CT perfusion imaging of the liver and the spleen can identify severe portal hypertension.

PURPOSE: To determine if hepatic and splenic perfusion parameters are useful in identifying severe portal hypertension (SPH). METHODS: The study enrolled 52 patients who underwent perfusion CT scan within one week before the hepatic venous pressure gradient (HVPG) measurement. A commercial software package was used for post-processing to generate hepatic and splenic perfusion parameters. Correlations were assessed using Pearson and Spearman rank correlation coefficients. Logistic regression was used to screen predictive parameters of SPH. The cut-off values of parameters for severe portal hypertension were calculated, as well as the sensitivity and specificity. RESULTS: There was a significant difference between SPH and non-severe portal hypertension (NSPH) in blood volume of liver (BVLiver), hepatic arterial fraction (HAF), hepatic arterial perfusion (HAP), portal venous perfusion (PVP), mean slope of increase in spleen (MSISpleen), BVSpleen, blood flow of spleen (BFSpleen), BVSpleen/Liver, and BVSpleen/Liver(P) (p < 0.05). The Spearman correlation coefficient was - 0.541 (p < 0.001) between BVSpleen/Live and HVPG and - 0.568 (p < 0.001) between BVSpleen/Liver(P) and HVPG. Using a BVSpleen/Liver value of 0.780 or BVSpleen/Liver(P) value of 1.061 as the cut-off value for the detection of SPH, the sensitivity and specificity were 94.7% and 72.7%, 100%, and 63.6% respectively. CONCLUSION: There was a moderate correlation between CT perfusion parameters BVSpleen/Liver, BVSpleen/Liver(P), and HVPG, which may be used to detect severe portal hypertension.

AAV1-hOTOF gene therapy for autosomal recessive deafness 9: a single-arm trial.

BACKGROUND: Autosomal recessive deafness 9, caused by mutations of the OTOF gene, is characterised by congenital or prelingual, severe-to-complete, bilateral hearing loss. However, no pharmacological treatment is currently available for congenital deafness. In this Article, we report the safety and efficacy of gene therapy with an adeno-associated virus (AAV) serotype 1 carrying a human OTOF transgene (AAV1-hOTOF) as a treatment for children with autosomal recessive deafness 9. METHODS: This single-arm, single-centre trial enrolled children (aged 1-18 years) with severe-to-complete hearing loss and confirmed mutations in both alleles of OTOF, and without bilateral cochlear implants. A single injection of AAV1-hOTOF was administered into the cochlea through the round window. The primary endpoint was dose-limiting toxicity at 6 weeks after injection. Auditory function and speech were assessed by appropriate auditory perception evaluation tools. All analyses were done according to the intention-to-treat principle. This trial is registered with Chinese Clinical Trial Registry, ChiCTR2200063181, and is ongoing. FINDINGS: Between Oct 19, 2022, and June 9, 2023, we screened 425 participants for eligibility and enrolled six children for AAV1-hOTOF gene therapy (one received a dose of 9 × 1011 vector genomes [vg] and five received 1·5 × 1012 vg). All participants completed follow-up visits up to week 26. No dose-limiting toxicity or serious adverse events occurred. In total, 48 adverse events were observed; 46 (96%) were grade 1-2 and two (4%) were grade 3 (decreased neutrophil count in one participant). Five children had hearing recovery, shown by a 40-57 dB reduction in the average auditory brainstem response (ABR) thresholds at 0·5-4·0 kHz. In the participant who received the 9 × 1011 vg dose, the average ABR threshold was improved from greater than 95 dB at baseline to 68 dB at 4 weeks, 53 dB at 13 weeks, and 45 dB at 26 weeks. In those who received 1·5 × 1012 AAV1-hOTOF, the average ABR thresholds changed from greater than 95 dB at baseline to 48 dB, 38 dB, 40 dB, and 55 dB in four children with hearing recovery at 26 weeks. Speech perception was improved in participants who had hearing recovery. INTERPRETATION: AAV1-hOTOF gene therapy is safe and efficacious as a novel treatment for children with autosomal recessive deafness 9. FUNDING: National Natural Science Foundation of China, National Key R&D Program of China, Science and Technology Commission of Shanghai Municipality, and Shanghai Refreshgene Therapeutics.

Preclinical evaluation of the efficacy and safety of AAV1-hOTOF in mice and nonhuman primates.

