All-RNA-mediated targeted gene integration in mammalian cells with rationally engineered R2 retrotransposons.

All-RNA-mediated targeted gene integration methods, rendering reduced immunogenicity, effective deliverability with non-viral vehicles, and a low risk of random mutagenesis, are urgently needed for next-generation gene addition technologies. Naturally occurring R2 retrotransposons hold promise in this context due to their site-specific integration profile. Here, we systematically analyzed the biodiversity of R2 elements and screened several R2 orthologs capable of full-length gene insertion in mammalian cells. Robust R2 system gene integration efficiency was attained using combined donor RNA and protein engineering. Importantly, the all-RNA-delivered engineered R2 system showed effective integration activity, with efficiency over 60% in mouse embryos. Unbiased high-throughput sequencing demonstrated that the engineered R2 system exhibited high on-target integration specificity (99%). In conclusion, our study provides engineered R2 tools for applications based on hit-and-run targeted DNA integration and insights for further optimization of retrotransposon systems.

Renal macrophages monitor and remove particles from urine to prevent tubule obstruction.

When the filtrate of the glomerulus flows through the renal tubular system, various microscopic sediment particles, including mineral crystals, are generated. Dislodging these particles is critical to ensuring the free flow of filtrate, whereas failure to remove them will result in kidney stone formation and obstruction. However, the underlying mechanism for the clearance is unclear. Here, using high-resolution microscopy, we found that the juxtatubular macrophages in the renal medulla constitutively formed transepithelial protrusions and "sampled" urine contents. They efficiently sequestered and phagocytosed intraluminal sediment particles and occasionally transmigrated to the tubule lumen to escort the excretion of urine particles. Mice with decreased renal macrophage numbers were prone to developing various intratubular sediments, including kidney stones. Mechanistically, the transepithelial behaviors of medulla macrophages required integrin ß1-mediated ligation to the tubular epithelium. These findings indicate that medulla macrophages sample urine content and remove intratubular particles to keep the tubular system unobstructed.

Genome-wide identification and expression characterization of the GH3 gene family of tea plant (Camellia sinensis).

To comprehensively understand the characteristics of the GH3 gene family in tea plants (Camellia sinensis), we identified 17 CsGH3 genes and analyzed their physicochemical properties, phylogenetic relationships, gene structures, promoters, and expression patterns in different tissues. The study showed that the 17 CsGH3 genes are distributed on 9 chromosomes, and based on evolutionary analysis, the CsGH3 members were divided into three subgroups. Gene duplication analysis revealed that segmental duplications have a significant impact on the amplification of CsGH3 genes. In addition, we identified and classified cis-elements in the CsGH3 gene promoters and detected elements related to plant hormone responses and non-biotic stress responses. Through expression pattern analysis, we observed tissue-specific expression of CsGH3.3 and CsGH3.10 in flower buds and roots. Moreover, based on predictive analysis of upstream regulatory transcription factors of CsGH3, we identified the potential transcriptional regulatory role of gibberellin response factor CsDELLA in CsGH3.14 and CsGH3.15. In this study, we found that CsGH3 genes are involved in a wide range of activities, such as growth and development, stress response, and transcription. This is the first report on CsGH3 genes and their potential roles in tea plants. In conclusion, these results provide a theoretical basis for elucidating the role of GH3 genes in the development of perennial woody plants and offer new insights into the synergistic effects of multiple hormones on plant growth and development in tea plants.

Optimizing Surface State Electrons of Topological Semi-Metal by Atomic Doping for Enhanced Hydrogen Evolution Reaction.

