Ratiometric CRISPR/Cas12a-Triggered CHA System Coupling with the MSRE to Detect Site-Specific DNA Methylation.

The precise determination of DNA methylation at specific sites is critical for the timely detection of cancer, as DNA methylation is closely associated with the initiation and progression of cancer. Herein, a novel ratiometric fluorescence method based on the methylation-sensitive restriction enzyme (MSRE), CRISPR/Cas12a, and catalytic hairpin assembly (CHA) amplification were developed to detect site-specific methylation with high sensitivity and specificity. In detail, AciI, one of the commonly used MSREs, was employed to distinguish the methylated target from nonmethylated targets. The CRISPR/Cas12a system was utilized to recognize the site-specific target. In this process, the protospacer adjacent motif and crRNA-dependent identification, the single-base resolution of Cas12a, can effectively ensure detection specificity. The trans-cleavage ability of Cas12a can convert one target into abundant activators and can then trigger the CHA reaction, leading to the accomplishment of cascaded signal amplification. Moreover, with the structural change of the hairpin probe during CHA, two labeled dyes can be spatially separated, generating a change of the Förster resonance energy transfer signal. In general, the proposed strategy of tandem CHA after the CRISPR/Cas12a reaction not only avoids the generation of false-positive signals caused by the target-similar nucleic acid but can also improve the sensitivity. The use of ratiometric fluorescence can eradicate environmental effects by self-calibration. Consequently, the proposed approach had a detection limit of 2.02 fM. This approach could distinguish between colorectal cancer and precancerous tissue, as well as between colorectal patients and healthy people. Therefore, the developed method can serve as an excellent site-specific methylation detection tool, which is promising for biological and disease studies.

One-step and label-free ratiometric fluorescence assay for the detection of plasma exosome towards cancer diagnosis.

Exosomes are closely associated with tumor development and are regarded as viable biomarkers for cancer. Here, a ratiometric fluorescence method was proposed for the one-step and label-free detection of plasma exosomes. A bicolor streptavidin magnetic beads were specifically created with an immobilized Cy5-labeled hairpin aptamer for CD63 (Cy5-Apt) on its surface to identify exosome, and a green color SYBR Green I (SGI) embedded in the stem of Cy5-Apt to respond to exosomes. After exosome capture, the Cy5-Apt could undergo a conformational shift and release the encapsulated SGI, allowing exosome measurement based on the fluorescence ratio of Cy5 and SGI. The enrichment, separation and detection of exosomes in proposed method could be completed in one step (30 min), which is a significant improvement over previous method. Furthermore, the use of ratiometric fluorescence and magnetic separation allows for exosome enrichment and interference elimination from complex matrices, improving accuracy and sensitivity. Particularly, the assay could detect exosomes in plasma and has potential to distinguish lung cancer patients from healthy volunteers with an area under the receiver operator characteristic curve of 0.85. Besides, the study provided an efficient method for analyzing the various divisions of exosomes by merely modifying the aptamer, which holds great promise for point-of-care applications.

A dual-model immobilization-free photoelectrochemical/visual colorimetric bioanalysis based on microemulsion self-assemblies mediated multifunctional signal amplification strategy.

Herein, a novel silver ion-loaded gold microemulsion assemblies (Au/Ag+ MAs) mediated multifunctional signal amplification strategy was proposed to construct a sensitive immobilization-free photoelectrochemical (PEC)/colorimetric biosensor for carcinoembryonic antigen (CEA) detection. Through the sandwiched reaction among CEA, the CEA aptamer (DNA1) loaded on the Au nanoparticles (NPs) functionalized iron oxide (Fe3O4) nanospheres and another CEA aptamer (DNA2) immobilized on Au/Ag+ MAs, a complex is formed and acquired by magnetic separation. Then, Au/Ag+ MAs of the complex are disassembled into Au NPs and Ag+ ions driven by an acetone response, and the obtained demulsification solution is transferred to the cadmium sulfide/cadmium telluride (CdS/CdTe) photoactive composites modified electrode. Based on the multiple inhibition functions (blocking effect of oleylamine; energy transfer effect of Au NPs; and electron snatching effect of Ag+), the photocurrent of the electrode decreases obviously, resulting in the ultrasensitive detection of CEA (a detection limit of 16 fg mL-1). Interestingly, the ion-exchange reactions between CdS/CdTe composites and Ag+ ions generate silver sulfide/silver telluride (Ag2S/Ag2Te) composites, and a color change of composites can be distinguished directly, leading to a quick visual detection of CEA. Compared with the traditional single-modal assay for CEA, such dual-modal PEC/colorimetric assay is a more accurate and reliable due to different mechanisms and independent signal conversion. This work will offer a new perspective for the applications of various self-assemblies in PEC bioanalysis.

