Engineering of a high-fidelity Cas12a nuclease variant capable of allele-specific editing.

CRISPR-Cas12a, often regarded as a precise genome editor, still requires improvements in specificity. In this study, we used a GFP-activation assay to screen 14 new Cas12a nucleases for mammalian genome editing, successfully identifying 9 active ones. Notably, these Cas12a nucleases prefer pyrimidine-rich PAMs. Among these nucleases, we extensively characterized Mb4Cas12a obtained from Moraxella bovis CCUG 2133, which recognizes a YYN PAM (Y = C or T). Our biochemical analysis demonstrates that Mb4Cas12a can cleave double-strand DNA across a wide temperature range. To improve specificity, we constructed a SWISS-MODEL of Mb4Cas12a based on the FnCas12a crystal structure and identified 8 amino acids potentially forming hydrogen bonds at the target DNA-crRNA interface. By replacing these amino acids with alanine to disrupt the hydrogen bond, we tested the influence of each mutation on Mb4Cas12a specificity. Interestingly, the F370A mutation improved specificity with minimal influence on activity. Further study showed that Mb4Cas12a-F370A is capable of discriminating single-nucleotide polymorphisms. These new Cas12a orthologs and high-fidelity variants hold substantial promise for therapeutic applications.

Nanopipette dynamic microscopy unveils nano coffee ring.

Liquid-phase electron microscopy (LP-EM) imaging has revolutionized our understanding of nanosynthesis and assembly. However, the current closed geometry limits its application for open systems. The ubiquitous physical process of the coffee-ring phenomenon that underpins materials and engineering science remains elusive at the nanoscale due to the lack of experimental tools. We introduce a quartz nanopipette liquid cell with a tunable dimension that requires only standard microscopes. Depending on the imaging condition, the open geometry of the nanopipette allows the imaging of evaporation-induced pattern formation, but it can also function as an ordinary closed-geometry liquid cell where evaporation is negligible despite the nano opening. The nano coffee-ring phenomenon was observed by tracking individual nanoparticles in an evaporating nanodroplet created from a thin liquid film by interfacial instability. Nanoflows drive the assembly and disruption of a ring pattern with the absence of particle-particle correlations. With surface effects, nanoflows override thermal fluctuations at tens of nanometers, in which nanoparticles displayed a "drunken man trajectory" and performed work at a value much smaller than kBT.

In Vivo Base Editing of Scn5a Rescues Type 3 Long QT Syndrome in Mice.

BACKGROUND: Pathogenic variants in SCN5A can result in long QT syndrome type 3, a life-threatening genetic disease. Adenine base editors can convert targeted A T base pairs to G C base pairs, offering a promising tool to correct pathogenic variants. METHODS: We generated a long QT syndrome type 3 mouse model by introducing the T1307M pathogenic variant into the Scn5a gene. The adenine base editor was split into 2 smaller parts and delivered into the heart by adeno-associated virus serotype 9 (AAV9-ABEmax) to correct the T1307M pathogenic variant. RESULTS: Both homozygous and heterozygous T1307M mice showed significant QT prolongation. Carbachol administration induced Torsades de Pointes or ventricular tachycardia for homozygous T1307M mice (20%) but not for heterozygous or wild-type mice. A single intraperitoneal injection of AAV9-ABEmax at postnatal day 14 resulted in up to 99.20% Scn5a transcripts corrected in T1307M mice. Scn5a mRNA correction rate >60% eliminated QT prolongation; Scn5a mRNA correction rate <60% alleviated QT prolongation. Partial Scn5a correction resulted in cardiomyocytes heterogeneity, which did not induce severe arrhythmias. We did not detect off-target DNA or RNA editing events in ABEmax-treated mouse hearts. CONCLUSIONS: These findings show that in vivo AAV9-ABEmax editing can correct the variant Scn5a allele, effectively ameliorating arrhythmia phenotypes. Our results offer a proof of concept for the treatment of hereditary arrhythmias.

Author Correction: Systems of mechanized and reactive droplets powered by multi-responsive surfactants.

In this Letter, the top structure of the right panel of Fig. 1a should be that of cis-9-octadecenoic acid, not trans-9-octadecenoic acid. Please see the accompanying Author Correction. This error has not been corrected online.

