Capillarity-powered and CRISPR/Cas12a-responsive DNA hydrogel distance sensor for highly sensitive visual detection of HPV DNA.

The rapid and specific identification and sensitive detection of human papillomavirus (HPV) infection is critical for preventing cervical cancer, particularly in resource-limited regions. In this work, we hope to propose a capillarity-powered and CRISPR/Cas12a-responsive DNA hydrogel distance sensor for point-of-care (POC) DNA testing. Using the thermal reversibility of DNA hydrogel and capillarity, the novel DNA hydrogel distance sensor can be rapidly and simply constructed by loading an ultra-thin CRISPR/Cas12a-responsive DNA-crosslinked hydrogel film at the end of the capillary tube. The target DNA-specific recombinase polymerase reaction (RPA) amplicons activate the trans-cleavage activity of the Cas12a enzyme, cleaving the crosslinked DNA in hydrogel film, and causing an increase of hydrogel's permeability. As a result, a sample solution containing target DNA travels into the capillary tube at a longer distance compared to the negative samples. Reading the solution traveling distance in capillary tubes, the novel sensor realizes target DNA detection without any special equipment. Benefiting from the exponential target amplification of RPA and multiple turnover response of trans-cleavage of CRISPR/Cas12a, the developed sensor can visually and specifically detect as low as 1 aM HPV 16 DNA within 30 min. These outstanding features, including exceptional sensitivity and specificity, simple and portable design, mild measurement conditions, quick turnaround time, and user-friendly read-out, make the novel distance sensor a promising option for POC diagnostic applications.

Single Methylation Sensitive Restriction Endonuclease-Based Cascade Exponential Amplification Assay for Visual Detection of DNA Methylation at Single-Molecule Level.

Function as a potential cancer biomarker, DNA methylation shows great significance in cancer diagnosis, prognosis, and treatment monitoring. While the lack of an ultrasensitive, specific, and accurate method at the single-molecule level hinders the analysis of the exceedingly low levels of DNA methylation. Herein, based on the outstanding recognition and digestion ability of methylation-sensitive restriction endonuclease (MSRE), we established a single MSRE-based cascade exponential amplification method, which requires only two ingeniously designed primers and only one recognition site of MSRE for the detection of DNA methylation. Differentiated by MSRE digestion, the cleaved unmethylated DNA is too short to induce any amplification reactions, while methylated DNA remains intact to trigger cascade exponential amplification and the subsequent CRISPR/Cas12a system. By integrating the two exponential amplification reactions, as low as 1 aM methylated DNA can be accurately detected, which corresponds to 6 molecules in a 10 µL system, indicating that our method is more sensitive than single amplification-based methods with the ability to detect DNA methylation at the single-molecule level. In addition, 0.1% methylated DNA can be effectively distinguished from large amounts of unmethylated DNA. Our method is further introduced to exploit the expression difference of DNA methylation among normal cells and cancer cells. Moreover, the visual detection of DNA methylation is also realized by the full hybridization between amplification products and the crRNA of CRISPR/Cas12a. Therefore, the proposed method has great potential to be a promising and robust bisulfite-free method for the detection of DNA methylation at the single-molecule level, which is of great importance for early diagnosis of cancer.

A Membrane-Confined Signal Amplification Strategy for Sensitive Monitoring of Extracellular Enzymatic Activity Upon Drug Stimulus.

Extracellular enzymes are not only strongly correlated with disease development but also play critical roles in modulating immune responses. Therefore, real-time monitoring of extracellular enzymatic activity can afford straightforward insights into their spatiotemporal dynamics upon drug stimulus, and provide promising tools to unravel their key roles in modulating the cell signaling. Although DNA-based sensing probes have been frequently developed for the detection of a variety of biomolecules, there still lacks a modular design strategy for amplified imaging of extracellular enzymatic activity associated with live cells. Herein, we developed an enzymatically triggerable signal amplification strategy for real-time dynamic imaging of extracellular enzyme activity through a cell membrane-confined hybrid chain reaction (HCR). We demonstrated that, by modifying the initiator DNA with enzyme-specific incision sites and cholesterol tail, extracellular enzyme-trigged HCR could be fulfilled on the surface of the cellular membrane, facilitating amplified detection of extracellular enzymatic activity. Dynamic monitoring of enzyme secretion of cancer cells upon stimulus or macrophage cells upon inflammation challenge has also been achieved. We envision that the design strategy could provide valuable information for dissecting the role of extracellular enzymes in modulating cell responses to drug treatment.

