A compact and high-performance setup of capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D).

Capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D) has the advantages of high throughput (simultaneous detection of multiple ions), high separation efficiency (higher than 105 theoretical plates) and rapid analysis capability (less than 5 min for common inorganic ions). A compact CE-C4D system is ideal for water quality control and on-site analysis. It is suitable not only for common cations (e.g. Na+, K+, Li+, NH4+, Ca2+, etc.) and anions (e.g. Cl-, SO42-, BrO3-, etc.) but also for some ions (e.g. lanthanide ions, Pb2+, Cd2+, etc.) that require complex derivatization procedures to be detected by ion chromatography (IC). However, an obvious limitation of the CE-C4D method is that its sensitivity (e.g. 0.3-1 µM for common inorganic ions) is often insufficient for trace analysis (e.g. 1 ppb or 20 nM level for common inorganic ions) without preconcentration. For this technology to become a powerful and routine analytical technique, the system should be made compact while maintaining trace analysis sensitivity. In this study, we developed an all-in-one version of the CE-C4D instrument with custom-made modular components to make it a convenient, compact and high-performance system. The system was designed using direct digital synthesis (DDS) technology to generate programmable sinusoidal waveforms with any frequency for excitation, a kilovolt high-voltage power supply for capillary electrophoresis separation, and an "effective" differential C4D cell with a low-noise circuitry for high-sensitivity detection. We characterized the system with different concentrations of Cs+, and even a low concentration of 20 nM was detectable without preconcentration. Moreover, the optimized CE-C4D setup was applied to analyse mixed ions at a trace concentration of 200 nM with excellent signal-to-noise ratios. In typical applications, the limits of detection based on the 3σ criterion (without baseline filtering) were 9, 10, 24, 5, and 12 nM for K+, Cs+, Li+, Ca2+, and Mg2+, respectively, and about 7, 6, 6 and 6 nM for Br-, ClO4-, BrO3- and SO42-, respectively. Finally, the setup was also applied for the analysis of all 14 lanthanide ions and rare-earth minerals, and it showed an improvement in sensitivity by more than 25 times.

FeP-Based Nanotheranostic Platform for Enhanced Phototherapy/Ferroptosis/Chemodynamic Therapy.

Ferroptosis is an iron-dependent and lipid peroxides (LPO)-overloaded programmed damage cell death, induced by glutathione (GSH) depletion and glutathione peroxide 4 (GPX4) inactivation. However, the inadequacy of endogenous iron and reactive oxygen species (ROS) restricts the efficacy of ferroptosis. To overcome this obstacle, a near-infrared photo-responsive FeP@PEG NPs is fabricated. Exogenous iron pool can enhance the effect of ferroptosis via the depletion of GSH and further regulate GPX4 inactivation. Generation of ·OH derived from the Fenton reaction is proved by increased accumulation of lipid peroxides. The heat generated by photothermal therapy and ROS generated by photodynamic therapy can enhance cell apoptosis under near-infrared (NIR-808 nm) irradiation, as evidenced by mitochondrial dysfunction and further accumulation of lipid peroxide content. FeP@PEG NPs can significantly inhibit the growth of several types of cancer cells in vitro and in vivo, which is validated by theoretical and experimental results. Meanwhile, FeP@PEG NPs show excellent T2-weighted magnetic resonance imaging (MRI) property. In summary, the FeP-based nanotheranostic platform for enhanced phototherapy/ferroptosis/chemodynamic therapy provides a reliable opportunity for clinical cancer theranostics.

Large-scale generation of IL-12 secreting macrophages from human pluripotent stem cells for cancer therapy.

