Association of SARS-CoV-2 viral load with abnormal laboratory characteristics and clinical outcomes in hospitalised COVID-19 patients.

We conducted a retrospective, analytical cross-sectional and single-centre study that included 190 hospitalised COVID-19 patients in the Fujian Provincial Hospital South Branch between December 2022 and January 2023 to analyse the correlation of viral loads of throat swabs with clinical progression and outcomes. To normalise the Ct value as quantification of viral loads, we used RNase P gene as internal control gene and subtracted the Ct value of SARS-CoV-2 N gene from the Ct value of RNase P gene, termed △Ct. Most patients were discharged (84.2%), and only 10 (5.6%) individuals who had a lower △Ct value died. The initial △Ct value of participants was also significantly correlated with some abnormal laboratory characteristics, and the duration time of SARS-CoV-2 was longer in patients with severe symptoms and a lower △Ct value at admission. Our study suggested that the △Ct value may be used as a predictor of disease progression and outcomes in hospitalised COVID-19 patients.

Efficacy and Safety of Repetitive Transcranial Magnetic Stimulation for Poststroke Memory Disorder: A Meta-Analysis and Systematic Review.

BACKGROUND: Patients with post-stroke memory disorder (PSMD) have poor quality of life and it is necessary to identify more beneficial stimulation protocols for treatment with repetitive transcranial magnetic stimulation (rTMS). This meta-analysis was conducted to investigate the efficacy and safety of rTMS for improving memory performance, global cognition, and activities of daily living (ADL) among patients with PSMD. METHODS: The PubMed, Embase, Cochrane Central Register of Controlled Trials (CENTRAL), Chinese Biomedical Literature Database, Chinese National Knowledge Infrastructure, China Science and Technology Journal Database, and Wanfang databases were screened to identify relevant randomized controlled trials. The primary outcome was memory performance; secondary outcomes included global cognition, ADL, and adverse events. STATA software was used to perform data synthesis. RESULTS: Five articles with a total of 192 participants were included. The results indicated that rTMS was superior to control treatments for improving memory performance (mean difference [MD] = 1.73, 95% CI [Confidence Interval] [0.85, 2.60], p < 0.001), global cognition (MD = 2.44, 95% CI [0.96, 3.93], p < 0.001), and ADL (MD = 10.29, 95% CI [5.10, 15.48], p < 0.001). No significant differences were found between the low-frequency (LF) and high-frequency (HF) rTMS subgroups (p = 0.47, I2 = 0.00%) or between the sham rTMS and non-rTMS subgroups (p = 0.94, I2 = 0.00%). Four studies did not reported adverse events. CONCLUSIONS: rTMS may improve memory function, global cognition, and the ability to perform ADL in patients with PSMD. LF-rTMS and HF-rTMS may have equal efficacy for treatment of PSMD. Future studies should consider extending the follow-up period to explore the safety and long-term efficacy of rTMS for treatment of PSMD and the appropriate choice of placebo for clinical trials of this treatment.

Effectiveness and safety of repetitive transcranial magnetic stimulation on memory disorder in stroke: A protocol for systematic review and meta-analysis.

BACKGROUND: Approximately 23% to 55% of patients have memory impairments with a greatly negative effect on daily life 3 months after stroke. Repetitive transcranial magnetic stimulation (rTMS) has been widely used in the rehabilitation of stroke as it is safe, painless, and noninvasive. Moreover, few studies have investigated the effect of rTMS on poststroke memory disorder (PSMD). However, the efficacy of rTMS is not consistent and the optional stimulation frequency is unclear. Therefore, this protocol aims to evaluate the clinical effect and safety of rTMS on PSMD by analyzing results from randomized controlled trials. METHODS: Search strategies will be performed on seven databases: PubMed, EMBASE, CENTRAL, Chinese Biomedical Literature Database (CBM), Chinese National Knowledge Infrastructure (CNKI), Wan Fang, and Technology Periodical Database (VIP). Only randomized controlled trials registered before August 2021 will be included. Additionally, the language will be limited to English or Chinese. For the outcome, we will focus on the Rivermead Behavioral Memory Test. Additionally, the Montreal Cognitive Assessment, Mini-mental State Examination, Modified Barthel Index, and advent events will be included. Two authors will independently select the study, extract data, and assess quality. Moreover, disagreements will be resolved by the third author. STATA 14 and Review Manager 5.4 will be used to perform the analysis. We will evaluate bias risk in accordance with the Cochrane Handbook for Systematic Reviews of Interventions. To assess the quality of evidence, the Grading of Recommendations Assessment, Development, and Evaluation method will be employed. RESULTS: This study will provide a comprehensive analysis of the current evidence on rTMS for PSMD. CONCLUSION: A reliable conclusion regarding whether rTMS is an effective and safe intervention for patients with PSMD and the effect of stimulation frequency and sham stimulation will be provided. This study will provide new insights for TMS in treating PSMD, and offer appropriate treatmentoptions to patients and clinicians. PROSPERO REGISTRATION NUMBER: CRD42021282439.