Topological materials carrying topological surface states (TSSs) have extraordinary carrier mobility and robustness, which provide a new platform for searching for efficient hydrogen evolution reaction (HER) electrocatalysts. However, the majority of these TSSs originate from the sp band of topological quantum catalysts rather than the d band. Here, based on the density functional theory calculation, it is reported a topological semimetal Pd3Sn carrying TSSs mainly derived from d orbital and proposed that optimizing surface state electrons of Pd3Sn by introduction heteroatoms (Ni) can promote hybridization between hydrogen atoms and electrons, thereby reducing the Gibbs free energy (ΔGH) of adsorbed hydrogen and improving its HER performance. Moreover, this is well verified by electrocatalytic experiment results, the Ni-doped Pd3Sn (Ni0.1Pd2.9Sn) show much lower overpotential (-29 mV vs RHE) and Tafel slope (17 mV dec-1) than Pd3Sn (-39 mV vs RHE, 25 mV dec-1) at a current density of 10 mA cm-2. Significantly, the Ni0.1Pd2.9Sn nanoparticles exhibit excellent stability for HER. The electrocatalytic activity of Ni0.1Pd2.9Sn nanoparticles is superior to that of commercial Pt. This work provides an accurate guide for manipulating surface state electrons to improve the HER performance of catalysts.

Inhibitory Activity Against Rhizoctonia Solani and Chemical Composition of Extract from Moutan Cortex.

Rice sheath blight (RSB), caused by Rhizoctonia solani, is a significant disease of rice. The negative effects of chemical fungicides have created an urgent need for low-toxicity botanical fungicides. Our previous research revealed that the ethanol crude extract of Moutan Cortex (MC) exhibited superior antifungal activity against R. solani at 1000 µg/mL, resulting in a 100 % inhibition rate. The antifungal properties were mainly found in the petroleum ether extract. However, the active ingredients of the extract are still unclear. In this study, gas chromatography-mass spectrometry (GC-MS) was utilised for the analysis of its chemical components. The mycelium growth rate method was utilized to detect the antifungal activity. The findings indicated that paeonol constituted the primary active component, with a content of more than 96 %. Meanwhile, paeonol was the most significant antifungal active ingredient, the antifungal activity of paeonol (EC50=44.83 µg/mL) was much higher than that of ß-sitosterol and ethyl propionate against R. solani. Observation under an optical microscope revealed that paeonol resulted in abnormal mycelial morphology. This study provided theoretical support for identifying monomer antifungal compounds and developing biological fungicides for R. solani.

Extracts of Knoxia roxburghii (Spreng.) M. A. Rau Induce Apoptosis in Human MCF-7 Breast Cancer Cells via Mitochondrial Pathways.

Knoxia roxburghii (Spreng.) M. A. Rau (KR) is a plant clinically used in traditional Chinese medicine (TCM) for the treatment of cancer. The study objectives were to examine the effects of KR extracts, petroleum ether (PET), ethyl acetate (EtoAc), butanol (n-BuOH), and H2O-soluble fractions (HSF) of the 75% EtOH extraction on A549 (non-small cell lung cancer), HepG2 (liver cancer), HeLa (cervical cancer), MCF-7 (breast cancer), and L02 (normal hepatocyte) cells. It was found that HSF exhibited the strongest cytotoxic activity against MCF-7 cells, and was accompanied by reduced mitochondrial transmembrane potential, increased levels of intra-cellular reactive oxygen species (ROS) and activated caspases, and upregulated pro-apoptotic and downregulated anti-apoptotic proteins. LC-MS analysis further showed that HSF primarily consisted of calycosin, aloe emodin, rein, maackiain, asperuloside, orientin, vicenin-2, and kaempferide, which have been mostly reported for anti-tumor activity in previous studies. In summary, the current study illustrated the effect, mechanism, and the potential major active components of KR against breast cancer.

Influences of standardized clinical probing on peri-implant soft tissue seal in a situation of peri-implant mucositis: A histomorphometric study in dogs.