A diagnostic classification of lung nodules using multiple-scale residual network.

Computed tomography (CT) scans have been shown to be an effective way of improving diagnostic efficacy and reducing lung cancer mortality. However, distinguishing benign from malignant nodules in CT imaging remains challenging. This study aims to develop a multiple-scale residual network (MResNet) to automatically and precisely extract the general feature of lung nodules, and classify lung nodules based on deep learning. The MResNet aggregates the advantages of residual units and pyramid pooling module (PPM) to learn key features and extract the general feature for lung nodule classification. Specially, the MResNet uses the ResNet as a backbone network to learn contextual information and discriminate feature representation. Meanwhile, the PPM is used to fuse features under four different scales, including the coarse scale and the fine-grained scale to obtain more general lung features of the CT image. MResNet had an accuracy of 99.12%, a sensitivity of 98.64%, a specificity of 97.87%, a positive predictive value (PPV) of 99.92%, and a negative predictive value (NPV) of 97.87% in the training set. Additionally, its area under the receiver operating characteristic curve (AUC) was 0.9998 (0.99976-0.99991). MResNet's accuracy, sensitivity, specificity, PPV, NPV, and AUC in the testing set were 85.23%, 92.79%, 72.89%, 84.56%, 86.34%, and 0.9275 (0.91662-0.93833), respectively. The developed MResNet performed exceptionally well in estimating the malignancy risk of pulmonary nodules found on CT. The model has the potential to provide reliable and reproducible malignancy risk scores for clinicians and radiologists, thereby optimizing lung cancer screening management.

HPIP is an essential scaffolding protein running through the EGFR-RAS-ERK pathway and drives tumorigenesis.

The EGFR-RAS-ERK pathway plays a key role in cancer development and progression. However, the integral assembly of EGFR-RAS-ERK signaling complexes from the upstream component EGFR to the downstream component ERK is largely unknown. Here, we show that hematopoietic PBX-interacting protein (HPIP) interacts with all classical components of the EGFR-RAS-ERK pathway and forms at least two complexes with overlapping components. Experiments of HPIP knockout or knockdown and chemical inhibition of HPIP expression showed that HPIP is required for EGFR-RAS-ERK signaling complex formation, EGFR-RAS-ERK signaling activation, and EGFR-RAS-ERK signaling-mediated promotion of aerobic glycolysis as well as cancer cell growth in vitro and in vivo. HPIP expression is correlated with EGFR-RAS-ERK signaling activation and predicts worse clinical outcomes in patients with lung cancer. These results provide insights into EGFR-RAS-ERK signaling complex formation and EGFR-RAS-ERK signaling regulation and suggest that HPIP may be a promising therapeutic target for cancer with dysregulated EGFR-RAS-ERK signaling.

A Direct-Contact Photocurrent-Direction-Switching Biosensing Platform Based on In Situ Formation of CN QDs/TiO2 Nanodiscs and Double-Supported 3D DNA Walking Amplification.

