Structural and functional characterizations of infectivity and immune evasion of SARS-CoV-2 Omicron.

The SARS-CoV-2 Omicron variant with increased fitness is spreading rapidly worldwide. Analysis of cryo-EM structures of the spike (S) from Omicron reveals amino acid substitutions forging interactions that stably maintain an active conformation for receptor recognition. The relatively more compact domain organization confers improved stability and enhances attachment but compromises the efficiency of the viral fusion step. Alterations in local conformation, charge, and hydrophobic microenvironments underpin the modulation of the epitopes such that they are not recognized by most NTD- and RBD-antibodies, facilitating viral immune escape. Structure of the Omicron S bound with human ACE2, together with the analysis of sequence conservation in ACE2 binding region of 25 sarbecovirus members, as well as heatmaps of the immunogenic sites and their corresponding mutational frequencies, sheds light on conserved and structurally restrained regions that can be used for the development of broad-spectrum vaccines and therapeutics.

Memory B cell repertoire from triple vaccinees against diverse SARS-CoV-2 variants.

Omicron (B.1.1.529), the most heavily mutated SARS-CoV-2 variant so far, is highly resistant to neutralizing antibodies, raising concerns about the effectiveness of antibody therapies and vaccines1,2. Here we examined whether sera from individuals who received two or three doses of inactivated SARS-CoV-2 vaccine could neutralize authentic Omicron. The seroconversion rates of neutralizing antibodies were 3.3% (2 out of 60) and 95% (57 out of 60) for individuals who had received 2 and 3 doses of vaccine, respectively. For recipients of three vaccine doses, the geometric mean neutralization antibody titre for Omicron was 16.5-fold lower than for the ancestral virus (254). We isolated 323 human monoclonal antibodies derived from memory B cells in triple vaccinees, half of which recognized the receptor-binding domain, and showed that a subset (24 out of 163) potently neutralized all SARS-CoV-2 variants of concern, including Omicron. Therapeutic treatments with representative broadly neutralizing monoclonal antibodies were highly protective against infection of mice with SARS-CoV-2 Beta (B.1.351) and Omicron. Atomic structures of the Omicron spike protein in complex with three classes of antibodies that were active against all five variants of concern defined the binding and neutralizing determinants and revealed a key antibody escape site, G446S, that confers greater resistance to a class of antibodies that bind on the right shoulder of the receptor-binding domain by altering local conformation at the binding interface. Our results rationalize the use of three-dose immunization regimens and suggest that the fundamental epitopes revealed by these broadly ultrapotent antibodies are rational targets for a universal sarbecovirus vaccine.

PIPdb: a comprehensive plasmid sequence resource for tracking the horizontal transfer of pathogenic factors and antimicrobial resistance genes.

Plasmids, as independent genetic elements, carrying resistance or virulence genes and transfer them among different pathogens, posing a significant threat to human health. Under the 'One Health' approach, it is crucial to control the spread of plasmids carrying such genes. To achieve this, a comprehensive characterization of plasmids in pathogens is essential. Here we present the Plasmids in Pathogens Database (PIPdb), a pioneering resource that includes 792 964 plasmid segment clusters (PSCs) derived from 1 009 571 assembled genomes across 450 pathogenic species from 110 genera. To our knowledge, PIPdb is the first database specifically dedicated to plasmids in pathogenic bacteria, offering detailed multi-dimensional metadata such as collection date, geographical origin, ecosystem, host taxonomy, and habitat. PIPdb also provides extensive functional annotations, including plasmid type, insertion sequences, integron, oriT, relaxase, T4CP, virulence factors genes, heavy metal resistance genes and antibiotic resistance genes. The database features a user-friendly interface that facilitates studies on plasmids across diverse host taxa, habitats, and ecosystems, with a focus on those carrying antimicrobial resistance genes (ARGs). We have integrated online tools for plasmid identification and annotation from assembled genomes. Additionally, PIPdb includes a risk-scoring system for identifying potentially high-risk plasmids. The PIPdb web interface is accessible at https://nmdc.cn/pipdb.