Genetically engineered macrophages (GEMs) have emerged as an appealing strategy to treat cancers, but they are largely impeded by the cell availability and technical challenges in gene transfer. Here, we develop an efficient approach to generate large-scale macrophages from human induced pluripotent stem cells (hiPSCs). Starting with 1 T150 dish of 106 hiPSCs, more than 109 mature macrophages (iMacs) could be generated within 1 month. The generated iMacs exhibit typical macrophage properties such as phagocytosis and polarization. We then generate hiPSCs integrated with an IL-12 expression cassette in the AAVS1 locus to produce iMacs secreting IL-12, a strong proimmunity cytokine. hiPSC-derived iMacs_IL-12 prevent cytotoxic T cell exhaustion and activate T cells to kill different cancer cells. Furthermore, iMacs_IL-12 display strong antitumor effects in a T cell-dependent manner in subcutaneously or systemically xenografted mice of human lung cancer. Therefore, we provide an off-the-shelf strategy to produce large-scale GEMs for cancer therapy.

Autophagy is essential for human myelopoiesis.

Emergency myelopoiesis (EM) is essential in immune defense against pathogens for rapid replenishing of mature myeloid cells. During the EM process, a rapid cell-cycle switch from the quiescent hematopoietic stem cells (HSCs) to highly proliferative myeloid progenitors (MPs) is critical. How the rapid proliferation of MPs during EM is regulated remains poorly understood. Here, we reveal that ATG7, a critical autophagy factor, is essential for the rapid proliferation of MPs during human myelopoiesis. Peripheral blood (PB)-mobilized hematopoietic stem/progenitor cells (HSPCs) with ATG7 knockdown or HSPCs derived from ATG7-/- human embryonic stem cells (hESCs) exhibit severe defect in proliferation during fate transition from HSPCs to MPs. Mechanistically, we show that ATG7 deficiency reduces p53 localization in lysosome for a potential autophagy-mediated degradation. Together, we reveal a previously unrecognized role of autophagy to regulate p53 for a rapid proliferation of MPs in human myelopoiesis.

Proteomics Sequencing Reveals the Role of TGF-ß Signaling Pathway in the Peripheral Blood of Offspring Rats Exposed to Fluoride.

The underlying mechanism of fluorosis has not been fully elucidated. The purpose of this study was to explore the mechanism of fluorosis induced by sodium fluoride (NaF) using proteomics. Six offspring rats exposed to fluoride without dental fluorosis were defined as group A, 8 offspring rats without fluoride exposure were defined as control group B, and 6 offspring rats exposed to fluoride with dental fluorosis were defined as group C. Total proteins from the peripheral blood were extracted and then separated using liquid chromatography-tandem mass spectrometry. The identified criteria for differentially expressed proteins were fold change > 1.2 or < 0.83 and P < 0.05. Gene Ontology function annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed using the oeCloud tool. The 177 upregulated and 22 downregulated proteins were identified in the A + C vs. B group. KEGG pathway enrichment analysis revealed that transforming growth factor-ß (TGF-ß) signaling pathway significantly enriched. PPI network constructed using Cytoscape confirmed RhoA may play a crucial role. The KEGG results of genes associated with fluoride and genes associated with both fluoride and inflammation in the GeneCards database also showed that TGF-ß signaling pathway was significantly enriched. The immunofluorescence in HPA database showed that the main expression sites of RhoA are plasma membrane and cytosol, while the main expression site of Fbn1 is the Golgi apparatus. In conclusion, long-term NaF intake may cause inflammatory response in the peripheral blood of rats by upregulating TGF-ß signaling pathway, in which RhoA may play a key role.

Differences in rice component distribution across layers and their relationship with taste.

BACKGROUND: Rice taste is closely associated with endosperm composition, which varies among different rice layers. Although clarifying the relationship between this difference and nutritional taste can guide rice breeding and cultivation practices, research on this topic is limited. RESULTS: Here, typical rice varieties having excellent and poor taste characteristics were selected to analyze the distribution characteristics and differences of their components. The varieties with excellent taste exhibited lower apparent amylose content (AAC) and protein content (PC), lesser short-chain (Fa) and long-chain (Fb3 ) amylopectin (AP) and more medium-chain (Fb1+2 ) AP, higher long-to-short chain ratio (Fa:Fb3 ), and higher nitrogen (N), magnesium (Mg) and calcium (Ca) content in layer 1 (L1) than the varieties with poor taste. Layer 2 (L2) played a key role in AAC and PC regulation in the varieties with excellent taste by reducing AAC and appropriately increasing PC, consequently improving rice taste. AP structure in layer 3 (L3) substantially affected the taste of the two types of varieties. The mineral content was the highest in L1, and increased potassium (K), Ca, and Mg content improved taste in all varieties. CONCLUSION: AAC in each layer contributes to rice taste. PC and minerals primarily act on L1 and L2, whereas AP acts on L2 and L3. Therefore, the endosperm formation process should be exploited for improving rice taste. Furthermore, key resources and cultivation should be identified and regulated, respectively, to improve rice taste. © 2023 Society of Chemical Industry.