Boundary guidance network for medical image segmentation.

Accurate segmentation of the tumor area is crucial for the treatment and prognosis of patients with bladder cancer. Cystoscopy is the gold standard for diagnosing bladder tumors. However, The vast majority of current work uses deep learning to identify and segment tumors from CT and MRI findings, and rarely involves cystoscopy findings. Accurately segmenting bladder tumors remains a great challenge due to their diverse morphology and fuzzy boundaries. In order to solve the above problems, this paper proposes a medical image segmentation network with boundary guidance called boundary guidance network. This network combines local features extracted by CNNs and long-range dependencies between different levels inscribed by Parallel ViT, which can capture tumor features more effectively. The Boundary extracted module is designed to extract boundary features and utilize the boundary features to guide the decoding process. Foreground-background dual-channel decoding is performed by boundary integrated module. Experimental results on our proposed new cystoscopic bladder tumor dataset (BTD) show that our method can efficiently perform accurate segmentation of tumors and retain more boundary information, achieving an IoU score of 91.3%, a Hausdorff Distance of 10.43, an mAP score of 85.3%, and a F1 score of 94.8%. On BTD and three other public datasets, our model achieves the best scores compared to state-of-the-art methods, which proves the effectiveness of our model for common medical image segmentation.

Specific quantitative detection of N6-methyladenosine at single-base resolution by extension-based isothermal exponential amplification reaction (E-IEXPAR).

BACKGROUND: N6-methyladenosine (m6A) is a common modification in RNA, crucial for various cellular functions and associated with human diseases. Quantification of m6A at single-base resolution is of great significance for exploring its biological roles and related disease research. However, existing analysis techniques, such as polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP), face challenges like the requirement for thermal cycling or intricate primer design. Therefore, it is urgent to establish a simple, non-thermal cycling and highly sensitive assay for m6A. RESULTS: Leveraging the inhibitory effect of m6A on primer elongation and uncomplicated feature of the isothermal exponential amplification reaction (IEXPAR), we have developed an extension-based IEXPAR (E-IEXPAR). This approach requires just a single extension primer and one template, simplifying the design process in comparison to the more complex primer requirements of the LAMP methods. The reactions are conducted at constant temperatures, therby elimiating the use of thermal cycling that needed in PCR methods. By combining IEXPAR with an extension reaction, E-IEXPAR can identify m6A in RNA concentrations as low as 4 fM. We have also introduced a new analytical model to process E-IEXPAR results, which can aid to minimize the impact of unmodified adenine (A) on m6A measurements, enabling accurate m6A quantification in small mixed samples and cellular RNA specimens. SIGNIFICANCE AND NOVELTY: E-IEXPAR streamlines m6A detection by eliminating the need for intricate primer design and thermal cycling, which are common in current analytical methods. Its utilization of an extension reaction for the initial identification of m6A, coupled with a novel calculation model tailored to E-IEXPAR outcomes, ensures accurate m6A selectivity in mixed samples. As a result, E-IEXPAR offers a reliable, straightforward, and potentially economical approach for specifically assaying m6A in both biological function studies and clinical research.

Plasma exosomal miR-1290 and miR-29c-3p as diagnostic biomarkers for lung cancer.

