF expression and 30 patients with low VEGF expression among the 124 HCC patients. The FS-T2WI, DCE-MRI and combined MRI radiomics models had AUCs of 0.8713, 0.7819, and 0.9191, respectively. There was no significant difference in the AUC between the FS-T2WI radiomics model and the DCE-MRI radiomics model (p > 0.05), but the AUC for the combined model was significantly greater than the AUCs for the other two models (p < 0.05) according to the DeLong test. The combined model had the greatest net benefit according to the DCA results. CONCLUSION: The radiomic model based on multisequence MR images has the potential to predict VEGF expression in HCC patients. The combined model showed the best performance.

Genomic epidemiology reveals multiple mechanisms of linezolid resistance in clinical enterococci in China.

BACKGROUND: Infections caused by linezolid-resistant enterococci (LRE) are clinically difficult to treat and threaten patient health. However, there is a lack of studies on long time-span LRE strains in China. For this reason, our study comprehensively revealed the resistance mechanisms of LRE strains collected in a Chinese tertiary care hospital from 2011 to 2022. METHODS: Enterococcal strains were screened and verified after retrospective analysis of microbial data. Subsequently, 65 LRE strains (61 Enterococcus faecalis and 4 Enterococcus faecium, MIC ≥ 8 µg/ml), 1 linezolid-intermediate Enterococcus faecium (MIC = 4 µg/ml) and 1 linezolid-susceptible Enterococcus faecium (MIC = 1.5 µg/ml) were submitted for whole-genome sequencing (WGS) analysis and bioinformatics analysis. RESULTS: The optrA gene was found to be the most common linezolid resistance mechanism in our study. We identified the wild-type OptrA and various OptrA variants in 98.5% of LRE strains (61 Enterococcus faecalis and 3 Enterococcus faecium). We also found one linezolid-resistant Enterococcus faecium strain carried both optrA and cfr(D) gene, while one linezolid-resistant Enterococcus faecium only harbored the poxtA gene. Most optrA genes (55/64) were located on plasmids, with impB-fexA-optrA, impB-fexA-optrA-erm(A), fexA-optrA-erm(A), and fexA-optrA segments. A minority of optrA genes (9/64) were found on chromosomes with the Tn6674-like platform. Besides, other possible linezolid resistance-associated mechanisms (mutations in the rplC and rplD genes) were also found in 26 enterococcal strains. CONCLUSIONS: Our study suggested that multiple mechanisms of linezolid resistance exist among clinical LRE strains in China.

Prognostic model on pregnancy outcomes for women with recurrent spontaneous abortions treated with cyclosporin A: A single-institution experience.

BACKGROUND: This study aimed to identify prognostic factors for pregnancy outcomes and construct a prognostic model for pregnancy outcomes in women with Recurrent Spontaneous Abortions (RSA) treated with cyclosporin A. METHODS: A total of 154 RSA patients treated with cyclosporin A between October 2016 and October 2018 were retrospectively recruited. Multivariate logistic regression was applied to identify the prognostic factors for pregnancy success in RSA women treated with cyclosporin A. The Receiver Operating Characteristic (ROC) curve was applied to construct prognostic value, and the prognostic performance was assessed using area under the ROC. RESULTS: After adjusting potential confounding factors, the authors noted increased age (OR = 0.771; 95 % CI 0.693‒0.858; p < 0.001) and positive antinuclear antibodies (OR = 0.204; 95 % CI 0.079‒0.526; p = 0.001) were associated with a reduced incidence of pregnancy success, while positive anti-ß2 glycoprotein-I-antibody (OR = 21.941; 95 % CI 1.176‒409.281; p = 0.039) was associated with an increased incidence of pregnancy success after treated with cyclosporin A. The AUC of combining these variables for predicting pregnancy failure was 0.809 (95 % CI 0.735‒0.880). CONCLUSIONS: This study systematically identified the prognostic factors for pregnancy success in women treated with cyclosporin A, and the constructed prognostic model based on these factors with relatively higher prognostic value. Further large-scale prospective studies should be performed to validate the prognostic value of the constructed model.

Unveiling the hidden risks: Pesticide residues in aquaculture systems.