BACKGROUND: Clinical probing is commonly recommended to evaluate peri-implant conditions. In a situation of peri-implant mucositis or peri-implantitis, the peri-implant seal healing from the disruption of soft tissue caused by probing has not yet been studied. This study aimed to investigate soft tissue healing after standardized clinical probing around osseointegrated implants with peri-implant mucositis in a dog model. METHODS: Three transmucosal implants in each hemi-mandible of six dogs randomly assigned to the peri-implant healthy group or peri-implant mucositis group were probed randomly in the mesial or distal site as probing groups (PH or PM), the cross-sectional opposite sites as unprobed control groups. Histomorphometric measurements of implant shoulder (IS)-most coronal level of alveolar bone contact to the implant surface (BCI), apical termination of the junctional epithelium (aJE)-BCI, mucosal margin (MM)-BCI, and MM-aJE were performed at 1 day, 1 week, and 2 weeks after probing. Apoptosis, proliferation, proinflammatory cytokines, and matrix metalloproteinases (MMPs) of peri-implant soft tissue were estimated by immunofluorescent analysis. RESULTS: In the PM group, apical migration of junctional epithelium was revealed by significantly decreased aJE-BCI from 1 day to 2 weeks in comparison to unprobed sites (p < 0.05), while no significant differences were found in the PH group. Immunofluorescent analysis showed higher levels of interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor-α (TNF-α), MMP-1, and MMP-8, together with exaggerated apoptosis and proliferation of peri-implant soft tissue in the PM group. CONCLUSION: Within the limitations, standardized clinical probing might lead to apical migration of the junctional epithelium in a situation of peri-implant mucositis.

Development and application of an intelligent nitrogen removal diagnosis and optimization framework for WWTPs: Low-carbon and stable operation.

Optimizing nitrogen removal is crucial for ensuring the efficient operation of wastewater treatment plants (WWTPs), but it is susceptible to variations in influent conditions and operational parameter constraints, and conflicts with the energy-saving and carbon emission reduction goals. To address these issues, this study proposes a hybrid framework integrating process simulation, machine learning, and multi-objective genetic algorithms for nitrogen removal diagnosis and optimization, aiming to predict the total nitrogen in effluent, diagnose nitrogen over-limit risks, and optimize the control strategies. Taking a full-scale WWTP as a case study, a process time-lag simulation-enhanced machine learning model (PTLS-ML) was developed, achieving R2 values of 0.94 and 0.79 for the training and testing sets, respectively. The proposed model successfully identified the potential reasons of nitrogen over-limit risks under different influent conditions and operational parameters, and accordingly provided optimization suggestions. In addition, the multi-objective optimization (MOO) algorithms analysis further demonstrated that maintaining 4-6 mg/L total nitrogen concentration in effluent by adjusting process operational parameters can effectively balance multiple objectives (i.e., effluent water quality, operating costs, and greenhouse gas emissions), achieving coordinated optimization. This framework can serve as a reference for stable operation, energy-saving, and emission reduction in the nitrogen removal of WWTPs.

Construction of an Engineered Escherichia coli with Efficient Chemotactic and Metabolizing Ability toward Tetrathionate.

The emergence of genetically engineered bacteria has provided a new means for the diagnosis and treatment of diseases. However, in vivo applications of these engineered bacteria are hindered by their inefficient accumulation in areas of inflammation. In this study, we constructed an engineered Escherichia coli (E. coli) for directional migration toward tetrathionate (a biomarker of gut inflammation), which is regulated by the TtrSR two-component system (TCS) from Shewanella baltica OS195 (S. baltica). Specifically, we removed endogenous cheZ to control the motility of E. coli. Moreover, we introduced the reductase gene cluster (ttrBCA) from Salmonella enterica serotype typhimurium (S. typhimurium), a major pathogen causing gut inflammation, into E. coli to metabolize tetrathionate. The resulting strain was tested for its motility along the gradients of tetrathionate; the engineered strain exhibits tropism to tetrathionate compared with the original strain. Furthermore, the engineered E. coli could only restore its smooth swimming ability when tetrathionate existed. With these modifications enabling tetrathionate-mediated chemotactic and metabolizing activity, this strategy with therapeutic elements will provide a great potential opportunity for target treatment of various diseases by swapping the corresponding genetic circuits.

