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Abstract Cas12j, a hypercompact and efficient Cas protein, has potential for use in CRISPR diagnostics, but has not yet been used because the trans‐cleavage activity of Cas12j is veiled. Here, the trans‐cleavage behavior of Cas12j1, 2, and 3 variants and evaluate their suitability for nucleic acid detection is unveiled. The target preferences and mismatch specificities of the Cas12j variants are precisely investigated and the optimal Cas12j reaction conditions are determined. As a result, the EXP‐J assay for miRNA detection by harnessing the robust trans‐cleavage activity of Cas12j on short ssDNA is developed. The EXP‐J method demonstrates exceptional detection capabilities for miRNAs, proving that Cas12j can be a pivotal component in molecular diagnostics. Furthermore, the translational potential of the EXP‐J assay is validated by detecting oncogenic miRNAs in plasma samples from lung cancer patients. This investigation not only elucidates the trans‐cleavage characteristics of Cas12j variants, but also advances the Cas12j‐based diagnostic toolkit.

Abstract Gastric cancer (GC) is recognized as the fifth most prevalent malignant tumor worldwide. It is characterized by diverse clinical symptoms, treatment responses, and prognoses. In GC prognosis, the promotion of epithelial–mesenchymal transition (EMT) fosters cancer cell invasion and metastasis, thereby triggering the dissemination of tumor cells. This study proposes a nucleic acid amplification circuit‐based hydrogel (NACH) assay for identifying exosomal miRNA derived from metastatic GC. The NACH assay employs the rolling circle amplification method and targets miRNA‐21, a tumor‐related oncogene, and miRNA‐99a, which promotes EMT. Specific amplification probes for each target are immobilized within the hydrogel, enabling a streamlined, one‐step amplification reaction. The NACH assay exhibits a detection limit of 1 fm for miRNA‐21 and miRNA‐99a, thereby enabling rapid and highly sensitive on‐site detection. Performance evaluation using exosomal miRNA extracted from cell culture media, mouse plasma, and human plasma revealed fluorescence intensity patterns similar to those obtained in qRT‐PCR. Furthermore, deploying a custom‐developed portable fluorometer for the NACH assay allows for diagnostic performance assessment and point‐of‐care testing using clinical samples from GC patients. These findings emphasize the potential of the NACH assay to be used as a robust tool for the genetic diagnosis of GC based on exosome detection.

Abstract Endonucleases have recently widely used in molecular diagnostics. Here, we report a strategy to exploit the properties of Argonaute (Ago) proteins for molecular diagnostics by introducing an artificial nucleic acid circuit with Ago protein (ANCA) method. The ANCA is designed to perform a continuous autocatalytic reaction through cross-catalytic cleavage of the Ago protein, enabling one-step, amplification-free, and isothermal DNA detection. Using the ANCA method, carbapenemase-producing Klebsiella pneumoniae (CPKP) are successfully detected without DNA extraction and amplification steps. In addition, we demonstrate the detection of carbapenem-resistant bacteria in human urine and blood samples using the method. We also demonstrate the direct identification of CPKP swabbed from surfaces using the ANCA method in conjunction with a three-dimensional nanopillar structure. Finally, the ANCA method is applied to detect CPKP in rectal swab specimens from infected patients, achieving sensitivity and specificity of 100% and 100%, respectively. The developed method can contribute to simple, rapid and accurate diagnosis of CPKP, which can help prevent nosocomial infections.