Herein, a direct-contact photocurrent-direction-switching photoelectrochemical (PEC) biosensing platform for the ultrasensitive and selective detection of soluble CD146 (sCD146) is reported for the first time via in situ formation of carbon nitride quantum dots (CN QDs)/titanium dioxide (TiO2 ) nanodiscs with the double-supported 3D DNA walking amplification. In this platform, metal organic frameworks (MOFs)-derived porous TiO2 nanodiscs exhibit excellent anodic photocurrent, whereas a single-stranded auxiliary DNA (ssDNA) as biogate is absorbed onto the TiO2 nanodiscs to block active sites. Subsequently, with the help of intermediate DNAs from target sCD146-induced double-supported 3D DNA walking signal amplification, the ssDNA can leave away from the surface of TiO2 nanodiscs due to the specific hybridization with intermediate DNAs. Afterward, the successful direct contact of CN QDs on TiO2 nanodiscs by porosity and electrostatic adsorption, leads to the effective photocurrent-direction switching from anodic to cathodic photocurrent. Based on direct-contact photocurrent-direction-switching CN QDs/TiO2 nanodiscs system and double-supported 3D DNA walking signal amplification, sCD146 is detected sensitively with a wide linear range (10 fg mL-1 to 5 ng mL-1 ) and a low limit of detection (2.1 fg mL-1 ). Also, the environmentally friendly and direct-contact photocurrent-direction-switching PEC biosensor has an application prospect for cancer biomarker detection.

Fluorescent Sensor Based on Magnetic Separation and Strand Displacement Amplification for the Sensitive Detection of Ochratoxin A.

Ochratoxin A (OTA) is a common mycotoxin, and it is a significant threat to human health throughout the food chain. In this study, a sensitive and specific fluorescent sensor based on magnetic separation technology combined with chain displacement amplification was developed for fast and easy detection of OTA in food. The designed strand displacement amplification can improve the sensitivity for the detection, and the magnetic nanomaterials can provide a large surface area, thus enhancing the capture efficiency of the target from the sample. Based on those designs, the experimental results showed that the proposed method displayed excellent performance. The linearity range was 0.5-128.0 ng/mL. The detection limit was 0.125 ng/mL; the relative standard deviations were 3.92-7.71%. Additionally, the developed method was satisfactorily applied to determine OTA in wheat, corn, and red wine samples at three spiked levels (1.0, 8.0, and 64.0 ng/mL). The recoveries ranged from 85.45 to 107.8% for wheat flour, 101.34 to 108.35% for corn flour, and 91.15 to 93.80% for red wine, respectively. Compared with high-performance liquid chromatography, the proposed method showed a lower limit of detection and equal recovery. Hence, the designed method is a potential and good detecting tool for OTA residue analysis in complex matrix samples.

HUNK inhibits epithelial-mesenchymal transition of CRC via direct phosphorylation of GEF-H1 and activating RhoA/LIMK-1/CFL-1.

Epithelial-mesenchymal transition (EMT) is associated with the invasive and metastatic phenotypes in colorectal cancer (CRC). However, the mechanisms underlying EMT in CRC are not completely understood. In this study, we find that HUNK inhibits EMT and metastasis of CRC cells via its substrate GEF-H1 in a kinase-dependent manner. Mechanistically, HUNK directly phosphorylates GEF-H1 at serine 645 (S645) site, which activates RhoA and consequently leads to a cascade of phosphorylation of LIMK-1/CFL-1, thereby stabilizing F-actin and inhibiting EMT. Clinically, the levels of both HUNK expression and phosphorylation S645 of GEH-H1 are not only downregulated in CRC tissues with metastasis compared with that without metastasis, but also positively correlated among these tissues. Our findings highlight the importance of HUNK kinase direct phosphorylation of GEF-H1 in regulation of EMT and metastasis of CRC.

A SERS bioassay based on vancomycin-modified PEI-interlayered nanocomposite and aptamer-functionalized SERS tags for synchronous detection of Acinetobacter baumannii and Klebsiella pneumoniae.

Klebsiella pneumoniae (KP) and Acinetobacter baumannii (AB) are two important gram-negative bacteria that cause pneumonia and have been recently known to be associated with food. The rapid detection of these pathogens in food is important to minimize their colonization of the gut and stop new threats of the disease from spreading across the food chain. Herein, a double-edged sword aptasensor was developed for the synchronous detection of KP and AB in food and clinical samples. A highly sensitive, selective, specific, and synchronous detection of the target bacteria was achieved, and the limit of detection (LOD) was 10 cells/mL with a liner range of 50 to 105 cells/mL. The total assay time was 1.5 h. This study does not only provide a new tool for the detection of the target bacteria, but also serves as a promising tool for food safety and pneumonia diagnosis.