Background: Enhancing the diagnostic efficacy of early-stage lung cancer is crucial for improving prognosis. The objective of this study was to ascertain dependable exosomal miRNAs as biomarkers for the diagnosis of lung cancer. Methods: Exosomal miRNA candidates were identified through miRNA sequencing and subsequently validated in various case-control sets using real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The correlation between the expression of exosomal miRNAs and the clinicopathological features of lung cancer was investigated. To assess the diagnostic efficacy of exosomal miRNAs for lung cancer, the receiver operating characteristic (ROC) curve analysis was conducted. The optimal cutoff value of exosomal miRNAs was determined in the testing cohort and subsequently confirmed in the validation cohort. Results: The results showed that the expression of exosomal miR-1290 was significantly elevated, while that of miR-29c-3p was significantly decreased in the plasma of lung cancer patients, especially in those with early-stage lung cancer, compared to individuals with benign lung conditions (P < 0.01). Exosomal miR-1290 and miR-29c-3p demonstrated superior diagnostic efficacy compared to conventional tumor biomarkers in distinguishing between lung cancer and benign lung diseases, as evidenced by their respective area under the curve (AUC) values of 0.934 and 0.868. Furthermore, exosomal miR-1290 and miR-29c-3p exhibited higher diagnostic efficiency in early-stage lung cancer than traditional tumor markers, with AUC values of 0.947 and 0.895, respectively. Notably, both exosomal miR-1290 and miR-29c-3p displayed substantial discriminatory capacity in distinguishing between non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), as indicated by their respective AUC values of 0.810 and 0.842. Conclusions: The findings of this study provided evidence that exosomal miR-1290 and miR-29c-3p hold significant potential as biomarkers for the early detection of lung cancer, as well as for differentiating between NSCLC and SCLC.

Amplification-free detection of telomerase activity at the single-cell level via Cas12a-lighting-up single microbeads (Cas12a-LSMBs).

Telomerase overexpresses in almost all cancer cells and has been deemed a universal biomarker for cancer diagnosis and therapy. However, simple and ultrasensitive detection of telomerase activity in single-cells is still a huge challenge. Herein, we wish to report Cas12a-lighting up single microbeads (Cas12a-LSMBs) for ultrasensitive detection of telomerase activity without nucleic acid amplification. In this platform, single-strand DNA reporter (ssDNA reporter)-functionalized single-microbeads (functionalized-SMBs) are employed as a reactor for the trans-cleavage of telomerase-activated CRISPR/Cas12a as well as a reporting unit for fluorescence signal enrichment and visualization. Due to the space-confined effect and signal enrichment mechanism on the surface of the functionalized SMBs, the Cas12a-LSMBs can accurately detect telomerase activity in crude cell lysates with high specificity. Importantly, we have demonstrated that the Cas12a-LSMBs are a reliable and practical tool to detect telomerase activity in single cells and investigate cellular heterogeneity of telomerase activity from cell-to-cell variations.

Postoperative urinary leakage after bilateral totally extraperitoneal herniorrhaphy in a patient with a healed cystostomy and appendectomy: A case report.

We report a case of postoperative urinary leakage after bilateral laparoscopic totally extraperitoneal (TEP) herniorrhaphy. A man in his upper 80s with a healed cystostomy and appendectomy underwent bilateral TEP herniorrhaphy. Urinary leakage was noted by ultrasound examination 4 days after bilateral TEP. Cystography and computed tomography conclusively confirmed a 6-mm extraperitoneal fistula at the site of the previous cystostomy. The fistula involved the anterior bladder wall and was associated with an extended urinoma. The patient was treated by indwelling catheterization using a Foley catheter and repeated ultrasound-guided puncture and aspiration of the inguinal effusion at the bedside. The patient was completely healed 69 days after the operation with no mesh infection or bladder dysfunction. We believe that urinary leakage is possible after TEP herniorrhaphy in patients with a healed suprapubic cystostomy. Therefore, indwelling catheterization using a Foley catheter should be implemented before surgery, and the Foley catheter can be removed within 1 week after surgery if no postoperative urinary leakage is observed. A history of suprapubic cystotomy should not be regarded as a contraindication for TEP surgery. This is the first report of urinary leakage after bilateral TEP herniorrhaphy in a patient with a healed cystostomy and appendectomy.

Double blocking gap-filling-ligation coupled with cascade isothermal amplification for ultrasensitive quantification of N6-methyladenosine.

We developed a method for quantifying N6-methyladenosine at one-nucleotide resolution based on double blocking gap-filling-ligation and cascade isothermal amplification. This proposed method can detect as low as 1 fM target RNA, achieving selectivity up to approximately 100-fold between m6A and A, and has been successfully applied to the analysis of m6A at specific sites in cell samples.

