Deep learning enables stochastic optical reconstruction microscopy-like superresolution image reconstruction from conventional microscopy.

Despite its remarkable potential for transforming low-resolution images, deep learning faces significant challenges in achieving high-quality superresolution microscopy imaging from wide-field (conventional) microscopy. Here, we present X-Microscopy, a computational tool comprising two deep learning subnets, UR-Net-8 and X-Net, which enables STORM-like superresolution microscopy image reconstruction from wide-field images with input-size flexibility. X-Microscopy was trained using samples of various subcellular structures, including cytoskeletal filaments, dot-like, beehive-like, and nanocluster-like structures, to generate prediction models capable of producing images of comparable quality to STORM-like images. In addition to enabling multicolour superresolution image reconstructions, X-Microscopy also facilitates superresolution image reconstruction from different conventional microscopic systems. The capabilities of X-Microscopy offer promising prospects for making superresolution microscopy accessible to a broader range of users, going beyond the confines of well-equipped laboratories.

Evaluation of guide-free Cas9-induced genomic damage and transcriptome changes in pig embryos.

Cas9 protein without sgRNAs can induce genomic damage at the cellular level in vitro. However, whether the detrimental effects occur in embryos after Cas9 treatment remains unknown. Here, using pig embryos as subjects, we observed that Cas9 protein transcribed from injected Cas9 mRNA can persist until at least the blastocyst stage. Cas9 protein alone can induce genome damage in preimplantation embryos, represented by the increased number of phosphorylated histone H2AX foci on the chromatin fiber, which led to apoptosis and decreased cell number of blastocysts. In addition, single-blastocyst RNA sequencing confirmed that Cas9 protein without sgRNAs can cause changes in the blastocyst transcriptome, depressing embryo development signal pathways, such as cell cycle, metabolism, and cellular communication-related signal pathways, while activating apoptosis and necroptosis signal pathways, which together resulted in impaired preimplantation embryonic development. These results indicated that attention should be given to the detrimental effects caused by the Cas9 protein when using CRISPR-Cas9 for germline genome editing, especially for the targeted correction of human pathological mutations using germline gene therapy.

Precise genome editing of the Kozak sequence enables bidirectional and quantitative modulation of protein translation to anticipated levels without affecting transcription.

None of the existing approaches for regulating gene expression can bidirectionally and quantitatively fine-tune gene expression to desired levels. Here, on the basis of precise manipulations of the Kozak sequence, which has a remarkable influence on translation initiation, we proposed and validated a novel strategy to directly modify the upstream nucleotides of the translation initiation codon of a given gene to flexibly alter the gene translation level by using base editors and prime editors. When the three nucleotides upstream of the translation initiation codon (named KZ3, part of the Kozak sequence), which exhibits the most significant base preference of the Kozak sequence, were selected as the editing region to alter the translation levels of proteins, we confirmed that each of the 64 KZ3 variants had a different translation efficiency, but all had similar transcription levels. Using the ranked KZ3 variants with different translation efficiencies as predictors, base editor- and prime editor-mediated mutations of KZ3 in the local genome could bidirectionally and quantitatively fine-tune gene translation to the anticipated levels without affecting transcription in vitro and in vivo. Notably, this strategy can be extended to the whole Kozak sequence and applied to all protein-coding genes in all eukaryotes.

Base editors: development and applications in biomedicine.

Base editor (BE) is a gene-editing tool developed by combining the CRISPR/Cas system with an individual deaminase, enabling precise single-base substitution in DNA or RNA without generating a DNA double-strand break (DSB) or requiring donor DNA templates in living cells. Base editors offer more precise and secure genome-editing effects than other conventional artificial nuclease systems, such as CRISPR/Cas9, as the DSB induced by Cas9 will cause severe damage to the genome. Thus, base editors have important applications in the field of biomedicine, including gene function investigation, directed protein evolution, genetic lineage tracing, disease modeling, and gene therapy. Since the development of the two main base editors, cytosine base editors (CBEs) and adenine base editors (ABEs), scientists have developed more than 100 optimized base editors with improved editing efficiency, precision, specificity, targeting scope, and capacity to be delivered in vivo, greatly enhancing their application potential in biomedicine. Here, we review the recent development of base editors, summarize their applications in the biomedical field, and discuss future perspectives and challenges for therapeutic applications.