Assessment of Efficacy and Accuracy of Cervical Cytology Screening With Artificial Intelligence Assistive System.

The role of artificial intelligence (AI) in pathology offers many exciting new possibilities for improving patient care. This study contributes to this development by identifying the viability of the AICyte assistive system for cervical screening, and investigating the utility of the system in assisting with workflow and diagnostic capability. In this study, a novel scanner was developed using a Ruiqian WSI-2400, trademarked AICyte assistive system, to create an AI-generated gallery of the most diagnostically relevant images, objects of interest (OOI), and provide categorical assessment, according to Bethesda category, for cervical ThinPrep Pap slides. For validation purposes, 2 pathologists reviewed OOIs from 32,451 cases of ThinPrep Paps independently, and their interpretations were correlated with the original ThinPrep interpretations (OTPI). The analysis was focused on the comparison of reporting rates, correlation between cytological results and histologic follow-up findings, and the assessment of independent AICyte screening utility. Pathologists using the AICyte system had a mean reading time of 55.14 seconds for the first 3000 cases trending down to 12.90 seconds in the last 6000 cases. Overall average reading time was 22.23 seconds per case compared with a manual reading time approximation of 180 seconds. Usage of AICyte compared with OTPI had similar sensitivity (97.89% vs 97.89%) and a statistically significant increase in specificity (16.19% vs 6.77%) for the detection of cervical intraepithelial neoplsia 2 and above lesions. When AICyte was run alone at a 50% negative cutoff value, it was able to read slides with a sensitivity of 99.30% and a specificity of 9.87%. When AICyte was run independently at this cutoff value, no sole case of high-grade squamous intraepithelial lesions/squamous cell carcinoma squamous lesion was missed. AICyte can provide a potential tool to help pathologists in both diagnostic capability and efficiency, which remained reliable compared with the baseline standard. Also unique for AICyte is the development of a negative cutoff value for which AICyte can categorize cases as "not needed for review" to triage cases and lower pathologist workload. This is the largest case number study that pathologists reviewed OOI with an AI-assistive system. The study demonstrates that AI-assistive system can be broadly applied for cervical cancer screening.

Systems of mechanized and reactive droplets powered by multi-responsive surfactants.

Although 'active' surfactants, which are responsive to individual external stimuli such as temperature, electric or magnetic fields, light, redox processes or chemical agents, are well known, it would be interesting to combine several of these properties within one surfactant species. Such multi-responsive surfactants could provide ways of manipulating individual droplets and possibly assembling them into larger systems of dynamic reactors. Here we describe surfactants based on functionalized nanoparticle dimers that combine all of these and several other characteristics. These surfactants and therefore the droplets that they cover are simultaneously addressable by magnetic, optical and electric fields. As a result, the surfactant-covered droplets can be assembled into various hierarchical structures, including dynamic ones, in which light powers the rapid rotation of the droplets. Such rotating droplets can transfer mechanical torques to their non-nearest neighbours, thus acting like systems of mechanical gears. Furthermore, droplets of different types can be merged by applying electric fields and, owing to interfacial jamming, can form complex, non-spherical, 'patchy' structures with different surface regions covered with different surfactants. In systems of droplets that carry different chemicals, combinations of multiple stimuli can be used to control the orientations of the droplets, inter-droplet transport, mixing of contents and, ultimately, sequences of chemical reactions. Overall, the multi-responsive active surfactants that we describe provide an unprecedented level of flexibility with which liquid droplets can be manipulated, assembled and reacted.

Retrospective study of 189 cases of acute perforated peptic ulcer: safety and efficacy of over-the-scope-clip based endoscopic closure.