Pathogenic mutations in the OTOF gene cause autosomal recessive hearing loss (DFNB9), one of the most common forms of auditory neuropathy. There is no biological treatment for DFNB9. Here, we designed an OTOF gene therapy agent by dual-adeno-associated virus 1 (AAV1) carrying human OTOF coding sequences with the expression driven by the hair cell-specific promoter Myo15, AAV1-hOTOF. To develop a clinical application of AAV1-hOTOF gene therapy, we evaluated its efficacy and safety in animal models using pharmacodynamics, behavior, and histopathology. AAV1-hOTOF inner ear delivery significantly improved hearing in Otof-/- mice without affecting normal hearing in wild-type mice. AAV1 was predominately distributed to the cochlea, although it was detected in other organs such as the CNS and the liver, and no obvious toxic effects of AAV1-hOTOF were observed in mice. To further evaluate the safety of Myo15 promoter-driven AAV1-transgene, AAV1-GFP was delivered into the inner ear of Macaca fascicularis via the round window membrane. AAV1-GFP transduced 60%-94% of the inner hair cells along the cochlear turns. AAV1-GFP was detected in isolated organs and no significant adverse effects were detected. These results suggest that AAV1-hOTOF is well tolerated and effective in animals, providing critical support for its clinical translation.

An efficient numerical method on modified space-time sparse grid for time-fractional diffusion equation with nonsmooth data.

In this paper, we focus on developing a high efficient algorithm for solving d-dimension time-fractional diffusion equation (TFDE). For TFDE, the initial function or source term is usually not smooth, which can lead to the low regularity of exact solution. And such low regularity has a marked impact on the convergence rate of numerical method. In order to improve the convergence rate of the algorithm, we introduce the space-time sparse grid (STSG) method to solve TFDE. In our study, we employ the sine basis and the linear element basis for spatial discretization and temporal discretization, respectively. The sine basis can be divided into several levels, and the linear element basis can lead to the hierarchical basis. Then, the STSG can be constructed through a special tensor product of the spatial multilevel basis and the temporal hierarchical basis. Under certain conditions, the function approximation on standard STSG can achieve the accuracy order O(2-JJ) with O(2JJ) degrees of freedom (DOF) for d=1 and O(2Jd) DOF for d>1, where J denotes the maximal level of sine coefficients. However, if the solution changes very rapidly at the initial moment, the standard STSG method may reduce accuracy or even fail to converge. To overcome this, we integrate the full grid into the STSG, and obtain the modified STSG. Finally, we obtain the fully discrete scheme of STSG method for solving TFDE. The great advantage of the modified STSG method can be shown in the comparative numerical experiment.

Improving adenine and dual base editors through introduction of TadA-8e and Rad51DBD.

Base editors, including dual base editors, are innovative techniques for efficient base conversions in genomic DNA. However, the low efficiency of A-to-G base conversion at positions proximal to the protospacer adjacent motif (PAM) and the A/C simultaneous conversion of the dual base editor hinder their broad applications. In this study, through fusion of ABE8e with Rad51 DNA-binding domain, we generate a hyperactive ABE (hyABE) which offers improved A-to-G editing efficiency at the region (A10-A15) proximal to the PAM, with 1.2- to 7-fold improvement compared to ABE8e. Similarly, we develop optimized dual base editors (eA&C-BEmax and hyA&C-BEmax) with markedly improved simultaneous A/C conversion efficiency (1.2-fold and 1.5-fold improvement, respectively) compared to A&C-BEmax in human cells. Moreover, these optimized base editors catalyze efficiently nucleotide conversions in zebrafish embryos to mirror human syndrome or in human cells to potentially treat genetic diseases, indicating their great potential in broad applications for disease modeling and gene therapy.

Intermittent versus continuous control of tracheal cuff pressure in patients undergoing mechanical ventilation: A systematic review and meta-analysis.

