One-Stage Anchor-Free Online Multiple Target Tracking with Deformable Local Attention and Task-Aware Prediction.

The tracking-by-detection paradigm currently dominates multiple target tracking algorithms. It usually includes three tasks: target detection, appearance feature embedding, and data association. Carrying out these three tasks successively usually leads to lower tracking efficiency. In this paper, we propose a one-stage anchor-free multiple task learning framework which carries out target detection and appearance feature embedding in parallel to substantially increase the tracking speed. This framework simultaneously predicts a target detection and produces a feature embedding for each location, by sharing a pyramid of feature maps. We propose a deformable local attention module which utilizes the correlations between features at different locations within a target to obtain more discriminative features. We further propose a task-aware prediction module which utilizes deformable convolutions to select the most suitable locations for the different tasks. At the selected locations, classification of samples into foreground or background, appearance feature embedding, and target box regression are carried out. Two effective training strategies, regression range overlapping and sample reweighting, are proposed to reduce missed detections in dense scenes. Ambiguous samples whose identities are difficult to determine are effectively dealt with to obtain more accurate feature embedding of target appearance. An appearance-enhanced non-maximum suppression is proposed to reduce over-suppression of true targets in crowded scenes. Based on the one-stage anchor-free network with the deformable local attention module and the task-aware prediction module, we implement a new online multiple target tracker. Experimental results show that our tracker achieves a very fast speed while maintaining a high tracking accuracy.

Exploration of the shared diagnostic genes and mechanisms between periodontitis and primary Sjögren's syndrome by integrated comprehensive bioinformatics analysis and machine learning.

BACKGROUND: Accumulating evidence has showed a bidirectional link between periodontitis (PD) and primary Sjögren's syndrome (pSS), but the mechanisms of their occurrence remain unclear. Hence, this study aimed to investigate the shared diagnostic genes and potential mechanisms between PD and pSS using bioinformatics methods. METHODS: Gene expression data for PD and pSS were acquired from the Gene Expression Omnibus (GEO) database. Differential expression genes (DEGs) analysis and weighted gene co-expression network analysis (WGCNA) were utilized to search common genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were conducted to explore biological functions. Three machine learning algorithms (least absolute shrinkage and selection operator (LASSO), support vector machine recursive feature elimination (SVM-RFE), and random forest (RF)) were used to further identify shared diagnostic genes, and these genes were assessed via receiver operating characteristic (ROC) curves in discovery and validation datasets. CIBERSORT was employed for immune cell infiltration analysis. Transcription factors (TFs)-genes and miRNAs-genes regulatory networks were conducted by NetworkAnalyst. Finally, relevant drug targets were predicted by DSigDB. RESULTS: Based on DEGs, 173 overlapping genes were obtained and primarily enriched in immune- and inflammation-related pathways. WGCNA revealed 34 common disease-related genes, which were enriched in similar biological pathways. Intersecting the DEGs with WGCNA results yielded 22 candidate genes. Moreover, three machine learning algorithms identified three shared genes (CSF2RB, CXCR4, and LYN) between PD and pSS, and these genes demonstrated good diagnostic performance (AUC>0.85) in both discovery and validation datasets. The immune cell infiltration analysis showed significant dysregulation in several immune cell populations. Regulatory network analysis highlighted that WRNIP1 and has-mir-155-5p might be pivotal co-regulators of the three shared gene expressions. Finally, the top 10 potential gene-targeted drugs were screened. CONCLUSION: CSF2RB, CXCR4, and LYN may serve as potential biomarkers for the concurrent diagnosis of PD and pSS. Additionally, we identified common molecular mechanisms, TFs, miRNAs, and candidate drugs between PD and pSS, which may provide novel insights and targets for future research on the pathogenesis, diagnosis, and therapy of both diseases.

Enhanced control of RNA modification and CRISPR-Cas activity through redox-triggered disulfide cleavage.

