Structural insights into curdlan degradation via a glycoside hydrolase containing a disruptive carbohydrate-binding module.

BACKGROUND: Degradation via enzymatic processes for the production of valuable ß-1,3-glucooligosaccharides (GOS) from curdlan has attracted considerable interest. CBM6E functions as a curdlan-specific ß-1,3-endoglucanase, composed of a glycoside hydrolase family 128 (GH128) module and a carbohydrate-binding module (CBM) derived from family CBM6. RESULTS: Crystallographic analyses were conducted to comprehend the substrate specificity mechanism of CBM6E. This unveiled structures of both apo CBM6E and its GOS-complexed form. The GH128 and CBM6 modules constitute a cohesive unit, binding nine glucoside moieties within the catalytic groove in a singular helical conformation. By extending the substrate-binding groove, we engineered CBM6E variants with heightened hydrolytic activities, generating diverse GOS profiles from curdlan. Molecular docking, followed by mutation validation, unveiled the cooperative recognition of triple-helical ß-1,3-glucan by the GH128 and CBM6 modules, along with the identification of a novel sugar-binding residue situated within the CBM6 module. Interestingly, supplementing the CBM6 module into curdlan gel disrupted the gel's network structure, enhancing the hydrolysis of curdlan by specific ß-1,3-glucanases. CONCLUSIONS: This study offers new insights into the recognition mechanism of glycoside hydrolases toward triple-helical ß-1,3-glucans, presenting an effective method to enhance endoglucanase activity and manipulate its product profile. Furthermore, it discovered a CBM module capable of disrupting the quaternary structures of curdlan, thereby boosting the hydrolytic activity of curdlan gel when co-incubated with ß-1,3-glucanases. These findings hold relevance for developing future enzyme and CBM cocktails useful in GOS production from curdlan degradation.

Profiling A-to-I RNA editing during mouse somatic reprogramming at the single-cell level.

Mouse somatic cells can be reprogrammed into induced pluripotent stem cells through a highly heterogeneous process regulated by numerous biological factors, including adenosine-to-inosine (A-to-I) RNA editing. In this study, we analyzed A-to-I RNA editing sites using a single-cell RNA sequencing (scRNA-seq) dataset with high-depth and full-length coverage. Our method revealed that A-to-I RNA editing frequency varied widely at the single-cell level and underwent dynamic changes. We also found that A-to-I RNA editing level was correlated with the expression of the RNA editing enzyme ADAR1. The analysis combined with gene ontology (GO) enrichment revealed that ADAR1-dependent A-to-I editing may downregulate the expression levels of Igtp, Irgm2, Mndal, Ifi202b, and Tapbp in the early stage, to inhibit the pathways of cellular response to interferon-beta and regulation of protein complex stability to promote mesenchymal-epithelial transition (MET). Notably, we identified a negative correlation between A-to-I RNA editing frequency and the expression of certain genes, such as Nras, Ube2l6, Zfp987, and Adsl.

A map of bat virus receptors derived from single-cell multiomics.

Bats are considered reservoirs of many lethal zoonotic viruses and have been implicated in several outbreaks of emerging infectious diseases, such as SARS-CoV, MERS-CoV, and SARS-CoV-2. It is necessary to systematically derive the expression patterns of bat virus receptors and their regulatory features for future research into bat-borne viruses and the prediction and prevention of pandemics. Here, we performed single-nucleus RNA sequencing (snRNA-seq) and single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq) of major organ samples collected from Chinese horseshoe bats (Rhinolophus affinis) and systematically checked the expression pattern of bat-related virus receptors and chromatin accessibility across organs and cell types, providing a valuable dataset for studying the nature of infection among bat-borne viruses.

High-resolution 3D spatiotemporal transcriptomic maps of developing Drosophila embryos and larvae.

Drosophila has long been a successful model organism in multiple biomedical fields. Spatial gene expression patterns are critical for the understanding of complex pathways and interactions, whereas temporal gene expression changes are vital for studying highly dynamic physiological activities. Systematic studies in Drosophila are still impeded by the lack of spatiotemporal transcriptomic information. Here, utilizing spatial enhanced resolution omics-sequencing (Stereo-seq), we dissected the spatiotemporal transcriptomic changes of developing Drosophila with high resolution and sensitivity. We demonstrated that Stereo-seq data can be used for the 3D reconstruction of the spatial transcriptomes of Drosophila embryos and larvae. With these 3D models, we identified functional subregions in embryonic and larval midguts, uncovered spatial cell state dynamics of larval testis, and revealed known and potential regulons of transcription factors within their topographic background. Our data provide the Drosophila research community with useful resources of organism-wide spatiotemporally resolved transcriptomic information across developmental stages.

Cas9-Based Local Enrichment and Genomics Sequence Revision of Megabase-Sized Shark IgNAR Loci.

The 0.8-Mb Ig new Ag receptor (IgNAR) region of the whitespotted bamboo shark (Chiloscyllium plagiosum) is incompletely assembled in Chr_44 of the reference genome. Here we used Cas9-assisted targeting of chromosome segments (CATCH) to enrich the 2 Mb region of the Chr_44 IgNAR loci and sequenced it by PacBio and next-generation sequencing. A fragment >3.13 Mb was isolated intact from the RBCs of sharks. The target was enriched 245.531-fold, and sequences had up to 94% coverage with a 255× mean depth. Compared with the previously published sequences, 20 holes were filled, with a total length of 3508 bp. In addition, we report five potential germline V alleles of IgNAR1 from six sharks that may belong to two clusters of the IgNAR. Our results provide a new method to research the germline of large Ig gene segments, as well as provide the enhanced bamboo shark IgNAR gene loci with fewer gaps.

Characterization of Residue Specific Protein Folding and Unfolding Dynamics in Cells.

In this work, we measured the millisecond residue specific protein folding and unfolding dynamics in E. coli cells for two protein GB3 mutants using NMR. The results show that the protein folding and unfolding dynamics in cells is different from that in buffer. Through a two-site exchange model, it is shown that both the population and the exchange rate are changed by the E. coli cellular environment. Further investigation suggests that the change is likely due to the quinary interaction with crowded molecules in the cell. Our work underlines the importance of cellular environment to protein folding kinetics and thermodynamics although this environmental effect may not be large enough to change the protein structure.