Endogenous Enzyme-Operated Spherical Nucleic Acids for Cell-Selective Protein Capture and Localization Regulation.

Precise regulation of protein activity and localization in cancer cells is crucial to dissect the function of the protein-involved cellular network in tumorigenesis, but there is a lack of suitable methodology. Here we report the design of enzyme-operated spherical nucleic acids (E-SNAs) for manipulation of the nucleocytoplasmic translocation of proteins with cancer-cell selectivity. The E-SNAs are constructed by programmable engineering of aptamer-based modules bearing enzyme-responsive units in predesigned sites and further combination with SNA nanotechnology. We demonstrate that E-SNAs are able to regulate cytoplasmic-to-nuclear shuttling of RelA protein efficiently and specifically in tumor cells, while they remain inactive in normal cells due to insufficient enzyme expression. We further confirmed the generality of this strategy by investigating the enzyme-modulated inhibition/activation of thrombin activity by varying the aptamer-based design.

An Enzymatically Gated Catalytic Hairpin Assembly Delivered by Lipid Nanoparticles for the Tumor-Specific Activation of Signal Amplification in miRNA Imaging.

MicroRNA (miRNA) imaging in disease sites is vital to elucidate their role in cancer progression. However, limited tumor specificity remains a major barrier for traditional amplification approaches due to associated background signal leakage. Here, we report a generalizable approach via the combination of enzymatically triggered catalytic hairpin assembly with lipid nanoparticles (LNPs)-based delivery strategy for tumor-specific activation of signal amplification and therefore sensitive miRNA imaging. The signal amplification is established via engineering of traditional catalytic hairpin assembly with enzymatically activated motifs to achieve triggable miRNA imaging in cancer cells. Furthermore, by the introduction of LNPs to combat biological barriers, we demonstrate that the system enables amplified miRNA imaging in vivo with reduced off-tumor signal, leading to enhanced tumor-to-background contrast compared with traditional methods. This approach that relies on specific triggers and controlled delivery to distinguish miRNA in cancer cells from normal cells should be useful in tumor diagnosis.

Specific detection of fusion transcripts based on a duplex-specific nuclease and isothermal exponential amplification reaction.

Fusion genes are mostly found in tumor tissues, but are in low expression levels in healthy tissues, making them good candidate biomarkers for tumor diagnosis and therapy. Here, we propose a duplex-specific nuclease-isothermal exponential amplification reaction (DSN-IEXPAR) method for the detection of fusion transcripts. A DNA probe is specifically designed for fusion transcript recognition and hybridization, and DSN cleavages the DNA probe in the DNA/RNA duplex. Through controlling the recognition and cleavage temperature, DSN can only cut the DNA probe fully matched with the target fusion transcript rather than other transcripts containing partial the same sequence, endowing the proposed method with high specificity to the fusion transcript in the presence of homologous sequences. The truncated DNA probe after cutting can subsequently trigger IEXPAR as a probe, so as low as 100 fM fusion transcript can be detected with the proposed DSN-IEXPAR. The evaluation of the analytical performance of DSN-IEXPAR demonstrates that it can provide an effective platform for fusion transcript detection in the ordinary laboratory and clinical diagnosis.

One-step and highly sensitive quantification of fusion genes with isothermal amplification initiated by a fusion-site anchored stem-loop primer.

As causative oncogenes and drug targets, fusion genes play critical roles in tumorigenesis, development, and treatment and thus are regarded as tumor-specific molecular biomarkers. Specific identification and sensitive quantification of fusion genes are of great significance in cancer diagnosis, classification, and prognosis as well as minimal residual disease (MRD) monitoring. Herein, we proposed a specific and sensitive method for the quantitative detection of fusion transcripts by designing stem-loop primers to directly track fusion junctions of fusion genes and subsequently initiate reverse transcript loop-mediated isothermal amplification (LAMP). Benefitting from the specific and direct mechanism of stem-loop primers and the high amplification efficiency of LAMP, the proposed method can sensitively measure fusion gene transcripts with a detection limit of 100 aM (1 zmol) and achieve a wide linear dynamic range spanning at least six orders of magnitude (100 aM-100 pM). Significantly, the whole fusion transcript assay can be accomplished in one step under isothermal conditions, greatly simplifying the operation and detection processes. Meanwhile, the one-step analysis method in one tube may effectively eliminate false-positive results from product cross-contamination during multiple experimental operations and cover-opening measurements. We have demonstrated that the proposed method is practical and accurate for the quantitation of fusion transcripts in biological samples. Owing to the outstanding features of high sensitivity, excellent specificity, and simple operation, the new strategy may provide a robust and attractive platform for the quantification of fusion genes.