Chemical RNA modification has emerged as a flexible approach for post-synthetic modifications in chemical biology research. Guide RNA (gRNA) plays a crucial role in the clustered regularly interspaced short palindromic repeats and associated protein system (CRISPR-Cas). Several toolkits have been developed to regulate gene expression and editing through modifications of gRNA. However, conditional regulation strategies to control gene editing in cells as required are still lacking. In this context, we introduce a strategy employing a cyclic disulfide-substituted acylating agent to randomly acylate the 2'-OH group on the gRNA strand. The CRISPR-Cas systems demonstrate off-on transformation activity driven by redox-triggered disulfide cleavage and undergo intramolecular cyclization, which releases the functionalized gRNA. Dithiothreitol (DTT) exhibits superior reductive capabilities in cleaving disulfides compared to glutathione (GSH), requiring fewer reductants. This acylation method with cyclic disulfides enables conditional control of CRISPR-Cas9, CRISPR-Cas13a, RNA hybridization, and aptamer folding. Our strategy facilitates precise in vivo control of gene editing, making it particularly valuable for targeted applications.

The polycaprolactone and silk fibroin nanofibers with Janus-structured sheaths for antibacterial and antioxidant by loading Taxifolin.

Electrospinning is a widely recognized method for producing Janus or core-shell nanofibers. In this study, nanofibrous membranes were fabricated through co-axial electrospinning utilizing polycaprolactone (PCL) and silk fibroin (SF) as the Janus shell, and taxifolin (TAX) and SF as the core. The resulting nanofibers had diameters of 816 ± 161 nm and core diameters of 73 ± 5 nm. The morphology and properties of the PCL-SF@SF/TAX nanofibers were subsequently analyzed. The results demonstrated that the nanofibrous membranes achieved physical and chemical characteristics potential for tissue engineering and drug delivery. Specifically, the membranes exhibited a Young's modulus of 9.64 ± 0.29 MPa, a water contact angle of 79.1 ± 1.3°, and a weight loss of 17.3 ± 1.0 % over a period of 28 days. The incorporation of TAX endowed the membranes with antibacterial properties, effectively combating Escherichia coli and Staphylococcus aureus. Furthermore, the membranes demonstrated antioxidant capabilities, with a DPPH radical scavenging efficiency of 38.5 ± 5.6 % and a Trolox-equivalent antioxidant capacity of 0.24 ± 0.01 mM. The release of the antioxidant was sustained over 28 days, following first-order release kinetics. The nanofibrous membranes, referred to as PSST, exhibit promising potential for use as biomaterials, characterized by their antibacterial activity, antioxidant and cytocompatibility.

Multifunctional bilayer nanofibrous membrane enhances periodontal regeneration via mesenchymal stem cell recruitment and macrophage polarization.

The continuous stimulation of periodontitis leads to a decrease in the number of stem cells within the lesion area and significantly impairing their regenerative capacity. Therefore, it is crucial to promote stem cell homing and regulate the local immune microenvironment to suppress inflammation for the regeneration of periodontitis-related tissue defects. Here, we fabricated a novel multifunctional bilayer nanofibrous membrane using electrospinning technology. The dense poly(caprolactone) (PCL) nanofibers served as the barrier layer to resist epithelial invasion, while the polyvinyl alcohol/chitooligosaccharides (PVA/COS) composite nanofiber membrane loaded with calcium beta-hydroxy-beta-methylbutyrate (HMB-Ca) acted as the functional layer. Material characterization tests revealed that the bilayer nanofibrous membrane presented desirable mechanical strength, stability, and excellent cytocompatibility. In vitro, PCL@PVA/COS/HMB-Ca (P@PCH) can not only directly promote rBMSCs migration and differentiation, but also induce macrophage toward pro-healing (M2) phenotype-polarization with increasing the secretion of anti-inflammatory and pro-healing cytokines, thus providing a favorable osteoimmune environment for stem cells recruitment and osteogenic differentiation. In vivo, the P@PCH membrane effectively recruited host MSCs to the defect area, alleviated inflammatory infiltration, and accelerated bone defects repair. Collectively, our data indicated that the P@PCH nanocomposite membrane might be a promising biomaterial candidate for guided tissue regeneration in periodontal applications.

