Prime editing (PE), as a "search-and-replace" genome editing technology, has shown the attractive potential of versatile genome editing ability, which is, in principle, currently superior to other ...well-established genome-editing technologies in the all-in-one operation scope. However, essential technological solutions of PE technology, such as the improvement of genome editing efficiency, the inhibition of potential off-targets and intended edits accounting for unexpected side-effects, and the development of effective delivery systems, are necessary to broaden its application. Since the advent of PE, many optimizations have been performed on PE systems to improve their performance, resulting in bright prospects for application in many fields. This review briefly discusses the development of PE technology, including its functional principle, noteworthy barriers restraining its application, current efforts in technical optimization, and its application directions and potential risks. This review may provide a concise and informative insight into the burgeoning field of PE, highlight the exciting prospects for this powerful tool, and provide clues for questions that may propel the field forward.
Pinene is a monoterpene, that is used in the manufacture of fragrances, insecticide, fine chemicals, and renewable fuels. Production of pinene by metabolic-engineered microorganisms is a sustainable ...method. Purple non-sulfur photosynthetic bacteria belong to photosynthetic chassis that are widely used to synthesize natural chemicals. To date, researches on the synthesis of pinene by purple non-sulfur photosynthetic bacteria has not been reported, leaving the potential of purple non-sulfur photosynthetic bacteria synthesizing pinene unexplored.
Rhodobacter sphaeroides strain was applied as a model and engineered to express the fusion protein of heterologous geranyl diphosphate synthase (GPPS) and pinene synthase (PS), hence achieving pinene production. The reaction condition of pinene production was optimized and 97.51 μg/L of pinene was yielded. Then, genes of 1-deoxy-D-xylulose 5-phosphate synthase, 1-deoxy-D-xylulose 5-phosphate reductoisomerase and isopentenyl diphosphate isomerase were overexpressed, and the ribosome binding site of GPPS-PS mRNA was optimized, improving pinene titer to 539.84 μg/L.
In this paper, through heterologous expression of GPPS-PS, pinene was successfully produced in R. sphaeroides, and pinene production was greatly improved by optimizing the expression of key enzymes. This is the first report on pinene produce by purple non-sulfur photosynthetic bacteria, which expands the availability of photosynthetic chassis for pinene production.
The convolutional neural network (CNN) is widely used in various computer vision problems such as image recognition and image classification because of its powerful ability to process image data. ...However, it is an end-to-end model that remains a "block box" for users. The internal logic of CNN is not explicitly known. Interpreting CNN can help us better understand neural networks and the various ways they benefit us as users. In this paper, we explain the contributions of the convolutional layer of CNN with a neuroscience experiment paradigm: the Ms. Pac-Man video game. Ms. Pac-Man is a popular game that provides a complex yet natural decision-making task rather than a laboratory artifact. An analysis of the game can thus intuitively reveal the complicated decision-making process in animal brains. We sought to (1) elucidate the role of the CNN convolutional layer and (2) analyze the low-level strategies in animal brains based on high-level decisions. We use recorded videos of monkeys playing the Ms. Pac-Man game to empirically demonstrate that our network is able to predict the moving direction of the Pac-Man at every time step. We further find that the decision-making process at work during gameplay is high-reward-driven. A heatmap of the weighted feature map at each convolutional layer shows that CNN makes predictions based on the most important input pattern, which in this case is the high reward entities in the game.
The pivotal roles of miRNAs in carcinogenesis, metastasis, and prognosis have been demonstrated recently in various cancers. This study intended to investigate the specific roles of hsa-miR-654-5p in ...lung cancer, which is, in general, rarely discussed. A series of closed-loop bioinformatic functional analyses were integrated with in vitro experimental validation to explore the overall biological functions and pan-cancer regulation pattern of miR-654-5p. We found that miR-654-5p abundance was significantly elevated in LUAD tissues and correlated with patients’ survival. A total of 275 potential targets of miR-654-5p were then identified and the miR-654-5p-RNF8 regulation axis was validated in vitro as a proof of concept. Furthermore, we revealed the tumor-suppressing roles of miR-654-5p and demonstrated that miR-654-5p inhibited the lung cancer cell epithelial-mesenchymal transition (EMT) process, cell proliferation, and migration using target-based, abundance-based, and ssGSEA-based bioinformatic methods and in vitro validation. Following the construction of a protein–protein interaction network, 11 highly interconnected hub genes were identified and a five-genes risk scoring model was developed to assess their potential prognostic ability. Our study does not only provide a basic miRNA-mRNA-phenotypes reference map for understanding the function of miR-654-5p in different cancers but also reveals the tumor-suppressing roles and prognostic values of miR-654-5p.