AIMS AND OBJECTIVES: To evaluate the effects and safety of intermittent versus continuous control of cuff pressure in patients with mechanical ventilation. BACKGROUND: Tracheal cuff pressure management is vital to the prognosis of patients with mechanical ventilation. DESIGN: A meta-analysis. METHODS: This meta-analysis was conducted and reported according to the PRISMA checklist. We searched Pubmed, Embase, The Cochrane Library, BMJ Best Practice, Web of Science, ProQuest Dissertations, as well as the Chinese Biomedical Literature Database, Wanfang, and China national knowledge infrastructure databases up to 5 August 2022 for randomised controlled trials (RCTs) on the intermittent versus continuous control of cuff pressure. Review Manager 5.3 software was used for relevant data analysis. RESULTS: A total of 18 RCTs involving 1998 patients with mechanical ventilation were included. The synthesised outcomes indicated that continuous control of cuff pressure is beneficial to reduce the incidence of ventilator-associated pneumonia (VAP) [RR = 0.41, 95%CI (0.35, 0.49)], aspiration [RR = 0.36, 95%CI (0.21, 0.63)], duration of mechanical ventilation [MD = -3.23, 95%CI (-4.66, -1.79)], length of ICU stay [MD = -4.12, 95%CI (-5.40, -2.83)], and increase the volume of subglottic drainage [MD = 18.54, 95%CI (16.50, 20.58)]. There was no significant difference in the mortality between two groups [RR = 1.01, 95%CI (0.84, 1.21)]. Egger regression analyses showed that there were no obvious publication biases in the synthesised results (all p > .05). CONCLUSIONS: Existing evidence shows that compared with intermittent monitoring of cuff pressure, continuous monitoring of cuff pressure can reduce the occurrence of aspiration and VAP, shorten the patient's duration of mechanical ventilation and length of ICU stay. RELEVANCE TO CLINICAL PRACTICE: Continuous monitoring of cuff pressure is more beneficial and should be promoted in clinical nursing care of patients undergoing mechanical ventilation.

Engineering a precise adenine base editor with minimal bystander editing.

Adenine base editors (ABEs) catalyze A-to-G transitions showing broad applications, but their bystander mutations and off-target editing effects raise safety concerns. Through structure-guided engineering, we found ABE8e with an N108Q mutation reduced both adenine and cytosine bystander editing, and introduction of an additional L145T mutation (ABE9), further refined the editing window to 1-2 nucleotides with eliminated cytosine editing. Importantly, ABE9 induced very minimal RNA and undetectable Cas9-independent DNA off-target effects, which mainly installed desired single A-to-G conversion in mouse and rat embryos to efficiently generate disease models. Moreover, ABE9 accurately edited the A5 position of the protospacer sequence in pathogenic homopolymeric adenosine sites (up to 342.5-fold precision over ABE8e) and was further confirmed through a library of guide RNA-target sequence pairs. Owing to the minimized editing window, ABE9 could further broaden the targeting scope for precise correction of pathogenic single-nucleotide variants when fused to Cas9 variants with expanded protospacer adjacent motif compatibility. bpNLS, bipartite nuclear localization signals.

Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing.

Cytosine base editors (CBEs) efficiently generate precise C·G-to-T·A base conversions, but the activation-induced cytidine deaminase/apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (AID/APOBEC) protein family deaminase component induces considerable off-target effects and indels. To explore unnatural cytosine deaminases, we repurpose the adenine deaminase TadA-8e for cytosine conversion. The introduction of an N46L variant in TadA-8e eliminates its adenine deaminase activity and results in a TadA-8e-derived C-to-G base editor (Td-CGBE) capable of highly efficient and precise C·G-to-G·C editing. Through fusion with uracil glycosylase inhibitors and further introduction of additional variants, a series of Td-CBEs was obtained either with a high activity similar to that of BE4max or with higher precision compared to other reported accurate CBEs. Td-CGBE/Td-CBEs show very low indel effects and a background level of Cas9-dependent or Cas9-independent DNA/RNA off-target editing. Moreover, Td-CGBE/Td-CBEs are more efficient in generating accurate edits in homopolymeric cytosine sites in cells or mouse embryos, suggesting their accuracy and safety for gene therapy and other applications.

Research on safety and compliance of imported microbial inoculants using high-throughput sequencing.

Microbial inoculants are widely used in wastewater treatment, soil remediation, and biological control. Safety and compliance for active constituents are considered to be the most important measures of imported microbial inoculants. Microbial inoculants composition was commonly identified by phenotypic culture, which is time-consuming and labor intense with occasionally false negative results provided, and can only be tested for specific species. High-throughput sequencing (HTS), known for its non-targeted detection of unknown species composition in samples, is suitable for composition consistency identification and biosafety analysis of imported microbial inoculants. In this study, the application of HTS for microflora distribution and resistance gene was verified in microbial inoculants for environmental protection and then applicated in imported microbial inoculants. Both Illumina- and Nanopore-based HTS methods identified the same dominant bacterial species successfully in the imported microbial inoculants. The main component of bacterial species was Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, and Enterococcus faecium, and further confirmed with traditional methods. The antibiotic resistance genes Bacillus subtilis mprF, bcrA, blt, lmrB, rphB, tet(L), tmrB, vmlR, ykkC, and ykkD were detected in all samples. Our results indicated that HTS processes the application potential to identify the active ingredients of microbial inoculants. Therefore, rapid and accurate identification of the microbial compositions in microbial formulation products is of high importance for port biosafety supervision.