The Dissemination of NDM-1 in Acinetobacter baumannii Strains.

Acinetobacter baumannii is a common pathogen in hospitals and usually causes bacteremia, pneumonia, meningitis, peritonitis and other diseases. Isolates carried NDM-1 gene can make several antibiotics such as carbapenems and other beta-lactams ineffective. Nowadays, the number of A. baumannii strains carrying NDM-1 has been climbing year by year in recent years. To characterise the transmission of NDM-1 in A. baumannii, we collected 2576 human-derived genomes of A. baumannii strains from NCBI database and found that 186 strains contained NDM-1 gene. The multi-locus sequence typing, phylogenetic tree, NDM-1 gene organization and the single nucleotide polymorphisms of NDM-1 were investigated. We hope that our work will provide a theoretical basis for the prevention of dissemination of NDM-1 in A. baumannii.

Semi-rational mutagenesis of an industrial Streptomyces fungicidicus strain for improved enduracidin productivity.

The biosynthesis of the valuable antibiotic enduracidin by Streptomyces fungicidicus TXX3120 is a complex multistep process. To identify the rate-limiting step of the entire biosynthetic process, we carried out a deep RNA sequencing towards the mycelia of TXX3120 at different fermentation stages. Comparative RNA-seq analysis indicated that the expression level of the endC gene during the enduracidin production phase was evidently lower than that of the other relevant genes to enduracidin biosynthesis. This result was further confirmed by quantitative RT-PCR, and the giant non-ribosomal peptide synthase (NRPS) encoded by endC was predicated to be the rate-limiting enzyme in enduracidin biosynthesis. To increase the expression of endC during the enduracidin production phase, a reporter-based selection system was developed by genetically replacing the initial part of the endC gene with a thiostrepton resistance gene (tsr), which will then act as a selectable marker to report the expression level of the rate-limiting gene endC, thereby facilitating the selection of enduracidin-overproducing mutants following random mutagenesis. After one round of mutagenesis, thiostrepton resistance selection, and restoration of the endC gene, three mutant strains with improved endC expression levels were obtained. Their highest enduracidin titers reached 9780.54, 9272.46, and 8849.06 U/mL, respectively representing 2.31-, 2.19-, and 2.09-fold of the initial industrial strain TXX3120. Our research provides a useful strategy for the rational breeding of industrial strains that synthesize complex natural products.

Adaptive Detection in Real-Time Gait Analysis through the Dynamic Gait Event Identifier.

The Dynamic Gait Event Identifier (DGEI) introduces a pioneering approach for real-time gait event detection that seamlessly aligns with the needs of embedded system design and optimization. DGEI creates a new standard for gait analysis by combining software and hardware co-design with real-time data analysis, using a combination of first-order difference functions and sliding window techniques. The method is specifically designed to accurately separate and analyze key gait events such as heel strike (HS), toe-off (TO), walking start (WS), and walking pause (WP) from a continuous stream of inertial measurement unit (IMU) signals. The core innovation of DGEI is the application of its dynamic feature extraction strategies, including first-order differential integration with positive/negative windows, weighted sleep time analysis, and adaptive thresholding, which together improve its accuracy in gait segmentation. The experimental results show that the accuracy rate of HS event detection is 97.82%, and the accuracy rate of TO event detection is 99.03%, which is suitable for embedded systems. Validation on a comprehensive dataset of 1550 gait instances shows that DGEI achieves near-perfect alignment with human annotations, with a difference of less than one frame in pulse onset times in 99.2% of the cases.

Spike N354 glycosylation augments SARS-CoV-2 fitness for human adaptation through structural plasticity.