Association between the infections of Trichomonas vaginalis and uterine cervical human papillomavirus: a meta-analysis.

Trichomonas vaginalis (TV) may have an impact on other reproductive tract infections. Studies on the connection between the infection of TV and human papillomavirus (HPV) have been inconsistent. We performed a systematic review of the relevant articles through keywords that satisfy the criteria and filtered the articles according to the inclusion and exclusion criteria. A total of 16 eligible studies were screened for the meta-analysis, involving a total of 150,605 women. RevMan 5.4 software was used for meta-analysis of the selected literatures. The results showed that the papers included in this study had good homogeneity and no significant publication bias was found in the current analysis. The pooled estimates using a fixed-effects model showed that TV was more prevalent in HPV-infected women than in non-infected women [odds ratio (OR): 1.51, 95% confidence interval (CI): 1.29-1.75]; In turn, HPV was more widespread in TV-infected women than in uninfected women (OR: 3.62, 95% CI: 2.71-4.85). Moreover, the interaction between TV and HPV infection was insensitive to the deletion of some studies and correlation coefficients, consequently, the results were robust and reliable. These results suggested that TV is positively associated with HPV infection, and HPV is also a risk factor for TV infection.

Prevalence and Genotype of Trichomonas vaginalis among Men in Xinxiang City, Henan Province, China.

Trichomonas vaginalis (T. vaginalis) could cause trichomoniasis through sexual transmission, which was globally distributed. In this study, the prevalence and phylogenetic analyses of T. vaginalis among men in Xinxiang were conducted. From October 2018 to December 2019, a total of 634 male clinical samples were collected, including 254 samples of semen, 43 samples of prostate fluid, and 337 samples of urine. These samples were examined by nested PCR and a total of 32 (5.05%) T. vaginalis-positive samples were detected. Among these samples, the positive rates of T. vaginalis in semen, prostate fluid, and urine were 7.87% (20/254), 4.65% (2/43), and 2.97% (10/337), respectively. Three actin genes were successfully isolated and sequenced from the 32 positive DNA samples, and the analysis of the sequence and phylogenetic tree showed that the three actin gene sequences exhibited 99.7%-100% homology to the published actin gene sequence (EU076580) in NCBI, and the T. vaginalis strains in the three positive samples were identified as genotype E. Our results demonstrate a notable genotype of T. vaginalis in the male population and provide insight into the performance of these genetic markers in the molecular epidemiology of trichomoniasis. However, further studies are needed to research the association between the genotype and the pathogenicity of T. vaginalis.

Excitatory Projections from the Prefrontal Cortex to Nucleus Accumbens Core D1-MSNs and κ Opioid Receptor Modulate Itch-Related Scratching Behaviors.