Isonitrile-Tetrazine Click-and-Release Chemistry for Controlling RNA-Guided Nucleic Acid Cleavage.

With the increasing demand for the regulation of CRISPR systems, a considerable number of studies have been conducted to control their excessive activity levels. In this context, we propose a method that involves a bioorthogonal cleavage reaction between isonitrile and tetrazine to modulate the cleavage activity of the CRISPR system. Importantly, isonitrile demonstrates significant potential for modifying sgRNAs, making it a promising candidate for bioorthogonal reactions, a phenomenon that has not been previously reported. Our approach utilizes the 3-isocyanopropyl-carbonate group as a caging group to deactivate the CRISPR systems, while tetrazine acts as an activator to restore their activities. Through the implementation of post-synthetic modifications and click-and-release chemistry, we have successfully achieved the regulation of RNA-guided nucleic acid cleavage, which holds great promise for controlling gene editing in human cells.

Mesoporous Metal-Organic Frameworks for Catalytic RNA Deprotection and Activation.

A metal-organic framework (MOF) with mespores (2 to 50 nm) allows the inclusion of large biomolecules, such as nucleic acids. However, chemical reaction on the nucleic acids, to further regulate their bioactivity, is yet to be demonstrated within MOF pores. Here, we report the deprotection of carbonate protected RNA molecules (21 to 102 nt) to restore their original activity using a MOF as a heterogeneous catalyst. Two MOFs, MOF-626 and MOF-636 are designed and synthesized, with mesopores of 2.2 and 2.8 nm, respectively, carrying isolated metal sites (Ni, Co, Cu, Pd, Rh and Ru). The pores favor the entrance of RNA, while the metal sites catalyze C-O bond cleavage at the carbonate group. Complete conversion of RNA is achieved by Pd-MOF-626, 90 times more efficiently than Pd(NO3 )2 . MOF crystals are also removable from the aqueous reaction media, leaving a negligible metal footprint, 3.9 ppb, only 1/55 of that using homogeneous Pd catalysts. These features make MOF potentially suited for bioorthogonal chemistry.

Narrowing the Gap: Improved Detector Training With Noisy Location Annotations.

Deep learning methods require massive of annotated data for optimizing parameters. For example, datasets attached with accurate bounding box annotations are essential for modern object detection tasks. However, labeling with such pixel-wise accuracy is laborious and time-consuming, and elaborate labeling procedures are indispensable for reducing man-made noise, involving annotation review and acceptance testing. In this paper, we focus on the impact of noisy location annotations on the performance of object detection approaches and aim to, on the user side, reduce the adverse effect of the noise. First, noticeable performance degradation is experimentally observed for both one-stage and two-stage detectors when noise is introduced to the bounding box annotations. For instance, our synthesized noise results in performance decrease from 38.9% AP to 33.6% AP for FCOS detector on COCO test split, and 37.8%AP to 33.7%AP for Faster R-CNN. Second, a self-correction technique based on a Bayesian filter for prediction ensemble is proposed to better exploit the noisy location annotations following a Teacher-Student learning paradigm. Experiments for both synthesized and real-world scenarios consistently demonstrate the effectiveness of our approach, e.g., our method increases the degraded performance of the FCOS detector from 33.6% AP to 35.6% AP on COCO.

PDNet: Towards Better One-stage Object Detection with Prediction Decoupling.