Activatable Fluorescence-Encoded Nanoprobes Enable Simple Multiplexed RNA Imaging in Live Cells.

Benefiting from superior programmable performance and flexible design of DNA technologies, a variety of single-molecule RNA fluorescence imaging methodologies have been reported. However, the multiplexing capability is restricted owing to the spectral overlap of fluorophores. To overcome this limitation, some inspiring multiplex imaging strategies have been developed, but in practice, it remains challenging to achieve convenient and rapid imaging in live cells due to complex designs and additional pretreatments to increase cell permeability. Here, we report an activatable fluorescence-encoded nanoprobe (AFENP) strategy, through which fluorescence-encoded functional modules for qualitative analysis and activated nucleic acid assemblies functional modules for quantitative testing enable simple multiplexed RNA imaging in single live cells. As a proof of principle, by two distinguishable fluorophores (fluorescein and rhodamine B) and their seven distinctly differentiated intensity levels, self-assembled AFENP enables simplified and quick simultaneous in situ detection and imaging of seven types of targets in live single cells because the fluorescent quantitative signal is activated only in the presence of target avoiding the washing procedures and additional pretreatment to increase cell permeability is undesired. We expect that this practical single-cell analysis platform will be adopted for multiple gene expression analysis and imaging in live cells on account of its simplicity and multiplex capability.

Relationships between sarcopenia, nutrient intake, and gut microbiota in Chinese community-dwelling older women.

BACKGROUND: The relationship among gut microbiota, sarcopenia components, and influencing factors in female sarcopenic patients has been poorly investigated. METHODS: Female participants completed questionnaires of physical activity and dietary frequency and were assessed for the presence of sarcopenia by the Asian Working Group of Sarcopenia 2019 (AWGS 2019) criteria. Fecal samples were collected from 17 sarcopenia and 30 non-sarcopenia subjects for 16S sequencing and short chain fatty acid (SCFA) detection. RESULTS: The prevalence of sarcopenia was 19.20% among 276 participants. The dietary protein, fat, dietary fiber, vitamin B1, niacin, vitamin E, phosphorus, magnesium, iron, zinc, and cooper intake of sarcopenia were all remarkably low. In addition, the richness of gut microbiota (Chao1 and ACE indexes) was considerably reduced in sarcopenic patients, and the sarcopenic gut microbiota and its metabolite were decreased in Firmicutes/Bacteroidetes, Agathobacter, Dorea and Butyrate and were enriched in Shigella and Bacteroides. Correlation analysis showed that Agathobacter and Acetate were positively correlated with grip strength and gait speed, respectively, and Bifidobacterium was negatively correlated with grip strength and appendicular skeletal muscle index (ASMI). Moreover, the protein intake was positively related to Bifidobacterium. CONCLUSIONS: This cross-sectional study revealed the alterations of gut microbiota composition, SCFA, and nutrient intake in women with sarcopenia and their relation to sarcopenic components. These results provide insights into further studies on the role of nutrition and gut microbiota in sarcopenia and its use as a therapeutic approach.

MiR-3180 inhibits hepatocellular carcinoma growth and metastasis by targeting lipid synthesis and uptake.

