Unveiling Cas12j Trans-Cleavage Activity for CRISPR Diagnostics: Application to miRNA Detection in Lung Cancer Diagnosis.

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.

Systematic review and comparative analysis of endovascular and microsurgical management of giant ruptured fusiform mca aneurysms with illustrative cases.

BACKGROUND: Despite advances in neurosurgical techniques and technology, the management of ruptured giant fusiform MCA aneurysms remains challenging. In the literature, microsurgical intervention is the most commonly described approach. However, recent advancements in endovascular techniques have expanded therapeutic options and as a result there is no consensus on the optimal management of these aneurysms. METHODS: A literature search was performed through the PubMed, Google Scholar, and Embase databases, for surgical and endovascular management of ruptured giant fusiform MCA aneurysms. Inclusion criteria included: fusiform morphology, hemorrhage, major diameter greater than 2.5 cm and located along the MCA. RESULTS: Literature review yielded 21 studies published from 1981 to 2023 and a total of 32 patients ages 33.40 ± 18.28. The male to female ratio was 1.9:1. The average Hunt and Hess score upon presentation in the total population was 2.78 ± 1.48, and the average pre-operative mRS of the total population was 2.75 ± 1.83. The average major diameter was 3.80 ± 1.85 cm. Average follow-up was 8.9 ± 9.74 months. There was no statistical difference in age (p = 0.5609), pre-operative mRS (p = 0.2355), Hunt and Hess scale (p = 0.183), aneurysm major diameter (p = 0.594) or follow-up (0.8922) between the two modalities. There was no significant difference in clinical outcome between microsurgical and endovascular intervention, nor was there a significant difference when stratified according to sex, major diameter, or location along the MCA. Two case examples are presented after management with cerebral revascularization. CONCLUSION: Our analysis underscores the absence of statistical differences in clinical outcomes between microsurgical and endovascular strategies for ruptured giant fusiform MCA aneurysms, which highlights the need for complex surgical revascularization as represented on the illustrative cases where no endovascular option was available.

Influence of Multiple rISC Channels on the Maximum Efficiency and Roll-Off of TADF OLEDs.

In this work, we look into the detailed photophysical characterization of a multidonor-acceptor (D-A) family of thermally activated delayed fluorescent (TADF) emitters to find correlations with their device performance. Increasing the number of closely packed Ds around the A core leads to changes in dihedral angles between Ds and A, affecting the highest occupied molecular orbital (HOMO)/lowest unpccupied molecualar orbital (LUMO) separation and impacting the singlet-triplet energy gaps. Moreover, D-A dihedral angles change molecular conjugation affecting the spread of charge-transfer state energies as well as the energy of D local triplet states. The coupling between these triplet states and the dispersion in CT states lead to the appearance of multiple rISC channels, a phenomenon that is host-dependent, i.e., hosts with different rigidities twist the dihedral angles differently. We show that different subsets of rISC rates directly impact device performance, where faster rISC leads to external quantum efficiencies above 20% while slower rISC rates act as parasitic traps, severely affecting device roll-off. This explains why emitters with excellent peak external quantum efficiencies can also present very poor roll-off.

Apurinic/apyrimidinic endonuclease 1 alleviates inflammation in fibroblast-like synoviocytes from patients with rheumatoid arthritis.

Introduction: Apurinic/apyrimidinic endonuclease 1 (APEX1) is a protein with elevated expression in synovial fluids from rheumatoid arthritis (RA) patients. However, its role in RA pathogenesis remains unexplored. This study investigated the influence of APEX1 on inflammatory pathways in fibroblast-like synoviocytes (FLS) isolated from RA patients. Material and methods: FLS from RA patients (n = 5) were stimulated with recombinant tumor necrosis factor α (TNF-α) and interleukin (IL)-17. Subsequently, cells were treated with recombinant APEX1, and assessments were made on reactive oxygen species (ROS) production and mitochondrial membrane potential. Additionally, mRNA levels of IL-1 family members were quantified. Cell migration was evaluated through Transwell chamber assays, and levels of key secreted inflammatory cytokines were measured via enzyme-linked immunosorbent assay (ELISA). Results: The results demonstrated that APEX1 significantly reduced mitochondrial-specific ROS expression and restored mitochondrial membrane potential in TNF-α/IL-17-stimulated RA FLS. Furthermore, APEX1 treatments attenuated TNF-α/IL-17-induced activation of p38 MAPK, NF-κB, and PI3K 110 δ signaling pathways. Similarly, APEX1 significantly diminished TNF-α/IL-17-induced expression of inflammatory cytokines, including IL-1 family members, IL-6, IL-8, and vascular endothelial growth factor (VEGF). Notably, APEX1 downregulated cell migration of TNF-α/IL-17-treated RA FLS via inhibition of matrix metalloproteinase 3 (MMP3). Conclusions: These findings collectively underscore the role of APEX1 as a key mediator of cytokine-amplified migration, modulating ROS and MMP3 in RA FLS, thus supporting its potential as a therapeutic target in RA treatment.