Selective pressures have given rise to a number of SARS-CoV-2 variants during the prolonged course of the COVID-19 pandemic. Recently evolved variants differ from ancestors in additional glycosylation within the spike protein receptor-binding domain (RBD). Details of how the acquisition of glycosylation impacts viral fitness and human adaptation are not clearly understood. Here, we dissected the role of N354-linked glycosylation, acquired by BA.2.86 sub-lineages, as a RBD conformational control element in attenuating viral infectivity. The reduced infectivity is recovered in the presence of heparin sulfate, which targets the 'N354 pocket' to ease restrictions of conformational transition resulting in a 'RBD-up' state, thereby conferring an adjustable infectivity. Furthermore, N354 glycosylation improved spike cleavage and cell-cell fusion, and in particular escaped one subset of ADCC antibodies. Together with reduced immunogenicity in hybrid immunity background, these indicate a single spike amino acid glycosylation event provides selective advantage in humans through multiple mechanisms.

Comparative genomic analysis of Fusobacterium nucleatum reveals high intra-species diversity and cgmlst marker construction.

BACKGROUND: Fusobacterium nucleatum is a one of the most important anaerobic opportunistic pathogens in the oral and intestinal tracts of human and animals. It can cause various diseases such as infections, Lemierre's syndrome, oral cancer and colorectal cancer. The comparative genomic studies on the population genome level, have not been reported. RESULTS: We analyzed all publicly available Fusobacterium nucleatums' genomic data for a comparative genomic study, focusing on the pan-genomic features, virulence genes, plasmid genomes and developed cgmlst molecular markers. We found the pan-genome shows a clear open tendency and most of plasmids in Fusobacterium nucleatum are mainly transmitted intraspecifically. CONCLUSIONS: Our comparative analysis of Fusobacterium nucleatum systematically revealed the open pan-genomic features and phylogenetic tree based on cgmlst molecular markers. What's more, we also identified common plasmid typing among genomes. We hope that our study will provide a theoretical basis for subsequent functional studies.

Plasmer: an Accurate and Sensitive Bacterial Plasmid Prediction Tool Based on Machine Learning of Shared k-mers and Genomic Features.

Identification of plasmids in bacterial genomes is critical for many factors, including horizontal gene transfer, antibiotic resistance genes, host-microbe interactions, cloning vectors, and industrial production. There are several in silico methods to predict plasmid sequences in assembled genomes. However, existing methods have evident shortcomings, such as unbalance in sensitivity and specificity, dependency on species-specific models, and performance reduction in sequences shorter than 10 kb, which has limited their scope of applicability. In this work, we proposed Plasmer, a novel plasmid predictor based on machine-learning of shared k-mers and genomic features. Unlike existing k-mer or genomic-feature based methods, Plasmer employs the random forest algorithm to make predictions using the percent of shared k-mers with plasmid and chromosome databases combined with other genomic features, including alignment E value and replicon distribution scores (RDS). Plasmer can predict on multiple species and has achieved an average the area under the curve (AUC) of 0.996 with accuracy of 98.4%. Compared to existing methods, tests of both sliding sequences and simulated and de novo assemblies have consistently shown that Plasmer has outperforming accuracy and stable performance across long and short contigs above 500 bp, demonstrating its applicability for fragmented assemblies. Plasmer also has excellent and balanced performance on both sensitivity and specificity (both >0.95 above 500 bp) with the highest F1-score, which has eliminated the bias on sensitivity or specificity that was common in existing methods. Plasmer also provides taxonomy classification to help identify the origin of plasmids. IMPORTANCE In this study, we proposed a novel plasmid prediction tool named Plasmer. Technically, unlike existing k-mer or genomic features-based methods, Plasmer is the first tool to combine the advantages of the percent of shared k-mers and the alignment score of genomic features. This has given Plasmer (i) evident improvement in performance compared to other methods, with the best F1-score and accuracy on sliding sequences, simulated contigs, and de novo assemblies; (ii) applicability for contigs above 500 bp with highest accuracy, enabling plasmid prediction in fragmented short-read assemblies; (iii) excellent and balanced performance between sensitivity and specificity (both >0.95 above 500 bp) with the highest F1-score, which eliminated the bias on sensitivity or specificity that commonly existed in other methods; and (iv) no dependency of species-specific training models. We believe that Plasmer provides a more reliable alternative for plasmid prediction in bacterial genome assemblies.