Breast cancer is the leading cause of cancer-associated deaths among females. In recent decades, microRNAs (miRNAs), a type of short non-coding RNA that regulates gene expression at the ...post-transcription level, have been reported to participate in the regulation of many hub genes associated with tumorigenesis, tumor progression, and metastasis. However, the precise mechanism by which miRNAs regulate breast cancer metastasis remains poorly discussed, which limits the opportunity for the development of novel, effective therapeutic targets. Here, we aimed to determine the miR-622-related principal regulatory mechanism in cancer. First, we found that miR-622 was significantly related to a poor prognosis in various cancers. By utilizing an integrated miRNA prediction process, we identified 77 promising targets and constructed a protein-protein interaction network. Furthermore, enrichment analyses, including GO and KEGG pathway analyses, were performed to determine the potential function of miR-622, which revealed regulation networks and potential functions of miR-622. Then, we identified a key cluster comprised of six hub genes in the protein-protein interaction network. These genes were further chosen for pan-cancer expression, prognostic and predictive marker analyses based on the TCGA and GEO datasets to mine the potential clinical values of these hub genes. To further validate our bioinformatic results, the regulatory axis of miR-622 and RNF8, one of the hub genes recently reported to promote breast cancer cell EMT process and breast cancer metastasis, was selected as
in vitro
proof of concept.
In vitro
, we demonstrated the direct regulation of RNF8 by miR-622 and found that the predicted miR-622-RNF8 axis could regulate RNF8-induced epithelial-mesenchymal transition, cell migration, and cell viability. These results were further demonstrated with rescue experiments. We established a closed-loop miRNA-target-phenotype research model that integrated the bioinformatic analysis of the miRNA target genes and experimental validation of the identified key miRNA-target-phenotype axis. We not only identified the hub target genes of miR-622
in silico
but also revealed the regulatory mechanism of miR-622 in breast cancer cell EMT process, viability, and migration
in vitro
for the first time.
RNF8 is an E3 ligase identified as a critical DNA damage-responsive protein. Recently, multiple reports have shown that RNF8 could be used as an important therapeutic target for cancer ...chemo/radiotherapy. However, the understanding of RNF8 remains limited due to the lack of its interactome reference map and comprehensive analysis of RNF8 in diverse cancers, which underscores the need to map the interactome of RNF8 via high-throughput methods. A two-way identification method based on LC-MS was designed for the identification of the RNF8 interactome with high-specificity. By in silico analysis and in vitro validation, we identified a new reference map of the RNF8 interactome network containing many new targets, such as YBX1, DNMT1, and HDCA1, new biological functions and the gene-disease associations of RNF8. Our results revealed a close relationship between RNF8 and neurodegenerative diseases or tumor-infiltrating immune cells using bulk RNA-seq and scRNA-seq datasets. As a proof of concept of our interactome map, we validated the direct binding between RNF8 and YBX1 and showed that RNF8 catalyzed the ubiquitination of YBX1. These results demonstrated that RNF8 might be a crucial regulator of YBX1. Our work provides a unique framework for researchers and clinicians who seek to better explore or understand RNF8-regulated biological functions in cancers. This study will hopefully facilitate the rational design and further development of anti-RNF8 therapy in cancers.