BACKGROUND: To compare the clinical outcomes in patients with acute perforated peptic ulcer (PPU) treated with over-the-scope clip (OTSC), non-surgical, and surgical interventions, and to explore the effectiveness and safety of OTSC closure. METHODS: Hospital stay, antibiotic use, diet resumption time, and mortality rate were analyzed retrospectively. Binary Logistic regression analysis was used to identify the risk factors influencing PPU complicated with sepsis. RESULTS: Patients were divided into three treatment groups: OTSC (n = 62), non-surgical (n = 72), and surgical (n = 55) groups. The median time (IQR) from symptom onset to admission was 9.0 (4-23) h. 88.71% (55/62) of the patients in In the OTSC group underwent OTSC closure within 24 h (median [IQR] time: 14.5 [7.00-30.25] h). The perforation diameters in the OTSC and surgical groups were 9.87 mm ± 5.97 mm and 8.55 mm ± 6.17 mm, respectively. The median (IQR) hospital stays in the OTSC (9.50 [7.00-12.25] days) and non-surgical group (9.00[7.00-13.00]days) were similar (p > 0.05), but shorter than that in surgical group (12.00[10.00-16.00]days), (p < 0.05). The median duration of antibiotic use was shorter in the OTSC group (7.00[3.00-10.00]) than in the non-surgical group (9.00[7.00-11.00]) and surgical group (11.00[9.00-13.00]) ( p < 0.05); and the time to resume oral feeding was shorter in the OTSC group (4.00[2.00-5.25]) than in the non-surgical group (7.00[6.13-9.00]) and surgical group (8.00[6.53-10.00]), respectively ( p < 0.05). No mortality difference among groups (p = 0.109) was found. Lower albumin level at admission, older age, and elevated creatinine levels were associated with increased sepsis risk, with OR(95%CI) of 0.826 (0.687-0.993), 1.077 (1.005-1.154), and 1.025 (1.006-1.043), respectively (all p < 0.05). CONCLUSION: OTSC closure improves clinical outcomes of acute PPU patients without sepsis. Age, hypoalbuminemia, and baseline renal dysfunction increase the risk of sepsis, while mortality was associated with sepsis and multiorgan dysfunction.

Genome editing with natural and engineered CjCas9 orthologs.

CjCas9 is one of the smallest CRISPR-associated (Cas9) nucleases for mammalian genome editing. However, it requires a long N4RYAC (R = A or G; Y = C or T) protospacer-adjacent motif (PAM), limiting its DNA targeting scope. In this study, we investigated the PAMs of three CjCas9 orthologs, including Hsp1Cas9, Hsp2Cas9, and CcuCas9, by performing a GFP-activation assay. Interestingly, Hsp1Cas9 and CcuCas9 recognized unique N4RAA and N4CNA PAMs, respectively. We further generated an Hsp1Cas9-Hsp2Cas9 chimeric Cas9 (Hsp1-Hsp2Cas9), which recognized a simple N4CY PAM. Genome-wide off-target analysis revealed that Hsp1-Hsp2Cas9 has very few off-targets compared to SpCas9. By analyzing the crystal structure of CjCas9, we identified eight mutations that can improve the specificity and generate a high-fidelity Hsp1-Hsp2Cas9-Y. Hsp1-Hsp2Cas9-Y enables the knockout of B4GALNT2 and CMAH in porcine fetal fibroblasts (PFFs). Moreover, we developed a high-fidelity Hsp1-Hsp2Cas9-KY which displayed undetectable off-targets revealed by GUIDE-seq at four tested loci. These natural and engineered Cas9 nucleases enabled efficient genome editing in multiple mammalian cells, expanding the DNA targeting scope.

Research Progress on the Experimental Model and Underlying Mechanistic Studies of Tension-Type Headaches.

PURPOSE OF REVIEW: Tension-type headaches (TTH) significantly diminish patients' quality of life and increase absenteeism, thereby imposing a substantial economic burden. Animal models are essential tools for studying disease mechanisms and drug development. However, until now, little focus has been placed on summarizing the animal models of TTH and associated mechanistic studies. This narrative review discusses the current animal models of TTH and related mechanistic studies to provide insights into the pathophysiological mechanisms of and treatments for TTH. RECENT FINDINGS: The primary method for constructing an animal model of TTH involves injecting a solution of pain relievers, such as adenosine triphosphate, nerve growth factor, or a high concentration of salt solution, into the neck to initiate harmful cervical muscle responses. This model enables the examination of the interaction between peripheral muscles and central sensitization, which is crucial for understanding the pathophysiology of TTH. Mechanistic studies based on this model have investigated the effect of the P2X receptor antagonist, P2X7 receptor blockade, the P2Y1 receptor agonist 2-MESADP, P2Y1 receptor antagonist MRS2179, nitric oxide synthase inhibitors, and acetylsalicylic acid. Despite notable advancements, the current model of TTH has limitations, including surgical complexity and the inability to replicate chronic tension-type headache (CTTH). To gain a more comprehensive understanding and develop more effective treatment methods, future studies should focus on simplifying surgical procedures, examining other predisposing factors, and establishing a model for chronic TTH. This will offer a deeper insight into the pathophysiological mechanism of TTH and pave the way for improved treatment approaches.