The present study systematically assessed the presence and ecological risks of 79 pesticides in various aquaculture systems, namely pond aquaculture (PA), greenhouse aquaculture (GA), and raceway aquaculture (RA) at different aquaculture stages, along with evaluating the pesticide removal of four tailwater treatment systems. Sixteen herbicides and two fungicides were identified, with the total concentrations ranging from 8.33 ng/L to 3248.45 ng/L. The PA system demonstrated significantly higher concentrations (p < 0.05) and a wider range of pesticide residues compared to the GA and RA systems. Prometryn, simetryn, atrazine, and thifluzamide were found to be the predominant pesticides across all three aquaculture modes, suggesting their significance as pollutants that warrant monitoring. Additionally, the findings indicated that the early aquaculture stage exhibits the highest levels of pesticide concentration, underscoring the importance of heightened monitoring and regulatory interventions during this phase. Furthermore, among the four tailwater treatment systems analyzed, the recirculating tailwater treatment system exhibited the highest efficacy in pesticide removal. A comprehensive risk assessment revealed minimal ecological risks in both the aquaculture and tailwater environments. However, the pesticide mixtures present high risks to algae and low to medium risks to aquatic invertebrates and fish, particularly during the early stages of aquaculture. Simetryn and prometryn were identified as high-risk pesticides. Based on the prioritization index, simetryn, prometryn, diuron, and ametryn are recommended for prioritization in risk assessment. This study offers valuable data for pesticide control and serves as a reference for the establishment of a standardized pesticide monitoring and management system at various stages of aquaculture.

Comparative antioxidant and metabolomic analysis for the identification of differential response of mussel (Mytilus coruscus) to four succinate dehydrogenase inhibitor fungicides.

Succinate dehydrogenase inhibitor fungicides (SDHIs) are frequently detected in the marine environment. However, studies on the toxicity of SDHIs to marine organisms, Mytilus coruscus (M. coruscus), are poorly reported. Therefore, the antioxidant activities and metabolomic response of four SDHIs, namely, boscalid (BC), thifluzamide (TF), fluopyram (FO), and bixafen (BIX), to (M. coruscus), were comprehensively investigated. The antioxidant activity of BC and TF was significantly increased (p<0.05), whereas those of FO and BIX were significantly decreased. Furthermore, metabolite discriminations among M. coruscus to four SDHIs were illustrated by an untargeted metabolomics approach. A total of 52, 50, 93, and 129 differential metabolites were obtained for BC, TF, FO, and BIX. KEGG of the different metabolites show that the four SDHIs had differential effects on the metabolic pathways of M. coruscus. The current study demonstrated four SDHIs triggered glucose metabolism, lipid metabolism, tricarboxylic acid cycle, and oxidative phosphorylation processes and caused the disruption of nutrient and energy conversion processes in mussels. Finally, five biomarkers were screened by analyzing common differential metabolites that emerged from the four SDHI exposures, which could be used for risk assessment of marine ecosystem exposure to SDHIs. Our results demonstrated the use of metabolomics to understand the potential mechanisms of toxicity of four SDHIs to mussels and to identify potential targets for future targeted risk assessment.

Hemimasticatory spasm caused by single venous compression of the root of the trigeminal nerve: An MRI study for a case report and review of literature.

Hemimasticatory spasm is a very rare disorder of the trigeminal nerve characterized by paroxysmal involuntary contraction of the jaw-closing muscles. Although its cause is not fully known, vascular compression of the trigeminal nerve is thought to be involved. Magnetic resonance imaging (MRI) can indicate continuing vascular compression for hemimasticatory spasm. Here, we report a case of hemimasticatory spasm that was caused by single venous compression of the trigeminal nerve root on MRI and was confirmed by microvascular decompression surgery.

Nickel-Catalyzed Reductive Alkene Cross-Dialkylation with Unactivated Alkyl Electrophiles.

A Ni-catalyzed reductive dialkylation of 8-aminoquinoline-tethered aliphatic alkenes with two unactivated alkyl electrophiles is disclosed here. Key to the development of this transformation is the combination of primary alkyl (pseudo)halides and secondary alkyl iodides that produce products in a single regioselective manner. The reaction exhibits good functional group compatibility, and its synthetic utility was demonstrated by the concise synthesis of the precursors of biologically relevant molecules.