Large-scale genomic survey and characterization of mcr genes carried by foodborne Cronobacter isolates.

IMPORTANCE: Cronobacter is an emerging foodborne opportunistic pathogen, which can cause neonatal meningitis, bacteremia, and NEC by contaminating food. However, the entire picture of foodborne Cronobacter carriage of the mcr genes is not known. Here, we investigated the mcr genes of Cronobacter isolates by whole-genome sequencing and found 133 previously undescribed Cronobacter isolates carrying mcr genes. Further genomic analysis revealed that these mcr genes mainly belonged to the mcr-9 and mcr-10. Genomic analysis of the flanking structures of mcr genes revealed that two core flanking structures were prevalent in foodborne Cronobacter isolates, and the flanking structure carrying IS1R was found for the first time in this study.

Monitoring microbial communities in intensive care units over one year in China.

Healthcare-associated infections (HAIs) seriously threaten patient health in intensive care units (ICUs). Profiling the microbial composition and diversity in ICU is important to prevent HAI-related spreading. Given that microbial communities vary across different environments, the time-scale characteristics of pathogens in ICUs have not been explored in China. In our study, to study the bacterial communities of two different ICUs in China, we proceeded dynamic monitoring using 16S rRNA sequencing for a whole year among the bed sheets, bed rails, shared pulse oximeters, bedside lockers, nurses' hands, floor, and carts. Our results showed that the microbial composition significantly changed within months. Significant differences in alpha and beta diversities were also observed among the 12 sampling months in each ICU. Additionally, we found the persistence of several HAI-related bacteria, including Acinetobacter, Pseudomonas, Staphylococcus, Escherichia, and Enterococcus. Source tracking analysis showed that most bacteria in both ICUs came from buildings or human skin. With deep investigations of hospital microbial surveillance on a long-term time-scale, we hope that these results will provide constructive guidelines to prevent the spread of HAIs in ICUs.

Genomic Characterization of a Uropathogenic Escherichia coli ST405 Isolate Harboring bla CTX-M-15-Encoding IncFIA-FIB Plasmid, bla CTX-M-24-Encoding IncI1 Plasmid, and Phage-Like Plasmid.

Escherichia coli sequence type 405 is an emerging antibiotic-resistant clonal group associated with the global dissemination of extended-spectrum ß-lactamase-producing E. coli. In this study, we report the genome assembly and characterization of a uropathogenic E. coli ST405 strain, SZESBLEC201, based on long and short reads obtained from the Nanopore and Illumina sequencing platforms, respectively. Whole-genome sequencing revealed that SZESBLEC201 harbors a 5,020,403 bp chromosome and three plasmids, namely, pSZESBLEC201-1, pSZESBLEC201-2, and pSZESBLEC201-3. pSZESBLEC201-1 (111,621 bp) belongs to the IncFIA-FIB type and harbors bla CTX-M-15. However, this plasmid does not harbor conjugative transfer-associated genes, rendering pSZESBLEC201-1 unable to be conjugatively transferred. pSZESBLEC201-2 (95,138 bp) is a phage-like plasmid that shows a strong genome synteny with Escherichia phage P1 but with the absence of mobile genetic elements and some regulatory genes. pSZESBLEC201-3 (92,865 bp) belongs to the IncI1 type and carries bla CTX-M-24. In contrast to pSZESBLEC201-1, pSZESBLEC201-3 retains its full active conjugation machinery and can be transferred via conjugation. The genetic features of the genome show that the SZESBLEC201 has a unique virulence pattern compared with genetically similar strains found in the same country (China). The plasmid backbones exhibit a high degree of similarity to those of geographically distant isolates, highlighting the global spread of bla CTX-M genes and the genome plasticity of this clonal group. The coexistence of two bla CTX-M variants in the same strain increases the risk of the emergence of new bla CTX-M variants. Further studies on phage-like plasmids are necessary to provide insights into their biological activities and clinical significance.