Differences in the effectiveness of the high-efficient concentrated pretreatment method on the norovirus detection in oysters and mussels.

Oysters and mussels are important vectors for norovirus (NoV). An efficient pretreatment method for NoV detection in oysters based on ISO 15216-2:2019 was established in our previous work, but its effectiveness for other types of shellfish remains unknown. Therefore, this study systematically compared the differences between the standard and modified ISO methods in detecting NoV for oysters and mussels. Using the standard ISO method, the recovery rates of NoV in oysters (2.10 ± 0.80 %) and mussels (2.39 ± 0.56 %) were comparable (p > 0.05, unpaired t-test). In contrast, the virus recovery rates in oysters (19.83 ± 3.64 %) and mussels (46.96 ± 3.55 %) were both significantly improved by the modified method. Also, a significant difference was found between the virus recovery rates in two shellfish (p < 0.05, unpaired t-test), resulting in a 2.09-fold difference in their virus concentrations. Additionally, the limits of detection at 95 % probability of the modified ISO method for oysters and mussels could both reach 3.33 × 103 copies/g of digestive glands. Finally, the modified ISO method has been successfully applied in commercial oysters (14/27, 51.85 %) and mussels (15/23, 65.22 %), and the results indicated a significant difference in NoV recovery rates between two shellfish (p < 0.05, one-way analysis of variance). In summary, the modified ISO method showed higher virus recovery rates than the standard ISO method, which would be used as an essential tool for NoV detection in oysters and mussels.

Multiplexed base editing through Cas12a variant-mediated cytosine and adenine base editors.

Cas12a can process multiple sgRNAs from a single transcript of CRISPR array, conferring advantages in multiplexed base editing when incorporated into base editor systems, which is extremely helpful given that phenotypes commonly involve multiple genes or single-nucleotide variants. However, multiplexed base editing through Cas12a-derived base editors has been barely reported, mainly due to the compromised efficiencies and restricted protospacer-adjacent motif (PAM) of TTTV for wild-type Cas12a. Here, we develop Cas12a-mediated cytosine base editor (CBE) and adenine base editor (ABE) systems with elevated efficiencies and expanded targeting scope, by combining highly active deaminases with Lachnospiraceae bacterium Cas12a (LbCas12a) variants. We confirm that these CBEs and ABEs can perform efficient C-to-T and A-to-G conversions, respectively, on targets with PAMs of NTTN, TYCN, and TRTN. Notably, multiplexed base editing can be conducted using the developed CBEs and ABEs in somatic cells and embryos. These Cas12a variant-mediated base editors will serve as versatile tools for multiplexed point mutation, which is notably important in genetic improvement, disease modeling, and gene therapy.

3DPro: Querying Complex Three-Dimensional Data with Progressive Compression and Refinement.

Large-scale three-dimensional spatial data has gained increasing attention with the development of self-driving, mineral exploration, CAD, and human atlases. Such 3D objects are often represented with a polygonal model at high resolution to preserve accuracy. This poses major challenges for 3D data management and spatial queries due to the massive amounts of 3D objects, e.g., trillions of 3D cells, and the high complexity of 3D geometric computation. Traditional spatial querying methods in the Filter-Refine paradigm have a major focus on indexing-based filtering using approximations like minimal bounding boxes and largely neglect the heavy computation in the refinement step at the intra-geometry level, which often dominates the cost of query processing. In this paper, we introduce 3DPro, a system that supports efficient spatial queries for complex 3D objects. 3DPro uses progressive compression of 3D objects preserving multiple levels of details, which significantly reduces the size of the objects and has the data fit into memory. Through a novel Filter-Progressive-Refine paradigm, 3DPro can have query results returned early whenever possible to minimize decompression and geometric computations of 3D objects in higher resolution representations. Our experiments demonstrate that 3DPro out-performs the state-of-the-art 3D data processing techniques by up to an order of magnitude for typical spatial queries.

Efficient 3D Spatial Queries for Complex Objects.