Principles of paralog-specific targeted protein degradation engaging the C-degron E3 KLHDC2.

PROTAC® (proteolysis-targeting chimera) molecules induce proximity between an E3 ligase and protein-of-interest (POI) to target the POI for ubiquitin-mediated degradation. Cooperative E3-PROTAC-POI complexes have potential to achieve neo-substrate selectivity beyond that established by POI binding to the ligand alone. Here, we extend the collection of ubiquitin ligases employable for cooperative ternary complex formation to include the C-degron E3 KLHDC2. Ligands were identified that engage the C-degron binding site in KLHDC2, subjected to structure-based improvement, and linked to JQ1 for BET-family neo-substrate recruitment. Consideration of the exit vector emanating from the ligand engaged in KLHDC2's U-shaped degron-binding pocket enabled generation of SJ46421, which drives formation of a remarkably cooperative, paralog-selective ternary complex with BRD3BD2. Meanwhile, screening pro-drug variants enabled surmounting cell permeability limitations imposed by acidic moieties resembling the KLHDC2-binding C-degron. Selectivity for BRD3 compared to other BET-family members is further manifested in ubiquitylation in vitro, and prodrug version SJ46420-mediated degradation in cells. Selectivity is also achieved for the ubiquitin ligase, overcoming E3 auto-inhibition to engage KLHDC2, but not the related KLHDC1, KLHDC3, or KLHDC10 E3s. In sum, our study establishes neo-substrate-specific targeted protein degradation via KLHDC2, and provides a framework for developing selective PROTAC protein degraders employing C-degron E3 ligases.

Significance as a Prognostic Factor of Eosinophil Count in Nasal Polyp Tissue in Patients with Chronic Rhinosinusitis Accompanied by Asthma.

Background/Objectives: Patients with chronic rhinosinusitis (CRS) accompanied by asthma often show poor prognoses and require continuous management. This study aimed to assess the prognostic value of eosinophil counts in nasal polyp tissue for selecting individuals who would benefit from ongoing management in CRS patients with asthma. Methods: Patients with asthma who underwent endoscopic sinus surgery for CRS with nasal polyps were included in the study. Eosinophil counts in nasal polyp tissues were quantified, and retrospective data were collected from laboratory and clinical findings, including endoscopic examinations, CT scans, and Japan Endoscopic Sinus Surgery Rating and Evaluation Committee (JESREC) scores. Disease control status was evaluated through endoscopic examination 6 months post-surgery. Results: A total of 42 patients were divided into two groups based on their disease management status 6 months post-operation: the well-control group (24 patients, 57.14%) and the poor-control group (18 patients, 42.86%). Demographics and laboratory findings did not show significant differences between the groups. However, the JESREC score (p = 0.04) and tissue eosinophil count (p = 0.02) were significantly different. Multivariate analysis identified tissue eosinophil count as the only risk factor associated with prognosis, with a cut-off value of 90/HPF. Conclusions: In CRS patients with asthma, high tissue eosinophil counts in nasal polyps were associated with poor disease control, which is the most potent predictor of prognosis. The assessment of eosinophil counts in nasal polyp tissue could aid in identifying patients who would benefit from continuous management and tailored interventions for improved outcomes.

Modeling Behavior of Salmonella spp. and Listeria monocytogenes in Raw and Processed Vegetables.