PURPOSE: Reprogrammed lipid metabolism is a hallmark of cancer that provides energy, materials, and signaling molecules for rapid cancer cell growth. Cancer cells acquire fatty acids primarily through de novo synthesis and uptake. Targeting altered lipid metabolic pathways is a promising anticancer strategy. However, their regulators have not been fully investigated, especially those targeting both synthesis and uptake. METHODS: Immunohistochemistry was performed on samples from patients with hepatocellular carcinoma (HCC) to establish the correlation between miR-3180, stearoyl-CoA desaturase-1 (SCD1), and CD36 expression, quantified via qRT-PCR and western blotting. The correlation was analyzed using a luciferase reporter assay. Cell proliferation, migration, and invasion were analyzed using CCK-8, wound healing, and transwell assays, respectively. Oil Red O staining and flow cytometry were used to detect lipids. Triglycerides and cholesterol levels were analyzed using a reagent test kit. CY3-labeled oleic acid transport was analyzed using an oleic acid transport assay. Tumor growth and metastasis were detected in vivo in a xenograft mouse model. RESULTS: MiR-3180 suppressed de novo fatty acid synthesis and uptake by targeting the key lipid synthesis enzyme SCD1 and key lipid transporter CD36. MiR-3180 suppressed HCC cell proliferation, migration, and invasion in an SCD1- and CD36-dependent manner in vitro. The mouse model demonstrated that miR-3180 inhibits HCC tumor growth and metastasis by inhibiting SCD1- and CD36-mediated de novo fatty acid synthesis and uptake. MiR-3180 expression was downregulated in HCC tissues and negatively correlated with SCD1 and CD36 levels. Patients with high miR-3180 levels showed better prognosis than those with low levels. CONCLUSIONS: Our investigation indicates that miR-3180 is a critical regulator involved in de novo fatty acid synthesis and uptake, which inhibits HCC tumor growth and metastasis by suppressing SCD1 and CD36. Therefore, miR-3180 is a novel therapeutic target and prognostic indicator for patients with HCC.

Magnetic-nanowaxberry-based microfluidic ExoSIC for affinity and continuous separation of circulating exosomes towards cancer diagnosis.

Exosomes are seen as promising biomarkers for minimally invasive liquid biopsies and disease surveillance. However, the complexity of body fluids, inherent heterogeneity, and tiny size of exosomes impede their extraction, consequently restricting their clinical application. In this study, in order to efficiently isolate exosomes from clinical samples, an irregular serpentine channel microfluidic chip (ExoSIC) was designed to continuously separate exosomes from plasma based on a magnetic-nanowaxberry (MNWB). In the ExoSIC, irregular serpentine microchannels are utilized to increase fluid chaotic mixing, hence improving exosome capture efficiency. In comparison to commonly used spherical magnetic particles, the designed MNWB can not only enhance the capture efficiency of exosomes, but also possess a size-exclusion effect to improve exosome purity. Consequently, the ExoSIC exhibited a large yield (24 times higher than differential centrifugation), optimum purity (greater than precipitation and similar to differential centrifugation), and high specificity. Furthermore, the ExoSIC was utilized for plasma-based cancer diagnosis by multiplex monitoring of five exosomal biomarkers (exosomal concentration, EGFR, EpCAM, SAA1 and FV), and the AUC reached 0.791. This work provides a comprehensive framework for exosome-based cancer diagnostics in order to meet clinical requirements for exosome isolation and downstream analysis.

A review of sensors for classification and subtype discrimination of cancer: Insights into circulating tumor cells and tumor-derived extracellular vesicles.

Liquid biopsy can reflect the state of tumors in vivo non-invasively, thus providing a strong basis for the early diagnosis, individualized treatment monitoring and prognosis of tumors. Circulating tumor cells (CTCs) and tumor-derived extracellular vesicles (tdEVs) contain information-rich components, such as nucleic acids and proteins, and they are essential markers for liquid biopsies. Their capture and analysis are of great importance for the study of disease occurrence and development and, consequently, have been the subject of many reviews. However, both CTCs and tdEVs carry the biological characteristics of their original tissue, and few reviews have focused on their function in the staging and classification of cancer. In this review, we focus on state-of-the-art sensors based on the simultaneous detection of multiple biomarkers within CTCs and tdEVs, with clinical applications centered on cancer classification and subtyping. We also provide a thorough discussion of the current challenges and prospects for novel sensors with the ultimate goal of cancer classification and staging. It is hoped that these most advanced technologies will bring new insights into the clinical practice of cancer screening and diagnosis.

Universal DNAzyme walkers-triggered CRISPR-Cas12a/Cas13a bioassay for the synchronous detection of two exosomal proteins and its application in intelligent diagnosis of cancer.

