Screening DNA aptamers that control the DNA cleavage, homology-directed repair, and transcriptional regulation of the CRISPR-(d)Cas9 system.

Accurate genome editing based on various molecular tools has always been the focus of gene-editing research and the primary goal for therapeutic application. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is a well-established gene-editing method that is preferred due to its simplicity and high efficiency. In this study, a group of single-stranded DNA aptamers with high affinity and high specificity for the Cas9 protein were obtained by the systematic evolution of ligands through the exponential enrichment method. Their binding affinity and possible binding domains to the Cas9 protein were analyzed. In addition, we demonstrated the effectiveness of aptamers in regulating dCas9-modulated gene transcription, in terms of both transcriptional activation and repression. Additionally, the aptamers successfully reduced the off-target effect and improved the efficiency of gene homologous recombination repair mediated by CRISPR-Cas9. The findings suggest a potential method to better control precise gene editing and enrich the diversity of modulating tools for the CRISPR-Cas9 system.

Efficient Cross-Modality Insulator Augmentation for Multi-Domain Insulator Defect Detection in UAV Images.

Regular inspection of the insulator operating status is essential to ensure the safe and stable operation of the power system. Unmanned aerial vehicle (UAV) inspection has played an important role in transmission line inspection, replacing former manual inspection. With the development of deep learning technologies, deep learning-based insulator defect detection methods have drawn more and more attention and gained great improvement. However, former insulator defect detection methods mostly focus on designing complex refined network architecture, which will increase inference complexity in real applications. In this paper, we propose a novel efficient cross-modality insulator augmentation algorithm for multi-domain insulator defect detection to mimic real complex scenarios. It also alleviates the overfitting problem without adding the inference resources. The high-resolution insulator cross-modality translation (HICT) module is designed to generate multi-modality insulator images with rich texture information to eliminate the adverse effects of existing modality discrepancy. We propose the multi-domain insulator multi-scale spatial augmentation (MMA) module to simultaneously augment multi-domain insulator images with different spatial scales and leverage these fused images and location information to help the target model locate defects with various scales more accurately. Experimental results prove that the proposed cross-modality insulator augmentation algorithm can achieve superior performance in public UPID and SFID insulator defect datasets. Moreover, the proposed algorithm also gives a new perspective for improving insulator defect detection precision without adding inference resources, which is of great significance for advancing the detection of transmission lines.

An Underwater Crack Detection System Combining New Underwater Image-Processing Technology and an Improved YOLOv9 Network.

Underwater cracks are difficult to detect and observe, posing a major challenge to crack detection. Currently, deep learning-based underwater crack detection methods rely heavily on a large number of crack images that are difficult to collect due to their complex and hazardous underwater environments. This study proposes a new underwater image-processing method that combines a novel white balance method and bilateral filtering denoising method to transform underwater crack images into high-quality above-water images with original crack features. Crack detection is then performed based on an improved YOLOv9-OREPA model. Through experiments, it is found that the new image-processing method proposed in this study significantly improves the evaluation indicators of new images, compared with other methods. The improved YOLOv9-OREPA also exhibits a significantly improved performance. The experimental results demonstrate that the method proposed in this study is a new approach suitable for detecting underwater cracks in dams and achieves the goal of transforming underwater images into above-water images.

Weather-Domain Transfer-Based Attention YOLO for Multi-Domain Insulator Defect Detection and Classification in UAV Images.

Insulator defect detection of transmission line insulators is an important task for unmanned aerial vehicle (UAV) inspection, which is of immense importance in ensuring the stable operation of transmission lines. Transmission line insulators exist in complex weather scenarios, with small and inconsistent shapes. These insulators under various weather conditions could result in low-quality images captured, limited data numbers, and imbalanced sample problems. Traditional detection methods often struggle to accurately identify defect information, resulting in missed or false detections in real-world scenarios. In this paper, we propose a weather domain synthesis network for extracting cross-modality discriminative information on multi-domain insulator defect detection and classification tasks. Firstly, we design a novel weather domain synthesis (WDSt) module to convert various weather-conditioned insulator images to the uniform weather domain to decrease the existing domain gap. To further improve the detection performance, we leverage the attention mechanism to construct the Cross-modality Information Attention YOLO (CIA-YOLO) model to improve the detection capability for insulator defects. Here, we fuse both shallow and deep feature maps by adding the extra object detection layer, increasing the accuracy for detecting small targets. The experimental results prove the proposed Cross-modality Information Attention YOLO with the weather domain synthesis algorithm can achieve superior performance in multi-domain insulator datasets (MD-Insulator). Moreover, the proposed algorithm also gives a new perspective for decreasing the multi-domain insulator modality gap with weather-domain transfer, which can inspire more researchers to focus on the field.

