Detection and Identification of Pesticides in Fruits Coupling to an Au-Au Nanorod Array SERS Substrate and RF-1D-CNN Model Analysis.

In this research, a method was developed for fabricating Au-Au nanorod array substrates through the deposition of large-area Au nanostructures on an Au nanorod array using a galvanic cell reaction. The incorporation of a granular structure enhanced both the number and intensity of surface-enhanced Raman scattering (SERS) hot spots on the substrate, thereby elevating the SERS performance beyond that of substrates composed solely of an Au nanorod. Calculations using the finite difference time domain method confirmed the generation of a strong electromagnetic field around the nanoparticles. Motivated by the electromotive force, Au ions in the chloroauric acid solution were reduced to form nanostructures on the nanorod array. The size and distribution density of these granular nanostructures could be modulated by varying the reaction time and the concentration of chloroauric acid. The resulting Au-Au nanorod array substrate exhibited an active, uniform, and reproducible SERS effect. With 1,2-bis(4-pyridyl)ethylene as the probe molecule, the detection sensitivity of the Au-Au nanorod array substrate was enhanced to 10-11 M, improving by five orders of magnitude over the substrate consisting only of an Au nanorod array. For a practical application, this substrate was utilized for the detection of pesticides, including thiram, thiabendazole, carbendazim, and phosmet, within the concentration range of 10-4 to 5 × 10-7 M. An analytical model combining a random forest and a one-dimensional convolutional neural network, referring to the important variable-one-dimensional convolutional neural network model, was developed for the precise identification of thiram. This approach demonstrated significant potential for biochemical sensing and rapid on-site identification.

MicroRNA Detection with CRISPR/Cas.

Low-cost detection of miRNAs has caught broad attention in recent years due to the potential application of these small noncoding RNAs for diagnostics and therapeutic purposes. Their small size and low abundance, however, derive challenges in engineering robust detection tools. To date, multiple detection assays have been developed to achieve highly specific recognition of trace amount of miRNA with state-of-the-art nucleic acid detection and signal amplification techniques. In this chapter we describe how isothermal amplification techniques and CRISPR/Cas-based techniques can be integrated to generate rationally designed miRNA detection systems for specific miRNA.

Comprehensive Analysis of hsa-miR-654-5p's Tumor-Suppressing Functions.

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.

The novel sandwich composite structure: a new detection strategy for the ultra-sensitive detection of cyclotrimethylenetrinitramine (RDX).

This study presents a novel sandwich composite structure that was designed for the ultra-sensitive detection of cyclotrimethylenetrinitramine (RDX). Au nanorod arrays (Au NRAs) were prepared and bound to 10-7M 6-MNA as adsorption sites for RDX, while Au nanorods (Au NRs) were modified using 10-5M 6-MNA as SERS probes. During detection, RDX molecules connect the SERS probe to the surface of the Au NRAs, forming a novel type of Au NRAs-RDX-Au NRs 'sandwich' composite structure. The electromagnetic coupling effect between Au NRs and Au NRAs is enhanced due to the molecular level of the connection spacing, resulting in new 'hot spots'. Meanwhile, Au NRAs and Au NRs have an auto-enhancement effect on 6-MNA. In addition, the presence of charge transfer in the formed 6-MNA-RDX complex induced chemical enhancement. The limits of detection of RDX evaluated by Raman spectroscopy using 6-MNA were as low as 10-12mg ml-1(4.5 × 10-15M) with good linear correlation between 10-12and 10-8mg ml-1(correlation coefficientR2 = 0.9985). This novel sandwich composite structure accurately detected RDX contamination in drinking water and on plant surfaces in an environment with detection limits as low as 10-12mg ml-1and 10-8mg ml-1.

A 'sandwich' structure for highly sensitive detection of TNT based on surface-enhanced Raman scattering.