Itch is an uncomfortable and complex sensation that elicits the desire to scratch. The nucleus accumbens (NAc) activity is important in driving sensation, motivation, and emotion. Excitatory afferents from the medial prefrontal cortex (mPFC), amygdala, and hippocampus are crucial in tuning the activity of dopamine receptor D1-expressing and D2-expressing medium spiny neurons (Drd1-MSN and Drd2-MSN) in the NAc. However, a cell-type and neural circuity-based mechanism of the NAc underlying acute itch remains unclear. We found that acute itch induced by compound 48/80 (C48/80) decreased the intrinsic membrane excitability in Drd1-MSNs, but not in Drd2-MSNs, in the NAc core of male mice. Chemogenetic activation of Drd1-MSNs alleviated C48/80-induced scratching behaviors but not itch-related anxiety-like behaviors. In addition, C48/80 enhanced the frequency of spontaneous EPSCs (sEPSCs) and reduced the paired-pulse ratio (PPR) of electrical stimulation-evoked EPSCs in Drd1-MSNs. Furthermore, C48/80 increased excitatory synaptic afferents to Drd1-MSNs from the mPFC, not from the basolateral amygdala (BLA) or ventral hippocampus (vHipp). Consistently, the intrinsic excitability of mPFC-NAc projecting pyramidal neurons was increased after C48/80 treatment. Chemogenetic inhibition of mPFC-NAc excitatory synaptic afferents relieved the scratching behaviors. Moreover, pharmacological activation of κ opioid receptor (KOR) in the NAc core suppressed C48/80-induced scratching behaviors, and the modulation of KOR activity in the NAc resulted in the changes of presynaptic excitatory inputs to Drd1-MSNs in C48/80-treated mice. Together, these results reveal the neural plasticity in synapses of NAc Drd1-MSNs from the mPFC underlying acute itch and indicate the modulatory role of the KOR in itch-related scratching behaviors.SIGNIFICANCE STATEMENT Itch stimuli cause strongly scratching desire and anxiety in patients. However, the related neural mechanisms remain largely unclear. In the present study, we demonstrated that the pruritogen compound 48/80 (C48/80) shapes the excitability of dopamine receptor D1-expressing medium spiny neurons (Drd1-MSNs) in the nucleus accumbens (NAc) core and the glutamatergic synaptic afferents from medial prefrontal cortex (mPFC) to these neurons. Chemogenetic activation of Drd1-MSNs or inhibition of mPFC-NAc excitatory synaptic afferents relieves the scratching behaviors. In addition, pharmacological activation of κ opioid receptor (KOR) in the NAc core alleviates C48/80-induced itch. Thus, targeting mPFC-NAc Drd1-MSNs or KOR may provide effective treatments for itch.

Structural Analyses of a Dominant Cryptosporidium parvum Epitope Presented by H-2Kb Offer New Options To Combat Cryptosporidiosis.

Cryptosporidium parvum has gained much attention as a major cause of diarrhea in the world, particularly in those with compromised immune systems. The data currently available on how the immune system recognizes C. parvum are growing rapidly, but we lack data on the interactions among host major histocompatibility complex (MHC) diversity and parasitic T-cell epitopes. To identify antigenic epitopes in a murine model, we performed systematic profiling of H-2Kb-restricted peptides by screening the dominant Cryptosporidium antigens. The results revealed that the glycoprotein-derived epitope Gp40/15-SVF9 induced an immunodominant response in C. parvum-recovered C57BL/6 mice, and injection of the cytotoxic-T-lymphocyte (CTL) peptide with the adjuvant activated peptide-specific CD8+ T cells. Notably, the SVF9 epitope was highly conserved across Cryptosporidium hominis, C. parvum, and many other Cryptosporidium species. SVF9 also formed stable peptide-MHC class I (MHC I) complexes with HLA-A*0201, suggesting cross-reactivity between H-2Kb and human MHC I specificities. Crystal structure analyses revealed that the interactions of peptide-MHC surface residues of H-2Kb and HLA-A*0201 are highly conserved. The hydrogen bonds of H-2Kb-SVF9 are similar to those of a dominant epitope presented by HLA-A*0201, which can be recognized by a public human T-cell receptor (TCR). Notably, we found double conformations in position 4 (P4), 5 (P5) of the SVF9 peptide, which showed high flexibility, and multiple peptide conformations generated more molecular surfaces that can potentially be recognized by TCRs. Our findings demonstrate that an immunodominant C. parvum epitope and its homologs from different Cryptosporidium species and subtypes can benefit vaccine development to combat cryptosporidiosis. IMPORTANCE Adaptive immune responses and T lymphocytes have been implicated as important mechanisms of parasite-induced protection. However, the role of CD8+ T lymphocytes in the resolution of C. parvum infection is largely unresolved. Our results revealed that the glycoprotein-derived epitope Gp40/15-SVF9 induced an immunodominant CD8+ T-cell response in C57BL/6 mice. Crystal structure analyses revealed that the interactions of the H-2Kb-SVF9 peptide are similar to those of a dominant epitope presented by HLA-A*0201, which can be recognized by human TCRs. In addition, we found double conformations of the SVF9 peptide, which showed high flexibility and multiple peptide conformations that can potentially be recognized by TCRs.

