Extrachromosomal circular DNA orchestrates genome heterogeneity in urothelial bladder carcinoma.

Rationale: Extrachromosomal circular DNA is a hallmark of cancer, but its role in shaping the genome heterogeneity of urothelial bladder carcinoma (UBC) remains poorly understood. Here, we comprehensively analyzed the features of extrachromosomal circular DNA in 80 UBC patients. Methods: We performed whole-genome/exome sequencing (WGS/WES), Circle-Seq, single-molecule real-time (SMRT) long-read sequencing of circular DNA, and RNA sequencing (RNA-Seq) on 80 pairs of tumor and AT samples. We used our newly developed circular DNA analysis software, Circle-Map++ to detect small extrachromosomal circular DNA from Circle-Seq data. Results: We observed a high load and significant heterogeneity of extrachromosomal circular DNAs in UBC, including numerous single-locus and complex chimeric circular DNAs originating from different chromosomes. This includes highly chimeric circular DNAs carrying seven oncogenes and circles from nine chromosomes. We also found that large tumor-specific extrachromosomal circular DNAs could influence genome-wide gene expression, and are detectable in time-matched urinary sediments. Additionally, we found that the extrachromosomal circular DNA correlates with hypermutation, copy number variation, oncogene amplification, and clinical outcome. Conclusions: Overall, our study provides a comprehensive extrachromosomal circular DNA map of UBC, along with valuable data resources and bioinformatics tools for future cancer and extrachromosomal circular DNA research.

Understanding the Role of Large Language Models in Personalizing and Scaffolding Strategies to Combat Academic Procrastination.

Traditional interventions for academic procrastination often fail to capture the nuanced, individual-specific factors that underlie them. Large language models (LLMs) hold immense potential for addressing this gap by permitting open-ended inputs, including the ability to customize interventions to individuals' unique needs. However, user expectations and potential limitations of LLMs in this context remain underexplored. To address this, we conducted interviews and focus group discussions with 15 university students and 6 experts, during which a technology probe for generating personalized advice for managing procrastination was presented. Our results highlight the necessity for LLMs to provide structured, deadline-oriented steps and enhanced user support mechanisms. Additionally, our results surface the need for an adaptive approach to questioning based on factors like busyness. These findings offer crucial design implications for the development of LLM-based tools for managing procrastination while cautioning the use of LLMs for therapeutic guidance.

Associations between body composition profile and hypertension in different fatty liver phenotypes.

Background: It is currently unclear whether and how the association between body composition and hypertension varies based on the presence and severity of fatty liver disease (FLD). Methods: FLD was diagnosed using ultrasonography among 6,358 participants. The association between body composition and hypertension was analyzed separately in the whole population, as well as in subgroups of non-FLD, mild FLD, and moderate/severe FLD populations, respectively. The mediation effect of FLD in their association was explored. Results: Fat-related anthropometric measurements and lipid metabolism indicators were positively associated with hypertension in both the whole population and the non-FLD subgroup. The strength of this association was slightly reduced in the mild FLD subgroup. Notably, only waist-to-hip ratio and waist-to-height ratio showed significant associations with hypertension in the moderate/severe FLD subgroup. Furthermore, FLD accounted for 17.26% to 38.90% of the association between multiple body composition indicators and the risk of hypertension. Conclusions: The association between body composition and hypertension becomes gradually weaker as FLD becomes more severe. FLD plays a significant mediating role in their association.

Circle-Seq reveals genomic and disease-specific hallmarks in urinary cell-free extrachromosomal circular DNAs.