Ultra-sensitive detection of 2,4,6-trinitrotoluene (TNT) plays an important role in society security and human health. The Raman probe molecule p-aminothiophenol (PATP) can interact with TNT in three ways to form a TNT-PATP complex. In this paper, a 'sandwich' structure was developed to detect TNT with high sensitivity. Au nano-pillar arrays (AuNPAs) substrates modified by low-concentration PATP through Au-S bonds were acted as capture probe for TNT. Meanwhile, Ag nano-particles (AgNPs) modified by PATP at higher concentration were employed as tags for surface-enhanced Raman scattering (SERS). The formation of the TNT-PATP complex is not only the means by which AuNPAs substrates recognize and capture TNT, but also links the SERS tags to TNT, forming an AuNPAs-TNT-AgNPs 'sandwich' structure. The Raman signal of PATP was greatly enhanced mainly because novel 'hot spots' formed between the AuNPAs and AgNPs of the 'sandwich' structure. The Raman signal of PATP was further amplified by the chemical enhancement effect induced by the TNT-PATP complex formation. Based on this mechanism, the limit of detection (LOD) of TNT was determined from the Raman signal of PATP. The LOD reached 10-9 mg/mL (4.4 × 10-12 M), much lower than that suggested by the US Environmental Protection Agency (88 nM). Moreover, TNT was selectively detected over several TNT analogues 2,4-dinitrotoluene (DNT), p-nitrotoluene (NT) and hexogen (RDX). Finally, the 'sandwich' structure was successfully applied to TNT detection in environmental water and sand.

Attenuation of Antiviral Immune Response Caused by Perturbation of TRIM25-Mediated RIG-I Activation under Simulated Microgravity.

Microgravity is a major environmental factor of space flight that triggers dysregulation of the immune system and increases clinical risks for deep-space-exploration crews. However, systematic studies and molecular mechanisms of the adverse effects of microgravity on the immune system in animal models are limited. Here, we establish a ground-based zebrafish disease model of microgravity for the research of space immunology. RNA sequencing analysis demonstrates that the retinoic-acid-inducible gene (RIG)-I-like receptor (RLR) and the Toll-like receptor (TLR) signaling pathways are significantly compromised by simulated microgravity (Sµg). TRIM25, an essential E3 for RLR signaling, is inhibited under Sµg, hampering the K63-linked ubiquitination of RIG-I and the following function-induction positive feedback loop of antiviral immune response. These mechanisms provide insights into better understanding of the effects and principles of microgravity on host antiviral immunity and present broad potential implications for developing strategies that can prevent and control viral diseases during space flight.

Fabrication of Ag@Au (core@shell) nanorods as a SERS substrate by the oblique angle deposition process and sputtering technology.

Gold (Au) and silver (Ag) are the main materials exhibiting strong Surface-Enhanced Raman Scattering (SERS) effects. The Ag nano-rods (AgNRs) and Au nano-rods (AuNRs) SERS substrates prepared using the technology of the oblique angle deposition (OAD) process have received considerable attention in recent years because of their rapid preparation process and good repeatability. However, AgNR substrates are unstable due to the low chemical stability of Ag. To overcome these limitations, an Ag@Au core-shell nano-rod (NR) array SERS substrate was fabricated using the OAD process and sputtering technology. Moreover, simulation analysis was performed using finite-difference time-domain calculations to evaluate the enhancement mechanism of the Ag@Au NR array substrate. Based on the simulation results and actual process conditions, the Ag@Au core-shell NR array substrate with the Au shell thickness of 20 nm was studied. To characterize the substrate's SERS performance, 1,2-bis(4-pyridyl)ethylene (BPE) was used as the Raman probe. The limit of detection of BPE could reach 10-12 M. The Ag@Au NR array substrate demonstrated uniformity with an acceptable relative standard deviation. Despite the strong oxidation of the hydrogen peroxide (H2O2) solution, the Ag@Au NR array substrate maintains good chemical stability and SERS performance. And long-term stability of the Ag@Au NR substrate was observed over 8 months of storage time. Our results show the successful preparation of a highly sensitive, repeatable and stable substrate. Furthermore, this substrate proves great potential in the field of biochemical sensing.

Novel nucleic acid detection strategies based on CRISPR-Cas systems: From construction to application.