Customizing biomimetic surface attributes of dendritic lipopeptide nanoplatforms for extended circulation.

The pressing demand for innovative approaches to create delivery systems with heightened drug loading and prolonged circulation has spurred numerous efforts, yielding some successes but accompanied by constraints. Our study proposes employing dendritic lipopeptide with precisely balanced opposing charges to extend blood residency for biomimetic nanoplatforms. Neutrally mixed-charged zwitterionic nanoparticles (NNPs) achieved a notable 19 % simvastatin loading content and kept stable even after one-month storage at 4 °C. These nanoplatforms demonstrated low cytotoxicity in NIH-3T3 and L02 cells and negligible hemolysis (<5 %). NNPs inhibited protein adhesion (>95 %) from positively and negatively charged sources through surface hydration. In comparison to positively charged CNPs, NNPs demonstrated an 86 % decrease in phagocytic rate by BMDMs, highlighting their efficacy. Importantly, NNPs showed prolonged circulation compared to CNPs and free simvastatin. These findings highlight the potential of this biomimetic nanoplatform for future therapeutic applications with enhanced drug loading and circulation traits.

Advances in Nano-Functional Materials in Targeted Thrombolytic Drug Delivery.

Thrombotic disease has been listed as the third most fatal vascular disease in the world. After decades of development, clinical thrombolytic drugs still cannot avoid the occurrence of adverse reactions such as bleeding. A number of studies have shown that the application of various nano-functional materials in thrombus-targeted drug delivery, combined with external stimuli, such as magnetic, near-infrared light, ultrasound, etc., enrich the drugs in the thrombus site and use the properties of nano-functional materials for collaborative thrombolysis, which can effectively reduce adverse reactions such as bleeding and improve thrombolysis efficiency. In this paper, the research progress of organic nanomaterials, inorganic nanomaterials, and biomimetic nanomaterials for drug delivery is briefly reviewed.

[Rules of prescriptions for heart failure of dilated cardiomyopathy based on latent structure model and association rules].

The latent structure model and association rules analysis were employed to explore the compatibility rules of prescriptions for heart failure of dilated cardiomyopathy, with a view to providing theoretical support for the clinical treatment of this disease based on syndrome differentiation and the formulation of guidelines. The articles about the treatment of heart failure of dilated cardiomyopathy were retrieved from CNKI, Wanfang, VIP, and SinoMed. The database was established in Microsoft Excel 2019. Lantern 5.0 and Rstudio were used to analyze the latent structure and association rules of Chinese medicine with the frequency greater than 4.00%. Furthermore, the frequency structure model was used to mine the rules of prescriptions for heart failure of dilated cardiomyopathy. The study included 175 traditional Chinese medicine(TCM) prescriptions, involving 128 Chinese medicines, with the cumulative frequency of 1 847. High-frequency medicines included Astragali Radix, Salviae Miltiorrhizae Radix et Rhizoma, Poria, Cinnamomi Ramulus, Glycyrrhizae Radix et Rhizoma, with the main effects of tonifying, activating blood, resolving stasis, and releasing exterior. A total of 17 hidden variables, 34 hidden categories, and 6 comprehensive cluster models, along with 15 core prescriptions, were obtained. According to the prescriptions, the patients mainly had the syndromes of heart-Yang and Qi deficiency, Qi deficiency and blood stasis, heart-kidney Yang deficiency or Qi-Yin deficiency. Fifty-four strong association rules were obtained through association rule analysis. The highest degree of support was observed for the combination of Salviae Miltiorrhizae Radix et Rhizoma-Astragali Radix, while the highest degree of confidence was found for the combination of Salviae Miltiorrhizae Radix et Rhizoma-Cinnamomi Ramulus-Ophiopogonis Radix-Astragali Radix. The heart failure of dilated cardiomyopathy, characterized by internal deficiency and excess manifestations, is attributed to deficiency, stasis, and water. These factors are closely associated with the heart, lung, and spleen. The treatment should follow the principle of invigorating Qi and warming Yang, and meanwhile the method of activating blood and resolve stasis or moving Qi and promoting urination can be adopted according to the specific syndrome of patients.