CRISPR applications in cancer diagnosis and treatment.

Cancer remains a significant global health challenge, necessitating the exploration of novel and more precise therapeutic options beyond conventional treatments. In this regard, clustered regularly interspaced short palindromic repeats (CRISPR) systems have emerged as highly promising tools for clinical gene editing applications. The CRISPR family encompasses diverse CRISPR-associated (Cas) proteins that possess the ability to recognize specific target sequences. The initial CRISPR system consisted of the Cas9 protein and a single-guide RNA, which guide Cas9 to the desired target sequence, facilitating precise double-stranded cleavage. In addition to the traditional cis-cleavage activity, the more recently discovered Cas12 and Cas13 proteins exhibit trans-cleavage activity, which expands their potential applications in cancer diagnosis. In this review, we provide an overview of the functional characteristics of Cas9, Cas12, and Cas13. Furthermore, we highlight the latest advancements and applications of these CRISPR systems in cancer gene therapy and molecular diagnosis. We also emphasize the importance of understanding the strengths and limitations of each CRISPR system to maximize their clinical utility. By providing a comprehensive overview of the current state of CRISPR technology in cancer research, we aim to inspire further exploration and innovation in this rapidly evolving field.

Fifteen-MiRNA-Based Signature Is a Reliable Prognosis-Predicting Tool for Prostate Cancer Patients.

Recurrence is a major problem for prostate cancer patients, thus, identifying prognosis-related markers to evaluate clinical outcomes is essential. Here, we established a fifteen-miRNA-based recurrence-free survival (RFS) predicting signature based on the miRNA expression profile extracted from The Cancer Genome Atlas (TCGA) database by the LASSO Cox regression analysis. The median risk score generated by the signature in both the TCGA training and the external Memorial Sloan-Kettering Cancer Center (MSKCC) validation cohorts was employed and the patients were subclassified into low- and high-risk subgroups. The Kaplan-Meier plot and log-rank analyses showed significant survival differences between low- and high-risk subgroups of patients (TCGA, log-rank P < 0.001 & MSKCC, log-rank P = 0.045). In addition, the receiver operating characteristic curves of both the training and external validation cohorts indicated the good performance of our model. After predicting the downstream genes of these miRNAs, the miRNA-mRNA network was visualized by Cytoscape software. In addition, pathway analyses found that the differences between two groups were mainly enriched on tumor progression and drug resistance-related pathways. Multivariate analyses revealed that the miRNA signature is an independent indicator of RFS prognosis for prostate cancer patients with or without clinicopathological features. In summary, our novel fifteen-miRNA-based prediction signature is a reliable method to evaluate the prognosis of prostate cancer patients.

N6-methyladenosine demethylase FTO suppresses clear cell renal cell carcinoma through a novel FTO-PGC-1α signalling axis.

The abundant and reversible N6-methyladenosine (m6A) RNA modification and its modulators have important roles in regulating various gene expression and biological processes. Here, we demonstrate that fat mass and obesity associated (FTO), as an m6A demethylase, plays a critical anti-tumorigenic role in clear cell renal cell carcinoma (ccRCC). FTO is suppressed in ccRCC tissue. The low expression of FTO in human ccRCC correlates with increased tumour severity and poor patient survival. The Von Hippel-Lindau-deficient cells expressing FTO restores mitochondrial activity, induces oxidative stress and ROS production and shows impaired tumour growth, through increasing expression of PGC-1α by reducing m6A levels in its mRNA transcripts. Our work demonstrates the functional importance of the m6A methylation and its modulator, and uncovers a critical FTO-PGC-1α axis for developing effective therapeutic strategies in the treatment of ccRCC.

A Method for Settlement Detection of the Transmission Line Tower under Wind Force.

In view of the settlement problem of transmission tower foundation, the vibration characteristics of transmission towers under wind force are measured experimentally. In this paper, the 110 kV cat head transmission tower of Xi'an Polytechnic University is measured and analyzed. Firstly, the acceleration sensor and meteorological sensor are installed on the tower to collect the vibration response and environment parameters of the tower in real time. Then, an experiment platform is built to simulate the tower settlement, and the vibration response of the tower after settlement is measured in time. Finally, the low-order modal frequencies of the transmission tower before and after settlement under wind force load are extracted by stochastic subspace identification (SSI), and the relationship between modal frequencies of different modes is analyzed via temperature correction. By comparison and analysis, it is obvious that the X-direction modal frequencies before and after settlement under natural wind load are changed, and the change rate increases with the increase of settlement displacement, which can be used as effective evidence for judging the settlement of transmission tower foundation.

