Phase-separated DropCRISPRa platform for efficient gene activation in mammalian cells and mice.

Liquid-liquid phase separation (LLPS) plays a critical role in regulating gene transcription via the formation of transcriptional condensates. However, LLPS has not been reported to be engineered as a tool to activate endogenous gene expression in mammalian cells or in vivo. Here, we developed a droplet-forming CRISPR (clustered regularly interspaced short palindromic repeats) gene activation system (DropCRISPRa) to activate transcription with high efficiency via combining the CRISPR-SunTag system with FETIDR-AD fusion proteins, which contain an N-terminal intrinsically disordered region (IDR) of a FET protein (FUS or TAF15) and a transcription activation domain (AD, VP64/P65/VPR). In this system, the FETIDR-AD fusion protein formed phase separation condensates at the target sites, which could recruit endogenous BRD4 and RNA polymerase II with an S2 phosphorylated C-terminal domain (CTD) to enhance transcription elongation. IDR-FUS9Y>S and IDR-FUSG156E, two mutants with deficient and aberrant phase separation respectively, confirmed that appropriate phase separation was required for efficient gene activation. Further, the DropCRISPRa system was compatible with a broad set of CRISPR-associated (Cas) proteins and ADs, including dLbCas12a, dAsCas12a, dSpCas9 and the miniature dUnCas12f1, and VP64, P65 and VPR. Finally, the DropCRISPRa system could activate target genes in mice. Therefore, this study provides a robust tool to activate gene expression for foundational research and potential therapeutics.

Short cell-penetration peptide conjugated bioreducible polymer enhances gene editing of CRISPR system.

CRISPR-based gene therapy offers precise targeting and specific editing of disease-related gene sequences, potentially yielding long-lasting treatment effects. However, efficient delivery remains a significant challenge for its widespread application. In this study, we design a novel short peptide-conjugated bioreducible polymer named TSPscp as a safe and effective delivery vector for the CRISPR system. Our results show that TSPscp markedly boosts transcriptional activation and genome editing activities of multiple CRISPR systems as confirmed by decomposition-seq and Deep-seq, which is resulted from its capability in facilitating delivery of plasmid DNA by promoting cellular uptake and lysosomal escape. Additionally, TSPscp further enhances genome editing of CRISPR by delivery of minicircle DNA, a condensed form of regular plasmid DNA. More importantly, TSPscp significantly improves delivery and genome editing of CRISPR system in vivo. In summary, our study highlights TSPscp as a promising delivery tool for CRISPR applications in vivo.

Policy implementation deviation of government purchase of old age care services in Jiangsu, China: based on empirical and policy analysis.

BACKGROUND: Government purchase of social forces to participate in old age care services can release the burden of social care. Current research on performance evaluation in this field mainly focussed on the establishment of appropriate evaluation indices. However, discussion on the policy implementation deviation is scarce. This study aimed to evaluate the performance of China's local government purchase of old age care services, analyse the characteristics of related policies and explore their deviation. METHODS: The persons who participated in the Training of the Trainer (ToT) organized by the Red Cross Society were enrolled. The policy documents were obtained from the official websites. The K-means cluster was used to determine the project performance grades. We compared the project performance grades between service objects and undertakers with different characteristics utilizing the non-parametric test. Based on the framework of 'Collaborative Participation - Project Performance Objective', we analysed the content of relevant policy aiding by NVivo 12. RESULTS: Data of project performance were collected from 306 participants. The standardized mean score of the efficiency dimension was the lowest (0.70 ± 0.24). The projects were divided into four grades: poor (17.0%), average (27.5%), good (12.4%) and excellent (43.1%). There were statistically significant differences in project performance grades only between advanced ageing groups (Z = 2.429, P = 0.015). As well, the policy also mentioned that the services focus should be tilted towards the oldest old. The purchasers mainly involved the Ministry of Civil Affairs and Health management departments in the policy. Respite services were less mentioned in the responsibilities of the undertakers. The requirement for efficiency and effectiveness was mentioned in less than half of the policy documents. CONCLUSION: Policy attention is needed for the responsibilities and functions of the intermediate purchasing force, as well as more precise directions and responsibilities of undertakers. The purchasers and undertakers should improve management abilities and capacity of old age care services and focus on associated factors to achieve the best marginal benefit. In addition, the embedded performance evaluation needs to be updated periodically to bridge the deviation between policy implementation and policy formulation.