Characterization of the enhanced infectivity and antibody evasion of Omicron BA.2.75.

Recently emerged SARS-CoV-2 Omicron subvariant, BA.2.75, displayed a growth advantage over circulating BA.2.38, BA.2.76, and BA.5 in India. However, the underlying mechanisms for enhanced infectivity, especially compared with BA.5, remain unclear. Here, we show that BA.2.75 exhibits substantially higher affinity for host receptor angiotensin-converting enzyme 2 (ACE2) than BA.5 and other variants. Structural analyses of BA.2.75 spike shows its decreased thermostability and increased frequency of the receptor binding domain (RBD) in the "up" conformation under acidic conditions, suggesting enhanced low-pH-endosomal cell entry. Relative to BA.4/BA.5, BA.2.75 exhibits reduced evasion of humoral immunity from BA.1/BA.2 breakthrough-infection convalescent plasma but greater evasion of Delta breakthrough-infection convalescent plasma. BA.5 breakthrough-infection plasma also exhibits weaker neutralization against BA.2.75 than BA.5, mainly due to BA.2.75's distinct neutralizing antibody (NAb) escape pattern. Antibody therapeutics Evusheld and Bebtelovimab remain effective against BA.2.75. These results suggest BA.2.75 may prevail after BA.4/BA.5, and its increased receptor-binding capability could support further immune-evasive mutations.

Selection for Cheaper Amino Acids Drives Nucleotide Usage at the Start of Translation in Eukaryotic Genes.

Coding regions have complex interactions among multiple selective forces, which are manifested as biases in nucleotide composition. Previous studies have revealed a decreasing GC gradient from the 5'-end to 3'-end of coding regions in various organisms. We confirmed that this gradient is universal in eukaryotic genes, but the decrease only starts from the ∼ 25th codon. This trend is mostly found in nonsynonymous (ns) sites at which the GC gradient is universal across the eukaryotic genome. Increased GC contents at ns sites result in cheaper amino acids, indicating a universal selection for energy efficiency toward the N-termini of encoded proteins. Within a genome, the decreasing GC gradient is intensified from lowly to highly expressed genes (more and more protein products), further supporting this hypothesis. This reveals a conserved selective constraint for cheaper amino acids at the translation start that drives the increased GC contents at ns sites. Elevated GC contents can facilitate transcription but result in a more stable local secondary structure around the start codon and subsequently impede translation initiation. Conversely, the GC gradients at four-fold and two-fold synonymous sites vary across species. They could decrease or increase, suggesting different constraints acting at the GC contents of different codon sites in different species. This study reveals that the overall GC contents at the translation start are consequences of complex interactions among several major biological processes that shape the nucleotide sequences, especially efficient energy usage.

Identifying Two Novel Clusters in Calcium Oxalate Stones With Urinary Tract Infection Using 16S rDNA Sequencing.

Urinary stones and urinary tract infection (UTI) are the most common diseases in urology and they are characterized by high incidence and high recurrence rate in China. Previous studies have shown that urinary stones are closely associated with gut or urine microbiota. Calcium oxalate stones are the most common type of urinary stones. However, the profile of urinary tract microorganisms of calcium oxalate stones with UTI is not clear. In this research, we firstly found two novel clusters in patients with calcium oxalate stones (OA) that were associated with the WBC/HP (white blood cells per high-power field) level in urine. Two clusters in the OA group (OA1 and OA2) were distinguished by the key microbiota Firmicutes and Enterobacteriaceae. We found that Enterobacteriaceae enriched in OA1 cluster was positively correlated with several infection-related pathways and negatively correlated with a few antibiotics-related pathways. Meantime, some probiotics with higher abundance in OA2 cluster such as Bifidobacterium were positively correlated with antibiotics-related pathways, and some common pathogens with higher abundance in OA2 cluster such as Enterococcus were positively correlated with infection-related pathways. Therefore, we speculated that as a sub-type of OA disease, OA1 was caused by Enterobacteriaceae and the lack of probiotics compared with OA2 cluster. Moreover, we also sequenced urine samples of healthy individuals (CK), patients with UTI (I), patients with uric acid stones (UA), and patients with infection stones (IS). We identified the differentially abundant taxa among all groups. We hope the findings will be helpful for clinical treatment and diagnosis of urinary stones.