Despite substantial advances that have been made in understanding the etiology of hepatocellular carcinoma (HCC), the early-stage diagnosis and treatment of advanced-stage HCC remain a major ...challenge. RNF8, an E3 ligase important for the DNA damage response, has been proven to facilitate the progression of breast and lung cancer, but its role in HCC remains unclear. In this study, we find that the expression of RNF8 is up-regulated in HCC tissues and positively correlated with poor prognosis of HCC. Furthermore, silencing RNF8 by siRNAs attenuates the migration of HCC cells and inhibits epithelial-mesenchymal transition (EMT) by regulating the expressions of proteins including N-cadherin, β-catenin, snail, and ZO-1. Moreover, Kaplan‒Meier survival analysis shows that high RNF8 expression predicts poor survival benefits from sorafenib. Finally, cell viability assay demonstrates that RNF8 depletion enhances the sensitivity of HCC cells to sorafenib and lenvatinib treatment. We hypothesize that the inhibitory role of RNF8 in EMT and its enhancing effects on anti-cancer drugs orchestrate the protective effects of RNF8 deficiency in HCC, which indicates its potential in clinical application.
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In the eukaryotic cellular milieu, proteins are continuously synthesized and degraded effectively via endogenous protein degradation machineries such as the ubiquitin–proteasome and ...lysosome pathways. By reengineering and repurposing these natural protein regulatory mechanisms, the targeted protein degradation (TPD) strategies are presenting biologists with powerful tools to manipulate the abundance of proteins of interest directly, precisely, and reversibly at the post-translational level. In recent years, TPD is gaining massive attention and is recognized as a paradigm shift both in basic research, application-oriented synthetic biology, and pioneering clinical work. In this review, we summarize the updated information, especially the engineering efforts and developmental route, of current state-of-the-art TPD technology such as Trim-Away, LYTACs, and AUTACs. Besides, the general design principle, benefits, problems, and opportunities to be addressed were further analyzed, with the aim of providing guidelines for exploration, discovery, and further application of novel TPD tools in the future.
Due to high incidence, poor prognosis, and easy transformation into pancreatic cancer (PC) with high mortality, early diagnosis and prevention of acute pancreatitis (AP) have become significant ...research focuses. In this work, we proposed a magnetic single-drop microextraction (SDME) system with spatial confinement to enhance the aggregation-induced emission (AIE) effect for simultaneous fluorescence detection of miRNA-155 (associated with AP) and miRNA-196a (associated with PC). The target miRNAs were selectively recognized by the hairpin probe and triggered the DNA amplification reaction; then, the DNA strands with two independent probes of G-quadruplex/TAIN and Cy5 were constructed on the surfaces of the magnetic beads. The SDME process, in which a drop containing the fluorescence probes was formed at the tip of the magnetic microextraction rod rapidly within 10 s, was performed by magnetic extraction. In this way, G-quadruplex/TAIN was enriched owing to the spatial confinement of the single-drop system, and the fluorescence signal given off (by G-quadruplex/TAIN) was highly enhanced (AIE effect). This was detected directly by fluorescence spectrophotometry. The approach achieved low limits of detection of 2.1 aM for miRNA-196a and 8.1 aM for miRNA-155 and wide linear ranges from 10 aM to 10 nM for miRNA-196a and from 25 aM to 10 nM for miRNA-155. This novel method was applied to the fluorescence detection of miRNAs in human serum samples. High relative recoveries from 95.6% to 104.8% were obtained.
MicroRNAs (miRNAs) are a class of endogenous, noncoding, single-stranded RNA molecules with an average length of ~22 nucleotides. Various miRNAs play important roles in the processes of cell ...differentiation, biological development, and disease development. More often, these roles are collectively played by a group of miRNAs rather than a single miRNA. Analytical tools that only quantify a single miRNA are inadequate in meeting the needs of miRNA research. Therefore, based on different signal differentiation techniques in conjunction with nucleic acid amplification strategies, a variety of methods for simultaneous detection of multiple miRNAs have been developed. This article summarizes the latest progress in the simultaneous detection of multiple miRNAs, and discusses the applications of these methods. This review also provides some ideas for the further development, focusing on the signal amplification and discrimination strategies demonstrated by some methods, as well as the application and future developmental trends of these methods.
•Advanced strategies for analysis of multiple microRNAs are reviewed.•Spatio-temporal signal separation methods such as chromatography are described.•Signal discrimination strategies coupled with amplification approaches are categorized.•Future trends and directions are discussed.
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