3D spatial data has been generated at an extreme scale from many emerging applications, such as high definition maps for autonomous driving and 3D Human BioMolecular Atlas. In particular, 3D digital pathology provides a revolutionary approach to map human tissues in 3D, which is highly promising for advancing computer-aided diagnosis and understanding diseases through spatial queries and analysis. However, the exponential increase of data at 3D leads to significant I/O, communication, and computational challenges for 3D spatial queries. The complex structures of 3D objects such as bifurcated vessels make it difficult to effectively support 3D spatial queries with traditional methods. In this article, we present our work on building an efficient and scalable spatial query system, iSPEED, for large-scale 3D data with complex structures. iSPEED adopts effective progressive compression for each 3D object with successive levels of detail. Further, iSPEED exploits structural indexing for complex structured objects in distance-based queries. By querying with data represented in successive levels of details and structural indexes, iSPEED provides an option for users to balance between query efficiency and query accuracy. iSPEED builds in-memory indexes and decompresses data on-demand, which has a minimal memory footprint. iSPEED provides a 3D spatial query engine that can be invoked on-demand to run many instances in parallel implemented with, but not limited to, MapReduce. We evaluate iSPEED with three representative queries: 3D spatial joins, 3D nearest neighbor query, and 3D spatial proximity estimation. The extensive experiments demonstrate that iSPEED significantly improves the performance of existing spatial query systems.

Rapid screening for antigenic characterization of GII.17 norovirus strains with variations in capsid gene.

The emergence of the novel GII.17 Kawasaki 2014 norovirus variant raising the interest of the public, has replaced GII.4 as the predominant cause of noroviruses outbreaks in East Asia during 2014-2015. Antigenic variation of the capsid protein is considered as one of the key mechanisms of norovirus evolution. In this study, we screened a panel of GII.17 mutants. First, we produced norovirus P proteins using cell-free protein synthesis (CFPS) system, comparing the results to pure proteins expressed in a cell-based system. Next, we determined the binding capability of specific monoclonal antibody (mAb) 2D11 using a unique set of wild-type GII.17 strains. Results of the EIA involving a panel of mutant cell-free proteins indicated that Q298 was the key residue within loop 1. These data highlighted the essential residues in the linear antibody binding characteristics of novel GII.17. Furthermore, it supported the CFPS as a promising tool for rapidly screening mutants via the scalable expression of norovirus P proteins.

AGBE: a dual deaminase-mediated base editor by fusing CGBE with ABE for creating a saturated mutant population with multiple editing patterns.

Establishing saturated mutagenesis in a specific gene through gene editing is an efficient approach for identifying the relationships between mutations and the corresponding phenotypes. CRISPR/Cas9-based sgRNA library screening often creates indel mutations with multiple nucleotides. Single base editors and dual deaminase-mediated base editors can achieve only one and two types of base substitutions, respectively. A new glycosylase base editor (CGBE) system, in which the uracil glycosylase inhibitor (UGI) is replaced with uracil-DNA glycosylase (UNG), was recently reported to efficiently induce multiple base conversions, including C-to-G, C-to-T and C-to-A. In this study, we fused a CGBE with ABE to develop a new type of dual deaminase-mediated base editing system, the AGBE system, that can simultaneously introduce 4 types of base conversions (C-to-G, C-to-T, C-to-A and A-to-G) as well as indels with a single sgRNA in mammalian cells. AGBEs can be used to establish saturated mutant populations for verification of the functions and consequences of multiple gene mutation patterns, including single-nucleotide variants (SNVs) and indels, through high-throughput screening.

Double knock-in pig models with elements of binary Tet-On and phiC31 integrase systems for controllable and switchable gene expression.

Inducible expression systems are indispensable for precise regulation and in-depth analysis of biological process. Binary Tet-On system has been widely employed to regulate transgenic expression by doxycycline. Previous pig models with tetracycline regulatory elements were generated through random integration. This process often resulted in uncertain expression and unpredictable phenotypes, thus hindering their applications. Here, by precise knock-in of binary Tet-On 3G elements into Rosa26 and Hipp11 locus, respectively, a double knock-in reporter pig model was generated. We characterized excellent properties of this system for controllable transgenic expression both in vitro and in vivo. Two attP sites were arranged to flank the tdTomato to switch reporter gene. Single or multiple gene replacement was efficiently and faithfully achieved in fetal fibroblasts and nuclear transfer embryos. To display the flexible application of this system, we generated a pig strain with Dox-inducing hKRASG12D expression through phiC31 integrase-mediated cassette exchange. After eight months of Dox administration, squamous cell carcinoma developed in the nose, mouth, and scrotum, which indicated this pig strain could serve as an ideal large animal model to study tumorigenesis. Overall, the established pig models with controllable and switchable transgene expression system will provide a facilitating platform for transgenic and biomedical research.