Recent one-stage object detectors follow a per-pixel prediction approach that predicts both the object category scores and boundary positions from every single grid location. However, the most suitable positions for inferring different targets, i.e., the object category and boundaries, are generally different. Predicting all these targets from the same grid location thus may lead to sub-optimal results. In this paper, we analyze the suitable inference positions for object category and boundaries, and propose a prediction-target-decoupled detector named PDNet to establish a more flexible detection paradigm. Our PDNet with the prediction decoupling mechanism encodes different targets separately in different locations. A learnable prediction collection module is devised with two sets of dynamic points, i.e., dynamic boundary points and semantic points, to collect and aggregate the predictions from the favorable regions for localization and classification. We adopt a two-step strategy to learn these dynamic point positions, where the prior positions are estimated for different targets first, and the network further predicts residual offsets to the positions with better perceptions of the object properties. Extensive experiments on the MS COCO benchmark demonstrate the effectiveness and efficiency of our method. With a single ResNeXt-64x4d-101-DCN as the backbone, our detector achieves 50.1 AP with single-scale testing, which outperforms the state-of-the-art methods by an appreciable margin under the same experimental settings. Moreover, our detector is highly efficient as a one-stage framework. Our code will be public.

ZIF-8 modified multifunctional injectable photopolymerizable GelMA hydrogel for the treatment of periodontitis.

Periodontitis is a chronic inflammatory disease caused by plaque that leads to alveolar bone resorption. In the treatment of periodontitis, it is necessary to reduce the bacterial load and promote alveolar bone regeneration. In this study, zeolitic imidazolate framework-8 (ZIF-8) is used in the treatment of periodontitis, and an injectable photopolymerizable ZIF-8/gelatin methacryloyl (GelMA) composite hydrogel (GelMA-Z) is constructed. We confirm that ZIF-8 nanoparticles are successfully loaded into GelMA, which demonstrates fluidity and photopolymerizability. GelMA-Z continuously releases Zn2+ and shows good cytocompatibility. In vitro, GelMA-Z can effectively upregulate the expression of osteogenesis-related genes and proteins, increase alkaline phosphatase activity, promote extracellular matrix mineralization by rat bone mesenchymal stem cells, and exert an obvious antibacterial effect against Porphyromonas gingivalis. In vivo, GelMA-Z reduces the bacterial load, relieves inflammation and promotes alveolar bone regeneration in a rat model. The above results show that GelMA-Z has potential prospects in the treatment of periodontitis. STATEMENT OF SIGNIFICANCE: Various methods have been explored for the treatment of periodontitis. However, current regiments have difficulty achieving ideal alveolar bone regeneration. In this study, we constructed a zeolitic imidazolate framework-8 (ZIF-8)/gelatin methacryloyl (GelMA) composite hydrogel (GelMA-Z). (1) The injectable and photopolymerizable GelMA-Z showed biocompatibility in vitro and in vivo. (2) GelMA-Z continually released zinc ions to promote the osteogenic differentiation of bone mesenchymal stem cells and kill bacteria in vitro. (3) In a rat model, the GelMA-Z pregel solution was used to fill the periodontal pocket and then crosslinked by UV exposure. GelMA-Z can stably remain in the periodontal pocket to reduce the bacterial load, relieve inflammation and promote alveolar bone regeneration. In conclusion, GelMA-Z has great potential for use in the treatment of periodontitis, especially in promoting alveolar bone regeneration.

Isoprenylcysteine carboxyl methyltransferase promotes the progression of tongue squamous cell carcinoma via the K-Ras and RhoA signaling pathways.

OBJECTIVE: This research investigated the biological role of isoprenylcysteine carboxyl methyltransferase (ICMT) in tongue squamous cell carcinoma (TSCC) progression meanwhile to explore the conceivable mechanism. METHODS: The mRNA and protein expression were measured using real-time PCR and Western blot. Cell proliferation, apoptosis, cycle distribution, migration and invasion were evaluated by CCK-8 assay, flow cytometry, wound-healing assay and transwell assay. The anti-tumor activity of ICMT silencing was observed in nude mice. RESULTS: Our results indicated that silencing of ICMT-mediated methylation effectively inhibited TSCC cells proliferation in vitro and reduced tumor growth in vivo. Moreover, ICMT knockdown also induced cell apoptosis and cell cycle arrest of both CAL-27 and SCC-4 cells. In addition, CAL-27 and SCC-4 cells migration and invasion were weakened by ICMT siRNA. Mechanistically, ICMT deficiency significantly decreased the K-Ras and RhoA membrane targeting localization, leading to the suppression of K-Ras- and RhoA-mediated downstream signaling in CAL-27 and SCC-4 cells. CONCLUSIONS: Altogether, our findings identified a crucial role played by ICMT in the progression of TSCC and the potential mechanisms by which exerted its effects, indicating that targeting ICMT may represent a promising therapeutic strategy for TSCC.