BACKGROUND: Extrachromosomal circular deoxyribonucleic acid (eccDNA) is evolving as a valuable biomarker, while little is known about its presence in urine. METHODS: Here, we report the discovery and analysis of urinary cell-free eccDNAs (ucf-eccDNAs) in healthy controls and patients with advanced chronic kidney disease (CKD) by Circle-Seq. RESULTS: Millions of unique ucf-eccDNAs were identified and comprehensively characterised. The ucf-eccDNAs are GC-rich. Most ucf-eccDNAs are less than 1000 bp and are enriched in four pronounced peaks at 207, 358, 553 and 732 bp. Analysis of the genomic distribution of ucf-eccDNAs shows that eccDNAs are found on all chromosomes but enriched on chromosomes 17, 19 and 20 with a high density of protein-coding genes, CpG islands, short interspersed transposable elements (SINEs) and simple repeat elements. Analysis of eccDNA junction sequences further suggests that microhomology and palindromic repeats might be involved in eccDNA formation. The ucf-eccDNAs in CKD patients are significantly higher than those in healthy controls. Moreover, eccDNA with miRNA genes is highly enriched in CKD ucf-eccDNA. CONCLUSIONS: This work discovers and provides the first deep characterisation of ucf-eccDNAs and suggests ucf-eccDNA as a valuable noninnvasive biomarker for urogenital disorder diagnosis and monitoring.

UNSUPERVISED DOMAIN ALIGNMENT BASED OPEN SET STRUCTURAL RECOGNITION OF MACROMOLECULES CAPTURED BY CRYO-ELECTRON TOMOGRAPHY.

Cellular cryo-Electron Tomography (cryo-ET) provides three-dimensional views of structural and spatial information of various macromolecules in cells in a near-native state. Subtomogram classification is a key step for recognizing and differentiating these macromolecular structures. In recent years, deep learning methods have been developed for high-throughput subtomogram classification tasks; however, conventional supervised deep learning methods cannot recognize macromolecular structural classes that do not exist in the training data. This imposes a major weakness since most native macromolecular structures in cells are unknown and consequently, cannot be included in the training data. Therefore, open set learning which can recognize unknown macromolecular structures is necessary for boosting the power of automatic subtomogram classification. In this paper, we propose a method called Margin-based Loss for Unsupervised Domain Alignment (MLUDA) for open set recognition problems where only a few categories of interest are shared between cross-domain data. Through extensive experiments, we demonstrate that MLUDA performs well at cross-domain open-set classification on both public datasets and medical imaging datasets. So our method is of practical importance.

Wnt/ß-catenin signaling pathway inhibits porcine reproductive and respiratory syndrome virus replication by enhancing the nuclear factor-κB-dependent innate immune response.

The Wnt/ß-catenin signaling pathway is an evolutionarily highly conserved signaling pathway related to the replication of various viruses. However, the interaction between the Wnt/ß-catenin pathway and porcine reproductive and respiratory syndrome virus (PRRSV) is unknown. In the present study, we showed that PRRSV-infected Marc-145 and PAM cells expressed high levels of c-myc and cyclinD1 mRNA and accumulation of ß-catenin in the nucleus. PRRSV nonstructural proteins (Nsps) 1α, 1ß, 3, 4, 7, 10, and 12, and proteins encoded by open reading frames (ORFs) 2b, 3, and 5 induced the activation of the Wnt pathway according to TOP/FOP luciferase reporter assay. But, Nsp5 inhibited the activation of the Wnt pathway. Pre-treatment with Wnt3a inhibited PRRSV replication in Marc-145 cells in a dose-dependent manner. Over-expression of ß-catenin also inhibited PRRSV replication, while silencing of ß-catenin by small hairpin RNA increased its replication in Marc-145 cells. Over-expression of ß-catenin increased interferon regulatory factor (IRF)-3 expression and nuclear factor (NF)-κB phosphorylation, NF-κB and interferon-stimulated response element promoter activities, and interferon-ß, DExD/H-box helicase 58 (DDX58), interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and IL-8 mRNA expression. Conversely, silencing ß-catenin decreased phosphorylated IRF-3 and NF-κB, NF-κB and IFIT1 promoter activities, and IFN-ß, DDX58, IFIT1, IL-1ß, TNF-α, and IL-8 mRNA levels in Marc-145 cells. Co-immunoprecipitation and immunofluorescence colocalization analyses confirmed that ß-catenin interacted with NF-κB in Marc-145 cells. In conclusion, PRRSV infection activates the Wnt/ß-catenin signaling pathway via Nsps 1α, 1ß, 3, 4, 7, 10, and 12, and proteins encoded by ORFs 2b, 3, and 5. The Wnt/ß-catenin pathway then inhibits PRRSV replication by enhancing the NF-κB-dependent innate immune response. These findings further our understanding of the role of the Wnt/ß-catenin signaling pathway in regulating PRRSV replication and provide new insights into virus-host interactions.