The VP2 protein exhibits cross-interaction to the VP1 protein in norovirus GII.17.

Norovirus is a major cause of acute gastroenteritis worldwide. Like the major capsid protein (VP1), the minor capsid protein (VP2) also contains a hypervariable domain. Generally, a hypervariable domain is functionally driven. However, many functions of VP2 remain unknown and worth exploring. Without sufficient sequences and an available crystallographic model, it is difficult to explore VP2's mysteries. As a helper of stabilizing and coordinating the formation of virus-like particles (VLPs), we asked whether VP2 interacted with the major capsid protein (VP1) in GII.17 and if so, what the key interaction residues were. Here, we reported cross-interaction among four strains represented four clusters of GII.17, and the VP1 interaction domain of VP2 (174-179aa) was found. However, the VP1 interaction domain of VP2 was not universal in different clusters of GII.17. VP2 might evolve in a different pattern from VP1. Additionally, in contrast to previous reports, we found that VP2 localized in the cytoplasm. More possibilities of VP2 should be further explored.

Receptor profile and immunogenicity of the non-epidemic norovirus GII.8 variant.

Noroviruses are causative agents of acute nonbacterial gastroenteritis epidemics worldwide. There are various genotypes, among which the non-epidemic genotype GII.8 can cause norovirus outbreaks. We previously demonstrated that the immunogenicity of GII.8 differed from that of epidemic variants. This study aimed to comprehensively compare the receptor profile and immunogenicity of the GII.8 variant with those of the epidemic variants. Using the baculovirus-insect cell expression system, we observed that recombinant capsid protein VP1 of the norovirus GII.8 GZ2017-L601 strain formed virus-like particles (VLPs) with a diameter of approximately 30 nm, as evidenced by transmission electron microscopy analysis. The GII.8 VLPs showed weak or moderate binding with all secretor histo-blood group antigens (HBGAs), but not with non-secretors, as evidenced by the HBGA-VLP binding test. The GII.8 VLP antiserum obtained from immunized BALB/c mice was tested for cross-reactivity with other norovirus genotypes (n = 28). The results showed that this antiserum demonstrated moderate cross-reactivity with GI.1, GII.3, and GII.15; however, no cross-reactivity with the epidemic variants of GII.2, GII.4, and GII.17 was observed. Additionally, the blocking-antibody activity of GII.8 antisera against GII.4 VLP-HBGAs and GII.17 VLP-HBGAs interactions and the cross-blocking of GII.8 VLP-HBGAs interactions by GI.1 and GII.4 antisera were evaluated using the HBGAs-VLP blocking test. However, no cross-blocking effect was observed. In summary, the characterization of norovirus GII.8 VLPs and derived antisera revealed that the GII.8 immunogenicity differed from that of epidemic variants.

Global prevalence of norovirus in cases of acute gastroenteritis from 1997 to 2021: An updated systematic review and meta-analysis.

BACKGROUND: The worldwide response towards the acute gastroenteritis epidemic was well known, but the absence of an updated systematic review of global norovirus epidemiology in cases of gastroenteritis existed. We aimed to conduct and update a systematic review and meta-analysis of studies assessing norovirus prevalence among gastroenteritis patients worldwide. METHODS: Four databases (PubMed, EMBASE, Cochrane Library, and Web of Science) were searched for epidemiological papers from 2014 to 2021 which applied the PCR method to access the prevalence of norovirus in acute gastroenteritis patients more than a full year. Statistical analysis was conducted using R-4.0.0 software. RESULTS: A total of 405 records with 842, 926 cases were included. The pooled prevalence of norovirus was 16% (95%CI 15, 17). Children under 5 years old were at a higher risk with norovirus. A higher prevalence was seen in South America (22%, 95% CI 18, 27), while other continents showed a similar result with the overall prevalence of norovirus. No association was found between national income level and norovirus prevalence. A gradient of decreasing prevalence was noticed from community (20%, 95% CI 16, 24) to outpatients (18%, 95% CI 16, 20) to hospital setting (included both in- and outpatients, 17%, 95% CI 16, 19) to inpatients (15%, 95% CI 13, 17). CONCLUSION: Norovirus were associated with 16% acute gastroenteritis globally. To fully understand the prevalence of norovirus worldwide, the continual surveillance of norovirus epidemics was required.

