Massively parallel CRISPR off-target detection enables rapid off-target prediction model building.

BACKGROUND: CRISPR (clustered regularly interspaced short palindromic repeats) genome editing holds tremendous potential in clinical translation. However, the off-target effect has always been a major concern. METHODS: Here, we have developed a novel sensitive and specific off-target detection method, AID-seq (adaptor-mediated off-target identification by sequencing), that can comprehensively and faithfully detect the low-frequency off targets generated by different CRISPR nucleases (including Cas9 and Cas12a). FINDINGS: Based on AID-seq, we developed a pooled strategy to simultaneously identify the on/off targets of multiple gRNAs, as well as using mixed human and human papillomavirus (HPV) genomes to screen the most efficient and safe targets from 416 HPV gRNA candidates for antiviral therapy. Moreover, we used the pooled strategy with 2,069 single-guide RNAs (sgRNAs) at a pool size of about 500 to profile the properties of our newly discovered CRISPR, FrCas9. Importantly, we successfully built an off-target detection model using these off-target data via the CRISPR-Net deep learning method (area under the receiver operating characteristic curve [AUROC] = 0.97, area under the precision recall curve [AUPRC] = 0.29). CONCLUSIONS: To our knowledge, AID-seq is the most sensitive and specific in vitro off-target detection method to date. And the pooled AID-seq strategy can be used as a rapid and high-throughput platform to select the best sgRNAs and characterize the properties of new CRISPRs. FUNDING: This work was supported by The National Natural Science Foundation of China (grant nos. 32171465 and 82102392), the General Program of Natural Science Foundation of Guangdong Province of China (grant no. 2021A1515012438), Guangdong Basic and Applied Basic Research Foundation (grant no. 2020A1515110170), and the National Ten Thousand Plan-Young Top Talents of China (grant no. 80000-41180002).

HPV integration generates a cellular super-enhancer which functions as ecDNA to regulate genome-wide transcription.

Human papillomavirus (HPV) integration is a critical step in cervical cancer development; however, the oncogenic mechanism at the genome-wide transcriptional level is still poorly understood. In this study, we employed integrative analysis on multi-omics data of six HPV-positive and three HPV-negative cell lines. Through HPV integration detection, super-enhancer (SE) identification, SE-associated gene expression and extrachromosomal DNA (ecDNA) investigation, we aimed to explore the genome-wide transcriptional influence of HPV integration. We identified seven high-ranking cellular SEs generated by HPV integration in total (the HPV breakpoint-induced cellular SEs, BP-cSEs), leading to intra-chromosomal and inter-chromosomal regulation of chromosomal genes. The pathway analysis revealed that the dysregulated chromosomal genes were correlated to cancer-related pathways. Importantly, we demonstrated that BP-cSEs existed in the HPV-human hybrid ecDNAs, explaining the above transcriptional alterations. Our results suggest that HPV integration generates cellular SEs that function as ecDNA to regulate unconstrained transcription, expanding the tumorigenic mechanism of HPV integration and providing insights for developing new diagnostic and therapeutic strategies.

VIS Atlas: A Database of Virus Integration Sites in Human Genome from NGS Data to Explore Integration Patterns.

Integration of oncogenic DNA viruses into the human genome is a key step in most virus-induced carcinogenesis. Here, we constructed a virus integration site (VIS) Atlas database, an extensive collection of integration breakpoints for three most prevalent oncoviruses, human papillomavirus, hepatitis B virus, and Epstein-Barr virus based on the next-generation sequencing (NGS) data, literature, and experimental data. There are 63,179 breakpoints and 47,411 junctional sequences with full annotations deposited in the VIS Atlas database, comprising 47 virus genotypes and 17 disease types. The VIS Atlas database provides (1) a genome browser for NGS breakpoint quality check, visualization of VISs, and the local genomic context; (2) a novel platform to discover integration patterns; and (3) a statistics interface for a comprehensive investigation of genotype-specific integration features. Data collected in the VIS Atlas aid to provide insights into virus pathogenic mechanisms and the development of novel antitumor drugs. The VIS Atlas database is available at https://www.vis-atlas.tech/.

Differential diagnosis and feature visualization for thyroid nodules using computer-aided ultrasonic diagnosis system: initial clinical assessment.