Effect of RhoE expression on the migration and invasion of tongue squamous cell carcinoma.

OBJECTIVES: This study aims to investigate the effect of RhoE expression on the migration and invasion of tongue squamous cell carcinoma (TSCC). METHODS: Forty-eight TSCC cases were selected from the Maxillofacial Surgery Center of Qingdao Municipal Hospital from 2017 to 2019. The expression of RhoE in the specimens (TSCC and adjacent tissues) was detected by immunohistochemistry, and RhoE mRNA and protein were extracted to further detect the expression of RhoE. SCC-4 and CAL-27 cells were selected for in vitro experiments. Transient transfection was used to overexpress RhoE. Real-time fluorescence quantitative PCR (qRT-PCR) and Western blot analyses were conducted to detect the overexpression efficiency. Scratch test and Transwell cell invasion tests were used to detect the migration and invasion ability of TSCC, respectively. The expression levels of Rho-associated coiled-coil-containing protein kinase 1 (ROCK1), matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase-9 (MMP-9) were detected by Western blot. Experimental data were analyzed by Graphpad prism 8.2.1 software. RESULTS: The expression level of RhoE in TSCC was significantly lower than that in adjacent tissues (P<0.05). The migration and invasion abilities of TSCC were significantly lower than those in the control group (P<0.05). The Western blot showed significantly lower expression levels of ROCK1, MMP-2, and MMP-9 in the experimental group than in the control group (P<0.05). CONCLUSIONS: RhoE expression is low in TSCC. Over expression RhoE in TSCC can significantly decrease its migration and invasion abilities. Hence, RhoE may play an important role in regulating the metastasis and invasion of TSCC and provide a new target for gene therapy.

Effects of isoprenylcysteine carboxylmethyltransferase silencing on the migration and invasion of tongue squamous cell carcinoma.

OBJECTIVES: The effect of isoprenylcysteine carboxymethyltransferase (ICMT) silencing on the migration and invasion of tongue squamous cell carcinoma was investigated by constructing the small interfering RNA (siRNA) of ICMT. METHODS: Through liposomal transfection, siRNA was transfected into human tongue squamous cell carcinoma CAL-27 and SCC-4 cells (ICMT-siRNA group) with a negative control group (transfected with NC-siRNA) and a blank control group (transfected with a transfection reagent but not with siRNA). Quantitative real-time polymerase chain reaction was performed to analyze the mRNA expression of ICMT and RhoA in each group of cells after transfection and to measure the silencing efficiency. Western blot was applied to examine the expression levels of ICMT, total RhoA, membrane RhoA, ROCK1, matrix metalloproteinase (MMP)-2, and MMP-9 proteins in each group. The migration and invasion abilities were evaluated via wound healing and Transwell motility assays. RESULTS: After CAL-27 and SCC-4 cells were transfected with ICMT-siRNA, the expression levels of ICMT genes and proteins decreased significantly in the experimental group compared with those in the negative and blank control groups (P<0.05). The mRNA and total protein expression levels of RhoA in the two groups were not significantly different (P>0.05). The expression levels of RhoA membrane protein, ROCK1, MMP-2, and MMP-9 decreased (P<0.05). The migration and invasion abilities were inhibited (P<0.05). CONCLUSIONS: The migration and invasion abilities of CAL-27 and SCC-4 cells were reduced significantly after the transfection of ICMT-siRNA, and the involved mechanism might be related to the RhoA-ROCK signaling pathway.