Detection of Broken Strands of Transmission Line Conductors Using Fiber Bragg Grating Sensors.

Transmission lines are affected by Aeolian vibration, which causes strands to break and eventually causes an entire line to break. In this paper, a method for monitoring strand breaking based on modal identification is proposed. First, the natural frequency variation of a conductor caused by strand breakage is analyzed, and a modal experiment of the LGJ-95/15 conductor is conducted. The measurement results show that the natural frequencies of the conductor decrease with an increasing number of broken strands. Next, a monitoring system incorporating a fiber Bragg grating (FBG)-based accelerometer is designed in detail. The FBG sensor is mounted on the conductor to measure the vibration signal. A wind speed sensor is used to measure the wind speed signal and is installed on the tower. An analyzer is also installed on the tower to calculate the natural frequencies, and the data are sent to the monitoring center via 3G. Finally, a monitoring system is tested on a 110 kV experimental transmission line, and the short-time Fourier transform (STFT) method and stochastic subspace identification (SSI) method are used to identify the natural frequencies of the conductor vibration. The experimental results show that SSI analysis provides a higher precision than does STFT and can extract the natural frequency under various wind speeds as an effective basis for discriminating between broken strands.

Experimental Study on the Icing Dielectric Constant for the Capacitive Icing Sensor.

The capacitive method is considered to be a suitable icing-detection technology, but the lack of fundamental parameters restricts the development of icing-detection sensors. In this paper, an artificial icing laboratory, a capacitive sensor, and some simulation conductors have been designed for obtaining the artificial icing samples. Subsequently, the same characteristic values of artificial icing have been measured by an LCR device, under a selected frequency. This research found that the value of the icing dielectric constant closely correlated with its density, internal sublayer, and the test temperature. Finally, a fitting formula has been presented for calculating the relative dielectric constant, which may provide some important reference value for the design of icing-detection sensors.

[Expression characteristics of the Daxx gene in the mouse testis during spermatogenesis].

OBJECTIVE: To investigate the expression characteristic of the Daxx gene in the mouse testis and its role in spermatogenesis. METHODS: Real-time PCR, Western blot and immunofluorescence were used in examining the expression characteristics of DAXX in the testis tissue from wild-type, Sertoli cell-specific androgen receptor knockout (SCARKO) and androgen receptor knockout (ARKO) mice at different postnatal weeks . RESULTS: The Daxx gene was highly expressed in the testis tissue and mainly in the nuclei of the wild-type mice at 4 postnatal weeks. Compared with the wild-type, the ARKO mice showed a markedly decreased expression of DAXX (0.299±0.026), which displayed a polar distribution in the spermatogenic cells (0.853±0.058) and exhibited no significant difference in the SCARKO mice (1.000±0.015). CONCLUSIONS: The Daxx gene expression is the highest in the middle-stage development of the mouse testis, significantly decreased in ARKO mice as compared with the wild-type, and its location influenced by specific AR knockout in Sertoli cells. DAXX may be involved in the regulation of spermatogenesis in mice.

Design of a Wireless Sensor Module for Monitoring Conductor Galloping of Transmission Lines.

Conductor galloping may cause flashovers and even tower collapses. The available conductor galloping monitoring methods often employ acceleration sensors to measure the conductor translations without considering the conductor twist. In this paper, a new sensor for monitoring conductor galloping of transmission lines based on an inertial measurement unit and wireless communication is proposed. An inertial measurement unit is used for collecting the accelerations and angular rates of a conductor, which are further transformed into the corresponding geographic coordinate frame using a quaternion transformation to reconstruct the galloping of the conductor. Both the hardware design and the software design are described in details. The corresponding test platforms are established, and the experiments show the feasibility and accuracy of the proposed monitoring sensor. The field operation of the proposed sensor in a conductor spanning 734 m also shows its effectiveness.

Regulatory genome annotation of 33 insect species.