BACKGROUND: To assess the diagnostic efficacy of the computer-aided ultrasonic diagnosis system (CAD system) in differentiating benign and malignant thyroid nodules. METHODS: The images of 296 thyroid nodules were included in validation sets. The diagnostic efficacy of the CAD system was compared with that of junior physicians and senior physicians, as well as that of the combination diagnosis of the CAD system with junior physicians. The diagnostic efficacy of the CAD system for different sizes of thyroid nodules was compared. RESULTS: The diagnostic sensitivity and accuracy of the CAD system were higher than those of junior physicians (83.4% vs. 72.2%, 73.0% vs. 69.6%), but the diagnostic specificity of the CAD system was lower than that of junior physicians (62.1% vs. 66.9%). The diagnostic accuracy of the CAD system was lower than that of senior physicians (73.0% vs. 83.8%). However, the combination diagnosis of the CAD system with junior physicians had higher accuracy (81.8%) and AUC (0.842) than those of either the CAD system or junior physicians alone, and comparable diagnostic performance with those of senior physicians. The Kappa was 0.635 in the combination diagnosis of the CAD system with junior physicians, showing good consistency with the pathological results. The accuracy (76.4%) of the CAD system was the highest for nodules of 1-2 cm. CONCLUSION: The CAD system can effectively assist physicians to identify malignant and benign thyroid nodules, reduce the overdiagnosis and overtreatment of thyroid nodules, avoid unnecessary invasive fine needle aspiration, and improve the diagnostic accuracy of junior physicians.

Global Trade Pattern of Medical Devices and China's Trade Position: Based on Data From 2001 to 2020.

To depict the evolution of the global trade of medical devices, this study analyzes the spatiotemporal evolution characteristics of global and China's trade patterns of medical devices from 2001 to 2020 based on data from the World Bank and United Nations Commodity Trade Statistics Database, and thereby investigates the status quo of global and China's medical device trade, as well as changes in China's position in the global medical device trade. The findings are as follows. First, the total global trade volume of medical devices is generally on the rise, showing closer network connections. Despite some changes in trade position, the core countries in the global medical device trade network are relatively fixed. The intermediate position of core trading countries has been weakened on the whole, whereas exporting countries have generally assumed an enhanced central position. Communities with geographical proximity have been formed in the global medical device trade network, including two large communities, the Asian-European countries and the Pacific Rim countries, and one small community, the South American countries. Second, with its rapidly growing trade volume of medical devices with other countries, China has now become the fourth largest medical device trading country in the world. Its number of import and export partners has remained relatively stable and continued to increase. Its export markets are relatively concentrated, and a tripartite pattern of import sources has been formed. China has established extensive interdependent relations and almost no one-way dependent relations in the medical device trade. Among its major trading partners for medical devices, the interdependence of China with developed countries/regions, such as European and American countries and Japan, has generally deepened.

Spatiotemporal evolution of online attention to vaccines since 2011: An empirical study in China.

Since the outbreak of Coronavirus Disease 2019 (COVID-19), the Chinese government has taken a number of measures to effectively control the pandemic. By the end of 2021, China achieved a full vaccination rate higher than 85%. The Chinese Plan provides an important model for the global fight against COVID-19. Internet search reflects the public's attention toward and potential demand for a particular thing. Research on the spatiotemporal characteristics of online attention to vaccines can determine the spatiotemporal distribution of vaccine demand in China and provides a basis for global public health policy making. This study analyzes the spatiotemporal characteristics of online attention to vaccines and their influencing factors in 31 provinces/municipalities in mainland China with Baidu Index as the data source by using geographic concentration index, coefficient of variation, GeoDetector, and other methods. The following findings are presented. First, online attention to vaccines showed an overall upward trend in China since 2011, especially after 2016. Significant seasonal differences and an unbalanced monthly distribution were observed. Second, there was an obvious geographical imbalance in online attention to vaccines among the provinces/municipalities, generally exhibiting a spatial pattern of "high in the east and low in the west." Low aggregation and obvious spatial dispersion among the provinces/municipalities were also observed. The geographic distribution of hot and cold spots of online attention to vaccines has clear boundaries. The hot spots are mainly distributed in the central-eastern provinces and the cold spots are in the western provinces. Third, the spatiotemporal differences in online attention to vaccines are the combined result of socioeconomic level, socio-demographic characteristics, and disease control level.