The complete mitogenome and phylogeny analysis of Pseudohemiculter hainanensis (Boulenger, 1900) (Cyprinidae: Cultrinae).

The Pseudohemiculter hainanensis (Boulenger, 1900) is a small Cyprinidae fish that has a wide distribution in China. In this study, we characterized the complete mitochondrial genome of P. hainanensis by the Illumina NovaSeq sequencing platform in Guangxi, China. The assembled mitogenome is 16,647 base pairs (bp) and consists of 13 protein-coding genes (PCGs), 22 transfer RNAs, two ribosomal RNAs, and a control region (D-loop). Nucleotide composition of the complete mitogenome is 29.69% (A), 24.82% (T), 27.97% (C), and 17.52% (G), with an A + T bias of 54.51%. The maximum-likelihood tree based on 13 PCGs showed that Pseudohemiculter hainanensis formed an independent lineage and P. hainanensis was closer to T. houdemeri.

Preparation of Degradable Antiwaxing Coating and a Novel Method for Quantifying Antiwaxing Property of Coatings.

The deposition of wax on the surface of the pipeline or the facilities is harmful to oil industries, which can cause serious safety problems and economic loss. To address this problem, a degradable glutinous rice/chitosan-phytic acid composite coating (GR/CS-PA) was prepared. The GR can serve as an adhesive, and it can also enhance the flexibility of CS-PA. The CS-PA component can be used to retain water to prevent the adhesion of crude oil and enhance the mechanical properties of the antiwaxing layer. With the hydration layer on the surface of the GR/CS-PA coating, which can prevent contact between the pollutants and the GR/CS-PA, the coating possesses excellent antifouling properties, especially excellent antiwaxing performance. In addition, an antiwaxing performance testing method was established to quantificationally evaluate the antiwaxing performance of the coating, and the antiwaxing performance (AWP) value was proposed to be the criterion of the antiwaxing performance of the coating. We hope that the green coating and the quantitative testing method can be applied in the petroleum industry to solve the wax deposition problem.

Signature of seven cuproptosis-related lncRNAs as a novel biomarker to predict prognosis and therapeutic response in cervical cancer.

Background: Given the high incidence and high mortality of cervical cancer (CC) among women in developing countries, identifying reliable biomarkers for the prediction of prognosis and therapeutic response is crucial. We constructed a prognostic signature of cuproptosis-related long non-coding RNAs (lncRNAs) as a reference for individualized clinical treatment. Methods: A total of seven cuproptosis-related lncRNAs closely related to the prognosis of patients with CC were identified and used to construct a prognostic signature via least absolute shrinkage and selection operator regression analysis in the training set. The predictive performance of the signature was evaluated by Kaplan-Meier (K-M) analysis, receiver operating characteristic (ROC) analysis, and univariate and multivariate Cox analyses. Functional enrichment analysis and single-sample gene set enrichment analysis were conducted to explore the potential mechanisms of the prognostic signature, and a lncRNA-microRNA-mRNA network was created to investigate the underlying regulatory relationships between lncRNAs and cuproptosis in CC. The associations between the prognostic signature and response to immunotherapy and targeted therapy were also assessed. Finally, the prognostic value of the signature was validated using the CC tissues with clinical information in my own center. Results: A prognostic signature was developed based on seven cuproptosis-related lncRNAs, including five protective factors (AL441992.1, LINC01305, AL354833.2, CNNM3-DT, and SCAT2) and two risk factors (AL354733.3 and AC009902.2). The ROC curves confirmed the superior predictive performance of the signature compared with conventional clinicopathological characteristics in CC. The ion transport-related molecular function and various immune-related biological processes differed significantly between the two risk groups according to functional enrichment analysis. Furthermore, we discovered that individuals in the high-risk group were more likely to respond to immunotherapy and targeted therapies including trametinib and cetuximab than those in the low-risk group. Finally, CC tissues with clinical data from my own center further verify the robustness of the seven-lncRNA risk signature. Conclusion: We generated a cuproptosis-related lncRNA risk signature that could be used to predict prognosis of CC patients. Moreover, the signature could be used to predict response to immunotherapy and chemotherapy and thus could assist clinicians in making personalized treatment plans for CC patients.