Accumulation and elimination properties and comparative toxicity of fluxapyroxad in juvenile and adult large yellow croaker (Larimichthys crocea).

Fluxapyroxad (FX), a succinate dehydrogenase inhibitor fungicide, has been detected in global marine and aquatic organisms. However, as a new pollutant, its biotoxicity and ecological risks to marine aquatic organisms are unclear. The accumulation and elimination processes and toxic effects of FX on Larimichthys crocea (L. crocea) at environmental concentrations were assessed. FX (1.0 µg/L) was rapidly enriched and persisted prolonged in L. crocea muscle and FX is highly toxic to juvenile L. crocea with the 96 h LC50 of 245.0 µg/L. Furthermore, the toxic effects of FX on juvenile L. crocea and adults L. crocea were compared and analyzed. In contrast to those of adult L. crocea, juvenile L. crocea showed a stronger oxidative stress response and rescued liver damage in terms of antioxidant enzyme activity, energy supply, and liver damage to FX. Transcriptomic analysis also showed that drug metabolism was activated. In the adult L. crocea, the disturbance of the energy metabolism, oxidative respiration, TCA cycle, and lipid metabolism genes were firstly found. The results revealed the accumulation and elimination pattern and ecotoxicological hazards of FX to L. crocea, which provided important theoretical basis for the study of environmental risks caused by new pollutants to marine organisms.

High-throughput analytical methodology of monoalkyl phthalate esters and the composite risk assessment with their parent phthalate esters in aquatic organisms and seawater.

A sensitive, robust, and highly efficient analytical methodology involving solid phase extraction coupled to ultra-high performance liquid chromatography tandem mass spectrometry was successfully established to detect 13 monoalkyl phthalate esters (MPAEs) in aquatic organisms and seawater. After the organisms were preprocessed using enzymatic deconjugation with ß-glucuronidase, extraction, purification, and qualitative and quantitative optimization procedures were performed. Under optimal conditions, the limits of detection varied from 0.07 to 0.88 µg/kg (wet weight) and 0.04-1.96 ng/L in organisms and seawater, respectively. Collectively, MPAEs achieved acceptable recovery values (91.0-102.7%) with relative standard deviations less than 10.4% and matrix effects ranging from 0.93 to 1.07 in the above matrix. Furthermore, MPAEs and phthalate esters were detected by the developed methodology and gas chromatography-triple quadrupole tandem mass spectrometer in practical samples, respectively. Mono-n-butyl phthalate and mono-iso-butyl phthalate were the most predominant congeners, accounting for 24.8-35.2% in aquatic organisms and seawater. Comprehensive health and ecological risks were higher after the MPAEs were incorporated than when phthalate esters were considered separately, and greater than their risk threshold. Therefore, the risks caused by substances and their metabolites in multiple media, with analogous structure-activity relationships, should be considered to ensure the safety of aquatic organisms and consumers.

Prognostic model on pregnancy outcomes for women with recurrent spontaneous abortions treated with cyclosporin A: A single-institution experience

Abstract Background: This study aimed to identify prognostic factors for pregnancy outcomes and construct a prognostic model for pregnancy outcomes in women with Recurrent Spontaneous Abortions (RSA) treated with cyclosporin A. Methods: A total of 154 RSA patients treated with cyclosporin A between October 2016 and October 2018 were retrospectively recruited. Multivariate logistic regression was applied to identify the prognostic factors for pregnancy success in RSA women treated with cyclosporin A. The Receiver Operating Characteristic (ROC) curve was applied to construct prognostic value, and the prognostic performance was assessed using area under the ROC. Results: After adjusting potential confounding factors, the authors noted increased age (OR = 0.771; 95 % CI 0.693‒0.858; p < 0.001) and positive antinuclear antibodies (OR = 0.204; 95 % CI 0.079‒0.526; p = 0.001) were associated with a reduced incidence of pregnancy success, while positive anti-β2 glycoprotein-I-antibody (OR = 21.941; 95 % CI 1.176‒409.281; p = 0.039) was associated with an increased incidence of pregnancy success after treated with cyclosporin A. The AUC of combining these variables for predicting pregnancy failure was 0.809 (95 % CI 0.735‒0.880). Conclusion: This study systematically identified the prognostic factors for pregnancy success in women treated with cyclosporin A, and the constructed prognostic model based on these factors with relatively higher prognostic value. Further large-scale prospective studies should be performed to validate the prognostic value of the constructed model.