Clinical Application of C-TIRADS Category and Contrast-Enhanced Ultrasound in Differential Diagnosis of Solid Thyroid Nodules Measuring ≥1 cm.

BACKGROUND The aim of this study was to investigate the performance of Chinese Thyroid Imaging Reporting and Data System (C-TIRADS) category combined with contrast-enhanced ultrasound (CEUS) in diagnosing thyroid cancer. MATERIAL AND METHODS From October 2020 to March 2021, 116 thyroid nodules from 113 patients who underwent conventional ultrasound and CEUS examinations at the General Hospital of Northern Theater Command were reviewed. In the conventional ultrasound examination, thyroid nodules were categorized by C-TIRADS. The nodules were reclassified based on CEUS scoring, then a combined diagnosis was made. The pathological results were taken as the criterion standard. To compare the diagnostic performance of the 3 methods according to the receiver operating characteristic curves produced for thyroid nodules. RESULTS After chi-square test, the 7 characteristics of enhancement patterns, internal homogeneity, wash-in, wash-out, ring enhancement, morphology, and contrast agent retention characteristics (CAR) were statistically different between malignant and benign tumors; A high diagnostic performance was demonstrated by C-TIRADS combined with CEUS in diagnosing thyroid cancer. The area under the curve (AUC), the sensitivity, and the specificity of C-TIRADS combined with CEUS for diagnosing thyroid cancer were 0.918 (95% CI: 0.852-0.961), 80.9% (95% CI: 69.1-89.8%), and 90.6% (95% CI: 79.3-96.9%), respectively. The AUC of C-TIRADS alone was significantly lower than that of C-TIRADS combined with CEUS (P=0.0056), while there was no significant difference between CEUS and C-TIRADS combined with CEUS (P=0.59). CONCLUSIONS The combined method of C-TIRADS and CEUS, with reduced rate of thyroid nodule biopsy and clinical application value, has higher diagnostic accuracy than the single diagnosis method.

Peripheral nerve defects repaired with autogenous vein grafts filled with platelet-rich plasma and active nerve microtissues and evaluated by novel multimodal ultrasound techniques.

BACKGROUND: Developing biocompatible nerve conduits that accelerate peripheral nerve regeneration, lengthening and functional recovery remains a challenge. The combined application of nerve microtissues and platelet-rich plasma (PRP) provides abundant Schwann cells (SCs) and various natural growth factors and can compensate for the deficiency of SCs in the nerve bridge, as well as the limitations of applying a single type of growth factor. Multimodal ultrasound evaluation can provide additional information on the stiffness and microvascular flow perfusion of the tissue. This study was designed to investigate the effectiveness of a novel tissue-engineered nerve graft composed of an autogenous vein, nerve microtissues and PRP in reconstructing a 12-mm tibial nerve defect and to explore the value of multimodal ultrasound techniques in evaluating the prognosis of nerve repair. METHODS: In vitro, nerve microtissue activity was first investigated, and the effects on SC proliferation, migration, factor secretion, and axonal regeneration of dorsal root ganglia (DRG) were evaluated by coculture with nerve microtissues and PRP. In vivo, seventy-five rabbits were equally and randomly divided into Hollow, PRP, Micro-T (Microtissues), Micro-T + PRP and Autograft groups. By analysing the neurological function, electrophysiological recovery, and the comparative results of multimodal ultrasound and histological evaluation, we investigated the effect of these new nerve grafts in repairing tibial nerve defects. RESULTS: Our results showed that the combined application of nerve microtissues and PRP could significantly promote the proliferation, secretion and migration of SCs and the regeneration of axons in the early stage. The Micro-T + PRP group and Autograft groups exhibited the best nerve repair 12 weeks postoperatively. In addition, the changes in target tissue stiffness and microvascular perfusion on multimodal ultrasound (shear wave elastography; contrast-enhanced ultrasonography; Angio PlaneWave UltrasenSitive, AngioPLUS) were significantly correlated with the histological results, such as collagen area percentage and VEGF expression, respectively. CONCLUSION: Our novel tissue-engineered nerve graft shows excellent efficacy in repairing 12-mm defects of the tibial nerve in rabbits. Moreover, multimodal ultrasound may provide a clinical reference for prognosis by quantitatively evaluating the stiffness and microvescular flow of nerve grafts and targeted muscles.