Profiling the impact of the promoters on CRISPR-Cas12a system in human cells.

The plasmid vector platform is the most commonly used vector for the expression of the versatile CRISPR-Cas technique and the promoter is a crucial element for the expression vector, thus profiling the impact of the promoters on CRISPR editors provides the basic information for the gene-editing toolkits and can be a guideline for its design. Herein, we made a parallel comparison among four commonly used promoters (CAG, ~ 1700 bp; EF1a core, ~ 210 bp; CMV, ~ 500 bp; and PGK, ~ 500 bp) in CRISPR-Cas12a system in mammalian cells to explore the impact of promoters on this powerful tool. We found that without badly damaging targeting specificity, the CAG promoter-driving Cas12a editor exhibited the most active (efficiency takes as 100%, specificity index = ~ 75%) in genomic cleavage, multiplex editing, transcriptional activation, and base editing, followed by promoter CMV (efficiency = 70 ~ 90% (vs CAG), specificity index = ~ 78%), and then EF1a core and PGK (both efficiency = 40-60%, vs CAG) but with higher specificity (specificity index = ~ 84% and ~ 82%, respectively). Therefore, CAG is recommended in the CRISPR-Cas12a system for the applications that need a robust editing activity but without size limitation, CMV mostly can be an alternative for CAG when requiring a smaller space, EF1a is similar to PGK with relatively high specificity, but has a smaller size, thus is more suitable for in vivo therapeutic applications. The data outlined the properties of the widely used promoters in the CRISPR-Cas12a system, which can be a guide for its applications and can be a useful resource for the gene-editing field.

Sensing mechanism of an Au-TiO2-Ag nanograting based on Fano resonance effects.

In recent years, with the development of nano-photonics, Fano resonance has gained increasing attention. Due to its high sensitivity, real-time detection, and label-free properties, the Fano resonance sensor has been widely applied in the fields of biochemistry and environmental detection. To improve the sensing characteristics of Fano resonance, an A u-T i O 2-A g grating structure is proposed in this paper, and the sensing performance is enhanced by a bi-metallic grating and deposited T i O 2. The characteristics of both sensing and field distribution of the model are accordingly analyzed using the finite-difference time-domain method. By varying the structural parameters such as grating period, grating height, silver film thickness, and T i O 2 layer thickness, the tuning of sensing characteristics can be realized, and afterwards, the sensing performance is improved; consequently, the Fano resonance reflection spectrum with high sensitivity and a high figure of merit (FOM) value is obtained. When the grating period P = 200 nm, grating height T1 = 90 nm, silver film thickness T2 = 20 nm, T i O 2 layer thickness T3 = 20 nm, and S i O 2 layer thickness T4 = 600 nm, such a structure indicates favorable sensing performance, and sensor detection accuracy can reach 10-3; maximum sensitivity is 1400 nm/RIU, and maximum FOM can reach 4212R I U -1. The results demonstrate that the designed Fano resonance sensing model has good potential for application.

MiniCAFE, a CRISPR/Cas9-based compact and potent transcriptional activator, elicits gene expression in vivo.

CRISPR-mediated gene activation (CRISPRa) is a promising therapeutic gene editing strategy without inducing DNA double-strand breaks (DSBs). However, in vivo implementation of these CRISPRa systems remains a challenge. Here, we report a compact and robust miniCas9 activator (termed miniCAFE) for in vivo activation of endogenous target genes. The system relies on recruitment of an engineered minimal nuclease-null Cas9 from Campylobacter jejuni and potent transcriptional activators to a target locus by a single guide RNA. It enables robust gene activation in human cells even with a single DNA copy and is able to promote lifespan of Caenorhabditis elegans through activation of longevity-regulating genes. As proof-of-concept, delivered within an all-in-one adeno-associated virus (AAV), miniCAFE can activate Fgf21 expression in the liver and regulate energy metabolism in adult mice. Thus, miniCAFE holds great therapeutic potential against human diseases.

Engineered Cas12a-Plus nuclease enables gene editing with enhanced activity and specificity.