Abstract Although activin receptor IIB (ACVR2B) is emerging as a novel pathogenic receptor, its ligand and assembled components (or assembly) are totally unknown in the context of osteoarthritis (OA) pathogenesis. The present results suggest that upregulation of ACVR2B and its assembly could affect osteoarthritic cartilage destruction. It is shown that the ACVR2B ligand, activin A, regulates catabolic factor expression through ACVR2B in OA development. Activin A Tg mice (Col2a1‐Inhba) exhibit enhanced cartilage destruction, whereas heterozygous activin A KO mice (Inhba+/−) show protection from cartilage destruction. In silico analysis suggests that the Activin A‐ACVR2B axis is involved in Nox4‐dependent ROS production. Activin A Tg:Nox4 KO (Col2a1‐Inhba:Nox4−/−) mice show inhibition of experimental OA pathogenesis. NOX4 directly binds to the C‐terminal binding site on ACVR2B‐ACVR1B and amplifies the pathogenic signal for cartilage destruction through SMAD2/3 signaling. Together, the findings reveal that the ACVR2B assembly, which comprises Activin A, ACVR2B, ACVR1B, Nox4, and AP‐1‐induced HIF‐2α, accelerates OA development. Furthermore, it is shown that shRNA‐mediated ACVR2B knockdown or trapping ligands of ACVR2B abrogate OA development by competitively disrupting the ACVR2B‐Activin A interaction. These results suggest that the ACVR2B assembly is required to amplify osteoarthritic cartilage destruction and could be a potential therapeutic target in efforts to treat OA.

Maintenance of homoeostasis in human body is a very important indicator in all cell activities. When exposed to a disease, various immune cells are activated due to the inflammatory response, and particularly T cells play a role in inducing apoptosis of mutated cells such as tumor cells. When the activity of T cells is very low, infection by external invasion is easy, and on the contrary, excessive activation leads to chronic inflammation caused by autoimmune diseases. Many clinical studies related to pulsed magnetic field (PMF) demonstrated its efficacy in reducing pain, improving blood circulation, as well as blood’s acid-base balance. Therefore, our study has tried to investigate the influence of PMF on the regulation of acid-base homeostasis in EL4 T lymphoma cell. In addition, we have tried to explain the role of PMF on immune cell activity by measuring the level of pro-inflammatory cytokine, TNF-α in culture supernatants. EL4 cells were cultured in a DMEM medium supplemented with 10% FBS and 1% penicillin in an incubator at 37 °C and 5% CO2 condition. Our PMF stimulator has the maximum strength of 4700 G at a transition time of 222 μs with pulse intervals of 1 Hz. The homoeostasis in pH was improved as PMF strength increases. Cell viability decreased by 32% after PMF stimulation of 4700 G. It was observed that the concentration of TNF-α, a cytokine related to inflammation, also decreased as the strength of PMF increased. These results suggest that PMF stimulation improves the anti-inflammatory effect, therefore, it is thought to affect the immune system by balancing the activation and suppression of immune cells. For clinical use, our study might suggest non-invasive PMF can be developed as a medical devices modulating immune system, although it is necessary to optimize the PMF conditions such as pulse shape, duration, or repetition rate.

Rapid detection of antiviral resistant strains is important for effective clinical treatment. Here the authors develop an antibody which specifically binds to multidrug-resistant influenza virus and demonstrated detection in a number of different systems using the antibody.

Korean ginseng (Panax ginseng) and American ginseng (Panax quinquefolius) are widely used medicinal plants with similar morphology but different medicinal efficacy. Roots, flowers, and processed products of Korean and American ginseng can be difficult to differentiate from each other, leading to illegal trade in which one species is sold as the other. This study was carried out to develop convenient and reliable chloroplast genome-derived DNA markers for authentication of Korean and American ginseng in commercial processed products. One codominant marker could reproducibly identify both species and intentional mixtures of the two species. We further developed a set of species-unique dominant DNA markers. Each species-specific dominant marker could detect 1% cross contamination with other species by low resolution agarose gel electrophoresis or quantitative polymerase chain reaction. Both markers were successfully applied to evaluate the original species from various processed ginseng products purchased from markets in Korea and China. We believe that high-throughput application of this marker system will eradicate illegal trade and promote confident marketing for both species to increase the value of Korean as well as American ginseng in Korea and worldwide.