Dual base editor catalyzes both cytosine and adenine base conversions in human cells.

Although base editors are useful tools for precise genome editing, current base editors can only convert either adenines or cytosines. We developed a dual adenine and cytosine base editor (A&C-BEmax) by fusing both deaminases with a Cas9 nickase to achieve C-to-T and A-to-G conversions at the same target site. Compared to single base editors, A&C-BEmax's activity on adenines is slightly reduced, whereas activity on cytosines is higher and RNA off-target activity is substantially decreased.

Increasing the efficiency and targeting range of cytidine base editors through fusion of a single-stranded DNA-binding protein domain.

Cytidine base editors are powerful genetic tools that catalyse cytidine to thymidine conversion at specific genomic loci, and further improvement of the editing range and efficiency is critical for their broader applications. Through insertion of a non-sequence-specific single-stranded DNA-binding domain from Rad51 protein between Cas9 nickase and the deaminases, serial hyper cytidine base editors were generated with substantially increased activity and an expanded editing window towards the protospacer adjacent motif in both cell lines and mouse embryos. Additionally, hyeA3A-BE4max selectively catalysed cytidine conversion in TC motifs with a broader editing range and much higher activity (up to 257-fold) compared with eA3A-BE4max. Moreover, hyeA3A-BE4max specifically generated a C-to-T conversion without inducing bystander mutations in the haemoglobin gamma gene promoter to mimic a naturally occurring genetic variant for amelioration of ß-haemoglobinopathy, suggesting the therapeutic potential of the improved base editors.

Correction to: Increasing targeting scope of adenosine base editors in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants.

In the original publication the grant number is incorrectly published. The correct grant number should be read as "17140901600". The corrected contents are provided in this correction article. This work was partially supported by grants from the National Natural Science Foundation of China (Nos. 81670470 and 81600149), a grant from the Shanghai Municipal Commission for Science and Technology (17140901600, 18411953500 and 15JC1400201) and a grant from National Key Research and Development Program (2016YFC0905100).

Arabidopsis ENOR3 regulates RNAi-mediated antiviral defense.

Viruses can infect host plants to cause severe diseases and substantial agricultural loss, while plants have evolved RNA interference (RNAi) strategy to defend against viral infection. Despite enormous efforts, only a few host proteins in RNAi pathway were shown to mediate antiviral defense, including RNA-dependent RNA polymerase 1 (RDR1), RDR6, DICER-LIKE 2 (DCL2) and DCL4. In this study, we carried out a genetic screen for antiviral factors of RNAi pathway in Arabidopsis rdr6 background via inoculation with a 2b-deficient Cucumber Mosaic Virus (CMV-Δ2b). We identified a mutant susceptible to CMV-Δ2b, referred to as enhancer of rdr6 (enor) 3-1 rdr6, and found that ENOR3 encodes a functionally unknown protein with high homology to the mammalian Non Imprinted in Prader-Willi/Angelman (NIPA) magnesium transporters. ENOR3 inhibits accumulation of CMV-Δ2b and acts additively with RDR1, RDR6, DCL2 and DCL4 in antiviral defense. These results uncover that ENOR3 is a key component in antiviral RNAi pathway, and provide new insights into antiviral immunity.

Arabidopsis ALA1 and ALA2 Mediate RNAi-Based Antiviral Immunity.

RNA intereferencing (RNAi) pathway regulates antiviral immunity and mediates plant growth and development. Despite considerable research efforts, a few components in RNAi pathway have been revealed, including ARGONAUTEs (AGOs), DICER-LIKEs (DCLs), RNA-dependent RNA polymerase 1 and 6 (RDR1/6), and ALTERED MERISTEM PROGRAM 1 (AMP1). In this study, we performed a forward genetic screening for enhancers of rdr6 via inoculation of CMV2aTΔ2b, a 2b-deficient Cucumber Mosaic Virus that is unable to suppress RNAi-mediated antiviral immunity. We uncover that the membrane-localized flippase Aminophospholipid ATPase 1 (ALA1) cooperates with RDR6 and RDR1 to promote antiviral immunity and regulate fertility in Arabidopsis. Moreover, we find that ALA2, a homolog of ALA1, also participates in antiviral immunity. Our findings suggest that ALA1 and ALA2 act as novel components in the RNAi pathway and function additively with RDR1 and RDR6 to mediate RNAi-based antiviral immunity and plant development.