Cueing effect of attention among nurses with different anxiety levels: an EEG study.

The attention to cueing among nurses with anxiety affects their nursing quality seriously. Nevertheless, the neural mechanism of attention under anxiety among nurses has not been revealed. In this study, we utilized the event-related potential (ERP) and functional brain networks to investigate the neural mechanism of the cueing attention differences between anxiety and non-anxiety nurse groups (AG-20 nurses; NAG-20 nurses) in the spatial cueing task. The results revealed that in the invalid cues (144 trials), longer reaction times, larger P2 amplitudes, and more linkages between the right frontal and parietal areas were found in AG compared to NAG. In the valid cues (288 trials), there were no significant behavioral and neural differences between the two groups. The AG in the invalid cues showed slower response times, larger P2 and N5 amplitudes, and denser linkages originating from the occipital cortex than those in the valid cues. The convolutional neural network was trained for discriminating between the anxiety nurses and the normal ones, with the average accuracy being 0.76. The findings provided a potential physiological biomarker to predict the anxiety group who need to give more psychological attention. Nurse leaders maybe get more information for offering solutions to retain mental health among nurses.

Trained immunity of alveolar macrophages enhances injury resolution via KLF4-MERTK-mediated efferocytosis.

Recent studies suggest that training of innate immune cells such as tissue-resident macrophages by repeated noxious stimuli can heighten host defense responses. However, it remains unclear whether trained immunity of tissue-resident macrophages also enhances injury resolution to counterbalance the heightened inflammatory responses. Here, we studied lung-resident alveolar macrophages (AMs) prechallenged with either the bacterial endotoxin or with Pseudomonas aeruginosa and observed that these trained AMs showed greater resilience to pathogen-induced cell death. Transcriptomic analysis and functional assays showed greater capacity of trained AMs for efferocytosis of cellular debris and injury resolution. Single-cell high-dimensional mass cytometry analysis and lineage tracing demonstrated that training induces an expansion of a MERTKhiMarcohiCD163+F4/80low lung-resident AM subset with a proresolving phenotype. Reprogrammed AMs upregulated expression of the efferocytosis receptor MERTK mediated by the transcription factor KLF4. Adoptive transfer of these trained AMs restricted inflammatory lung injury in recipient mice exposed to lethal P. aeruginosa. Thus, our study has identified a subset of tissue-resident trained macrophages that prevent hyperinflammation and restore tissue homeostasis following repeated pathogen challenges.

CDetection.v2: One-pot assay for the detection of SARS-CoV-2.

Introduction: The ongoing 2019 coronavirus disease pandemic (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants, is a global public health threat. Early diagnosis and identification of SARS-CoV-2 and its variants plays a critical role in COVID-19 prevention and control. Currently, the most widely used technique to detect SARS-CoV-2 is quantitative reverse transcription real-time quantitative PCR (RT-qPCR), which takes nearly 1 hour and should be performed by experienced personnel to ensure the accuracy of results. Therefore, the development of a nucleic acid detection kit with higher sensitivity, faster detection and greater accuracy is important. Methods: Here, we optimized the system components and reaction conditions of our previous detection approach by using RT-RAA and Cas12b. Results: We developed a Cas12b-assisted one-pot detection platform (CDetection.v2) that allows rapid detection of SARS-CoV-2 in 30 minutes. This platform was able to detect up to 5,000 copies/ml of SARS-CoV-2 without cross-reactivity with other viruses. Moreover, the sensitivity of this CRISPR system was comparable to that of RT-qPCR when tested on 120 clinical samples. Discussion: The CDetection.v2 provides a novel one-pot detection approach based on the integration of RT-RAA and CRISPR/Cas12b for detecting SARS-CoV-2 and screening of large-scale clinical samples, offering a more efficient strategy for detecting various types of viruses.

Genome assembly and annotation of the king ratsnake, Elaphe carinata.