Lb2Cas12a and its engineered variants mediate genome editing in human cells.

Cas12a-mediated targeted genome engineering strategies have enabled a broad range of research and clinical applications. However, the limited target-selection spectrum and low activity/fidelity remain a bottleneck for its widespread application in precision site-specific human genome editing. Therefore, there exists an acute need to identify novel Cas12a nucleases with improved features for genome editing. By screening a range of candidate Cas12a nucleases, here we demonstrate that Lb2Cas12a possesses genome editing activity in human cells and it has greater flexibility in PAM (5'-BYYV-3') selection. Furthermore, we engineered Lb2Cas12a to generate variants (Lb2Cas12a-RVR and Lb2Cas12a-RR), which greatly expands the target-selection spectrum. Our study illustrated that Lb2Cas12a could be harnessed as additional genome editing tool for the manipulation of human genome.

Engineered FnCas12a with enhanced activity through directional evolution in human cells.

Clustered regularly interspaced short palindromic repeat-Cas12a has been harnessed to manipulate the human genome; however, low cleavage efficiency and stringent protospacer adjacent motif hinder the use of Cas12a-based therapy and applications. Here, we have described a directional evolving and screening system in human cells to identify novel FnCas12a variants with high activity. By using this system, we identified IV-79 (enhanced activity FnCas12a, eaFnCas12a), which possessed higher DNA cleavage activity than WT FnCas12a. Furthermore, to widen the target selection spectrum, eaFnCas12a was engineered through site-directed mutagenesis. eaFnCas12a and one engineered variant (eaFnCas12a-RR), used for correcting human RS1 mutation responsible for X-linked retinoschisis, had a 3.28- to 4.04-fold improved activity compared with WT. Collectively, eaFnCas12a and its engineered variants can be used for genome-editing applications that requires high activity.

Learning a Single Model with a Wide Range of Quality Factors for JPEG Image Artifacts Removal.

Lossy compression brings artifacts into the compressed image and degrades the visual quality. In recent years, many compression artifacts removal methods based on convolutional neural network (CNN) have been developed with great success. However, these methods usually train a model based on one specific value or a small range of quality factors. Obviously, if the test images quality factor does not match to the assumed value range, then degraded performance will be resulted. With this motivation and further consideration of practical usage, a highly robust compression artifacts removal network is proposed in this paper. Our proposed network is a single model approach that can be trained for handling a wide range of quality factors while consistently delivering superior or comparable image artifacts removal performance. To demonstrate, we focus on the JPEG compression with quality factors, ranging from 1 to 60. Note that a turnkey success of our proposed network lies in the novel utilization of the quantization tables as part of the training data. Furthermore, it has two branches in parallel-i.e., the restoration branch and the global branch. The former effectively removes the local artifacts, such as ringing artifacts removal. On the other hand, the latter extracts the global features of the entire image that provides highly instrumental image quality improvement, especially effective on dealing with the global artifacts, such as blocking, color shifting. Extensive experimental results performed on color and grayscale images have clearly demonstrated the effectiveness and efficacy of our proposed single-model approach on the removal of compression artifacts from the decoded image.

High-fidelity SaCas9 identified by directional screening in human cells.

CRISPR-Staphylococcus aureus Cas9 (CRISPR-SaCas9) has been harnessed as an effective in vivo genome-editing tool to manipulate genomes. However, off-target effects remain a major bottleneck that precludes safe and reliable applications in genome editing. Here, we characterize the off-target effects of wild-type (WT) SaCas9 at single-nucleotide (single-nt) resolution and describe a directional screening system to identify novel SaCas9 variants with desired properties in human cells. Using this system, we identified enhanced-fidelity SaCas9 (efSaCas9) (variant Mut268 harboring the single mutation of N260D), which could effectively distinguish and reject single base-pair mismatches. We demonstrate dramatically reduced off-target effects (approximately 2- to 93-fold improvements) of Mut268 compared to WT using targeted deep-sequencing analyses. To understand the structural origin of the fidelity enhancement, we find that N260, located in the REC3 domain, orchestrates an extensive network of contacts between REC3 and the guide RNA-DNA heteroduplex. efSaCas9 can be broadly used in genome-editing applications that require high fidelity. Furthermore, this study provides a general strategy to rapidly evolve other desired CRISPR-Cas9 traits besides enhanced fidelity, to expand the utility of the CRISPR toolkit.