Effects of isoprenylcysteine carboxyl methyltransferase silencing on the proliferation and apoptosis of tongue squamous cell carcinoma.

OBJECTIVES: This study aimed to explore the effects of silencing isoprenylcysteine carboxyl methyltransfe-rase (Icmt) through small interfering RNA (siRNA) interference on the proliferation and apoptosis of tongue squamous cell carcinoma (TSCC). METHODS: Three siRNA were designed and constructed for the Icmt gene sequence and were then transfected into TSCC cells CAL-27 and SCC-4 to silence Icmt expression. The tested cells were divided as follows: RNA interference groups Icmt-siRNA-1, Icmt-siRNA-2, and Icmt-siRNA-3, negative control group, and blank control group. The transfection efficiency of siRNA was detected by the fluorescent group Cy3-labeled siRNA, and the expression of Icmt mRNA was screened by quantitive real-time polymerase chain reaction (qRT-PCR) selected the experimental group for subsequent experiments. The expression of Icmt, RhoA, Cyclin D1, p21, extracellular regulated protein kinases (ERK), and phospho-extracellular regulated protein kinases (p-ERK) were analyzed by Western blot. The proliferation abilities of TSCC cells were determined by cell counting kit-8 assay. The change in apoptosis was detected by AnnexinV-APC/propidium staining (PI) assay. Cell-cycle analysis was conducted by flow cytometry. RESULTS: The expression of Icmt mRNA and protein in TSCC cells significantly decreased after Icmt-siRNA transfection (P<0.05). No significant difference in RhoA mRNA and protein expression was detected (P>0.05), but the expression of RhoA membrane protein decreased compared with the negative control group and blank control groups (P<0.05). Cyclin D1 expression decreased, whereas p21 expression significantly increased and the relative expression of ERK protein in the experimental group did not significantly different that in the control group (P>0.05). However, the phosphorylation level of ERK was significantly reduced (P<0.05). The cell cycles of TSCC CAL-27 and SCC-4 were altered in G1/S, cell proliferation activity was inhibited, and apoptosis was induced (P<0.05). CONCLUSIONS: Silencing Icmt can effectively downregulate its expression in TSCC cells, reduce the RhoA membrane targeting localization and cell proliferation, and induce apoptosis. Thus, Icmt may be a potential gene therapy target for TSCC.

The Manipulation of RNA-Guided Nucleic Acid Cleavage with Ninhydrin Chemistry.

CRISPR (clustered regularly interspaced short palindromic repeats) systems have been established as valuable genome-editing tools. Controlling CRISPR systems has high biological significance and this field has garnered intense interest. There is a considerable need for simple approaches with no need for protein engineering. The CRISPR systems usually require a guide RNA (gRNA) moiety to recruit and direct the nuclease complexes. In this respect, the ninhydrin (1,2,3-indantrione monohydrate) seems to have considerable potential, as yet unexploited, for modifying gRNA. In this study, ninhydrin chemistry is explored for reversible postsynthetic modification of gRNA molecules. It is further shown that ninhydrin chemistry is efficient in modulating two important CRISPR systems. Thus, ninhydrin chemistry exhibits potential applications in future chemical biology studies.

[Effect of human bone marrow mesenchymal stem cells on invasion of tongue squamous cell carcinoma cell line Cal-27].