General Label-Free Fluorescent Aptamer Binding Assay Using Cationic Conjugated Polymers.

With more and more new aptamers being reported, a general, cost-effective yet reliable aptamer binding assay is still needed. Herein, we studied cationic conjugated polymer (CCP)-based binding assays taking advantage of the conformational change of aptamer after binding with a target, which is reflected by the fluorescence change of the CCP. Poly(3-(3'-N,N,N-triethylamino-1'-propyloxy)-4-methyl-2,5-thiophene hydrochloride) (PMNT) was used as a model CCP in this study, and the optimal buffer was close to physiological conditions with 100 mM NaCl and 10 mM MgCl2. We characterized four aptamers for K+, adenosine, cortisol, and caffeine. For cortisol and caffeine, the drop in the 580 nm peak intensity was used for quantification, whereas for K+ and adenosine, the fluorescence ratio at 580 over 530 nm was used. The longer stem of the stem-loop structured aptamer facilitated binding of the target and enlarged the detection signal. High specificity was achieved in differentiating targets with analogues. Compared with the SYBR Green I dye-based staining method, our method achieved equal or even higher sensitivity. Therefore, this assay is practicable as a general aptamer binding assay. The simple, label-free, quick response, and cost-effective features will make it a useful method to evaluate aptamer binding. At the same time, this system can also serve as label-free biosensors for target detection.

Enzymatically Controlled Nanoflares for Specific Molecular Recognition and Biosensing.

In situ sensing of physiological and pathological species in cancer cells is of great importance to unravel their molecular and cellular processes. However, the biosensing with conventional probes is often limited by the undesired on-target off-tumor interference. Here, we report a novel strategy to design enzymatically controlled nanoflares for sensing and imaging molecular targets in tumor cells. The triggerable nanoflare was designed via rational engineering of structure-switching aptamers with the incorporation of an enzyme-activatable site and further conjugation on gold nanoparticles. The nanoflare sensors did not respond to target molecules in normal cells, but they could be catalytically activated by specific enzymes in cancer cells, thereby enabling cancer-specific sensing and imaging in vitro and in vivo with improved tumor specificity. Considering that diverse aptamers were selected, we expect that this strategy would facilitate the precise detection of a broad range of targets in tumors and may promote the development of smart probes for cancer diagnosis.

Multiple stem-loop primers induced cascaded loop-mediated isothermal amplification for direct recognition and specific detection of circular RNAs.

Circular RNAs (circRNAs), as a novel class of endogenous noncoding RNAs, play important roles in many biological functions, such as acting as microRNA sponges and cancer biomarkers. Rapid and sensitive detection of circRNAs will be of great benefit to better understand their biological functions especially in clinical diagnosis using circRNAs as biomarkers. Herein, by using multiple stem-loop primers (SLPs) to specifically recognize the unique junction site of circRNAs, we demonstrate a multiple SLPs-induced cascaded loop-mediated isothermal amplification (LAMP) assay which has the ability to directly and specifically discriminate circRNAs from homologous linear RNAs without any linear RNA removal procedure. Taking advantage of the displacement activity of Bst DNA polymerase, the rationally designed SLPs can directly recognize the target circRNA and generate a large amount of double stem-loop DNAs which can initiate a cascaded LAMP reaction with improved efficiency. The use of additional SLPs can accelerate the reaction rate; as a result, the LAMP reaction can be finished within 35 min which is much quicker than traditional LAMP. The proposed method can detect as low as 1 fM circRNA and has been successfully applied to the detection of circRNAs in total RNA samples extracted from cell lines.

Sensitive detection of fusion transcripts with padlock probe-based continuous cascade amplification (P-CCA).