[Serological feature and molecular mechanism for a case with A307 subgroup].

OBJECTIVE: To explore the serological feature and molecular mechanism for a case with A307 subgroup of the ABO blood group system. METHODS: Serological assay was carried out to determine the ABO blood group of the proband and his family members. Genotypes for exons 1 to 7 of the ABO gene were determined with sequence-specific primer polymerase chain reaction (SSP-PCR) and direct sequencing. The impact of the variant on the stability of alpha-1,3-N-acetylgalactosaminyltransferase (GTA) was predicted through construction of a 3D molecular model. RESULTS: The proband, his brother and daughter were diagnosed with Aend phenotype by serological analysis. Their ABO genotype was determined as A307/O02, with heterozygous c.467C>T (p.P156L) and c.745C>T (p.R249W) variants identified in exon 7 of the ABO gene. Molecular modeling suggested that the p.R249W variant may alter the number of hydrogen bonds between the amino acids. The protein was predicted to have a decreased Δ Δ G value of thermodynamic stability. CONCLUSION: The p.R249W variant may give rise to the A307 subgroup by reducing the stability of the GTA enzyme, leading to serological features of Aend phenotype.

Efficient cleavage resolves PAM preferences of CRISPR-Cas in human cells.

Clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) of bacterial adaptive immunity have been adopted as a powerful and versatile tool for manipulation of the genome. This paradigm has been widely applied in biological research and treatments of animal or cellular disease models. A critical feature of CRISPR-Cas is the protospacer adjacent motif (PAM), which dictates the DNA target recognition mechanism of Cas proteins. While, direct identifying functional PAM sequences in human cells remains a challenge. Here, we developed a positive screen system termed PAM-DOSE (PAM Definition by Observable Sequence Excision) to delineate the functional PAMs in human cells. Specifically, the PAM libraries for CRISPR-Cas (SpCas9, SpCas9-NG, FnCas12a, AsCas12a, LbCas12a and MbCas12a) were generated and the corresponding CRISPR-Cas mediated cleaved fragments with functional PAM in human cells were harvested for DNA sequencing, which could be tracked and visualized with either florescence microscopy or flow cytometry analysis. With this system, we identified the functional PAMs of CRISPR-Cas members. We also found that spacer sequence affects the PAM preference of Cas proteins. This method will facilitate identification of functional PAMs for Cas-mediated human genome editing applications.

Boosting activity of high-fidelity CRISPR/Cas9 variants using a tRNAGln-processing system in human cells.

CRISPR/Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. High-fidelity Cas9 variants have been identified; however, they often have reduced activity, constraining their utility, which presents a major challenge for their use in research applications and therapeutics. Here we developed a tRNAGln-processing system to restore the activity of multiple high-fidelity Cas9 variants in human cells, including SpCas9-HF1, eSpCas9, and xCas9. Specifically, acting on previous observations that small guide RNAs (sgRNAs) harboring an extra A or G (A/G) in the first 5' nucleotide greatly affect the activity of high-fidelity Cas9 variants and that tRNA-sgRNA fusions improve Cas9 activity, we investigated whether a GN20 sgRNA fused to different tRNAs (G-tRNA-N20) could restore the activity of SpCas9 variants in human cells. Using flow cytometry, a T7E1 assay, deep sequencing-based DNA cleavage activity assays, and HEK-293 cells, we observed that a tRNAGln-sgRNA fusion system enhanced the activity of Cas9 variants, which could be harnessed for efficient correction of a pathogenic mutation in the retinoschisin 1 (RS1) gene, resulting in 6- to 8-fold improved Cas9 activity. We propose that the tRNA-processing system developed here specifically for human cells could facilitate high-fidelity Cas9-mediated human genome-editing applications.

Efficient Human Genome Editing Using SaCas9 Ribonucleoprotein Complexes.

Genome editing using RNA-guided nucleases in their ribonucleoprotein (RNP) form represents a promising strategy for gene modification and therapy because they are free of exogenous DNA integration and have reduced toxicity in vivo and ex vivo. However, genome editing by Cas9 nuclease from Staphylococcus aureus (SaCas9) has not been reported in its RNP form, which recognizes a longer protospacer adjacent motif (PAM), 5'-NNGRRT-3', compared with Streptococcus pyogenes Cas9 (SpCas9) of 5'-NGG-3' PAM. Here, SaCas9-RNP-mediated genome editing is reported in human cells. The SaCas9-RNP displayed efficient genome editing activities of enhanced green fluorescent protein (EGFP) coding gene as well as three endogenous genes (OPA1, RS1, and VEGFA). Further, SaCas9-RNP is successfully implemented to correct a pathogenic RS1 mutation for X-linked juvenile retinoschisis. It is also shown that off-target effects triggered by SaCas9-RNP are undetectable by targeted deep sequencing. Collectively, this study demonstrates the potential of SaCas9-RNP-mediated genome editing in human cells, which could facilitate genome-editing-based therapy.