Discovery of potent BET bromodomain 1 stereoselective inhibitors using DNA-encoded chemical library selections.

BRDT, BRD2, BRD3, and BRD4 comprise the bromodomain and extraterminal (BET) subfamily which contain two similar tandem bromodomains (BD1 and BD2). Selective BD1 inhibition phenocopies effects of tandem BET BD inhibition both in cancer models and, as we and others have reported of BRDT, in the testes. To find novel BET BD1 binders, we screened >4.5 billion molecules from our DNA-encoded chemical libraries with BRDT-BD1 or BRDT-BD2 proteins in parallel. A compound series enriched only by BRDT-BD1 was resynthesized off-DNA, uncovering a potent chiral compound, CDD-724, with >2,000-fold selectivity for inhibiting BRDT-BD1 over BRDT-BD2. CDD-724 stereoisomers exhibited remarkable differences in inhibiting BRDT-BD1, with the R-enantiomer (CDD-787) being 50-fold more potent than the S-enantiomer (CDD-786). From structure­activity relationship studies, we produced CDD-956, which maintained picomolar BET BD1 binding potency and high selectivity over BET BD2 proteins and had improved stability in human liver microsomes over CDD-787. BROMOscan profiling confirmed the excellent pan-BET BD1 affinity and selectivity of CDD-787 and CDD-956 on BD1 versus BD2 and all other BD-containing proteins. A cocrystal structure of BRDT-BD1 bound with CDD-956 was determined at 1.82 Å and revealed BRDT-BD1­specific contacts with the αZ and αC helices that explain the high affinity and selectivity for BET BD1 versus BD2. CDD-787 and CDD-956 maintain cellular BD1-selectivity in NanoBRET assays and show potent antileukemic activity in acute myeloid leukemia cell lines. These BET BD1-specific and highly potent compounds are structurally unique and provide insight into the importance of chirality to achieve BET specificity.

Guazia, the earliest ovule without cupule but with unique integumentary lobes.

The earliest ovules in the Late Devonian (Famennian) are surrounded by a cupule that is involved in both protection and pollination, and generally have free integumentary lobes. Here we report a new taxon from the Famennian of China, Guazia dongzhiensis gen. et sp. nov. The terminally borne ovule is apparently acupulate (without cupule) and has four radially arranged wing-like integumentary lobes that are extensively fused, and folded lengthwise and inwards. Guazia provides evidence that not all Devonian seeds possess a cupule and it increases their diversity in integumentary lobes. This genus also suggests that the integuments develop new functions, probably including wind dispersal at the expense of the cupules.

FrCas9 is a CRISPR/Cas9 system with high editing efficiency and fidelity.

Genome editing technologies hold tremendous potential in biomedical research and drug development. Therefore, it is imperative to discover gene editing tools with superior cutting efficiency, good fidelity, and fewer genomic restrictions. Here, we report a CRISPR/Cas9 from Faecalibaculum rodentium, which is characterized by a simple PAM (5'-NNTA-3') and a guide RNA length of 21-22 bp. We find that FrCas9 could achieve comparable efficiency and specificity to SpCas9. Interestingly, the PAM of FrCas9 presents a palindromic sequence, which greatly expands its targeting scope. Due to the PAM sequence, FrCas9 possesses double editing-windows for base editor and could directly target the TATA-box in eukaryotic promoters for TATA-box related diseases. Together, our results broaden the understanding of CRISPR/Cas-mediated genome engineering and establish FrCas9 as a safe and efficient platform for wide applications in research, biotechnology and therapeutics.

Targeting Diagnosis of High-Risk Papillary Thyroid Carcinoma Using Ultrasound Contrast Agent With the BRAFV600E Mutation: An Experimental Study.