Given the persistent occurrence of foodborne illnesses linked to both raw and processed vegetables, understanding microbial behavior in these foods under distribution conditions is crucial. This study aimed to develop predictive growth models for Salmonella spp. and Listeria monocytogenes in raw (mung bean sprouts, onion, and cabbage) and processed vegetables (shredded cabbage salad, cabbage and onion juices) at various temperatures, ranging from 4 to 36 °C. Growth models were constructed and validated using isolated strains of Salmonella spp. (S. Bareilly, S. Enteritidis, S. Typhimurium) and L. monocytogenes (serotypes 1/2a and 1/2b) from diverse food sources. The minimum growth temperatures for Salmonella varied among different vegetable matrices: 8 °C for mung bean sprouts, 9 °C for both onion and cabbage, and 10 °C for ready-to-eat (RTE) shredded cabbage salad. Both pathogens grew in cabbage juice at temperatures above 17 °C, while neither demonstrated growth in onion juice, even at 36 °C. Notably, Salmonella spp. exhibited faster growth than L. monocytogenes in all tested samples. At 8 °C, the lag time (LT) and specific growth rate (SGR) for Salmonella spp. in mung bean sprouts were approximately tenfold longer and threefold slower, respectively, compared to those at 10 °C. A decrease in refrigerator storage temperature by 1 or 2 degrees significantly prevented the growth of Salmonella in raw vegetables. These findings offer valuable insights into assessing the risk of foodborne illness associated with the consumption of raw and processed vegetables and inform management strategies in mitigating these risks.

Enhanced Minor Ginsenoside Contents of Nano-Sized Black Korean Ginseng through Hot Melt Extrusion.

Black ginseng (BG), a traditional medicinal herb produced through a nine-stage steaming and drying process, exhibits stronger pharmacological efficacy, including antioxidant, anti-inflammatory, and anti-cancer properties, when compared to white and red ginseng. The ginsenosides in BG are classified as major and minor types, with minor ginsenosides demonstrating superior pharmacological properties. However, their low concentrations limit their availability for research and clinical applications. In this study, hot melt extrusion (HME) was utilized as an additional processing technique to enhance the content of minor ginsenoside in BG, and the physicochemical properties of the formulation were analyzed. Ginsenoside content in BG and HME-treated BG (HME-BG) was analyzed using high-performance liquid chromatography (HPLC), while their physicochemical properties were evaluated through dynamic light scattering (DLS), electrophoretic light scattering (ELS), X-ray diffraction (XRD), thermogravimetric analysis (TGA), and Fourier-transform infrared spectroscopy (FT-IR). HME treatment resulted in a significant increase in minor ginsenosides Rg3 and compound K (CK) by 330% and 450%, respectively, while major ginsenosides Rg1 and Rb1 decreased or were not detected. Additionally, HME-BG demonstrated reduced particle size, improved PDI, and decreased crystallinity. HME treatment effectively converts major ginsenosides in BG into minor ginsenosides, enhancing its pharmacological efficacy and showing great potential for research and development applications.

Performance of ChatGPT-3.5 and GPT-4 in national licensing examinations for medicine, pharmacy, dentistry, and nursing: a systematic review and meta-analysis.

BACKGROUND: ChatGPT, a recently developed artificial intelligence (AI) chatbot, has demonstrated improved performance in examinations in the medical field. However, thus far, an overall evaluation of the potential of ChatGPT models (ChatGPT-3.5 and GPT-4) in a variety of national health licensing examinations is lacking. This study aimed to provide a comprehensive assessment of the ChatGPT models' performance in national licensing examinations for medical, pharmacy, dentistry, and nursing research through a meta-analysis. METHODS: Following the PRISMA protocol, full-text articles from MEDLINE/PubMed, EMBASE, ERIC, Cochrane Library, Web of Science, and key journals were reviewed from the time of ChatGPT's introduction to February 27, 2024. Studies were eligible if they evaluated the performance of a ChatGPT model (ChatGPT-3.5 or GPT-4); related to national licensing examinations in the fields of medicine, pharmacy, dentistry, or nursing; involved multiple-choice questions; and provided data that enabled the calculation of effect size. Two reviewers independently completed data extraction, coding, and quality assessment. The JBI Critical Appraisal Tools were used to assess the quality of the selected articles. Overall effect size and 95% confidence intervals [CIs] were calculated using a random-effects model. RESULTS: A total of 23 studies were considered for this review, which evaluated the accuracy of four types of national licensing examinations. The selected articles were in the fields of medicine (n = 17), pharmacy (n = 3), nursing (n = 2), and dentistry (n = 1). They reported varying accuracy levels, ranging from 36 to 77% for ChatGPT-3.5 and 64.4-100% for GPT-4. The overall effect size for the percentage of accuracy was 70.1% (95% CI, 65-74.8%), which was statistically significant (p < 0.001). Subgroup analyses revealed that GPT-4 demonstrated significantly higher accuracy in providing correct responses than its earlier version, ChatGPT-3.5. Additionally, in the context of health licensing examinations, the ChatGPT models exhibited greater proficiency in the following order: pharmacy, medicine, dentistry, and nursing. However, the lack of a broader set of questions, including open-ended and scenario-based questions, and significant heterogeneity were limitations of this meta-analysis. CONCLUSIONS: This study sheds light on the accuracy of ChatGPT models in four national health licensing examinations across various countries and provides a practical basis and theoretical support for future research. Further studies are needed to explore their utilization in medical and health education by including a broader and more diverse range of questions, along with more advanced versions of AI chatbots.