Role of Glycolysis/Gluconeogenesis and HIF-1 Signaling Pathways in Rats with Dental Fluorosis Integrated Proteomics and Metabolomics Analysis.

Fluoride is widely distributed, and excessive intake will lead to dental fluorosis. In this study, six offspring rats administrated 100 mg/L sodium fluoride were defined as the dental fluorosis group, and eight offspring rats who received pure water were defined as the control group. Differentially expressed proteins and metabolites extracted from peripheral blood were identified using the liquid chromatography tandem mass spectrometry and gas chromatography mass spectrometry, with the judgment criteria of fold change >1.2 or <0.83 and p < 0.05. A coexpression enrichment analysis using OmicsBean was conducted on the identified proteins and metabolites, and a false discovery rate (FDR) < 0.05 was considered significant. Human Protein Atlas was used to determine the subcellular distribution of hub proteins. The Gene Cards was used to verify results. A total of 123 up-regulated and 46 down-regulated proteins, and 12 up-regulated and 2 down-regulated metabolites were identified. The significant coexpression pathways were the HIF-1 (FDR = 1.86 × 10−3) and glycolysis/gluconeogenesis (FDR = 1.14 × 10−10). The results of validation analysis showed the proteins related to fluorine were mainly enriched in the cytoplasm and extrinsic component of the cytoplasmic side of the plasma membrane. The HIF-1 pathway (FDR = 1.01 × 10−7) was also identified. Therefore, the HIF-1 and glycolysis/gluconeogenesis pathways were significantly correlated with dental fluorosis.

Copper-catalyzed C2 alkenylation of pyridine-N-oxides with alkynes.

A general and expedient method to construct the scaffolds of 2-alkenylpyridines, through copper-catalyzed C2 alkenylation of pyridine-N-oxides with alkynes, has been disclosed. This protocol shows operational simplicity, good functional group compatibility and broad substrate scope.

Copper-Catalyzed Hydrogen Atom Transfer and Aryl Migration Strategy for the Arylalkylation of Activated Alkenes.

Herein, we describe the copper-catalyzed arylalkylation of activated alkenes via hydrogen-atom transfer and aryl migration strategy. The reaction was carried out through a radical-mediated continuous migration pathway using N-fluorosulfonamides as the alkyl source. The primary, secondary, and tertiary alkyl radicals formed by intramolecular hydrogen-atom transfer proceeded smoothly. This methodology is an efficient approach for the synthesis of various amide derivatives possessing a quaternary carbon center with good yields and high regioselectivity.

GFI1 regulates chromatin state essential in human endothelial-to-haematopoietic transition.

OBJECTIVES: During embryonic haematopoiesis, haematopoietic stem/progenitor cells (HSPCs) develop from hemogenic endothelial cells (HECs) though endothelial to haematopoietic transition (EHT). However, little is known about how EHT is regulated in human. Here, we report that GFI1 plays an essential role in enabling normal EHT during haematopoietic differentiation of human embryonic stem cells (hESCs). RESULTS: GFI1 deletion in hESCs leads to a complete EHT defect due to a closed chromatin state of hematopoietic genes in HECs. Mechanically, directly regulates important signaling pathways essential for the EHT such as PI3K signaling.etc. CONCLUTIONS: Together, our findings reveal an essential role of GFI1 mediated epigenetic mechanism underlying human EHT during hematopoiesis.

CTCF functions as an insulator for somatic genes and a chromatin remodeler for pluripotency genes during reprogramming.