Exosomal proteins are considered to be promising indicators of cancer. Herein, a novel DNAzyme walkers-triggered CRISPR-Cas12a/Cas13a strategy was proposed for the synchronous determination of exosomal proteins: serum amyloid A-1 protein (SAA1) and coagulation factor V (FV). In this design, the paired antibodies were used to recognize targets, thereby ensuring the specificity. DNAzyme walkers were employed to convert the contents of SAA1 and FV into activators (P1 and P2), and one target can produce abundant activators, thus achieving an initial amplification of signal. Furthermore, the P1 and P2 can activate CRISPR-Cas12a/Cas13a system, which in turn trans-cleaves the reporters, enabling a second amplification and generating two fluorescent signals. The assay is highly sensitive (limits of detection as low as 30.00 pg/mL for SAA1 and 200.00 pg/mL for FV), highly specific and ideally accurate. More importantly, it is universal and can be used to detect both non-membrane and membrane proteins in exosome. Besides, the method can be successfully applied to detect SAA1 and FV in plasma exosomes to differentiate between lung cancer patients and healthy individuals. To explore the application of the developed method in tumor diagnosis, a deep learning model based on the expressions of SAA1 and FV was developed. The accuracy of this model can achieve 86.96%, which proves that it has a promising practical application capacity. Thus, this study does not only provide a new tool for the detection of exosomal proteins and cancer diagnosis, but also propose a new strategy to detect non-nucleic acid analytes for CRISPR-Cas system.

Correction to "Fluorescent Sensor Based on Magnetic Separation and Strand Displacement Amplification for the Sensitive Detection of Ochratoxin A".

[This corrects the article DOI: 10.1021/acsomega.3c01408.].

Accurate classification of lung nodules on CT images using the TransUnet.

Background: Computed tomography (CT) is an effective way to scan for lung cancer. The classification of lung nodules in CT screening is completely doctor dependent, which has drawbacks, including difficulty classifying tiny nodules, subjectivity, and high false-positive rates. In recent years, deep convolutional neural networks, a deep learning technology, have been shown to be effective in medical imaging diagnosis. Herein, we propose a deep convolutional neural network technique (TransUnet) to automatically classify lung nodules accurately. Methods: TransUnet consists of three parts: the transformer, the Unet, and global average pooling (GAP). The transformer encodes discriminative features via global self-attention modeling on CT image patches. The Unet, which collects context by constricting route, enables exact lunge nodule localization. The GAP categorizes CT images, assigning each sample a score. Python was employed to pre-process all CT images in the LIDI-IDRI, and the obtained 8,474 images (3,259 benign and 5,215 lung nodules) were used to evaluate the method's performance. Results: The accuracies of TransUnet in the training and testing sets were 87.90 and 84.62%. The sensitivity, specificity, and AUC of the proposed TransUnet on the testing dataset were 70.92, 93.17, and 0.862%, respectively (0.844-0.879). We also compared TransUnet to three well-known methods, which outperformed these methods. Conclusion: The experimental results on LIDI-IDRI demonstrated that the proposed TransUnet has a great performance in classifying lung nodules and has a great potential application in diagnosing lung cancer.

Transcriptional Repression of Aerobic Glycolysis by OVOL2 in Breast Cancer.

Aerobic glycolysis (Warburg effect), a hallmark of cancer, plays a critical role in cancer cell growth and metastasis; however, direct inhibition of the Warburg effect remains largely unknown. Herein, the transcription factor OVO-like zinc finger 2 (OVOL2) is demonstrated to directly repress the expression of several glycolytic genes, blocking the Warburg effect and breast tumor growth and metastasis in vitro and in vivo. OVOL2 inhibits glycolysis by recruiting the nuclear receptor co-repressor (NCoR) and histone deacetylase 3 (HDAC3). The tumor suppressor p53, a key regulator of cancer metabolism, activates OVOL2 by binding to the oncoprotein mouse double minute 2 homolog (MDM2) and inhibiting MDM2-mediated ubiquitination and degradation of OVOL2. OVOL2 expression is negatively correlated with glycolytic gene expression and can be a good predictor of prognosis in patients with breast cancer. Therefore, targeting the p53/MDM2/OVOL2 axis provides a potential avenue for cancer treatment, especially breast cancer.