Annotation of newly sequenced genomes frequently includes genes, but rarely covers important non-coding genomic features such as the cis-regulatory modules-e.g., enhancers and silencers-that regulate gene expression. Here, we begin to remedy this situation by developing a workflow for rapid initial annotation of insect regulatory sequences, and provide a searchable database resource with enhancer predictions for 33 genomes. Using our previously developed SCRMshaw computational enhancer prediction method, we predict over 2.8 million regulatory sequences along with the tissues where they are expected to be active, in a set of insect species ranging over 360 million years of evolution. Extensive analysis and validation of the data provides several lines of evidence suggesting that we achieve a high true-positive rate for enhancer prediction. One, we show that our predictions target specific loci, rather than random genomic locations. Two, we predict enhancers in orthologous loci across a diverged set of species to a significantly higher degree than random expectation would allow. Three, we demonstrate that our predictions are highly enriched for regions of accessible chromatin. Four, we achieve a validation rate in excess of 70% using in vivo reporter gene assays. As we continue to annotate both new tissues and new species, our regulatory annotation resource will provide a rich source of data for the research community and will have utility for both small-scale (single gene, single species) and large-scale (many genes, many species) studies of gene regulation. In particular, the ability to search for functionally related regulatory elements in orthologous loci should greatly facilitate studies of enhancer evolution even among distantly related species.

Synthetic Circular gRNA Mediated Biological Function of CRISPR-(d)Cas9 System.

Ever since the gene editing function was discovered in the CRISPR-Cas9 system, numerous applications and utilities were investigated in order to apply this technique to medical use. However, the clinical practice was limited by unsatisfactory efficiency and unacceptable off-target editing. Modifications from different aspects of the Cas9 protein and gRNAs were published that aimed to improve its function in one way or another. Under the inspiration of Jacob L. Litke and Samie R. Jaffrey, we propose a novel gRNA design that could achieve rapid circular gRNA assembly inside the cells. This circular design consists of the gRNA of interested flanked by Twister ribozymes. The function of this circular gRNA was proved in vitro in both CRISPR-dCas9 and CRISPR-Cas9 systems. It presented a remarkable reduction in the off-target rate in accompany with reduced efficiency. With future improvement in its efficiency, this tool broadens our understanding and possibility of the CRISPR application.

Synthesis of RNA-based gene regulatory devices for redirecting cellular signaling events mediated by p53.

Rationale: The p53 gene is a well-known tumor suppressor, and its mutation often contributes to the occurrence and development of tumors. Due to the diversity and complexity of p53 mutations, there is still no effective p53 gene therapy. In this study, we designed and constructed an aptazyme switch that could effectively sense cellular wild-type p53 protein and regulate downstream gene function flexibly. The application of this artificial device in combination with Cre-LoxP and dCas9-VP64 tools achieved a precisely targeted killing effect on tumor cells. Methods: The affinity of the aptamer to p53 protein was verified by SPR. p53 aptazyme and gene circuits were chemically synthesized. The function of the gene circuit was detected by cell proliferation assay, apoptosis assay and Western blot. The nude mouse transplantation tumor experiment was used to evaluate the inhibitory effect of gene circuits on tumor cells in vivo. Results: The results of the SPR experiment showed that the p53 aptamer RNA sequence had a robust binding effect with p53 protein. The p53 aptazyme could efficiently sense wild-type p53 protein and initiate self-cleavage in cells. The Cre-p53 aptazyme gene circuit and dCas9-VP64/sgRNA mediated gene circuit designed based on p53 aptazyme significantly inhibited the growth and promoted the apoptosis of wild-type p53-deficient cancer cells in vitro. In addition, the gene circuits also had a significant inhibitory effect on tumors in vivo. Conclusion: The study developed a novel and efficient ribozyme switch for p53-specific recognition and provided a modular strategy for aptazyme binding to cellular proteins. In addition, the p53 aptazyme successfully inhibited tumor growth through a combined application with other synthetic biological tools, providing a new perspective for cancer therapy.

Targeted Demethylation of the PLOD2 mRNA Inhibits the Proliferation and Migration of Renal Cell Carcinoma.

N6-methyladenosine (m6A) RNA modification is the most common internal mRNA modification in mammals and has been reported to play a key role in gene expression regulation. In this study, we detected a high level of m6A methylation of the PLOD2 3'-untranslated regions (3'UTR) in renal cell carcinoma (RCC). Furthermore, we found that the high expression level of PLOD2 was a prognostic indicator for patients with RCC. A dm6ACRISPR demethylation system was performed to accurately and specifically demethylate 3'UTR of PLOD2 and caused an inactivation of PLOD2 expression. Furthermore, we also performed many in vitro experiments to confirm that PLOD2 exerted tumor promoter effects by promoting tumor proliferation and migration. In conclusion, PLOD2 mRNA demethylated by dCas13b-ALKBH5 might provide a new light on the treatment for RCC.