CTCF mediates chromatin insulation and long-distance enhancer-promoter (EP) interactions; however, little is known about how these regulatory functions are partitioned among target genes in key biological processes. Here, we show that Ctcf expression is progressively increased during induced pluripotency. In this process, CTCF first functions as a chromatin insulator responsible for direct silencing of the somatic gene expression program and, interestingly, elevated Ctcf expression next ensures chromatin accessibility and contributes to increased EP interactions for a fraction of pluripotency-associated genes. Therefore, CTCF functions in a context-specific manner to modulate the 3D genome to enable cellular reprogramming. We further discover that these context-specific CTCF functions also enlist SMARCA5, an imitation switch (ISWI) chromatin remodeler, together rewiring the epigenome to facilitate cell-fate switch. These findings reveal the dual functions of CTCF in conjunction with a key chromatin remodeler to drive reprogramming toward pluripotency.

Eliminating predictable DNA off-target effects of cytosine base editor by using dual guiders including sgRNA and TALE.

Predictable DNA off-target effect is one of the major safety concerns for the application of cytosine base editors (CBEs). To eliminate Cas9-dependent DNA off-target effects, we designed a novel effective CBE system with dual guiders by combining CRISPR with transcription activator-like effector (TALE). In this system, Cas9 nickase (nCas9) and cytosine deaminase are guided to the same target site to conduct base editing by single-guide RNA (sgRNA) and TALE, respectively. However, if nCas9 is guided to a wrong site by sgRNA, it will not generate base editing due to the absence of deaminase. Similarly, when deaminase is guided to a wrong site by TALE, base editing will not occur due to the absence of single-stranded DNA. In this way, Cas9- and TALE-dependent DNA off-target effects could be completely eliminated. Furthermore, by fusing TALE with YE1, a cytidine deaminase with minimal Cas9-independent off-target effect, we established a novel CBE that could induce efficient C-to-T conversion without detectable Cas9- or TALE-dependent DNA off-target mutations.

Role of the hippo signaling pathway in the extracellular matrix degradation of chondrocytes induced by fluoride exposure.

To identify the role of the Hippo signaling pathway in the extracellular matrix degradation of chondrocytes induced by fluoride exposure. Environmental response genes (ERGs) of bone injury induced by fluoride exposure were obtained from the Comparative Toxicogenomics Database, and annotated by STRING for KEGG pathway enrichment analysis. The CCK-8 kit was used to measure the proliferation of ATDC5 cells. The malondialdehyde (MDA), total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), and glutathione peroxidase (GSH-PX) levels in ATDC5 cells were measured using oxidative stress detection kit. Western blot analysis was used to measure the p-MST1/2, p-LATS1/2, and p-YAP/YAP1 expression levels in the Hippo pathway and the COL2A1, ACAN and MMP13 expression levels in the cartilage matrix. Localizations of YAP1 and COL2A1 proteins in chondrocytes were performed using cell immunofluorescence. Continuous data from the multiple groups were compared using one-way analysis of variance, and then the differences between groups were tested with Dunnett's t-test, with the test level α = 0.05. The 145 ERGs of bone injury induced by fluoride exposure were identified, and KEGG enrichment analysis revealed Hippo signaling pathways significantly related to bone injury. A CCK-8 assay revealed that the viability of the ATDC5 cells was significantly decreased with increased fluorine concentration. The MDA content in 20 mg/L sodium fluoride (NaF) exposure group was significantly higher than that in the control group, the T-SOD, T-AOC and GSH-PX activities in 15 and 20 mg/L NaF exposure groups were significantly lower than those in the control group (P < 0.05). Western blot results showed the protein levels of p-MST1/2, p-LATS1/2 and p-YAP1 in 15 and 20 mg/L NaF exposure groups were significantly lower than those in the control group, while the YAP1 protein level in 20 mg/L NaF group was significantly higher than that in the control group. The COL2A1 and ACAN proteins in 20 mg/L NaF group were significantly decreased, while the MMP13 protein level in 15 and 20 mg/L NaF groups were significantly increased (P < 0.05). It was observed that the expression of YAP1 protein expression level in the cytoplasm decreased with the increased fluoride exposure, whereas that the expression level of YAP1 protein in the nucleus increased. Fluoride inhibited the proliferation of ATDC5 cells, induced oxidative stress, inhibited the activity of the Hippo pathway, and eventually led to cartilage matrix degradation.