[Application and optimization of CRISPR/Cas system in bacteria].

Clustered regular interspaced short palindromic repeats (CRISPR) system has been widely used in recent years. Compared with traditional genome editing technology, CRISPR/Cas system has notable advantages, including high editing efficiency, high specificity, low cost and the convenience for manipulation. Type Ⅱ and Ⅴ CRISPR/Cas system only requires a single Cas9 protein or a single Cpf1 protein as effector nucleases for cutting double-stranded DNA, developed as genome editing tools. At present, CRISPR/Cas9 technology has been successfully applied to the genome editing of eukaryotes such as zebrafish, mice and human cells, whereas limited progress has been made in the genome editing of bacteria. In our review, we describe CRISPR/Cas system, its mechanism and summarize the optimization and progress of genome editing in bacteria.

Basic and Clinical Application of Adeno-Associated Virus-Mediated Genome Editing.

Traditional gene therapy (gene replacement) has made a breakthrough in treating inherited diseases. Adeno-associated virus (AAV) has emerged as a highly promising vector with innate ability, boosting the development of gene replacement and gene targeting. With the recent advance of engineered nucleases that work efficiently in human cells, AAV mediated-genome editing with nucleases has raised hopes for in situ gene therapy of inherited and non-inherited diseases. Here, the applications of AAV-mediated genome editing are highlighted, and the prospect of AAV and nucleases that will render extension of such success in clinical gene therapy is discussed.

[Identification and pedigree analysis of ABO subgroup B303].

OBJECTIVE: To explore the molecular mechanism of a case of ABO discrepancies based on the results of blood group serology. METHODS: Five cases of the two-generation pedigrees were analyzed. ABO genotypes were determined using serological tests. DNA sequence analysis was performed on exon 6, exon 7 and intron 3 of the 5 cases to confirm the genotypes of a proband with B subgroup and 4 family members. RESULTS: There were 3 cases of subgroup AB3 and 1 case of subgroup B3 among the 5 family members. The genotypes were identified as A102/B303 and O02/B303, respectively. B303 differed from B101 by intron 3 point mutation (intron3 + 5G>A). CONCLUSION: The point mutation of intron 3 (intron 3+5G>A) is specific in B303.

Compounds with inhibitory activity on peristalsis from the seeds of Holarrhena antidysenterica.

Eight compounds were isolated from the seeds of Holarrhena antidysenterica Wall.ex A.DC. On the basis of physico-chemical properties and spectroscopic data, holarrhenanan (1) was identified as a new compound, compounds 2-3 were isolated from H. antidysenterica for the first time, and five known compounds were also obtained. Inhibitory effects of some compounds and extracts to the intestinal peristalsis were evaluated. Results showed that the extracts and compounds 4, 6 exhibited remarkable inhibitory effects with tension inhibition rate of 32.77, 32.77% and amplitude inhibition rate of 59.51, 55.98%, respectively on the vitro rabbit intestinal peristalsis.

SaCas9 Requires 5'-NNGRRT-3' PAM for Sufficient Cleavage and Possesses Higher Cleavage Activity than SpCas9 or FnCpf1 in Human Cells.

CRISPR/Cas9-mediated gene therapy holds great promise for the treatment of human diseases. The protospacer adjacent motif (PAM), the sequence adjacent to the target sequence, is an essential targeting component for the design of CRISPR/Cas9-mediated gene editing. However, currently, very few studies have attempted to directly study the PAM sequence in human cells. To address this issue, the authors develop a dual fluorescence reporter system that could be harnessed for identifying functional PAMs for genome editing endonuclease, including Cas9. With this system, the authors investigate the effects of different PAM sequences for SaCas9, which is small and has the advantage of allowing in vivo genome editing, and found only 5'-NNGRRT-3' PAM could induced sufficient target cleavage with multi-sites. The authors also found SaCas9 possesses higher activity than SpCas9 or FnCpf1 via plasmids (episomal) and chromosomes with integrated eGFP-based comparison. Taken together, the authors show that a dual fluorescence reporter system is a means to identifying a functional PAM and quantitatively comparing the efficiency of different genome editing endonucleases with the similar or identical target sequence in human cells.