OBJECTIVE: High-risk papillary thyroid carcinoma (PTC) patients with BRAF mutation have lymph node and distant metastases and poor prognosis. Therefore, this study aims to develop a targeted ultrasound contrast agent for the BRAFV600E mutation to screen high-risk PTC at early stage. METHODS: The targeted lipid nanobubbles carrying BRAFV600E antibody were prepared using thin film hydration-sonication and avidin-biotin binding methods. The physicochemical properties and stability of the targeted nanobubbles were detected by transmission electron microscopy, atomic force microscopy, and confocal laser scanning microscopy. The target binding abilities of the targeted nanobubbles in the PTC cells (B-CPAP) overexpressed mutant BRAFV600E were evaluated by immunofluorescence staining, quantitative real-time polymerase chain reaction, western blot, and fluorescence microscopy. After PTC tumor models overexpressed mutant BRAFV600E were established, the enhanced images of targeted lipid nanobubbles and untargeted lipid nanobubbles on PTC tumors in nude mice were observed using contrast-enhanced ultrasound imaging. RESULTS: The targeted lipid nanobubbles revealed uniform, round morphology, and good stability with a nanoscale size. Besides, BRAFV600E monoclonal antibody was observed to be combined on the surface of lipid nanobubbles. Furthermore, the targeted nanobubbles had a good targeting diagnosis ability in PTC cells with BRAFV600E overexpression. Moreover, the targeted nanobubbles had better ultrasound enhancement and peak intensity of the time-intensity curve (P < .001) in PTC tumors with BRAFV600E overexpression as compared to the untargeted lipid nanobubbles. CONCLUSION: The targeted lipid nanobubbles carrying BRAFV600E antibody could be regarded as a potential targeted ultrasound contrast agent for the diagnosis of high-risk PTC.

Influence of Rural Social Capital and Production Mode on the Subjective Well-Being of Farmers and Herdsmen: Empirical Discovery on Farmers and Herdsmen in Inner Mongolia.

Rural areas are crucial to the realization of sustainable development goals (SDGs). Rural social capital is indispensable for these areas to fulfil the SDGs. As China pursues rural revitalization, it is essential to achieve the sustainable development of rural areas within the agropastoral transition zone (APTZ) in northern China. The same applies to the SDGs' realization in other APTZs across the globe. From the micro perspective of individual farmers and herdsmen, this article collected 732 microscopic datapoints through repeated rural surveys, and adopted the multivariate ordered probit model to empirically analyze how the subjective well-being of farmers and herdsmen in northern China's APTZ was affected by the individual (person) and collective (community) layers of rural social capital. Specifically, the production mode was introduced to study the relationship between social capital and subjective well-being, and social capital was measured by a self-designed theoretical analysis framework, which covered six dimensions and two layers (person and community). It was verified that the individual social capital and collective social capital were mutually replaceable in terms of the effect on the subjective well-being of farmers and herdsmen. Additionally, this article examined the influence of different production modes on the social capital and subjective well-being of farmers and herdsmen, and discussed how the same amount of social capital contributes differently to the subjective well-being of farmers and herdsmen under different production models. The results showed that: (1) Social capital significantly promoted the subjective well-being of farmers and herdsmen, and social network was the leading contributor among the dimensions of individual social capital, while social trust was the leading contributor among the dimensions of collective social capital. By the contribution to the subjective well-being of farmers and herdsmen, the six dimensions of social capital can be ranked as social network > social trust > social participation > social standard > social fame > common vision. (2) Individual social capital and collective social capital were mutually replaceable in terms of enhancing the subjective well-being of farmers and herdsmen; when the individual social capital was insufficient, the collective social capital would exert a much greater influence on the subjective well-being of farmers and herdsmen; when the individual social capital grows, the farmers and herdsmen would depend less on collective social capital. (3) The same amount of social capital contributes differently to the subjective well-being of farmers and herdsmen under different production models; by contribution strength, the production models can be ranked as pure herdsmen (PH) > pure farmers (PF) > non-farmers/herdsmen (NFH) > farmers + herdsmen (FH). Finally, several policy suggestions were provided to improve the subjective well-being of farmers and herdsmen in APTZ. The results show that collective social capital eliminates the negative effect of individual social capital shortage on the subjective well-being of farmers and herdsmen. Thus, it is suggested to consider not only the construction of macroscopic hardware environment, but also to roll out policies and measures to improve the subjective well-being from the micro perspective of farmers and herdsmen. These suggestions are concentrated on the implementation of China's rural revitalization strategy, and the creation of relevant institutions and cultural environments, as well as the optimization of the internal hierarchy of farmers and herdsmen's social capital. The research results may help to promote the subjective well-being of farmers and herdsmen in rural areas within the APTZs of China and the world, and provide a reference and a path to realize SDGs in China and similar places across the globe.