Thin, Uniform, and Highly Packed Multifunctional Structural Carbon Fiber Composite Battery Lamina Informed by Solid Polymer Electrolyte Cure Kinetics.

Multifunctional structural batteries promise advancements in structural energy storage technologies by seamlessly integrating load-bearing and energy-storage functions within a single material, reducing weight, and enhancing safety. Yet, commercialization faces challenges in materials processing, assembly, and design optimization. Here, we report a systematic approach to develop a carbon fiber (CF)-based structural battery impregnated with epoxy-based solid polymer electrolyte (SPE) via robust vacuum-assisted compression molding (VACM). Informed by cure kinetics, SPE processing enhances the multifunctional performance with no fillers or additives. The thin flexible CF-based laminae impregnated under high pressure achieved a substantial enhancement of ∼160% in the fiber volume fraction (FVF) as although thin and strip-shaped, the fibers were optimally packed with low void. A CF/SPE-based battery was fabricated, with a hybrid layered ionic liquid (IL)/ carbonate electrolyte (CE) showing enhanced safety and multifunctional performance. Enhanced by thin, uniform, and stiff CF-based composites, this study propels the development of advanced multifunctional structures, thereby expediting sustainable commercialization.

Next-Generation Frozen Elephant Trunk Technique in the Era of Precision Medicine.

The frozen elephant trunk (FET) technique can be applied to extensive aortic pathology, including lesions in the aortic arch and proximal descending thoracic aorta. FET is useful for tear-oriented surgery in dissections, managing malperfusion syndrome, and promoting positive aortic remodeling. Despite these benefits, complications such as distal stent-induced new entry and spinal cord ischemia can pose serious problems with the FET technique. To prevent these complications, careful sizing and planning of the FET are crucial. Additionally, since the FET technique involves total arch replacement, meticulous surgical skills are essential, particularly for young surgeons. In this article, we propose several techniques to simplify surgical procedures, which may lead to better outcomes for patients with extensive aortic pathology. In the era of precision medicine, the next-generation FET device could facilitate the treatment of complex aortic diseases through a patient-tailored approach.

Improved Efficiency in WSe2 Solar Cells Using Amorphous InOx Heterocontacts.

van der Waals (vdW) layered materials have been shown to have excellent optoelectronic properties relevant to photovoltaics. Despite their promise, the demonstrated efficiencies of vdW material solar cells remain low and are seldom supported by statistics or spectral quantum efficiency analysis. In this study, we utilize a p-type WSe2 absorber, forming a solar cell with a transparent front InOx electron contact, and a rear Pd reflector/hole contact. We fabricate multiple devices providing statistics for 10 devices with an average 1 sun conversion efficiency above 5%, among which a champion efficiency of 6.37% is achieved. This is the highest AM 1.5G 1 sun efficiency reported for a vdW material solar cell, with a current density supported by external quantum efficiency analysis. This cell is also shown to have near unity quantum efficiency around λ = 600 nm. This work provides support to vdW materials being considered as serious candidates for future thin-film solar cells.

The dopamine analogue CA140 alleviates AD pathology, neuroinflammation, and rescues synaptic/cognitive functions by modulating DRD1 signaling or directly binding to Abeta.