Development and Application of a Novel Rapid and Throughput Method for Broad-Spectrum Anti-Foodborne Norovirus Antibody Testing.

Foodbone norovirus (NoV) is the leading cause of acute gastroenteritis worldwide. Candidate vaccines are being developed, however, no licensed vaccines are currently available for managing NoV infections. Screening for stimulated antibodies with broad-spectrum binding activities can be performed for the development of NoV polyvalent vaccines. In this study, we aimed to develop an indirect enzyme-linked immunosorbent assay (ELISA) for testing the broad spectrum of anti-NoV antibodies. Capsid P proteins from 28 representative NoV strains (GI.1-GI.9 and GII.1-GII.22 except GII.11, GII.18, and GII.19) were selected, prepared, and used as coating antigens on one microplate. Combined with incubation and the horseradish peroxidase chromogenic reaction, the entire process for testing the spectrum of unknown antibodies required 2 h for completion. The intra-assay and inter-assay coefficients of variation were less than 10%. The new method was successfully performed with monoclonal antibodies and polyclonal antibodies induced by multiple antigens. In conclusion, the indirect ELISA assay developed in this study had a good performance of reliability, convenience, and high-throughput screening for broad-spectrum antibodies.

Development of a High-Efficiency Immunomagnetic Enrichment Method for Detection of Human Norovirus via PAMAM Dendrimer/SA-Biotin Mediated Cascade-Amplification.

Human norovirus is a common cause of acute gastroenteritis worldwide, and oysters have been found to be the main carriers for its spread. The lack of efficient pre-treatment methods has been a major bottleneck limiting the detection of viruses in oysters. In this study, we established a novel immunomagnetic enrichment method using polyamidoamine (PAMAM) dendrimer/SA-biotin-mediated cascade amplification for reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) detection. We compared the capture efficiency of traditional immunomagnetic enrichment, biotin-amplified immunomagnetic enrichment, and PAMAM dendrimer/SA-biotin-mediated cascade-amplification immunomagnetic enrichment. The optimal capture efficiency of the novel method was 44.26 ± 1.45%, which increased by 183.17% (P < 0.01) and 18.09% (P < 0.05) compared with the first two methods, respectively. Three methods were all applied in detecting norovirus in 44 retail oysters, the detection rate of the PAMAM dendrimer/SA-biotin-mediated method was 25.0%, which was higher than those of traditional IME (15.90%) and SA-biotin-amplified IME (18.80%) by 9.1 and 6.2%, respectively. In conclusion, the novel method can be applied for the rapid detection of norovirus in oysters, which can help reduce the cost and time of detection and improve detection rates.

Antigenic Diversity of Human Norovirus Capsid Proteins Based on the Cross-Reactivities of Their Antisera.

Human norovirus (HuNoV), which is the major causative agent of acute gastroenteritis, has broad antigenic diversity; thus, the development of a broad-spectrum vaccine is challenging. To establish the relationship between viral genetic diversity and antigenic diversity, capsid P proteins and antisera of seven GI and 16 GII HuNoV genotypes were analyzed. Enzyme-linked immunosorbent assays showed that HuNoV antisera strongly reacted with the homologous capsid P proteins (with titers > 5 × 104). However, 17 (73.9%) antisera had weak or no cross-reactivity with heterologous genotypes. Interestingly, the GII.5 antiserum cross-reacted with seven (30.4%) capsid P proteins (including pandemic genotypes GII.4 and GII.17), indicating its potential use for HuNoV vaccine development. Moreover, GI.2 and GI.6 antigens reacted widely with heterologous antisera (n ≥ 5). Sequence alignment and phylogenetic analyses of the P proteins revealed conserved regions, which may be responsible for the immune crossover reactivity observed. These findings may be helpful in identifying broad-spectrum epitopes with clinical value for the development of a future vaccine.