XAF1 promotes anti-RNA virus immune responses by regulating chromatin accessibility.

A rapid induction of antiviral genes is critical for eliminating viruses, which requires activated transcription factors and opened chromatins to initiate transcription. However, it remains elusive how the accessibility of specific chromatin is regulated during infection. Here, we found that XAF1 functioned as an epigenetic regulator that liberated repressed chromatin after infection. Upon RNA virus infection, MAVS recruited XAF1 and TBK1. TBK1 phosphorylated XAF1 at serine-252 and promoted its nuclear translocation. XAF1 then interacted with TRIM28 with the guidance of IRF1 to the specific locus of antiviral genes. XAF1 de-SUMOylated TRIM28 through its PHD domain, which led to increased accessibility of the chromatin and robust induction of antiviral genes. XAF1-deficient mice were susceptible to RNA virus due to impaired induction of antiviral genes. Together, XAF1 acts as an epigenetic regulator that promotes the opening of chromatin and activation of antiviral immunity by targeting TRIM28 during infection.

The Effects of Nebulized Inhaled Triptolide on Airway Inflammation in a Mouse Model of Asthma.

Inhalation of nebulized TP has received little attention in the past. Here, we intend to investigate the effect of nebulized inhaled TP on airway inflammation in a mouse model of asthma. 29 SPF BALB/c mice were divided into four groups: blank control (Blk, n = 5), normal saline (NS, n = 8), dexamethasone (Dex, n = 8), and TP (n = 8). During the process of sensitization, mice in the three intervention groups were treated with nebulized NS, an injection of Dex, and nebulized triptolide, respectively. Then bronchoalveolar lavage fluid (BALF), peripheral blood, and lung tissue were collected. Relevant cytokines, transcriptional factors, and CD4+Th17+ T cell proportions were assessed and compared. IL-6, IL-17, IL-23, and TGF-ß1 demonstrated a significant difference between groups in the following order: Dex < TP < NS (P ≤ 0.001), while IL-10 changed in the opposite direction (P < 0.001). At the transcriptional level in lung tissue, the Ct value of IL-17 in the Dex group was significantly higher than in the NS and TP groups (P < 0.001). Meanwhile, it was higher in the TP group than in the NS group (P < 0.001). The Ct value of RORγt demonstrated a significant difference among three groups in the following order: Dex > TP > NS (P < 0.001). An opposite trend of FoxP3 Ct value was revealed in the order: NS > TP > Dex. The proportion of CD4+Th17+ cells was 9.53 ± 2.74% in the NS group, 4.23 ± 2.26% in the Dex group, and 6.76 ± 2.99% in the TP group, which shows significant differences between the NS and Dex (P < 0.001) or NS and TP groups (P < 0.05). Inhalation of nebulized triptolide can play a role in suppressing airway inflammation with inflammatory cytokines and transcriptional factors reduced and CD4+Th17+ T cells dampened, also in a manner less than injected dexamethasone.

Environmental profile, potential sources, and ecological risk of polycyclic aromatic hydrocarbons in a typical coastal bay and outer bay area.

As a class of persistent organic pollutants, polycyclic aromatic hydrocarbons (PAHs) are widely present and accumulate in multimedia environments. The pollution characteristics, spatiotemporal distribution, potential sources, influencing factors, and ecological risks of 16 PAHs were investigated in the water-sediment system of the Hangzhou Bay and outer bay area (HZB and OBA, respectively). The total concentrations of 16 PAHs (∑PAHs) were 220 ± 97.0 and 130 ± 36.0 ng/L in the seawater and 343 ± 179 and 505 ± 415 µg/kg (dry weight) in the sediments of the HZB and OBA, respectively. The pollution level of PAHs in the HZB seawater was higher than that in the OBA seawater, but the opposite result was found in the sediments. Moreover, ∑PAHs exhibited high temporal variability in the HZB seawater (rainy season > dry season), whereas ∑PAHs in the sediments showed no significant difference between seasons. The molecular diagnostic ratio method was used to identify pollution sources and showed that the PAHs in seawater came from different pollution sources (fuel combustion and petroleum), whereas the PAHs in the sediments originated from coincident sources (mixed combustion). Correlation analysis revealed that temperature was positively related to ∑PAHs, whereas salinity was negatively related to seawater ∑PAHs values. Ecological risk assessment demonstrated that the potential for adverse ecological effects was low to moderate in seawater but moderate to high in the sediments.