BACKGROUND: The CRISPR-Cas12a (formerly Cpf1) system is a versatile gene-editing tool with properties distinct from the broadly used Cas9 system. Features such as recognition of T-rich protospacer-adjacent motif (PAM) and generation of sticky breaks, as well as amenability for multiplex editing in a single crRNA and lower off-target nuclease activity, broaden the targeting scope of available tools and enable more accurate genome editing. However, the widespread use of the nuclease for gene editing, especially in clinical applications, is hindered by insufficient activity and specificity despite previous efforts to improve the system. Currently reported Cas12a variants achieve high activity with a compromise of specificity. Here, we used structure-guided protein engineering to improve both editing efficiency and targeting accuracy of Acidaminococcus sp. Cas12a (AsCas12a) and Lachnospiraceae bacterium Cas12a (LbCas12a). RESULTS: We created new AsCas12a variant termed "AsCas12a-Plus" with increased activity (1.5~2.0-fold improvement) and specificity (reducing off-targets from 29 to 23 and specificity index increased from 92% to 94% with 33 sgRNAs), and this property was retained in multiplex editing and transcriptional activation. When used to disrupt the oncogenic BRAFV600E mutant, AsCas12a-Plus showed less off-target activity while maintaining comparable editing efficiency and BRAFV600E cancer cell killing. By introducing the corresponding substitutions into LbCas12a, we also generated LbCas12a-Plus (activity improved ~1.1-fold and off-targets decreased from 20 to 12 while specificity index increased from 78% to 89% with 15 sgRNAs), suggesting this strategy may be generally applicable across Cas12a orthologs. We compared Cas12a-Plus, other variants described in this study, and the reported enCas12a-HF, enCas12a, and Cas12a-ultra, and found that Cas12a-Plus outperformed other variants with a good balance for enhanced activity and improved specificity. CONCLUSIONS: Our discoveries provide alternative AsCas12a and LbCas12a variants with high specificity and activity, which expand the gene-editing toolbox and can be more suitable for clinical applications.

Greatly Enhanced Plasmon-Exciton Coupling in Si/WS2/Au Nanocavities.

A strong light-matter interaction is highly desirable from the viewpoint of both fundamental research and practical application. Here, we propose a dielectric-metal hybrid nanocavity composed of a silicon (Si) nanoparticle and a thin gold (Au) film and investigate numerically and experimentally the coupling between the plasmons supported by the nanocavity and the excitons in an embedded tungsten disulfide (WS2) monolayer. When a Si/WS2/Au nanocavity is excited by the surface plasmon polariton generated on the surface of the Au film, greatly enhanced plasmon-exciton coupling originating from the hybridization of the surface plasmon polariton, the mirror-image-induced magnetic dipole, and the exciton modes is clearly revealed in the angle- or size-resolved scattering spectra. A Rabi splitting as large as ∼240 meV is extracted by fitting the experimental data with a coupled harmonic oscillator model containing three oscillators. Our findings open new horizons for constructing nanoscale photonic devices by exploiting dielectric-metal hybrid nanocavities.

The post-translational modification of Fascin: impact on cell biology and its associations with inhibiting tumor metastasis.

The post-translational modifications (PTMs), which are crucial in the regulation of protein functions, have great potential as biomarkers of cancer status. Fascin (Fascin actin-bundling protein 1, FSCN1), a key protein in the formation of filopodia that is structurally based on actin filaments (F-actin), is significantly associated with tumor invasion and metastasis. Studies have revealed various regulatory mechanisms of human Fascin, including PTMs. Although a number of Fascin PTM sites have been identified, their exact functions and clinical significance are much less explored. This review explores studies on the functions of Fascin and briefly discusses the regulatory mechanisms of Fascin. Next, to review the role of Fascin PTMs in cell biology and their associations with metastatic disease, we discuss the advances in the characterization of Fascin PTMs, including phosphorylation, ubiquitination, sumoylation, and acetylation, and the main regulatory mechanisms are discussed. Fascin PTMs may be potential targets for therapy for metastatic disease.

[Study on Parametric Release of Ethylene Oxide Sterilization of Medical Devices].

