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.

Extracellular Vesicles, as Drug-Delivery Vehicles, Improve the Biological Activities of Astaxanthin.

Astaxanthin (AST) exhibits potent antioxidant and anti-inflammatory activities but poor stability and biological efficacy, which limit its application in the food and medical industries. In the present study, a new strategy was proposed to enhance the biological activities of AST using fetal bovine serum-derived extracellular vesicles (EVs). Saponin-assisted incubation was used to load AST owing to its high encapsulation efficiency and loading capacity. AST-incorporated EVs (EV-ASTs) maintained their original EV morphology and showed high stability at 4 °C, 25 °C, and 37 °C over a 28-day period, which was attributed to the protective environment provided by the phospholipid bilayer membrane of the EVs. Additionally, the EV-ASTs exhibited excellent antioxidant and anti-inflammatory activities in HaCaT keratinocytes and RAW 264.7 macrophage cells, respectively; these were significantly higher than those of free AST. Furthermore, the mechanism associated with the enhanced biological activities of EV-ASTs was evaluated by analyzing the expression of genes involved in antioxidation and anti-inflammation, in parallel with cellular in vitro assays. These results provide insights into methods for improving the performance of hydrophobic drugs using nature-derived EVs and will contribute to the development of novel drug-delivery systems.

Loop-mediated isothermal amplification-based nucleic acid lateral flow assay for the specific and multiplex detection of genetic markers.

In this study, a loop-mediated isothermal amplification-based nucleic acid lateral flow assay (LAMP-NALFA) system was developed for the specific and multiplex detection of genetic markers at a low cost. In principle, the LAMP reaction was optimized to generate a single-stranded sequence in the LAMP product, which was designed to serve as a barcode sequence and to specifically bind to the DNA capture on a NALFA strip. As a target genetic marker, the Salmonella enterotoxin (stn) gene was chosen and determined down to 9 aM (∼5.44 copies/µL). Importantly, the proposed system clearly discriminated the specific target amplification products from non-specific amplification products resulting from primers or non-target nucleic acids, proving the high selectivity of the LAMP-NALFA system. Furthermore, the practical applicability of the system was demonstrated by detecting Salmonella bacteria in Luria-Bertani medium, drinking water, and eggshells, with a limit of detection of 1.6 CFU. Finally, two different bacteria (Salmonella and Staphylococcus) were simultaneously determined by the multiplex LAMP-NALFA system. It is expected that the LAMP-NALFA system could be used in a point-of-care setting for the detection of bacteria or viruses, consequently improving both individual and public health.

Split T7 promoter-based isothermal transcription amplification for one-step fluorescence detection of SARS-CoV-2 and emerging variants.

The negative global impact of the coronavirus disease pandemic has highlighted the crucial need for a rapid and convenient method of viral RNA detection. In this study, we report a novel method, termed as the split T7 promoter-based isothermal transcription amplification with light-up RNA aptamer (STAR), for one-pot detection of viral RNA. STAR uses a split T7 promoter that is applied to a three-way junction to mediate the selective transcription by the T7 RNA polymerase in the presence of target RNA. In addition, a light-up RNA aptamer is used for signal amplification. STAR can detect viral RNA in less than 30 min with high specificity and sensitivity. By testing of 60 nasopharyngeal SARS-CoV-2 samples, the STAR assay demonstrates an excellent sensitivity and specificity of 96.7% and 100%, respectively. Moreover, we provide experimental evidence of the broad applicability of this assay through the multiplex detection of SARS-CoV-2 variants (D614G mutation) and direct detection of bacterial 16S rRNA.

Surveillance of Shiga toxin-producing Escherichia coli and Campylobacter spp. in wild Japanese deer (Cervus nippon) and boar (Sus scrofa).

Increasing game meat consumption in Japan requires the dissemination of safety information regarding the presence of human pathogens in game animals. Health information regarding the suitability of these animals as a meat source is not widely available. In this study, we aimed to evaluate the safety of game meat and detect potential human pathogens in wild deer (Cervus nippon) and boar (Sus scrofa) in Japan. Fecal samples from 305 wild deer and 248 boars of Yamaguchi, Kagoshima, and Tochigi prefectures collected monthly for 2 years were examined for the prevalence of Shiga toxin-producing Escherichia coli (STEC) and Campylobacter spp. STEC was isolated from 51 deer consistently throughout the year and from three boars; O-antigen genotype O146, the expression of stx2b, and eaeA absence (n=33) were the major characteristics of our STEC isolates. Other serotypes included the medically important O157, stx2b or stx2c, and eaeA-positive (n=4) and O26, stx1a, and eaeA-positive strains (n=1). Campylobacter spp. were isolated from 17 deer and 31 boars. Campylobacter hyointestinalis was the most common species isolated from 17 deer and 25 boars, whereas Campylobacter lanienae and Campylobacter coli were isolated from three and two boars, respectively. Seasonal trends for the isolation of these bacteria were not significant. This study demonstrates that wild game animals carry human pathogens; therefore, detailed knowledge of the safe handling of game meat is needed to prevent foodborne infections.

Increased Salmonella Schwarzengrund prevalence and antimicrobial susceptibility of Salmonella enterica isolated from broiler chickens in Kagoshima Prefecture in Japan between 2013 and 2016.

This study aimed to analyze the Salmonella serovars, measure the minimum inhibitory concentration of antimicrobials, and examine the antimicrobial resistance genes of Salmonella isolated from 192 broiler flocks in Kagoshima Prefecture in Japan, from 2013 to 2016. We found that all Salmonella isolates belonged to three serovars: Salmonella Manhattan, S. Infantis, and S. Schwarzengrund. Among them, S. Schwarzengrund prevalence has recently increased annually making the main serovar. Most recovered isolates were highly resistant to streptomycin, sulfamethoxazole, and oxytetracycline. We saw the reduction of third-generation cephalosporin resistance and identified the reason of increased kanamycin resistance to be the increased number of S. Schwazengrund isolates. Among the kanamycin-resistant Salmonella isolates, aphA1 constituted the main resistance gene detected.

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.

Fluorescence, turn-on detection of melamine based on its dual functions as fluorescence enhancer of DNA-AgNCs and Hg(II)-scavenger.

A new method has been developed for the simple, fluorescence, turn-on detection of melamine, which utilizes DNA-templated silver nanoclusters (DNA-AgNCs) as a key component. In the sensor, melamine exhibits the dual functions: one is to enhance the fluorescence signal of DNA-AgNCs by its specific interaction with thymine residues in DNA template, and the other is to prevent Hg(II)-induced fluorescence quenching of DNA-AgNCs via its strong coordination with Hg(II). These consequently enable the sensitive and selective detection of melamine. By exploiting such novel features of melamine, we significantly increased the fluorescence response up to 360%, compared to the previous counterpart that relies on DNA-AgNCs only, and successfully determined melamine down to ca. 49 nM, a value that is 400 times lower than the safety level of 20 µM set by the US Food and Drug Administration. In addition, it was confirmed that the proposed approach works fine even in the real milk samples without any additional pre-treatment steps.

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.