The king ratsnake (Elaphe carinata) of the genus Elaphe is a common large, non-venomous snake widely distributed in Southeast and East Asia. It is an economically important farmed species. As a non-venomous snake, the king ratsnake predates venomous snakes, such as cobras and pit vipers. However, the immune and digestive mechanisms of the king ratsnake remain unclear. Despite their economic and research importance, we lack genomic resources that would benefit toxicology, phylogeography, and immunogenetics studies. Here, we used single-tube long fragment read sequencing to generate the first contiguous genome of a king ratsnake from Huangshan City, Anhui province, China. The genome size is 1.56 GB with a scaffold N50 of 6.53M. The total length of the genome is approximately 621 Mb, while the repeat content is 42.26%. Additionally, we predicted 22,339 protein-coding genes, including 22,065 with functional annotations. Our genome is a potentially useful addition to those available for snakes.

NetBID2 provides comprehensive hidden driver analysis.

Many signaling and other genes known as "hidden" drivers may not be genetically or epigenetically altered or differentially expressed at the mRNA or protein levels, but, rather, drive a phenotype such as tumorigenesis via post-translational modification or other mechanisms. However, conventional approaches based on genomics or differential expression are limited in exposing such hidden drivers. Here, we present a comprehensive algorithm and toolkit NetBID2 (data-driven network-based Bayesian inference of drivers, version 2), which reverse-engineers context-specific interactomes and integrates network activity inferred from large-scale multi-omics data, empowering the identification of hidden drivers that could not be detected by traditional analyses. NetBID2 has substantially re-engineered the previous prototype version by providing versatile data visualization and sophisticated statistical analyses, which strongly facilitate researchers for result interpretation through end-to-end multi-omics data analysis. We demonstrate the power of NetBID2 using three hidden driver examples. We deploy NetBID2 Viewer, Runner, and Cloud apps with 145 context-specific gene regulatory and signaling networks across normal tissues and paediatric and adult cancers to facilitate end-to-end analysis, real-time interactive visualization and cloud-based data sharing. NetBID2 is freely available at https://jyyulab.github.io/NetBID .

Temporal transcriptomic analysis using TrendCatcher identifies early and persistent neutrophil activation in severe COVID-19.

Studying temporal gene expression shifts during disease progression provides important insights into the biological mechanisms that distinguish adaptive and maladaptive responses. Existing tools for the analysis of time course transcriptomic data are not designed to optimally identify distinct temporal patterns when analyzing dynamic differentially expressed genes (DDEGs). Moreover, there are not enough methods to assess and visualize the temporal progression of biological pathways mapped from time course transcriptomic data sets. In this study, we developed an open-source R package TrendCatcher (https://github.com/jaleesr/TrendCatcher), which applies the smoothing spline ANOVA model and break point searching strategy, to identify and visualize distinct dynamic transcriptional gene signatures and biological processes from longitudinal data sets. We used TrendCatcher to perform a systematic temporal analysis of COVID-19 peripheral blood transcriptomes, including bulk and single-cell RNA-Seq time course data. TrendCatcher uncovered the early and persistent activation of neutrophils and coagulation pathways, as well as impaired type I IFN (IFN-I) signaling in circulating cells as a hallmark of patients who progressed to severe COVID-19, whereas no such patterns were identified in individuals receiving SARS-CoV-2 vaccinations or patients with mild COVID-19. These results underscore the importance of systematic temporal analysis to identify early biomarkers and possible pathogenic therapeutic targets.

Immunotoxicity In Vitro Assays for Environmental Pollutants under Paradigm Shift in Toxicity Tests.