BACKGROUND: We recently reported that the dopamine (DA) analogue CA140 modulates neuroinflammatory responses in lipopolysaccharide-injected wild-type (WT) mice and in 3-month-old 5xFAD mice, a model of Alzheimer's disease (AD). However, the effects of CA140 on Aß/tau pathology and synaptic/cognitive function and its molecular mechanisms of action are unknown. METHODS: To investigate the effects of CA140 on cognitive and synaptic function and AD pathology, 3-month-old WT mice or 8-month-old (aged) 5xFAD mice were injected with vehicle (10% DMSO) or CA140 (30 mg/kg, i.p.) daily for 10, 14, or 17 days. Behavioral tests, ELISA, electrophysiology, RNA sequencing, real-time PCR, Golgi staining, immunofluorescence staining, and western blotting were conducted. RESULTS: In aged 5xFAD mice, a model of AD pathology, CA140 treatment significantly reduced Aß/tau fibrillation, Aß plaque number, tau hyperphosphorylation, and neuroinflammation by inhibiting NLRP3 activation. In addition, CA140 treatment downregulated the expression of cxcl10, a marker of AD-associated reactive astrocytes (RAs), and c1qa, a marker of the interaction of RAs with disease-associated microglia (DAMs) in 5xFAD mice. CA140 treatment also suppressed the mRNA levels of s100ß and cxcl10, markers of AD-associated RAs, in primary astrocytes from 5xFAD mice. In primary microglial cells from 5xFAD mice, CA140 treatment increased the mRNA levels of markers of homeostatic microglia (cx3cr1 and p2ry12) and decreased the mRNA levels of a marker of proliferative region-associated microglia (gpnmb) and a marker of lipid-droplet-accumulating microglia (cln3). Importantly, CA140 treatment rescued scopolamine (SCO)-mediated deficits in long-term memory, dendritic spine number, and LTP impairment. In aged 5xFAD mice, these effects of CA140 treatment on cognitive/synaptic function and AD pathology were regulated by dopamine D1 receptor (DRD1)/Elk1 signaling. In primary hippocampal neurons and WT mice, CA140 treatment promoted long-term memory and dendritic spine formation via effects on DRD1/CaMKIIα and/or ERK signaling. CONCLUSIONS: Our results indicate that CA140 improves neuronal/synaptic/cognitive function and ameliorates Aß/tau pathology and neuroinflammation by modulating DRD1 signaling in primary hippocampal neurons, primary astrocytes/microglia, WT mice, and aged 5xFAD mice.

Green Synthesis of Silver Nanoparticle Using Black Mulberry and Characterization, Phytochemical, and Bioactivity.

Synthesis of silver nanoparticles (AgNPs) using plant extracts has been proposed as a more advantageous and environmentally friendly alternative compared to existing physical/chemical methods. In this study, AgNPs were synthesized from silver nitrate using black mulberry (BM) extract. The biosynthesized AgNPs were characterized through an UV-visible spectrometer, X-ray diffraction, and transmission electron microscopy. Additionally, BM-AgNPs were subjected to antioxidant, antibacterial, anti-inflammatory, and anticancer activities. AgNPs biosynthesized from BM extract were dark brown in color and showed a strong peak at 437 nm, confirming that AgNPs were successfully synthesized. The size of AgNPs was 170.17 ± 12.65 nm, the polydispersity index was 0.281 ± 0.07, and the zeta potential value was -56.6 ± 0.56 mV, indicating that the particles were stable. The higher total phenol, flavonoid, and anthocyanin content of BM-AgNPs compared to BM extract indicates that the particles contain multiple active substances due to the formation of AgNPs. The DPPH and ABTS assays showed decreased IC50 values compared to BM extract, demonstrating improved antioxidant activity. AgNPs inhibited the growth of S. aureus and E. coli at 600 µg/mL, with minimum bactericidal concentrations determined to be 1000 and 1200 µg/mL, respectively. The anti-inflammatory activity was 64.28% at a BM-AgNPs concentration of 250 µg/mL. As the concentration increased, the difference from the standard decreased, indicating the inhibitory effect of AgNPs on bovine serum albumin denaturation. The viability of MCF-7 cells treated with BM-AgNPs was found to be significantly lower than that of cells treated with BM extract. The IC50 value of BM-AgNPs was determined to be 96.9 µg/mL. This study showed that BM-AgNPs have the potential to be used in the pharmaceutical industry as antioxidant, antibacterial, anti-inflammatory, and anticancer agents.

Spin-Flip-Restricted Multiple-Resonance Emitters for Extended Device Lifetime in Indolocarbazole-Based Blue Organic Light-Emitting Diodes.