Development of a Dual Fluorescent Microsphere Immunological Assay for Detection of Pseudorabies Virus gE and gB IgG Antibodies.

Pseudorabies, also known as Aujezsky's disease, is an acute viral infection caused by pseudorabies virus (PRV). Swine are one of the natural hosts of pseudorabies and the disease causes huge economic losses in the pig industry. The establishment of a differential diagnosis technique that can distinguish between wild-type infection and vaccinated responses and monitor vaccine-induced immunoglobulin G(IgG) is crucial for the eventual eradication of pseudorabies. The aim of this study was to develop a rapid dual detection method for PRV gE and gB protein IgG antibodies with high specificity and sensitivity. PRV gE codons at amino acid residues (aa) 52-238 and gB codons at aa 539-741 were expressed to obtain recombinant PRV gE and gB proteins via a pMAL-c5x vector. After purification with Qiagen Ni-nitrilotriacetic acid (NTA) agarose affinity chromatography, the two proteins were analyzed via SDS-PAGE and immunoblotting assays. Two single fluorescent-microsphere immunoassays (FMIAs) were established by coupling two recombinant proteins (gE and gB) to magnetic microbeads, and an effective dual FMIA was developed by integrating the two single assays. Optimal serum dilution for each assay, correlation with other common swine virus-positive sera, and comparison with ELISA for two PRV antigens were tested for validation. Compared with ELISA, the specificity and sensitivity were 99.26% and 92.3% for gE IgG antibody detection, and 95.74% and 96.3% for the gB IgG antibody detection via dual FMIA. We provide a new method for monitoring PRV protective antibodies in vaccinated pigs and differentiating wild-type PRV infection from vaccinated responses simultaneously.

Phages bearing specific peptides with affinity for porcine reproductive and respiratory syndrome virus GP4 protein prevent cell penetration of the virus.

Porcine reproductive and respiratory syndrome (PRRS) has caused significant economic losses to the pig industry worldwide over the last 30 years. GP4 is a minor highly glycosylated structural protein composed of 187 and 183 amino acids in types I and II porcine reproductive and respiratory syndrome virus (PRRSV), respectively. The GP4 protein co-localizes with cluster of differentiation 163 (CD163), the major receptor on the target cell membrane, to mediate PRRSV internalization and disassembly. However, it remains to be established whether blocking interactions between GP4 and host cells can inhibit viral proliferation. In the present study, recombinant GP4 protein prepared and purified using the Escherichia coli system effectively recognized PRRSV-positive serum. Phage display biopanning on GP4 protein showed that the specific phages obtained could distinguish PRRSV from the other viruses. The exogenous peptide WHEYPLVWLSGY displayed on one of the candidate phages showed high affinity for GP4 protein and exerted a significant inhibitory effect on PRRSV penetration in vitro. Moreover, the N-terminus of GP4 was predicted as the critical receptor binding site and the beginning of the fifth scavenger receptor cysteine-rich domain of CD163 as the critical ligand recognition site based on sequence alignment and model prediction analyses. The current study expands our understanding of PRRSV GP4 and its receptor CD163 and provides a fresh perspective for the development of novel peptide-based viral inhibition reagents.