Beyond their widespread application as genome-editing and regulatory tools, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems also play a critical role in nucleic acid detection due to their high sensitivity and specificity. Recently developed Cas family effectors have opened the door to the development of new strategies for detecting different types of nucleic acids for a variety of purposes. Precise and efficient nucleic acid detection using CRISPR-Cas systems has the potential to advance both basic and applied biological research. In this review, we summarize the CRISPR-Cas systems used for the recognition and detection of specific nucleic acids for different purposes, including the detection of genomic DNA, nongenomic DNA, RNA, and pathogenic microbe genomes. Current challenges and further applications of CRISPR-based detection methods will be discussed according to the most recent developments.

Bioinformatic Identification of miR-622 Key Target Genes and Experimental Validation of the miR-622-RNF8 Axis in Breast Cancer.

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.

Avenues Toward microRNA Detection In Vitro: A Review of Technical Advances and Challenges.

Over the decades, the biological role of microRNAs (miRNAs) in the post-transcriptional regulation of gene expression has been discovered in many cancer types, thus initiating the tremendous expectation of their application as biomarkers in the diagnosis, prognosis, and treatment of cancer. Hence, the development of efficient miRNA detection methods in vitro is in high demand. Extensive efforts have been made based on the intrinsic properties of miRNAs, such as low expression levels, high sequence homology, and short length, to develop novel in vitro miRNA detection methods with high accuracy, low cost, practicality, and multiplexity at point-of-care settings. In this review, we mainly summarized the newly developed in vitro miRNA detection methods classified by three key elements, including biological recognition elements, additional micro-/nano-materials and signal transduction/readout elements, their current challenges and further applications are also discussed.

Assembly of TALE-based DNA scaffold for the enhancement of exogenous multi-enzymatic pathway.

Synthetic scaffold systems, which exhibit enzyme clustering effect, have been considered as an important parallel approach for metabolic flux control and pathway enhancement. Here, we described an improved DNA-based scaffold system for synthetic tri-enzymatic pathway in Escherichia coli. With plasmid DNA serving as scaffold and exogenous enzymes fused with rationally designed transcription activator-like effectors (TALEs), our approach successfully clustered three TALE-fused enzymes and significantly increased the production of a mevalonate-producing tri-enzymatic pathway with the optimized scaffold structure and plasmid copy number. These results further suggested the scalability and robustness of the TALE-based scaffold system, and we can assume that it can be used on numerous multi-enzyme metabolic pathways due to its programmable features.

Highly Effective and Low-Cost MicroRNA Detection with CRISPR-Cas9.

MicroRNAs have been reported as related to multiple diseases and have potential applications in diagnosis and therapeutics. However, detection of miRNAs remains improvable, given their complexity, high cost, and low sensitivity as of currently. In this study, we attempt to build a novel platform that detects miRNAs at low cost and high efficacy. This detection system contains isothermal amplification, detecting and reporting process based on rolling circle amplification, CRISPR-Cas9, and split-horseradish peroxidase techniques. It is able to detect trace amount of miRNAs from samples with mere single-base specificity. Moreover, we demonstrated that such scheme can effectively detect target miRNAs in clinical serum samples and significantly distinguish patients of non-small cell lung cancer from healthy volunteers by detecting the previously reported biomarker: circulating let-7a. As the first to use CRISPR-Cas9 in miRNA detection, this method is a promising approach capable of being applied in screening, diagnosing, and prognosticating of multiple diseases.

Spatial organization of heterologous metabolic system in vivo based on TALE.

For years, prokaryotic hosts have been widely applied in bio-engineering. However, the confined in vivo enzyme clustering of heterologous metabolic pathways in these organisms often results in low local concentrations of enzymes and substrates, leading to a low productive efficacy. We developed a new method to accelerate a heterologous metabolic system by integrating a transcription activator-like effector (TALE)-based scaffold system into an Escherichia coli chassis. The binding abilities of the TALEs to the artificial DNA scaffold were measured through ChIP-PCR. The effect of the system was determined through a split GFP study and validated through the heterologous production of indole-3-acetic acid (IAA) by incorporating TALE-fused IAA biosynthetic enzymes in E. coli. To the best of our knowledge, we are the first to use the TALE system as a scaffold for the spatial organization of bacterial metabolism. This technique might be used to establish multi-enzymatic reaction programs in a prokaryotic chassis for various applications.