Simultaneous determination and dietary intake risk assessment of 60 herbicide residues in aquatic products.

A sensitive and rugged analytical method was first established to simultaneously determine 60 herbicides in aquatic products with gas chromatography-tandem mass spectroscopy (GC-MS/MS). After extraction with acetonitrile (MeCN), NaCl and anhydrous Na2SO4 were added, concentrated supernatants were directly passed through the Carb/NH2 solid phase extraction column. Then, the cartridge was rinsed with elution solution (MeCN/toluene, 3:1, v/v), followed by GC-MS/MS analysis with multiple reaction monitoring. An excellent linearity (1.0-100.0 µg/L) with R2 value of ≥0.9991 was obtained, and the limits of quantification were 0.018-3.852 µg/kg. Satisfactory recoveries (70.8 %-117.6 %) with RSDs below 11.0 % of herbicide residues were obtained at spiked levels of 0.010-0.050 mg/kg. Furthermore, herbicide residues in actual aquatic products were analyzed, and the acute/chronic risk assessment of dietary exposure was carried out. The wide use of herbicides for controlling weed and removing moss and harmful algae may obviously increase the risk of contamination of the aquaculture environment and fishery products. Therefore, considerable attention and more research are necessary to monitor residue levels for herbicides in aquatic products and ensure the quality of marine products and consumer safety.

The collateral activity of RfxCas13d can induce lethality in a RfxCas13d knock-in mouse model.

BACKGROUND: The CRISPR-Cas13 system is an RNA-guided RNA-targeting system and has been widely used in transcriptome engineering with potentially important clinical applications. However, it is still controversial whether Cas13 exhibits collateral activity in mammalian cells. RESULTS: Here, we find that knocking down gene expression using RfxCas13d in the adult brain neurons caused death of mice, which may result from the collateral activity of RfxCas13d rather than the loss of target gene function or off-target effects. Mechanistically, we show that RfxCas13d exhibits collateral activity in mammalian cells, which is positively correlated with the abundance of target RNA. The collateral activity of RfxCas13d could cleave 28s rRNA into two fragments, leading to translation attenuation and activation of the ZAKα-JNK/p38-immediate early gene pathway. CONCLUSIONS: These findings provide new mechanistic insights into the collateral activity of RfxCas13d in mammalian cells and warn that the biosafety of the CRISPR-Cas13 system needs further evaluation before application to clinical treatments.

Establishment of an Integrated CRISPR/Cas9 Plasmid System for Simple and Efficient Genome Editing in Medaka In Vitro and In Vivo.

Although CRISPR/Cas9 has been used in gene manipulation of several fish species in vivo, its application in fish cultured cells is still challenged and limited. In this study, we established an integrated CRISPR/Cas9 plasmid system and evaluated its efficiency of gene knock-out or knock-in at a specific site in medaka (Oryzias latipes) in vitro and in vivo. By using the enhanced green fluorescent protein reporter plasmid pGNtsf1, we demonstrate that pCas9-U6sgRNA driven by endogenous U6 promoter (pCas9-mU6sgRNA) mediated very high gene editing efficiency in medaka cultured cells, but not by exogenous U6 promoters. After optimizing the conditions, the gene editing efficiencies of eight sites targeting for four endogenous genes were calculated, and the highest was up to 94% with no detectable off-target. By one-cell embryo microinjection, pCas9-mU6sgRNA also mediated efficient gene knock-out in vivo. Furthermore, pCas9-mU6sgRNA efficiently mediated gene knock-in at a specific site in medaka cultured cells as well as embryos. Collectively, our study demonstrates that the genetic relationship of U6 promoter is critical to gene editing efficiency in medaka cultured cells, and a simple and efficient system for medaka genome editing in vitro and in vivo has been established. This study provides an insight into other fish genome editing and promotes gene functional analysis.