Gene knock-out chain reaction enables high disruption efficiency of HPV18 E6/E7 genes in cervical cancer cells.

A genome editing tool targeting the high-risk human papillomavirus (HPV) oncogene is a promising therapeutic strategy to treat HPV-related cervical cancer. To improve gene knockout efficiency, we developed a gene knockout chain reaction (GKCR) method for continually generating mutagenic disruptions and used this method to disrupt the HPV18 E6 and E7 genes. We verified that the GKCR Cas9/guide RNA (gRNA) cassettes could integrated into the targeted loci via homology-independent targeted insertion (HITI). The qPCR results revealed that the GKCR method enabled a relatively higher Cas9/gRNA cassette insertion rate than a control method (the common CRISPR-Cas9 strategy). Tracking of Indels by DEcomposition (TIDE) assay results showed that the GKCR method produced a significantly higher percentage of insertions or deletions (indels) in the HPV18 E6 and E7 genes. Furthermore, by targeting the HPV18 E6/E7 oncogenes, we found that the GKCR method significantly upregulated the P53/RB proteins and inhibited the proliferation and motility of HeLa cells. The GKCR method significantly improved the gene knockout efficiency of the HPV18 E6/E7 oncogenes, which might provide new insights into treatment of HPV infection and related cervical cancer.

Non-homologous dsODN increases the mutagenic effects of CRISPR-Cas9 to disrupt oncogene E7 in HPV positive cells.

Genome editing tools targeting high-risk human papillomavirus (HPV) oncogene could be a promising therapeutic strategy for the treatment of HPV-related cervical cancer. We aimed to improve the editing efficiency and detect off-target effects concurrently for the clinical translation strategy by using CRISPR-Cas9 system co-transfected with 34nt non-homologous double-stranded oligodeoxynucleotide (dsODN). We firstly tested this strategy on targeting the Green Fluorescent Protein (GFP) gene, of which the expression is easily observed. Our results showed that the GFP+ cells were significantly decreased when using GFP-sgRNAs with dsODN, compared to using GFP-sgRNAs without donors. By PCR and Sanger sequencing, we verified the dsODN integration into the break sites of the GFP gene. And by amplicon sequencing, we observed that the indels% of the targeted site on the GFP gene was increased by using GFP-sgRNAs with dsODN. Next, we went on to target the HPV18 E7 oncogene by using single E7-sgRNA and multiplexed E7-sgRNAs respectively. Whenever using single sgRNA or multiplexed sgRNAs, the mRNA expression of HPV18 E7 oncogene was significantly decreased when adding E7-sgRNAs with dsODN, compared to E7-sgRNAs without donor. And the indels% of the targeted sites on the HPV18 E7 gene was markedly increased by adding dsODN with E7-sgRNAs. Finally, we performed GUIDE-Seq to verify that the integrated dsODN could serve as the marker to detect off-target effects in using single or multiplexed two sgRNAs. And we detected fewer on-target reads and off-target sites in multiplexes compared to the single sgRNAs when targeting the GFP and the HPV18 E7 genes. Together, CRISPR-Cas9 system co-transfected with 34nt dsODN concurrently improved the editing efficiency and monitored off-target effects, which might provide new insights in the treatment of HPV infections and related cervical cancer.

The comparison of ZFNs, TALENs, and SpCas9 by GUIDE-seq in HPV-targeted gene therapy.