Gene fusion, resulting from chromosomal rearrangements, is the juxtaposition of two or more original genes into the same set to form a functional gene. The significant specificity of fusion genes for tumor cells makes them promising candidates for diagnostic biomarkers and therapeutic targets. The sensitive detection of fusion transcripts is of great significance in biological research and disease diagnosis. Here, we propose a method for the sensitive detection of PML-RARα gene fusion transcripts by the direct ligation of the padlock probe at the junction site, and the cyclized DNA then triggers a continuous cascade amplification of two subsequent amplification reactions: rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP). Due to the ability of the ligation reaction to differentiate mismatched sequences and the high amplification efficiency of continuous cascade amplification reactions, the proposed method can detect as low as 1 fM targets with high specificity, and has been successfully applied to real samples. Through a facile design of the triggering sequence in padlock probes, the cascade RCA and LAMP can be integrated into one-tube isothermal reactions with a simple one-step operation. Therefore, this work provides a convenient padlock probe-based continuous cascade amplification (P-CCA) method for the detection of fusion transcripts, and offers a fast and reliable platform for the early clinical diagnosis of gene fusion-related cancers.

Multivalent Engineering of Exosomes with Activatable Aptamer Probes for Specific Regulation and Monitoring of Cell Targeting.

Reconstituting and probing exosome-cell interactions is critical for elucidating exosome-related cell biology and advancing their diagnostic and therapeutic potential. We report here an exosomal engineering strategy to achieve controlled regulation of exosome-cell interactions with activatable sensing capability. The approach relies on membrane-protein directed, programmable DNA self-assembly to construct a DNA polymeric scaffold with multivalent display of structure-switchable aptamer sensing probes on exosome surfaces. The engineered exosomes exhibit enhanced cancer cell targeting ability compared to exosomes modified with monovalent aptamers. Furthermore, the anchored aptamer probes could be activated by specific membrane protein targeting, followed by structural switching to report an output fluorescence signal, thus allowing dynamic monitoring of exosome-cell interactions both in vitro and in vivo. We envision this will provide a complementary tool for specific regulation and monitoring of exosome-cell docking interactions and will advance the development of exosome-based biomedical applications.

Ultrasensitive homogeneous detection of microRNAs in a single cell with specifically designed exponential amplification.

We firstly developed an ultrasensitive method based on specifically designed exponential amplification for miRNA detection with simple operation in homogeneous solutions. The proposed assay can detect miRNAs at a concentration as low as 1 aM and can be successfully applied for routine miRNA detection in a single cell.

Ultrasensitive multiplexed detection of miRNA targets of interest based on encoding probe extension in improved cDNA library.

MicroRNAs (miRNAs) are a class of regulatory small RNA molecules that play critical roles in a wide variety of biological processes. Abnormally expressed miRNAs have been increasingly utilized as biomarkers for cancer diagnosis. Generally, a specific cancer is associated with expression alterations of several species of miRNAs and different types of cancers are related to different miRNA species. Therefore, a universal method for multiplexed detection of miRNA targets of interest is now desirable for cancer diagnosis. In this paper, by adding an enzymatic digestion step to reduce the nonspecific adaptor dimers, we firstly improved the method to construct cDNA library of all miRNAs, which greatly increased the cDNA yield. By specifically designing DNA probes to hybridize with the cDNAs at key positions and doubly encoding DNA probes with different lengths and different fluorophores during single-base extension, each miRNA could produce a unique product, which could be separated and detected by capillary electrophoresis. Thus, miRNA targets of interest could be simultaneously detected with great specificity at single-base resolution. By using seventeen randomly selected miRNAs as the model, as low as 1.0 fM of each miRNA target could be simultaneously determined. Furthermore, we had achieved accurate analysis of multiple miRNAs in real biological RNA samples and found that several miRNAs expressed differently between cancer cells and normal cells, indicating that the proposed method had the ability to pick out aberrant expression miRNAs in real biological samples. Compared with high-throughput sequencing methods, the proposed method is simpler and specific, and very suitable for the detection of specific miRNAs associated with a disease, which shows great potential for cancer diagnosis.