This study briefly introduces the basic theory of sterilization, the characteristics of ethylene oxide sterilization for medical devices and the key factors about sterilization effectiveness, analyzes and compares three methods used in the product release of medical devices sterilized by ethylene oxide: test for sterility, traditional release and parametric release, and focuses on the theoretical basis, feasibility, validation requirements, advantages and disadvantages of parametric release.

Hepatitis B Virus Virions Produced Under Nucleos(t)ide Analogue Treatment Are Mainly Not Infectious Because of Irreversible DNA Chain Termination.

Nucleos(t)ide analogues (NAs) have been widely used for the treatment of chronic hepatitis B (CHB). Because viral DNA polymerase lacks proofreading function (3' exonuclease activity), theoretically, the incorporated NAs would irreversibly terminate viral DNA synthesis. This study explored the natures of nascent hepatitis B virus (HBV) DNA and infectivity of progeny virions produced under NA treatment. HBV infectivity was determined by infection of HepG2-NTCP cells and primary human hepatocytes (PHHs). Biochemical properties of HBV DNA in the progeny virions were investigated by qPCR, northern blotting, or Southern blotting hybridization, sucrose gradient centrifugation, and in vitro endogenous DNA polymerase assay. Progeny HBV virions produced under NA treatment were mainly not infectious to HepG2-NTCP cells or PHHs. Biochemical analysis revealed that under NA treatment, HBV DNA in nucleaocapsids or virions were predominantly short minus-strand DNA with irreversible termination. This finding was supported by the observation of first disappearance of relaxed circular DNA and then the proportional decline of HBV-DNA levels corresponding to the regions of PreC/C, S, and X genes in serial sera of patients receiving NA treatment. Conclusion: HBV virions produced under NA treatment are predominantly replication deficient because the viral genomes are truncated and elongation of DNA chains is irreversibly terminated. Clinically, our results suggest that the viral loads of CHB patients under NA therapy vary with the different regions of genome being detected by qPCR assays. Our findings also imply that NA prevention of perinatal and sexual HBV transmission as well as infection of transplanted livers works not only by reducing viral loads, but also by producing noninfectious virions.

A novel diagnostic method for pituitary adenoma based on magnetic resonance imaging using a convolutional neural network.

PURPOSE: This study was designed to develop a computer-aided diagnosis (CAD) system based on a convolutional neural network (CNN) to diagnose patients with pituitary tumors. METHODS: We included adult patients clinically diagnosed with pituitary adenoma (pituitary adenoma group), or adult individuals without pituitary adenoma (control group). After pre-processing, all the MRI data were randomly divided into training or testing datasets in a ratio of 8:2 to create or evaluate the CNN model. Multiple CNNs with the same structure were applied for different types of MR images respectively, and a comprehensive diagnosis was performed based on the classification results of different types of MR images using an equal-weighted majority voting strategy. Finally, we assessed the diagnostic performance of the CAD system by accuracy, sensitivity, specificity, positive predictive value, and F1 score. RESULTS: We enrolled 149 participants with 796 MR images and adopted the data augmentation technology to create 7960 new images. The proposed CAD method showed remarkable diagnostic performance with an overall accuracy of 91.02%, sensitivity of 92.27%, specificity of 75.70%, positive predictive value of 93.45%, and F1-score of 92.67% in separate MRI type. In the comprehensive diagnosis, the CAD achieved better performance with accuracy, sensitivity, and specificity of 96.97%, 94.44%, and 100%, respectively. CONCLUSION: The CAD system could accurately diagnose patients with pituitary tumors based on MR images. Further, we will improve this CAD system by augmenting the amount of dataset and evaluate its performance by external dataset.

HBV Genome and Life Cycle.

Chronic hepatitis B virus (HBV) infection remains to be a serious threat to public health and is associated with many liver diseases including chronic hepatitis B (CHB), liver cirrhosis, and hepatocellular carcinoma. Although nucleos(t)ide analogues (NA) and pegylated interferon-α (Peg-IFNα) have been confirmed to be efficient in inhibiting HBV replication, it is difficult to eradicate HBV and achieve the clinical cure of CHB. Therefore, long-term therapy has been recommended to CHB treatment under the current antiviral therapy. In this context, the new antiviral therapy targeting one or multiple critical steps of viral life cycle may be an alternative approach in future. In the last decade, the functional receptor [sodium-taurocholate cotransporting polypeptide (NTCP)] of HBV entry into hepatocytes has been discovered, and the immature nucleocapsids containing the non- or partially reverse-transcribed pregenomic RNA, the nucleocapsids containing double-strand linear DNA (dslDNA), and the empty particles devoid of any HBV nucleic acid have been found to be released into circulation, which have supplemented the life cycle of HBV. The understanding of HBV life cycle may offer a new instruction for searching the potential antiviral targets, and the new viral markers used to monitor the efficacy of antiviral therapy for CHB patients in the future.

