Tetrahedral DNA nanostructures enhance transcription isothermal amplification for multiplex detection of non-coding RNAs.

This study introduces an innovative detection system for multiple cancer biomarkers, employing transcription isothermal amplification methods in conjunction with a tetrahedral DNA nanostructure (TDN). We demonstrate that TDN enhances various transcription isothermal amplification methods by placing DNA probes in proximity. Notably, the TDN-enhanced split T7 promoter-based isothermal transcription amplification with light-up RNA aptamer (STAR) system stands out for its optimal performance and operational simplicity, especially in identifying non-coding RNAs such as microRNAs and long non-coding RNAs (lncRNAs). Multiplex detection of lncRNAs was also achieved by generating distinct light-up RNA aptamers, each emitting unique fluorescence signals. The system effectively identified the target lncRNAs, demonstrating high sensitivity and selectivity in both cell lines and clinical samples. The system, utilizing the single enzyme T7 RNA polymerase, can be easily tailored for alternative targets by substituting target-specific sequences in DNA probes and seamlessly integrated with other isothermal amplification methods for greater sensitivity and accuracy in the detection of multiple cancer biomarkers.

Development of a Novel DNA Aptamer Targeting Colorectal Cancer Cell-Derived Small Extracellular Vesicles as a Potential Diagnostic and Therapeutic Agent.

Colorectal cancer (CRC) as the second leading cause of global cancer deaths poses critical challenges in clinical settings. Cancer-derived small extracellular vesicles (sEVs), which are secreted by cancer cells, have been shown to mediate tumor development, invasion, and even metastasis, and have thus received increasing attention for the development of cancer diagnostic or therapeutic platforms. In the present study, the sEV-targeted systematic evolution of ligands by exponential enrichment (E-SELEX) is developed to generate a high-quality aptamer (CCE-10F) that recognizes and binds to CRC-derived sEVs. Via an in-depth investigation, it is confirmed that this novel aptamer possesses high affinity (Kd = 3.41 nm) for CRC-derived sEVs and exhibits a wide linear range (2.0 × 104 -1.0 × 106 particles µL-1 ) with a limit of detection (LOD) of 1.0 × 103 particles µL-1 . Furthermore, the aptamer discriminates CRC cell-derived sEVs from those derived from normal colon cell, human serum, and other cancer cells, showing high specificity for CRC cell-derived sEVs and significantly suppresses the critical processes of metastasis, including cellular migration, invasion, and angiogenesis, which are originally induced by sEVs themselves. These findings are highly encouraging for the potential use of the aptamer in sEV-based diagnostic and therapeutic applications.

Cas12a/blocker DNA-based multiplex nucleic acid detection system for diagnosis of high-risk human papillomavirus infection.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) proteins are an innovative tool in molecular diagnostics owing to their high specificity and modularity for target nucleic acid sequences. However, the sequence-indiscriminate trans-cleavage activity of the Cas protein renders multiplex detection challenging. In this study, we developed a Cas12a-based multiplex detection system by designing blocker DNA complementary to reporter DNA, which enables the simultaneous detection of two genes with a single Cas protein in a single reaction. As a proof of concept, we chose high-risk human papillomavirus (HPV) 16 and 18 as the model targets and incorporated recombinase polymerase amplification (RPA) and transcription reactions to achieve high accuracy and sensitivity. Using the proposed system, we detected the genes of both HPV 16 and 18 down to 1 aM within 80 min under isothermal conditions. We validated the performance of the system in detecting genomic DNA from various cell lines and clinical samples from cervical cancer patients with high specificity. The proposed system facilitated rapid multiplex detection of high-risk HPVs in a single reaction tube with only Cas12a, thus representing a more user-friendly and economical alternative to previous Cas protein-based multiplex detection assays. The proposed system has considerable potential for point-of-care testing and could be expanded to detect various nucleic acid biomarkers.

Organotypic tumor slice cultures provide a versatile platform for immuno-oncology and drug discovery.

Organotypic tumor slices represent a physiologically-relevant culture system for studying the tumor microenvironment. Systematic characterization of the tumor slice culture system will enable its effective application for translational research. Here, using flow cytometry-based immunophenotyping, we performed a comprehensive characterization of the immune cell composition in organotypic tumor slices prepared from four syngeneic mouse tumor models and a human liver tumor. We found that the immune cell compositions of organotypic tumor slices prepared on the same day as the tumor cores were harvested are similar. Differences were primarily observed in the lymphocyte population of a clinical hepatocellular carcinoma case. Viable populations of immune cells persisted in the tumor slices for 7 days. Despite some changes in the immune cell populations, we showed the utility of mouse tumor slices for assessing responses to immune-modulatory agents. Further, we demonstrated the ability to use patient-derived xenograft tumor slices for assessing responses to targeted and cytotoxic drugs. Overall, tumor slices provide a broadly useful platform for studying the tumor microenvironment and evaluating the preclinical efficacy of cancer therapeutics.

Associations between dietary patterns and hypertension among Korean adults: the Korean National Health and Nutrition Examination Survey (2008-2010).

The objective of this study is to identify the dietary patterns associated with the risk of hypertensions among Korean adults using data from the Korean National Health and Nutrition Examination Survey (KNHANES, 2008-2010). This study analyzes data from 11,883 subjects who participated in the health and nutrition survey, aging from 20 to 64 years. We performed factor analysis based on the weekly mean intake frequencies of 36 food groups to identify major dietary patterns. We identified three major dietary patterns in both sexes, namely "traditional", "western" and "dairy and carbohydrate" patterns. Participants in the highest quartile of western pattern scores had significantly higher blood pressure, serum total cholesterol, and triglyceride levels than those in the lowest quartile. Although not statistically significant, a trend (P for trend = 0.0732) toward a positive association between the western dietary pattern and hypertension risk was observed after adjustments for age, sex, education, income, body mass index (BMI), smoking, physical activity, and energy intake. The dairy and carbohydrate pattern was inversely related with BMI and blood pressures and positively associated with serum high-density lipoprotein (HDL)-cholesterol. After adjusting the age, sex, education, income, BMI, smoking, physical activity and energy intake, the dairy and carbohydrate pattern showed inverse associations with hypertension prevalence (OR = 0.64, 95% CI = 0.55-0.75; P for trend < 0.0001). Intakes of fiber, sodium, and antioxidant vitamins were significantly higher in the top quartile for the traditional pattern than in the lowest quartile for the traditional pattern (P for trend < 0.0001). Intakes of fiber (P for trend < 0.0001), calcium (P for trend < 0.0001), retinol (P for trend = 0.0164), vitamin B1 (P for trend = 0.001), vitamin B2 (P for trend < 0.0001), niacin (P for trend = 0.0025), and vitamin C (P for trend < 0.0001) were significantly increased across quartiles for the dairy and carbohydrate pattern whereas sodium (P for trend < 0.0001) intake was decreased for this pattern. In conclusion, the dairy and carbohydrate pattern may be associated with a reduced risk of hypertension whereas the western pattern may be associated with an increased risk of hypertension among Korean adults.