Wearable devices are becoming widespread in a wide range of applications, from healthcare to biomedical monitoring systems, which enable continuous measurement of critical biomarkers for medical diagnostics, physiological health monitoring and evaluation. Especially as the elderly population grows globally, various chronic and acute diseases become increasingly important, and the medical industry is changing dramatically due to the need for point-of-care (POC) diagnosis and real-time monitoring of long-term health conditions. Wearable devices have evolved gradually in the form of accessories, integrated clothing, body attachments and body inserts. Over the past few decades, the tremendous development of electronics, biocompatible materials and nanomaterials has resulted in the development of implantable devices that enable the diagnosis and prognosis through small sensors and biomedical devices, and greatly improve the quality and efficacy of medical services. This article summarizes the wearable devices that have been developed to date, and provides a review of their clinical applications. We will also discuss the technical barriers and challenges in the development of wearable devices, and discuss future prospects on wearable biosensors for prevention, personalized medicine and real-time health monitoring.

We designed a high-sensitivity magnetic resonance imaging contrast agent that could be used to diagnose diseases. First, magnetic nanocrystals were synthesized by a thermal decomposition method on an organic solvent to obtain a high magnetism and methoxy poly(ethylene glycol)-poly(lactic acid) as an amphiphilic polymer using the ring-opening polymerization method to stably disperse the magnetic nanocrystals in an aqueous phase. Subsequently, the magnetic nanoclusters simultaneously self-assembled with methoxy poly(ethylene glycol)-poly(lactic acid) using the nano-emulsion method to form magnetic nanoclusters. Because their shape was similar to a raspberry, they were named PEGylated magnetic nano-assemblies. The PEGylated magnetic nano-assemblies were dispersed stably in the aqueous phase with a uniform size of approximately 65–70 nm for an extended period (0 days: 68.8 ± 5.1 nm, 33 days: 69.2 ± 2.0 nm, and 44 days: 63.2 ± 5.6). They exhibited both enough of a magnetic resonance (MR) contrast effect and biocompatibility. In an in vivo study, the PEGylated magnetic nano-assemblies provided a high contrast effect for magnetic resonance images for a long time after one treatment, thereby improving the diagnostic visibility of the disease site.

We suggest a convenient nanoemulsion fabrication method to create hyaluronan (HA)-based nanohydrogels for effective transdermal delivery. First, hyaluronan-conjugated dodecylamine (HA–Do) HA-based polymers to load the lipophilic agents were synthesized with hyaluronan (HA) and dodecylamine (Do) by varying the substitution ratio of Do to HA. The synthetic yield of HA–Do was more than 80% (HA–Do (A): 82.7 ± 4.7%, HA–Do (B): 87.1 ± 3.9% and HA–Do (C): 81.4 ± 4.5%). Subsequently, nanohydrogels were fabricated using the nanoemulsion method. Indocyanine green (ICG) simultaneously self-assembled with HA–Do, and the size depended on the substitution ratio of Do in HA–Do (nanohydrogel (A): 118.0 ± 2.2 nm, nanohydrogel (B): 121.9 ± 11.4 nm, and nanohydrogel (C): 142.2 ± 3.8 nm). The nanohydrogels were delivered into cells, and had excellent biocompatibility. Especially, nanohydrogel (A) could deliver and permeate ICG into the deep skin layer, the dermis. This suggests that nanohydrogels can be potent transdermal delivery systems.