In this study, a multiple-resonance (MR) core structure is developed with a spin-flip-restricted emission mechanism based on a fused indolo[3,2,1-jk]carbazole (ICz) framework as emitters to improve the lifetime of blue organic light-emitting diodes. The molecular skeleton modulation approach applied to the conjugated π-system effectively stabilizes the triplet energy of the fused ICz emitters and narrows the full-width-at-half maximum (<20 nm). In addition, the emitters exhibit higher exciton stability than conventional boron-based MR emitters. The fused ICz-based blue fluorescent device exhibits a high external quantum efficiency of 7.2%, a blue index of 68.6 cd A-1 at a Commission internationale de l'éclairage y coordinate (CIEy) of 0.075, and a device lifetime 1.8 times longer than that of a boron-based emitter. In addition, a phosphor-sensitized fluorescent device based on the ICz emitter exhibited an improved external quantum efficiency of 20.6% with a CIEy coordinate of 0.076.

A dose-dependent bimodal switch by homologous Aux/IAA transcriptional repressors.

Combinatorial interactions between different regulators diversify and enrich the chance of transcriptional regulation in eukaryotic cells. However, a dose-dependent functional switch of homologous transcriptional repressors has rarely been reported. Here, we show that SHY2, an auxin/indole-3-acetic acid (Aux/IAA) repressor, exhibits a dose-dependent bimodal role in auxin-sensitive root-hair growth and gene transcription in Arabidopsis, whereas other Aux/IAA homologs consistently repress the auxin responses. The co-repressor (TOPLESS [TPL])-binding affinity of a bimodal Aux/IAA was lower than that of a consistently repressing Aux/IAA. The switch of a single amino acid residue in the TPL-binding motif between the bimodal form and the consistently repressing form switched their TPL-binding affinity and transcriptional and biological roles in auxin responses. Based on these data, we propose a model whereby competition between homologous repressors with different co-repressor-binding affinities could generate a bimodal output at the transcriptional and developmental levels.

High vacancy formation energy boosts the stability of structurally ordered PtMg in hydrogen fuel cells.

Alloys of platinum with alkaline earth metals promise to be active and highly stable for fuel cell applications, yet their synthesis in nanoparticles remains a challenge due to their high negative reduction potentials. Herein, we report a strategy that overcomes this challenge by preparing platinum-magnesium (PtMg) alloy nanoparticles in the solution phase. The PtMg nanoparticles exhibit a distinctive structure with a structurally ordered intermetallic core and a Pt-rich shell. The PtMg/C as a cathode catalyst in a hydrogen-oxygen fuel cell exhibits a mass activity of 0.50 A mgPt-1 at 0.9 V with a marginal decrease to 0.48 A mgPt-1 after 30,000 cycles, exceeding the US Department of Energy 2025 beginning-of-life and end-of-life mass activity targets, respectively. Theoretical studies show that the activity stems from a combination of ligand and strain effects between the intermetallic core and the Pt-rich shell, while the stability originates from the high vacancy formation energy of Mg in the alloy.

CRISPR/Cas9-mediated knock-in of a fluorescent reporter into the target locus of interest in human pluripotent stem cells.

The method presented herein is associated with the Lab Resource article titled "Generation of αMHC-EGFP knock-in in human pluripotent stem cell line, SNUe003-A-3, using CRISPR/CAS9-based gene targeting" [1]. The cardiac muscle-specific protein, α-myosin heavy chain (αMHC), is encoded by the human MYH6 gene, which is expressed in both the atria and ventricles during embryonic development and is predominantly expressed in the atria after birth [2]. Herein, the methods used to achieve CRISPR/SpCas9-mediated introduction of an EGFP reporter into αMHC, the target locus in human pluripotent stem cells (hPSCs) for cardiac lineage tracing and clinical cell sorting are described. The CRISPR-Cas9 system enables efficient replacement of the stop codon in the last exon of αMHC with a 2A non-joining peptide (T2A)-EGFP cassette. First, hPSCs are transfected with the donor construct and Cas9/sgRNA plasmids via electroporation and selected with neomycin for approximately 3 weeks. Thereafter, the established cell line exhibits typical characteristics of human embryonic stem cells (hESCs). When these cells differentiate into cardiomyocytes, the expression of EGFP is confirmed using confocal microscopy, flow cytometry analysis, and immunostaining.•The line enables monitoring of cell maturation events during human cardiac development.•The line is a valuable platform for cardiotoxicity tests and drug screening.•This method has already been employed in two original studies, as previously reported for reporter cell line generation using CRISPR/Cas9.