With the outbreak of COVID-19, increasingly more attention has been paid to the effects of environmental factors on the immune system of organisms, because environmental pollutants may act in synergy with viruses by affecting the immunity of organisms. The immune system is a developing defense system formed by all metazoans in the course of struggling with various internal and external factors, whose damage may lead to increased susceptibility to pathogens and diseases. Due to a greater vulnerability of the immune system, immunotoxicity has the potential to be the early event of other toxic effects, and should be incorporated into environmental risk assessment. However, compared with other toxicity endpoints, e.g., genotoxicity, endocrine toxicity, or developmental toxicity, there are many challenges for the immunotoxicity test of environmental pollutants; this is due to the lack of detailed mechanisms of action and reliable assay methods. In addition, with the strong appeal for animal-free experiments, there has been a significant shift in the toxicity test paradigm, from traditional animal experiments to high-throughput in vitro assays that rely on cell lines. Therefore, there is an urgent need to build high-though put immunotoxicity test methods to screen massive environmental pollutants. This paper reviews the common methods of immunotoxicity assays, including assays for direct immunotoxicity and skin sensitization. Direct immunotoxicity mainly refers to immunosuppression, for which the assays mostly use mixed immune cells or isolated single cells from animals with obvious problems, such as high cost, complex experimental operation, strong variability and so on. Meanwhile, there have been no stable and standard cell lines targeting immune functions developed for high-throughput tests. Compared with direct immunotoxicity, skin sensitizer screening has developed relatively mature in vitro assay methods based on an adverse outcome pathway (AOP), which points out the way forward for the paradigm shift in toxicity tests. According to the experience of skin sensitizer screening, this paper proposes that we also should seek appropriate nodes and establish more complete AOPs for immunosuppression and other immune-mediated diseases. Then, effective in vitro immunotoxicity assay methods can be developed targeting key events, simultaneously coordinating the studies of the chemical immunotoxicity mechanism, and further promoting the paradigm shift in the immunotoxicity test.

Anti-Fungal Activity of Moutan cortex Extracts against Rice Sheath Blight (Rhizoctonia solani) and Its Action on the Pathogen's Cell Membrane.

Rice sheath blight (RSB) caused by Rhizoctonia solani is one of the most destructive diseases of rice (Oryza sativa). Although chemical fungicides are the most important control methods, their long-term unreasonable application has brought about problems such as environmental pollution, food risks, and non-target poisoning. Therefore, considering the extraction of fungistatic substances from plants may be an alternative in the future. In this study, we found that the Moutan cortex ethanol extract has excellent antifungal activity against R. solani, with a 100% inhibition rate at 1000 µg/mL, which aroused our great exploration interest. In-depth exploration found that the antifungal active ingredients of M. cortex were mainly concentrated in the petroleum ether extract of the M. cortex ethanol extract, which still maintained a 100% inhibition rate with 250 µg/mL, and its effective medium concentration (EC50) was 145.33 µg/mL against R. solani. Through the measurement of extracellular relative conductivity and OD260, the petroleum ether extract induced leakage of intracellular electrolytes and nucleic acids, indicating that the cell membrane was ruined. Therefore, we preliminarily determined that the cell membrane may be the target of the petroleum ether extract. Moreover, we found that petroleum ether extract reduced the content of ergosterol, a component of the cell membrane, which may be one of the reasons for the cell membrane destruction. Furthermore, the increase of MDA content would lead to membrane lipid peroxidation, further aggravating membrane damage, resulting in increased membrane permeability. Also, the destruction of the cell membrane was observed by the phenomenon of the mycelium being transparent and broken. In conclusion, this is the first report of the M. cortex petroleum ether extract exhibiting excellent antifungal activity against R. solani. The effect of the M. cortex petroleum ether extract on R. solani may be on the cell membrane, inducing the disorder of intracellular substances and metabolism, which may be one of the antifungal mechanisms against R. solani.

Novel Pyrazole Carboxamide Containing a Diarylamine Scaffold Potentially Targeting Fungal Succinate Dehydrogenase: Antifungal Activity and Mechanism of Action.