Circular RNA hsa_circ_0075828 promotes bladder cancer cell proliferation through activation of CREB1.

Circular RNAs (circRNAs), one kind of non-coding RNA, have been reported as critical regulators for modulating gene expression in cancer. In this study, microarray analysis was used to screen circRNA expression profiles of bladder cancer (BC) 5637 cells, T24 cells and normal control SV-HUC-1 cells. The data from the microarray showed that hsa_circ_0075828 (named circCASC15) was most highly expressed in 5637 and T24 cells. circCASC15 was highly expressed in BC tissues and cells. Overexpression of circCASC15 was closely associated with BC tumor stage and promoted cell proliferation significantly in vitro and in vivo. Mechanistically, circCASC15 could act as miR-1224-5p sponge to activate the expression of CREB1 to promote cell proliferation in BC. In short, circCASC15 promotes cell proliferation in BC, which might be a new molecular target for BC diagnosis and therapy. [BMB Reports 2020; 53(2): 82-87].

RNAi-mediated control of CRISPR functions.

CRISPR-Cas9 has become a versatile tool for genome editing and regulation, and strategies to effectively control its activity have attracted much attention. RNAi, also a gene-regulating tool, is used as another mechanism by which eukaryotes resist the invasion of foreign genetic material. Methods: In this study, we analyzed the quantitative inhibition of the CRISPR system by using artificial miRNAs (amiRNAs) combined with the RNAi enhancer enoxacin to improve the targeting specificity of the CRISPR system. Furthermore, we examined the feasibility of improving the efficiency of gene editing and regulation by blocking the effects of natural intracellular miRNAs on sgRNAs. Results: amiRNAs targeting the sgRNA were used to control its expression, and the small molecule drug denoxacin was utilized to enhance this effect, especially in the presence of Cas9. amiRNA/enoxacin inhibited CRISPR-mediated gene editing and regulation both in vitro and in vivo and could tune sgRNA-targeting specificity. Furthermore, CRISPR efficiency was increased by blocking the effects of endogenous miRNAs. Conclusion: Our study provides an efficient molecular switch for conditional regulation of CRISPR activities in mammalian cells and also presents potentially useful approaches for solving current key issues of off-target effects and low targeting efficiency.

A Light-Inducible Split-dCas9 System for Inhibiting the Progression of Bladder Cancer Cells by Activating p53 and E-cadherin.

Optogenetic systems have been increasingly investigated in the field of biomedicine. Previous studies had found the inhibitory effect of the light-inducible genetic circuits on cancer cell growth. In our study, we applied an AND logic gates to the light-inducible genetic circuits to inhibit the cancer cells more specifically. The circuit would only be activated in the presence of both the human telomerase reverse transcriptase (hTERT) and the human uroplakin II (hUPII) promoter. The activated logic gate led to the expression of the p53 or E-cadherin protein, which could inhibit the biological function of tumor cells. In addition, we split the dCas9 protein to reduce the size of the synthetic circuit compared to the full-length dCas9. This light-inducible system provides a potential therapeutic strategy for future bladder cancer.

LncRNA NRON promotes the proliferation, metastasis and EMT process in bladder cancer.

Background: Bladder cancer (BC) is one of the most common malignancies world-wide with high morbidity and mortality. Long noncoding RNAs (lncRNAs) are thought to play a critical role in cancer development. LncRNA NRON, a repressor of activated T-cell nuclear factor (NFAT), has been shown to be dysregulated in many cancer types. However, the clinical significance and molecular mechanism of NRON in bladder cancer is still unknown. Methods: The expression levels of NRON in BC tissues and cell lines were tested by RT-qPCR. Survival analysis was performed to detect the correlation between NRON expression and clinical outcomes in patients with BC. The biological role of NRON in BC cells proliferation and metastasis was examined in vitro and in vivo. Results: The expression of NRON was significantly upregulated in BC specimens and cell lines compared with paired adjacent normal tissues and normal cell lines. The upregulation of NRON in bladder cancer patients was significantly associated with the depth of bladder tumor invasion and poor prognosis. Knockdown of NRON inhibited BC cells proliferation, migration, invasion and tumorigenicity. Furthermore, NRON promoted epithelial-mesenchymal transition (EMT) progression, and NRON-induced EZH2 expression contributed to this process. Conclusion: In conclusion, our results suggested that NRON acted as an oncogene and tumor biomarker for BC.