PURPOSE: To investigate the effect of human bone marrow mesenchymal stem cells (hBM-MSCs) on invasion of tongue squamous cell carcinoma cell line Cal-27 and its mechanism. METHODS: hBM-MSCs and Cal-27 were cultured respectively, and the morphology of the cells was observed under an inverted microscope. The co-cultured Cal-27 cells were obtained by co-culture of hBM-MSCs and Cal-27. The migration area of Cal-27 was observed by scratch test;transwell migration and invasion experiments were performed to observe migration and invasion of Cal-27, and a bar graph was then drawn. Fluorescence quantitative PCR was used to observe the effect of hBM-MSCs on gene expression of the tumor markers E-cadherin, twist, slug, snail, MMP-2 and MMP-9. Western blot was used to observe the effect of hBM-MSCs on protein expression of MMP-2 and MMP-9, related to the invasion of Cal-27. SPSS 19.0 software package was used for statistical analysis of the data. RESULTS: Under the influence of hBM-MSCs, the invasion of Cal-27 was promoted, accompanied by down-regulation of E-cadherin, up-regulation of twist, slug, snail, MMP-2, MMP-9 and up-regulation of MMP-2 and MMP-9 expression. CONCLUSIONS: hBM-MSCs can promote invasion of Cal-27 cells, which may be related to up-regulation of the expression of tumor markers related to invasion of Cal-27 cells.

Light-Driven Activation of RNA-Guided Nucleic Acid Cleavage.

As one of the most favorable stimuli, photoactivation provides an advantageous way to manipulate biological objects. In the current study, we have successfully demonstrated the use of light activation guide RNA (gRNA) strategy for controlling CRISPR systems. By conjugating photolabile protecting groups, the CRISPR functions became minimal, but exposure of acylated gRNAs to 365 nm light triggers the removal of masking groups, leading to the rescue of CRISPR functions. Furthermore, our strategy has been successfully used to control gene editing in human cells. This proof-of-concept study therefore demonstrates the promising potential of our strategy to versatile applications in chemical biology.

[Effects of farnesyltransferase silencing on the migration and invasion of tongue squamous cell carcinoma].

OBJECTIVE: This study aimed to explore the effects of silencing farnesyltransferase (FTase) on the migration and invasion of tongue squamous cell carcinoma (TSCC) through RNA interference. METHODS: TSCC cells (CAL27 and SCC-4) were cultured in vitro and then transfected with siRNA to silence FTase expression. The tested cells were categorized as follows: experimental group (three RNA interference groups), negative control group, and blank control group. mRNA expression of FTase and HRAS in each group was analyzed by quantitative real-time polymerase chain reaction. On the basis of FTase mRNA expression, the optimum interference group (highest silencing efficiency) was selected as the experimental group for further study. The protein expression of FTase, HRAS, p65, p-p65(S536), matrix metalloprotein-9 (MMP-9), hypoxia inducible factor-1α (HIF-1α), and vascular endothelial growth factor (VEGF) was analyzed by Western blot. The invasion and migration abilities of TSCC cells were determined by Transwell invasion assay and cell wound healing assay. RESULTS: The mRNA and protein expression of FTase in the experimental group decreased compared with that in the negative control and blank control groups (P<0.05). The mRNA and protein expression of HRAS was not significantly different among the groups (P>0.05). In the experimental group, the protein expression of p-p65(S536), MMP-9, HIF-1α, and VEGF decreased (P<0.05), whereas that of p65 had no significant change (P>0.05). The migration and invasion abilities of the experimental group were inhibited significantly (P<0.05). CONCLUSIONS: Silencing FTase in vitro could effectively downregulate its expression in TSCC cell lines and reduce the migration and invasion abilities to a certain extent. FTase could be a new gene therapy target of TSCC, and this research provided a new idea for the clinical treatment of TSCC.

Conditional control of RNA-guided nucleic acid cleavage and gene editing.

Prokaryotes use repetitive genomic elements termed CRISPR (clustered regularly interspaced short palindromic repeats) to destroy invading genetic molecules. Although CRISPR systems have been widely used in DNA and RNA technology, certain adverse effects do occur. For example, constitutively active CRISPR systems may lead to a certain risk of off-target effects. Here, we introduce post-synthetic masking and chemical activation of guide RNA (gRNA) to controlling CRISPR systems. An RNA structure profiling probe (2-azidomethylnicotinic acid imidazolide) is used. Moreover, we accomplish conditional control of gene editing in live cells. This proof-of-concept study demonstrates promising potential of chemical activation of gRNAs as a versatile tool for chemical biology.