Succinate dehydrogenase (SDH) is known as an ideal target for the development of novel fungicides. Over the years, a series of novel pyrazole carboxamides containing a diarylamine scaffold have been reported as potent SDH inhibitors (SDHIs) in our laboratory. Among them, compound SCU3038 (EC50 = 0.016 mg/L) against in vitro Rhizoctonia solani was better than fluxapyroxad (EC50 = 0.033 mg/L). However, its mechanism of action is still unclear. In this paper, in pot tests, bioactivity evaluation indicated that in vivo antifungal activity of compound SCU3038 (EC50 = 0.95 mg/L) against R. solani was better than that of fluxapyroxad (EC50 = 2.29 mg/L) and thifluzamide (EC50 = 1.88 mg/L). In field trials, control efficacy of compound SCU3038 (74.10%) at 200 g ai/ha against rice sheath blight was better than that of thifluzamide (71.40%). Furthermore, target evaluation showed that compound SCU3038 could inhibit the fungal SDH from R. solani and fix in the binding site of SDH by molecular docking, thereby it could dissolve and reduce mitochondria of R. solani as observed by electron microscopy. In addition, transcriptome results showed that compound SCU3038 affected the TCA cycle pathway in mitochondria, and this was manifested in the downregulation of eight genes and upregulation of one gene. The most important phenomenon was the repressed expression of SDH2 confirmed by qRT-PCR. It was observed that compound SCU3038 was a potent SDHI, and these results afforded further research on pyrazole carboxamides.

Asperuloside Prevents Peri-Implantitis via Suppression of NF-κB and ERK1/2 on Rats.

Peri-implantitis is characterized by inflammatory cell infiltration and hyperactivation of the osteoclasts surrounding dental implants which can result in bone resorption and ultimately implant failure. Therefore, coordinating the activity of inflammatory response and bone-resorbing osteoclasts is crucial for the prevention of peri-implantitis. Asperuloside (ASP), an iridoid glycoside, has significant anti-inflammatory activities, suggesting the great potential in attenuating peri-implantitis bone resorption. A ligature-induced peri-implantitis model in the maxilla of rats was established, and the effects of ASP on preventing peri-implantitis were evaluated after four weeks of ligation using micro-CT and histological staining. RT-PCR, western blotting, tartrate-resistant acid phosphatase (TRAP), and immunofluorescent staining were conducted on osteoclasts to confirm the mechanisms of ASP on osteoclastogenesis. The results show that ASP could lead to attenuation of alveolar bone resorption in peri-implantitis by inhibiting osteoclast formation and decreasing pro-inflammatory cytokine levels in vivo. Furthermore, ASP could inhibit osteoclastogenesis by downregulating expression levels of transcription factors nuclear factor of activated T-cell (NFATc1) via restraining the activations of nuclear factor kappa beta (NF-κB) and the phosphorylation of extracellular signal-related kinase 1/2 (ERK1/2). In conclusion, ASP could significantly attenuate bone resorption in peri-implantitis via inhibition of osteoclastogenesis by suppressing NF-κB and ERK1/2 signaling pathways activations.

Synergistic engineering of CRISPR-Cas nucleases enables robust mammalian genome editing.

The naturally occurring prokaryotic CRISPR-Cas systems provide valuable resources for the development of new genome-editing tools. However, the majority of prokaryotic Cas nucleases exhibit poor editing efficiency in mammalian cells, which significantly limits their utility. Here, we have developed a method termed Improving Editing Activity by Synergistic Engineering (MIDAS). This method exerts a synergistic effect to improve mammalian genome-editing efficiency of a wide range of CRISPR-Cas systems by enhancing the interactions between Cas nuclease with the protospacer adjacent motif (PAM) and the single-stranded DNA (ssDNA) substrate in the catalytic pocket simultaneously. MIDAS robustly and significantly increased the gene-editing efficiency of Cas12i, Cas12b, and CasX in human cells. Notably, a Cas12i variant, Cas12i Max , exhibited robust activity with a very broad PAM range (NTNN, NNTN, NAAN, and NCAN) and higher efficiency than the current widely used Cas nucleases. A high-fidelity version of Cas12i Max (Cas12i HiFi ) has been further engineered to minimize off-target effects. Our work provides an expandable and efficacious method for engineering Cas nucleases for robust mammalian genome editing.