CD146 coordinates brain endothelial cell-pericyte communication for blood-brain barrier development.

The blood-brain barrier (BBB) establishes a protective interface between the central neuronal system and peripheral blood circulation and is crucial for homeostasis of the CNS. BBB formation starts when the endothelial cells (ECs) invade the CNS and pericytes are recruited to the nascent vessels during embryogenesis. Despite the essential function of pericyte-EC interaction during BBB development, the molecular mechanisms coordinating the pericyte-EC behavior and communication remain incompletely understood. Here, we report a single cell receptor, CD146, that presents dynamic expression patterns in the cerebrovasculature at the stages of BBB induction and maturation, coordinates the interplay of ECs and pericytes, and orchestrates BBB development spatiotemporally. In mouse brain, CD146 is first expressed in the cerebrovascular ECs of immature capillaries without pericyte coverage; with increased coverage of pericytes, CD146 could only be detected in pericytes, but not in cerebrovascular ECs. Specific deletion of Cd146 in mice ECs resulted in reduced brain endothelial claudin-5 expression and BBB breakdown. By analyzing mice with specific deletion of Cd146 in pericytes, which have defects in pericyte coverage and BBB integrity, we demonstrate that CD146 functions as a coreceptor of PDGF receptor-ß to mediate pericyte recruitment to cerebrovascular ECs. Moreover, we found that the attached pericytes in turn down-regulate endothelial CD146 by secreting TGF-ß1 to promote further BBB maturation. These results reveal that the dynamic expression of CD146 controls the behavior of ECs and pericytes, thereby coordinating the formation of a mature and stable BBB.

Friend or Foe? Evidence Indicates Endogenous Exosomes Can Deliver Functional gRNA and Cas9 Protein.

The clustered regularly interspaced short palindromic repeats (CRISPR)/associated nuclease (Cas) system is an efficient gene editing tool. In this study, it is found that both single guide RNA (gRNA) and Cas9 protein could be exported from the CRISPR/Cas9-expressing cells by endogenous exosomes independently. Further experiments demonstrate that these naturally produced endogenous exosomes could be used as a vehicle to deliver the functional Cas9 and hepatitis B virus (HBV)-specific gRNA to cut HBV DNA transfected in HuH7 cells or human papilloma virus (HPV)-specific gRNA to cut the integrated HPV DNA in HeLa cells, respectively. In conclusion, this study indicates the potential of endogenous exosomes as a safe and effective delivery vehicle of the functional gRNA and Cas9 protein. Meanwhile, the endogenous exosomes-mediated delivery of gene editing activity to adjacent and distant cells or tissues may further complicate the off-target and safety concerns about the CRISPR/Cas9 system.

Aberration-resistible topological charge determination of annular-shaped optical vortex beams using Shack-Hartmann wavefront sensor.

Determining topological charge (TC) of optical vortex (OV) beams is important for many applications, such as optical measurement and information transmission through long-distance propagation. In this application, the OV beams usually have an annular intensity profile at the receiving end and are inevitably distorted by aberrations during propagation. In this paper, we propose a simple, direct, and aberration-resistible method to determine the TC value of this annular-shaped OV beam with a Shack-Hartmann wavefront sensor (SH-WFS). Our approach involves finding a closed-path along the annular intensity distribution ridge, and then determining the TC value by a discrete circulation calculation with only the phase-slope vectors along the closed-path. Verification experiments were performed using a phase-only spatial light modulator to generate OV beams and aberrations, while SH-WFS to measure the intensity profile and phase-slopes. The results show that our method can determine the TC value of up to ± 20. The robustness against wavefront aberration and simulated atmospheric turbulence was evaluated. We also verified the proposed closed-path's superiority to other circular closed-paths for TC determination.