Zinc-finger nucleases (ZFNs), transcription activator-like endonucleases (TALENs), and CRISPR-associated Cas9 endonucleases are three major generations of genome editing tools. However, no parallel comparison about the efficiencies and off-target activity of the three nucleases has been reported, which is critical for the final clinical decision. We for the first time developed the genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) method in ZFNs and TALENs with novel bioinformatics algorithms to evaluate the off-targets. By targeting human papillomavirus 16 (HPV16), we compared the performance of ZFNs, TALENs, and SpCas9 in vivo. Our data showed that ZFNs with similar targets could generate distinct massive off-targets (287-1,856), and the specificity could be reversely correlated with the counts of middle "G" in zinc finger proteins (ZFPs). We also compared the TALENs with different N-terminal domains (wild-type [WT]/αN/ßN) and G recognition modules (NN/NH) and found the design (αN or NN) to improve the efficiency of TALEN inevitably increased off-targets. Finally, our results showed that SpCas9 was more efficient and specific than ZFNs and TALENs. Specifically, SpCas9 had fewer off-target counts in URR (SpCas9, n = 0; TALEN, n = 1; ZFN, n = 287), E6 (SpCas9, n = 0; TALEN, n = 7), and E7 (SpCas9, n = 4; TALEN, n = 36). Taken together, we suggest that for HPV gene therapies, SpCas9 is a more efficient and safer genome editing tool. Our off-target data could be used to improve the design of ZFNs and TALENs, and the universal in vivo off-target detection pipeline for three generations of artificial nucleases provided useful tools for genome engineering-based gene therapy.

Diagnosis of thyroid cancer using a TI-RADS-based computer-aided diagnosis system: a multicenter retrospective study.

OBJECTIVE: The purpose of this study was to use a computer-aided diagnosis (CAD) system based on the Thyroid Imaging, Reporting, and Data System (TI-RADS) to improve the diagnostic performance of thyroid cancer by analyzing clinical ultrasound imaging data. METHODS: A retrospective diagnostic study of ultrasound image sets was conducted at five hospitals in China. A CAD system based on TI-RADS was applied in this study, and the diagnostic performance of CAD system was tested through multi-center data. The performance of the CAD system was compared with the consensus of three experienced radiologists. The interobserver agreement for cancer diagnosis was calculated between the CAD system and the consensus of the three experienced radiologists. RESULTS: The CAD system performed well in the diagnosis of thyroid cancer, with an area under the curve (AUC) value of 0.902 (95% CI: 0.884-0.918), and obtained results similar to those of the three experienced radiologists. The CAD system performed better in the internal test set than in the external test set (AUC: 0.930 vs 0.877, respectively). The performance of the CAD system in the diagnosis of thyroid cancer for nodules of different sizes (<1 cm, 1-2 cm and ≥2 cm) was basically similar (accuracy: 84.6% vs 85% vs 84.2%). The CAD system can recognize 15 ultrasound features of thyroid nodules, most of which reached the level of 3 experienced radiologists (12/15, 85%). CONCLUSION: The CAD system achieved an improved AUC and similar sensitivity and specificity in the diagnosis of thyroid cancer compared with the consensus of experienced radiologists.

Contrast agent retention features in contrast-enhanced ultrasound: diagnostic performance for the prediction of papillary thyroid carcinoma.

PURPOSE: To report a new feature of contrast-enhanced ultrasound (CEUS) and its diagnostic performance for the prediction of papillary thyroid carcinoma (PTC). METHODS: This retrospective study was conducted from October 2018 to March 2019, including 276 patients with 308 thyroid nodules who underwent CEUS examinations prior to surgery (90 patients, 122 nodules) or fine needle aspiration (186 patients, 186 nodules). Quantitative analysis of CEUS features was performed using time-intensity curves. After surgery, tissue sections stained with HE and an anti-CD34 primary antibody were used to characterize the cell number and microvessel density. The nodules were divided into retention and non-retention groups. RESULTS: There were 168 malignant nodules and 140 benign nodules. The contrast-agent retention (CAR) feature was only observed in 52 papillary carcinomas. The CAR feature showed the sensitivity of 30.9% albeit the high specificity of 100%, for the diagnosis of thyroid cancers. The maximum slope coefficient of the washout index was significantly lower in the retention group than in the non-retention group (P < 0.001). The enhancement intensity during the late stage of enhancement index was significantly higher in the retention group than in the non-retention group (P < 0.001). The cell number and microvessel density in nodules with CAR features were higher (P < 0.001, P = 0.004). CONCLUSION: The combination of the retention pattern of the CEUS observed herein with other CEUS features may be a useful tool to improve the diagnostic of the PTC.