[Network Meta-analysis of Chinese patent medicines in treatment of coronary heart disease complicated with heart failure].

The efficacy and safety of different Chinese patent medicines in the treatment of coronary heart disease complicated with heart failure were evaluated by network Meta-analysis. The randomized controlled trial(RCT) of Chinese patent medicines for coronary heart disease complicated with heart failure was retrieved from CNKI, Wanfang, VIP, SinoMed, PubMed, Web of Science, EMbase, and Cochrane Library with the time interval from inception to July 5, 2023. The quality of the included RCT was evaluated by the Cochrane's risk of bias assessment tool, and a network Meta-analysis was performed in Stata 16.0. Finally, a total of 82 RCTs were included, involving 9 298 patients and 11 Chinese patent medicines. Network Meta-analysis yielded the following results based on the surface under the cumulative ranking curve(SUCRA).(1)In terms of improving the clinical response rate, the top three interventions were Qishen Yiqi Dripping Pills + conventional western medicine, Zhenyuan Capsules + conventional western medicine, and Tongxinluo Capsules + conventional western medicine.(2) In terms of increasing left ventricular ejection fraction(LVEF), the top three interventions were Shexiang Baoxin Pills + conventional western medicine, Compound Danshen Dripping Pills + conventional western medicine, and Tongxinluo Capsules + conventional western medicine.(3) In terms of reducing left ventricular end-diastolic diameter(LVEDD), the top three interventions were Shexiang Tongxin Dripping Pills + conventional western medicine, Tongxinluo Capsules + conventional western medicine, and Shexiang Baoxin Pills + conventional western medicine.(4) In terms of reducing N-terminal pro-brain natriuretic peptide(NT-proBNP), the top three interventions were Shexiang Baoxin Pills + conventional western medicine, Qi-shen Yiqi Dripping Pills + conventional western medicine, and Compound Danshen Dripping Pills + conventional western medicine.(5) In terms of reducing hyper-sensitive C-reactive protein(hs-CRP), the top three interventions were Naoxintong Capsules + conventional western medicine, Shexiang Baoxin Pills + conventional western medicine, and Compound Danshen Dripping Pills + conventional western medicine.(6) In terms of increasing the distance of the six-minute walking trail(6MWT), the top three interventions were Zhen-yuan Capsules + conventional western medicine, Qili Qiangxin Capsules + conventional western medicine, and Qishen Yiqi Dripping Pills + conventional western medicine. The results showed that Chinese patent medicines combined with conventional western medicine can effectively improve the clinical response rate, LVEF, and 6MWT and reduce LVEDD, NT-proBNP, and hs-CRP. However, due to the overall low quality of the articles included and the few articles of some Chinese patent medicines, direct comparison between diffe-rent Chinese patent medicines remains to be carried out and the results need to be further verified.

Large Optical Asymmetry in Silver Nanoparticle Assemblies Enabled by CH-π Interaction-Mediated Chirality Transfer.

Transfer of asymmetry from the molecular system to the other distinct system requires appropriate chemical interactions. Here, we show how the CH-π interaction, one of the weakest hydrogen bonds, can be applied to transfer the asymmetry from π-conjugated chiral molecules to the assemblies of plasmonic Ag nanoparticles, where the aliphatic chains of chiral molecules and the polystyrene chains grafted on Ag nanoparticles are served as the hydrogen donor and acceptor, respectively. The optical asymmetry g-factor of the chiral assemblies of plasmonic nanoparticles is strongly dependent on the molecular weight of the polystyrene ligand, the core structure of the molecule, and the aliphatic chain length of the chiral molecule. Importantly, we explore a molecular mixing strategy to enhance the asymmetry g-factor of chiral molecular assemblies, which consequently promotes the g-factor of chiral plasmonics efficiently, reaching a high value of ∼0.05 under optimal conditions. Overall, we rationalize the chirality transfer from chiral molecules to inorganic nanoparticles, providing the guidance for structural design of chiral nanocomposites with a high g-factor.

Controlled Hierarchical Self-Assembly of Nanoparticles and Chiral Molecules into Tubular Nanocomposites.