Improved 93-11 Genome and Time-Course Transcriptome Expand Resources for Rice Genomics.

In 2002, the first crop genome was published using the rice cultivar 93-11, which is the progenitor of the first super-hybrid rice. The genome sequence has served as a reference genome for the indica cultivars, but the assembly has not been updated. In this study, we update the 93-11 genome assembly to a gap-less sequence using ultra-depth single molecule real-time (SMRT) reads, Hi-C sequencing, reference-guided, and gap-closing approach. The differences in the genome collinearity and gene content between the 93-11 and the Nipponbare reference genomes confirmed to map the indica cultivar sequencing data to the 93-11 genome, instead of the reference. Furthermore, time-course transcriptome data showed that the expression pattern was consistently correlated with the stages of seed development. Alternative splicing of starch synthesis-related genes and genomic variations of waxy make it a novel resource for targeted breeding. Collectively, the updated high quality 93-11 genome assembly can improve the understanding of the genome structures and functions of Oryza groups in molecular breeding programs.

5-Hydroxymethylcytosine signatures in circulating cell-free DNA as diagnostic and predictive biomarkers for coronary artery disease.

BACKGROUND: The 5-hydroxymethylcytosine (5hmC) DNA modification is an epigenetic marker involved in a range of biological processes. Its function has been studied extensively in tumors, neurodegenerative diseases, and atherosclerosis. Studies have reported that 5hmC modification is closely related to the phenotype transformation of vascular smooth muscle cells and endothelial dysfunction. However, its role in coronary artery disease (CAD) has not been fully studied. RESULTS: To investigate whether 5hmC modification correlates with CAD pathogenesis and whether 5hmC can be used as a biomarker, we used a low-input whole-genome sequencing technology based on selective chemical capture (hmC-Seal) to firstly generate the 5hmC profiles in the circulating cell-free DNA(cfDNA) of CAD patients, including stable coronary artery disease (sCAD) patients and acute myocardial infarction (AMI) patients. We detected a significant difference of 5hmC enrichment in gene bodies from CAD patients compared with normal coronary artery (NCA) individuals. Our results showed that CAD patients can be well separated from NCA individuals by 5hmC markers. The prediction performance of the model established by differentially regulated 5hmc modified genes were superior to common clinical indicators for the diagnosis of CAD (AUC = 0.93) and sCAD (AUC = 0.93). Specially, we found that 5hmC markers in cfDNA showed prediction potential for AMI (AUC = 0.95), which was superior to that of cardiac troponin I, muscle/brain creatine kinase, and myoglobin. CONCLUSIONS: Our results suggest that 5hmC markers derived from cfDNA can serve as effective epigenetic biomarkers for minimally noninvasive diagnosis and prediction of CAD.

Spatial organization of heterologous metabolic system in vivo based on TALE.

For years, prokaryotic hosts have been widely applied in bio-engineering. However, the confined in vivo enzyme clustering of heterologous metabolic pathways in these organisms often results in low local concentrations of enzymes and substrates, leading to a low productive efficacy. We developed a new method to accelerate a heterologous metabolic system by integrating a transcription activator-like effector (TALE)-based scaffold system into an Escherichia coli chassis. The binding abilities of the TALEs to the artificial DNA scaffold were measured through ChIP-PCR. The effect of the system was determined through a split GFP study and validated through the heterologous production of indole-3-acetic acid (IAA) by incorporating TALE-fused IAA biosynthetic enzymes in E. coli. To the best of our knowledge, we are the first to use the TALE system as a scaffold for the spatial organization of bacterial metabolism. This technique might be used to establish multi-enzymatic reaction programs in a prokaryotic chassis for various applications.