Embryonic stem cell (ESC)-based therapies open new possibilities as regenerative medicine for the treatment of human disease, but the presence of small numbers of undifferentiated ESCs within the transplant could lead to the development of tumors. The safety of ESC transplants would be enhanced if uncontrolled cell growth could be suppressed, using external stimuli. A lentiviral vector carrying the herpes simplex virus thymidine kinase (HSVtk) and green fluorescent protein (GFP) genes was used to genetically modify murine ESCs (HSVtkGFPESCs). In the presence of ganciclovir (GCV), 100 of HSVtkGFPESCs were killed in vitro, and 100 of flank tumors derived from HSVtkGFPESCs were eliminated. When CNS tumors were produced by the HSVtkGFPESCs, the tumor mass was completely eliminated on GCV treatment for 1 week. After GCV treatment for 3 weeks, histologic analysis showed no residual tumor cells and TaqMan realtime polymerase chain reaction analysis showed no genomic HSVtk copies or HSVtk mRNA. These data demonstrate that it is possible to use ex vivogene transfer to modify ESCs with conditional genetic elements that can be activated in vivoto control undifferentiated ESC outgrowth and to eliminate transduced ESCs that have escaped growth control after ESC-mediated therapy to the CNS. [ABSTRACT FROM AUTHOR]

For the electrical detection of target DNA (partial avian influenza virus/H1N1/HA sequence) prepared viaasymmetric PCR, we fabricated DNA-templated conducting gold nanowire bridges on planar nanogap electrodes using positively charged gold nanoparticles. [ABSTRACT FROM AUTHOR]

Based on their capacity for self-renewal and pluripotency, embryonic stem cells (ESC) are being developed as regenerative medicines, but transplantation protocols using differentiated ESC have been hindered by the fact that even small numbers of undifferentiated ESC within a transplant can lead to the development of teratomas. Within the limitations of current technology, it has not been possible to ensure that a population of differentiated ESC is entirely free of undifferentiated ESC. In this context, the safety profile of ESC transplants would be enhanced if uncontrolled cell growth could be suppressed using external stimuli. To address this issue, we hypothesized that gene transfer to the ESC of conditional elements that could kill the transduced cells if activated, would permit control of inappropriate outgrowth of ESC in vivo. To assess this strategy, we developed a replication-defective recombinant lentiviral gene transfer vector containing the herpes simplex virus thymidine kinase (HSVtk) gene (a suicide gene able to convert the nontoxic prodrug nucleotide analog ganciclovir (GCV) to a toxic form), an internal ribosomal entry site (IRES) and green fluorescent protein (GFP) driven by the cytomegalovirus immediate early promoter, and used this vector to genetically modify murine ESC (CGR8 strain). Fluorescence microscopy and FACS analysis demonstrated that 100% of these cells were killed in the presence of GCV (50 μM) in vitro. To assess the ability of the HSVtk suicide strategy to eliminate ESC outgrowth, cloned undifferentiated ESC (105 cells) carrying the HSVtk-IRES-GFP transgene were administered subcutaneously into SCID mice to induce teratomas. Teratoma formation was verified by the histological identification of primitive ectoderm, endoderm and mesoderm within the tumor mass. On day 14, when the tumor was 261±52 mm3 in size, intra- peritoneal GCV administration was initiated and continued for 4 wk (30 mg/kg, twice per day), while a control group received no GCV. Reduction of tumor mass in the group receiving GCV was evident by 2 wk and the tumors were completely eliminated by 4 wk (10/10 treated, 0/3 untreated). Residual fibrous, fat and cartilage tissue were observed at the site of the tumor in the GCV group, but histologic analysis showed no live tumor cells. Mice followed after cessation of GCV treatment did not show any recurrence of tumor growth, suggesting that the tumor-forming cells were killed and not simply arrested. The HSVtk gene is small (1.3 kb) and the use of an IRES avoids the need for a separate promoter, leaving sufficient space for most transgenes. These data demonstrate that this strategy can be used to deliver a therapeutic gene with additional conditional genetic elements that can be activated to control undifferentiated ESC outgrowth and transduced ESC that have escaped growth control due to insertional mutagenesis associated with the use of integrating vectors.Molecular Therapy (2006) 13, S172–S172; doi: 10.1016/j.ymthe.2006.08.516 [ABSTRACT FROM AUTHOR]