In this work, we show how the kinetics of molecular self-assembly can be coupled with the kinetics of the colloidal self-assembly of inorganic nanoparticles, which in turn drives the formation of several distinct hierarchically assembled tubular nanocomposites with lengths over tens of micrometers. These colloidal nanoparticles primarily serve as "artificial histones," around which the as-assembled supramolecular fibrils are wound into deeply kinetically trapped single-layered nanotubes, which leads to the formation of tubular nanocomposites that are resistant to supramolecular transformation thermally. Alternatively, when these nanoparticles are aggregated prior to the event of molecular self-assembly, these as-formed nanoparticle "oligomers" would be encapsulated into the thermodynamically favored double-layer supramolecular nanotubes, which enables the non-close-packing of nanoparticles inside the nanotubes and results in the nanoparticle superlattices with an open channel. Furthermore, increasing the amounts of nanoparticles enables the assembly of nanoparticles into pseudohexagonal superlattices at the external surface in a sequential fashion, which ultimately drives the formation of triple-layered hierarchically assembled tubular nanocomposites. Importantly, the sense of helicity transfers from the supramolecular nanotubes to the pseudo nanoparticle superlattices with a chiral vector of (2, 9). Our findings represent a strategy for controlling the hierarchical assembly bridging supramolecular chemistry to the inorganic solids to realize the complexity by design.

Small Molecules Mediated the Chirality Transfer in Self-Assembled Nanocomposites with Strong Circularly Polarized Luminescence.

Manipulation of the chirality at all scales has a cross-disciplinary importance and may address key challenges at the heart of physical sciences. One critical question in this field is how the chirality of one entity can be transferred to the asymmetry of another entity. Here, we find that small molecules play a crucial role in the chirality transfer from chiral organic molecules to CdSe/CdS nanorods, where the handedness of the nanorod assemblies either agrees or disagrees with that of the molecular assemblies, leading to the positive or inverse chirality transfer. The assembling mode of nanorods on the molecular assemblies, where the nanorods are either lying or standing, is closely associated with the handedness of the nanorod assemblies, resulting in opposite chirality. Furthermore, we have found that circularly polarized emission from chiral assemblies of nanorods is dependent on molecular additives. The promoted luminescence dissymmetry factor (glum) of the nanocomposites with a high value of ∼0.3 could be attained under optimal conditions.

Biomacromolecular Characterizations Using State-of-the-Art Quartz Crystal Microbalance with Dissipation.

Biomolecular characterization is essential in fields such as drug discovery, glycomics, and cell biology. This feature article focuses on the experimental use of quartz crystal microbalance with dissipation (QCM-D) as a powerful analytical technique to probe biological events ranging from biomacromolecular interactions and conformational changes of biomacromolecules to surface immobilization of biomacromolecules and cell morphological changes.

Afterglow Electrochemiluminescence from Nitrogen-Deficient Graphitic Carbon Nitride.

Almost all current electrochemiluminescent reagents require real-time electrochemical stimulation to emit light. Here, we report a novel electrochemiluminescent reagent, nitrogen-deficient graphitic carbon nitride (CNx), that can emit afterglow electrochemiluminescence (ECL) after cessation of electric excitation. CNx obtained by post-thermal treatment of graphitic carbon nitride (CN) with KSCN has a cyanamide group and a nitrogen vacancy, which created defects to trap electrically injected electrons. The trapped electrons can slowly release and react with coreactants to emit light with longevity. The cathodic afterglow ECL lasts for 70 s after pulsing the CNx nanosheet (CNxNS-1.6)-modified glassy carbon electrode at -1.0 V for 20 s in 2.0 M PBS containing 1 mM K2S2O8. The afterglow ECL mechanism is revealed by investigation of its influencing factors and ECL wavelength. The discovery of afterglow ECL may open a new doorway for new significant applications of the ECL technique and provide a deeper understanding of the structure-property relationships of CN.

Efficient Correction of a Hypertrophic Cardiomyopathy Mutation by ABEmax-NG.

[Figure: see text].

Using a molecular additive to control chiral supramolecular assembly and the subsequent chirality transfer process.

Hierarchical assembly of chiral molecules is achieved through the introduction of molecular additives, which enables the chiral assembly of nanosheets into helical nanorods with inverted chirality. Moreover, the hierarchical assembly of chiral molecules in the presence of a molecular additive can lead to the subsequent chirality transfer from a molecular system to nanoparticle assemblies.