QTL mapping of cucumber fruit flesh thickness by SLAF-seq.

Cucumber is an agriculturally and economically important vegetable crop worldwide. Fruit flesh thickness is an important trait for cucumber and also a central determinant of yield, yet little is known about the underlying mechanism of this trait. In this study, bulked segregant analysis (BSA) combined with specific length amplified fragment sequencing (SLAF-seq) was applied to finely map the gene that underlies fruit flesh thickness in cucumber. A 0.19-Mb-long quantitative trait locus on chromosome 2 controlling fruit flesh thickness (QTL fft2.1) was identified and further confirmed by simple sequence repeat (SSR) marker-based classical QTL mapping in 138 F2 individuals. Gene prediction of this 0.19-Mb region identified 20 genes. Quantitative RT-PCR revealed higher expression levels of Csa2 M058670.1 (SET domain protein-lysine methyltransferase) in D8 (thick fruit flesh parent) compared with that in XUE1 (thin fruit flesh parent) during fruit development. Sequence alignment analysis of Csa2M058670.1 from thick and thin fruit flesh cucumber lines revealed a 4-bp deletion mutation in the promoter region of this candidate gene, which may result in the loss of Csa2M058670.1 activation in thin fruit flesh lines. The data presented herein suggest that Csa2M058670.1 is a possible candidate gene for controlling flesh thickness in cucumber.

The development and evaluation of a computerized diagnosis scheme for pneumoconiosis on digital chest radiographs.

PURPOSE: To diagnose pneumoconiosis using a computer-aided diagnosis system based on digital chest radiographs. METHODS: Lung fields were first extracted by combining the traditional Otsu-threshold method with a morphological reconstruction on digital radiographs (DRs), and then subdivided into six non-overlapping regions (region (a-f)). Twenty-two wavelet-based energy texture features were calculated exclusively from each region and selected using a decision tree algorithm. A support vector machine (SVM) with a linear kernel was trained using samples with texture features to classify an individual region of a healthy subject or a pneumoconiosis patient. The final classification results were obtained by integrating these individual classifiers with the weighted voting method. All models were developed on a dataset of 85 healthy controls and 40 stage I or II pneumoconiosis patients and validated by using the bootstrap resampling with replacement method. RESULTS: The areas under receiver operating characteristic curves (AUCs) of regions (c) and (f) were 0.688 and 0.563, which were worse than those of the other four regions. Region (c) and (f) were both excluded from the individual classifiers that were going to be assembled further. When built on the selected texture features, each individual SVM showed a higher diagnostic performance for the training set and the test set. The classification performance after an ensemble was 0.997 and 0.961 of the AUC value for the training and test sets, respectively. The final results were 0.974 ± 0.018 for AUC value and 0.929 ± 0.018 for accuracy. CONCLUSION: The integrated SVM model built on the selected feature set showed the highest diagnostic performance among all individual SVM models. The model has good potential in diagnosing pneumoconiosis based on digital chest radiographs.

Diagnosis of pancreatic carcinoma based on combined measurement of multiple serum tumor markers using artificial neural network analysis.

BACKGROUND: Artificial neural network (ANN) has demonstrated the ability to assimilate information from multiple sources to enable the detection of subtle and complex patterns. In this research, we evaluated an ANN model in the diagnosis of pancreatic cancer using multiple serum markers. METHODS: In this retrospective analysis, 913 serum specimens collected at the Department of General Surgery of Beijing Friendship Hospital were analyzed for carbohydrate antigen 19-9 (CA19-9), carbohydrate antigen 125 (CA125), and carcinoembryonic antigen (CEA). The three tumor marker values were used as inputs into an ANN and randomized into a training set of 658 (70.31% were malignant) and a test set of the remaining 255 samples (70.69% were malignant). The samples were also evaluated using a Logistic regression (LR) model. RESULTS: The ANN-derived composite index was superior to each of the serum tumor markers alone and the Logistic regression model. The areas under receiver operating characteristic curves (AUROC) was 0.905 (95% confidence Interval (CI) 0.868-0.942) for ANN, 0.812 (95% CI 0.762-0.863) for the Logistic regression model, 0.845 (95% CI 0.798-0.893) for CA19-9, 0.795 (95% CI 0.738-0.851) for CA125, and 0.800 (95% CI 0.746-0.854) for CEA. ANN analysis of multiple markers yielded a high level of diagnostic accuracy (83.53%) compared to LR (74.90%). CONCLUSION: The performance of ANN model in the diagnosis of pancreatic cancer is better than the single tumor marker and LR model.