Efficacy of Mobile App-Based Cognitive Behavioral Therapy for Insomnia: Multicenter, Single-Blind Randomized Clinical Trial.

BACKGROUND: Cognitive behavioral therapy for insomnia (CBTi) is the first-line therapy for chronic insomnia. Mobile app-based CBTi (MCBTi) can enhance the accessibility of CBTi treatment; however, few studies have evaluated the effectiveness of MCBTi using a multicenter, randomized controlled trial design. OBJECTIVE: We aimed to assess the efficacy of Somzz, an MCBTi that provides real-time and tailored feedback to users, through comparison with an active comparator app. METHODS: In our multicenter, single-blind randomized controlled trial study, participants were recruited from 3 university hospitals and randomized into a Somzz group and a sleep hygiene education (SHE) group at a 1:1 ratio. The intervention included 6 sessions for 6 weeks, with follow-up visits over a 4-month period. The Somzz group received audiovisual sleep education, guidance on relaxation therapy, and real-time feedback on sleep behavior. The primary outcome was the Insomnia Severity Index score, and secondary outcomes included sleep diary measures and mental health self-reports. We analyzed the outcomes based on the intention-to-treat principle. RESULTS: A total of 98 participants were randomized into the Somzz (n=49, 50%) and SHE (n=49, 50%) groups. Insomnia Severity Index scores for the Somzz group were significantly lower at the postintervention time point (9.0 vs 12.8; t95=3.85; F2,95=22.76; ηp2=0.13; P<.001) and at the 3-month follow-up visit (11.3 vs 14.7; t68=2.61; F2,68=5.85; ηp2=0.03; P=.01) compared to those of the SHE group. The Somzz group maintained their treatment effect at the postintervention time point and follow-ups, with a moderate to large effect size (Cohen d=-0.62 to -1.35; P<.01 in all cases). Furthermore, the Somzz group showed better sleep efficiency (t95=-3.32; F2,91=69.87; ηp2=0.41; P=.001), wake after sleep onset (t95=2.55; F2,91=51.81; ηp2=0.36; P=.01), satisfaction (t95=-2.05; F2,91=26.63; ηp2=0.20; P=.04) related to sleep, and mental health outcomes, including depression (t95=2.11; F2,94=29.64; ηp2=0.21; P=.04) and quality of life (t95=-3.13; F2,94=54.20; ηp2=0.33; P=.002), compared to the SHE group after the intervention. The attrition rate in the Somzz group was 12% (6/49). CONCLUSIONS: Somzz outperformed SHE in improving insomnia, mental health, and quality of life. The MCBTi can be a highly accessible, time-efficient, and effective treatment option for chronic insomnia, with high compliance. TRIAL REGISTRATION: Clinical Research Information Service (CRiS) KCT0007292; https://cris.nih.go.kr/cris/search/detailSearch.do?seq=22214&search_page=L.

CRISPR/Cas12a Collateral Cleavage-Driven Transcription Amplification for Direct Nucleic Acid Detection.

The clustered regularly interspaced short palindromic repeat/Cas (CRISPR/Cas) system is a powerful tool for nucleic acid detection owing to specific recognition as well as cis- and trans-cleavage capabilities. However, the sensitivity of CRISPR/Cas-based diagnostic approaches is determined by nucleic acid preamplification, which has several limitations. Here, we present a method for direct nucleic acid detection without preamplification, by combining the CRISPR/Cas12a system with signal enhancement based on light-up RNA aptamer transcription. We first designed two DNA templates to transcribe the light-up RNA aptamer and kleptamer (Kb) RNA: the first DNA template encodes a Broccoli RNA aptamer for fluorescence signal generation, and the Kb DNA template comprises a dsDNA T7 promoter sequence and an ssDNA sequence that encodes an antisense strand for the Broccoli RNA aptamer. Hepatitis B virus (HBV) target recognition activates a CRISPR/Cas12a complex, leading to the catalytic cleavage of the ssDNA sequence. Transcription of the added Broccoli DNA template can then produce several Broccoli RNA aptamer transcripts for fluorescence enhancement. The proposed strategy exhibited excellent sensitivity and specificity with 22.4 fM detection limit, good accuracy, and stability for determining the target HBV dsDNA in human serum samples. Overall, this newly designed signal enhancement strategy can be employed as a universal sensing platform for ultrasensitive nucleic acid detection.